PROCEEDINGS OF
INTERNATIONAL SYMPOSIUM
ON A NEW ERA
IN FOOD SCIENCE AND TECHNOLOGY

2019-PART 1INTERNATIONAL SYMPOSIUM ON A NEW ERA
IN FOOD SCIENCE AND TECHNOLOGY
2019
ORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE, GIFU UNIVERSITY-PART 2UGSAS-GU & BWEL JOINT POSTER SESSIONON AGRICULTURAL
AND BASIN WATER ENVIRONMENTAL SCIENCES
2019
ORGANIZERS:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE, GIFU UNIVERSITY
GIFU UNIVERSITY REARING PROGRAM
FOR BASIN WATER ENVIRONMENTAL LEADERS

DATE: OCTOBER 9-10, 2019
VENUE: GIFU UNIVERSITY, JAPANInternational Symposium
on a New Era in Food Science and Technology 2019

Organized by The United Graduate School of Agricultural Science, Gifu University (UGSAS-GU)PROGRAM–PART 1DAY ONE: Wednesday, October 9
Time: 9:00-16:45
Time Table

Venue: 6F in UGSAS Building, Gifu University

9:00-9:30

Registration

9:30-9:35

Opening Remarks
Prof. Masateru Senge (Dean of UGSAS-GU)

Kyenote Speeches

Venue: 6F in UGSAS Building, Gifu University
Session Chair: Prof. Tomio Yabe (Gifu University)

9:35-10:05

Keynote Speech 01
Assoc. Prof. Nozomu Sakurai (National Institute of Genetics)

10:05-10:35

Keynote Speech 02
Dr. Shirley C. Agrupis (President of Mariano Marcos State University)

10:35-10:55

Coffee Break

10:55-11:25

Keynote Speech 03
Prof. Utpal Bora (Indian Institute of Technology Guwahati)

11:25-11:55

Keynote Speech 04
Assoc. Prof. Mohamad Yusof Maskat (Universiti Kebansaan Malaysia)

11:55-12:10

Photo Shoot

12:10-13:00

Lunch BreakSpecial Lecture

Venue: 6F in UGSAS Building, Gifu University

Venue: Room 100, Engineering Building, Gifu University

13:00-14:00

Special Lecture
Prof. Gary D. Christian (University of Washington)

14:00-14:30

BreakScientific Sessions

Venue: 6F in UGSAS Building, Gifu University

Session 1 ―Bioactives and Health / Prebiotics and Probiotics―
Session Chair: Prof. Tomoyuki Nakagawa (Gifu University)
14:30-14:45

01. Prof. Das Shonkor Kumar (Bangladesh Agricultural University)

14:45-15:00

02. Prof. Chen-jian Liu (Kunming University of Science and Technology)

15:00-15:15

03. Prof. Xiaoxiong Zeng (Nanjing Agricultural University)

15:15-15:45

Coffee BreakSession 2 ―Physical Properties of Food―
Session Chair: Assis. Prof. Teppei Imaizumi (Gifu University)
15:45-16:00

01. Assoc. Prof. Md. Sultan Mahomud(Hajee Mohammad Danesh Science and Technology University)

16:00-16:15

02. Assoc. Prof. Pongphen Jitareerat(King Mongkut’s University of Technology Thonburi)

16:15-16:30

03. Dr. Maharani Pertiwi Koentjoro

16:30-16:45

04. Dr. Achmad Ridwan Ariyantoro (Sebelas Maret University)(Nahdlatul Ulama University of Surabaya)

DAY TWO: Thursday, October 10
Time: 9:00-15:00
Time Table
9:00-9:30

Registration

Scientific Sessions

Venue: 6F in UGSAS Building, Gifu University

Session 3 ―Comprehensive Food Science and Others―
Session Chair: Prof. Takahisa Nishizu (Gifu University)
9:30-9:45

01. Assoc. Prof. Nancy Dewi Yuliana (Bogor Agricultural University)

9:45-10:00

02. Prof. Irmanida Batubara (Bogor Agricultural University)

10:00-10:15

03. Prof. A.H.M. Nurun Nabi (University of Dhaka)

10:15-10:30

04. Dr. Komariah (Sebelas Maret University)

10:30-10:45

05. Assis. Prof. Gang Ma (Shizuoka University)

10:45-11:00

Coffee BreakSession 4 ―Quality Control of Food―

Session Chair: Prof. Kohei Nakano (Gifu University)

11:00-11:15

01. Prof. Sanjib Kumar Panda (Central University of Rajasthan)

11:15-11:30

02. Assoc. Prof. Khandra Fahmy (Andalas University)

11:30-11:45

03. Assoc. Prof. Md. Harun Ar Rashid (Bangladesh Agricultural University)

11:45-12:00

04. Dr. Abdullah Iqbal (Food Research Institute, NARO)

12:00-12:15

05. Assis. Prof. Teppei Imaizumi (Gifu University)

12:15-12:20

Closing Remarks
Dr. Fumiaki Suzuki(Vice President / Director of International Affairs & Public Relations)

12:20-13:00

Lunch Break

13:00-15:00-PART 2-

Venue: 2F Conference Room, UGSAS Building, Gifu University*Please refer to the next page for details.

Venue: 6F in UGSAS Building, Gifu University
UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water
Environmental Sciences 2019

UGSAS-GU & BWEL Joint Poster Session on Agricultural andBasin Water Environmental Sciences 2019
Organized by The United Graduate School of Agricultural Science, Gifu University (UGSAS-GU)
Gifu University Rearing Program for Basin Water Environmental Leaders (BWEL)PROGRAM–PART 2DAY TWO: Thursday, October 10

Time: 13:00-15:00
Venue: 6F in UGSAS Building, Gifu University
Time Table
13:00-14:40

Poster Presentation

14:40-14:55

Best Presentation Award ceremony

14:55-15:00

Closing Remarks
Prof. Fusheng Li (Head of the Promotion Office of Gifu University Rearing
Program for Basin Water Environmental Leaders)Presenters
P01: Nayla Majeda Alfarafisa (UGSAS-GU)
P02: Putri Wulandari Zainal (UGSAS-GU)
P03: Eko Andrianto (UGSAS-GU)
P04: Fakfan Luangapai (Graduate School of Natural Science and Technology, Gifu University)
P05: Yolani Syaputri (UGSAS-GU)
P06: Raj Kishan Agrahari (UGSAS-GU)
P07: Panyapon Pumkaeo (UGSAS-GU)
P08: Hiroto Yamshita (UGSAS-GU)
P09: Annisyia Zarina Putri (UGSAS-GU)
P10: Arif Delviawan (UGSAS-GU)
P11: Maihemuti Mijiti (UGSAS-GU)
P12: Rachmad Adi Riyanto (UGSAS-GU)
P13: Vany Fadhilah(Graduate School of Integrated Science and Technology, Shizuoka University)
P14: Devendra Kumar Maravi (Indian Institute of Technology Guwahati)
P15: Tabli Ghosh (Indian Institute of Technology Guwahati)
P16: Purabi Bhagabati (Indian Institute of Technology Guwahati)
P17: Neha Mulchandani (Indian Institute of Technology Guwahati)
P18: Kona Mondal (Indian Institute of Technology Guwahati)
P19: Le Anh Tuan (UGSAS-GU; BWEL)
P20: Ruoming Cao (UGSAS-GU; BWEL)
P21: Cahyo Wisnu Rubiyanto (UGSAS-GU; BWEL)
P22: Islam Md Rashidul(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P23: Tahmidul Ashik(Graduate School of Natural Science and Technology, Gifu University; BWEL)P24: Ryota Matsuura(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P25: Akihiro Hayano(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P26: Masaya Toyoda (Graduate School of Engineering, Gifu University; BWEL)
P27: Takanori Ishii(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P28: Faisal Arsyad(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P29: Daichi Iijima(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P30: Hiroki Maruyama(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P31: Naoki Akiyama(Graduate School of Natural Science and Technology, Gifu University; BWEL)
P32: Wenjiao Li (Graduate School of Engineering, Gifu University; BWEL)
P33: Shiamita Kusuma Dewi
(Graduate School of Natural Science and Technology, Gifu University; BWEL)CONTENTS― PART 1 ―KEYNOTE SPEECHES
01: Construction of a metabolome database for uncovering the chemical world in foods
Nozomu Sakurai・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 2
02: Philippine Black Garlic (PhBG): Its physico-chemical properties and potential for foodand pharmaceutical application
Shirley C. Agrupis, Milen Fileza M. Inocencioi, Aira Cassandra S. Castro1,
Dionisio S. Bucao ・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 3
03: Sustainable food security: Importance of policy and technology
Utpal Bora・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 4
04: Deodourization of noni extract: A food processing approach
Mohamad Yusof Maskat, Haslaniza Hashim, Nur Hafiza Zairuh・・・・・・・・p. 5

SPECIAL LECTURE
The ethics of scientific writing: How to write and how not to write a paper
Gary D. Christian・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 8

SCIENTIFIC SESSIONSESSION 1 ―Bioactives and Health / Prebiotics and Probiotics―
01: Green tea departs baleful effects on health in Swiss albino mice
Das Shonkor Kumar, Kundu Swarup Kumar・・・・・・・・・・・・・・・・p. 10
02: Gut microbiota alterations from different lactobacillus probiotic-fermented yoghurt
treatments in slow-transit constipation
Chen-Jian Liu, Xiao-Ran Li, Xiao-Dan Tang, Jie Yu, Hai-Yan Zhang・・・・・・p.14
03: Effects of food polysaccharides on host’s health associated with the modulation of gut
microbiota
Dan Chen, Guijie Chen, Yu Ding, Hong Ye, Xiaoxiong Zeng・・・・・・・・・・p. 20
SESSION 2 ―Physical Properties of Food―
01: Effect of micellar κ-casein dissociation on the formation of soluble protein complexes andacid gel properties
Md. Sultan Muhomud, Nakako Katsuno, Takahisa Nishizu・・・・・・・・・・p. 26
02: Impacts of anti-browning agents and blanching against browning and fungal crowth intrimmed aromatic coconut fruit
Kittiya Payuhamayatakul, Apiradee Uthairatanakij, Varit Srilaong,
Panida Renumarn, Kanlaya Sripong, Pongphen Jitareerat・・・・・・・・・・p. 31
03: Effect of edible laccase on Chinese bread textureMaharani Pertiwi Koentjoro, Desy Lailatul Rachmah, Endry Nugroho Prasetyo
・・・・・・・・・p.37
04: Physical, chemical and sensory properties of the white bread from acetylated jack bean
flour
Achmad Ridwan Ariyantoro, Bambang Sigit Amanto, Rizki Novitasari・・・・・・p.43
SESSION 3 ―Comprehensive Food Science and Others―
01: Metabolomics and bioactivity profiles of torbangun (Plectranthus amboinicus) grown inJapan and in Indonesia
Nancy Dewi Yuliana, Muhammad Anwari Sugiharto, Hanifah Nuryani Lioe,
Masao Goto, Yuko Takano Ishikawa ・・・・・・・・・・・・・・・・・・・p. 47
02: Evolving potential functional foods to meet modern needs in commercial product
Irmanida Batubara, Susi Indariani, C. Hanny Wijaya, Katrin Roosita・・・・・p. 50
03: Evaluation of whole mitochondrial DNA sequence in healthy and type 2 diabetic
individuals with reference to Bangladeshi population
Sajoy Kanti Saha, Md Hasib, Ashish Das, Nafiul Huda, Tahirah Yasmin,
Md Ismail Hosen, A.H.M. Nurun Nabi ・・・・・・・・・・・・・・・・・・p. 56
04: Rice quality under ratooning farming system
Komariah, Rahajeng Putu Widiani Priswita ・・・・・・・・・・・・・・・・p. 62
05: Elucidating the molecular mechanism of β-citraurin accumulation in citrus fruit
Gang Ma, Lancui Zhang, Masaya Kato ・・・・・・・・・・・・・・・・・・p. 66
SESSION 4 ―Quality Control of Food―
01: Metabolite fingerprinting of assam tea (Camellia sinensis L.(O) Kuntze) under alumnium
stressSanjenbam Sanjibia Devi, Sanjib Kumar Panda・・・・・・・・・・・・・・p. 72
02: Improving postharvest handling of ‘Merah Delima’ papaya to reduce quality loss duringtransportation and storage
Khandra Fahmy・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 79
03: Effects of maturity stages and organic treatments to control postharvest fungal infection,
shelf life extension and quality retention of papaya for nutritional food safety
Md. Harun Ar Rashid, Md. Ahsan Habib・・・・・・・・・・・・・・・・・・p. 83
04: Extraction of chitosan from shrimp and crab shell and its effect on the quality of
pineapple juice
Sharmin Sultana, Md. Sajjad Hossain, Abdullah Iqbal・・・・・・・・・・・・p. 89
05: Electrical impedance analysis for evaluating tissue conditions of processed vegetables
Teppei Imaizumi, Haruki Ando, Satoshi Iwamoto・・・・・・・・・・・・・・p. 94

― PART 2 ―UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water
Environmental Sciences 2019
P01: The effect of Japanese persimmon (Diospyros kaki) extract on the prevention of
sarcopenia
Nayla Majeda Alfarafisa, Kohji Kitaguchi and Tomio Yabe・・・・・・・・・・p. 98
P02: Lipidomic profiling of cabbage stored at low temperature by liquid
chromatography-tandem mass spectroscopy
Putri Wulandari Zainal, Daimon Syukri, Teppei Imaizumi, Masayasu Nagata andKohei Nakano・・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 100
P03: First record of wolbachia infection in camellia spiny whitefly, Aleurocanthus camelliae(Hemiptera: Aleyrodidae), during their invasion stages in JapanEko Andrianto and Atsushi Kasai・・・・・・・・・・・・・・・・・・・・・p. 102
P04: Physicochemical properties of chitosan films contained curcumin nanoemulsion
Fakfan Luangapai, Methavee Peanparkdee and Satoshi Iwamoto・・・・・・・p. 104
P05: Identification of Plantaricin A on Lactobacillus plantarum IYO1511
Yolani Syaputri, Masanori Horie and Hitoshi Iwahashi・・・・・・・・・・・・p. 106
P06: Genome-wide association study for understanding aluminum signaling genes expression
in the shoots of Arabidopsis thaliana
Raj Kishan Agrahari, Yuriko Kobayashi, Ayan Sadhukhan, Sanjib Kumar Panda and
Hiroyuki Koyama・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 108
P07: Annual characterization and variations of airborne insect
Panyapon Pumkaeo, Junko Takahashi, and Hitoshi Iwahashi・・・・・・・・・p. 110
P08: The relationship between major chemical components and genomic structures in tea
accessions
Hiroto Yamshita, Tomoki Uchida, Hideyuki Katai, Lina Kawaguchi,
Atsushi J. Nagano, Akio Morita and Takashi Ikka・・・・・・・・・・・・・・p.112
P09: Screening and molecular breeding of amylose utilizing yeast for bioethanol production
Annisyia Zarina Putri and Tomoyuki Nakagawa・・・・・・・・・・・・・・・p. 114
P10: The influence of particle size distribution on the mechanical properties of wood plastic
composite
Arif Delviawan, Yoichi Kojima, Hikaru Kobori, Shigehiko Suzuki
and Kenji Aoki・・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 116
P11: A novel peptide derived from rice α-globulin decreased cholesterol micellar solubility invitro and inhibited cholesterol absorption in rats
Maihemuti Mijiti, Wang Jilite, Huang Bingyu and Satoshi Nagaoka
・・・・・・・・・・・・p. 118

P12: The effect of frozen storage on textural quality of cooked pasta
Rachmad Adi Riyanto and Takahisa Nishizu・・・・・・・・・・・・・・・・p. 120
P13: Effect of forest age on slope stability evaluated by a distributed landslide model
Vany Fadhilah, Al Nuriza Rahmadania and Fumitoshi Imaizumi・・・・・・・p. 122
P14: Evaluation and characterization of carotenoids and β-carotene in rice landraces from
Northeast India
Devendra Kumar Maravi, Muthuvel Jothi, Gundimeda Jwala Narasimha Rao andLingaraj Sahoo・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 124
P15: Magnetic cellulose nanofibers/chitosan based edible nano-coating with curcumin loading:
Influence of filler materials on perishable food products
Tabli Ghosh, Yoshikuni Teramoto, Kohei Nakano and Vimal Katiyar
・・・・・・・・・・p. 126
P16: Poly (L-lactic acid)/modified gum arabic biocomposites for targeted food packaging
applications
Purabi Bhagabati, Tabli Ghosh and Vimal Katiyar ・・・・・・・・・・・・・p. 128
P17: Controlling the molecular architecture to tailor the thermal and mechanical properties
and water vapor transmission rate of the biodegradable block copolymers: Versatilematerials for food packaging
Neha Mulchandani, Kazunari Masutani, Shinichi Sakurai, Yoshiharu Kimura
and Vimal Katiyar ・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 130
P18: Fabrication of nano cellulose from marine green algae residue and their effects as filler indeveloping biodegradable polymer composite
Kona Mondal, Shinichi Sakurai, Purabi Bhagabati, Vaibhav. V. Goud and Vimal
Katiyar ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 132
P19: A sensitivity analysis of Piano Key Weir discharge capacity based on 3D numericalmodeling
Le Anh Tuan and Ken Hiramatsu・・・・・・・・・・・・・・・・・・・・・p. 134
P20: Nitrogen deposition of bulk precipitation, throughfall and stemflow in a Lucidophyllous
forest near Gifu Park in Central Japan
Ruoming Cao, Siyu Chen, Shinpei Yoshitake and Toshiyuki Ohtsuka・・・・・・p. 136
P21: Various swidden activities play complementary roles for local food security in montane
northern Laos
Cahyo Wisnu Rubiyanto and Isao Hirota・・・・・・・・・・・・・・・・・p. 138
P22: Dynamics of dissolved organic carbon (DOC) and soil carbon sequestration in a
deciduous forest
Islam Md Rashidul, Yasuo Iimura, Takeo Onishi, Shinpei Yoshitake, Ashik Tahmidul
and Toshiyuki Ohtsuka・・・・・・・・・・・・・・・・・・・・・・・・・p. 140
P23: A paired catchment study of nitrogen dynamics in a cool-temperate mixed deciduous
broad-leaved and coniferous evergreen forest, central Japan
Tahmidul Ashik, Takeo Onishi, Rashidul Islam, Cao Ruoming and Toshiyuki Ohtsuka

・・・・・・・・・・・p. 142
P24: Evaluation of wave power generation potential in coastal area using wave model
Ryota Matsuura, Tomonao Kobayashi and Jun Yoshino・・・・・・・・・・・・p. 144
P25: A new Pythium species causing lettuce crown rot
Akihiro Hayano, Kensuke Yamada, Ayaka Hieno, Haruhisa Suga and Koji Kageyama
・・・・・・・・・・・・p. 146
P26: Future climate projections for storm surge associated with typhoon Jebi (2018) by apseudo global warming downscaling
Masaya Toyoda, Jun Yoshino and Tomonao Kobayashi・・・・・・・・・・・・p. 148
P27: Prediction of object-movement by image processing withspectrum analysis
Takanori Ishii and Rina Takada・・・・・・・・・・・・・・・・・・・・・p. 150
P28: A new idea of energy-saving activated sludge wastewater treatment process by promoting
co-growth of algae and bacteria
Faisal Arsyad and Fusheng Li・・・・・・・・・・・・・・・・・・・・・・p. 152
P29: A new species of Pythium with spiny oogonia isolated from forest soil in Japan
Daichi Iijima, Ayaka Hieno, Haruhisa Suga and Koji Kageyama・・・・・・・p. 154
P30: Bacterial community structure in fixed-bed activated carbon adsorbers treating Nagara
River Water
Hiroki Maruyama, Wenjiao Li, Yasushi Isiguro, Toshiro Yamada andFusheng Li・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・p. 156
P31: Efficient multi-site operation of optical communication between satellite and ground
Naoki Akiyama and Akane Kusabuka・・・・・・・・・・・・・・・・・・p. 158
P32: Vermicomposting of fruit and vegetable waste with the addition of excess activatedsludge: A feasible approach leading to final products of higher utilization value
Wenjiao Li and Fusheng Li・・・・・・・・・・・・・・・・・・・・・・・p. 160
P33: Inhibition of Arsenic transfer from contaminated soil to vegetation bywood ash
Shiamita Kusuma Dewi, Huijuan Shao, Shunichiro Mitsunaga and Yongfen Wei
・・・・・・・・・・・p. 162

-PART 1INTERNATIONAL SYMPOSIUM ON A NEW ERA
IN FOOD SCIENCE AND TECHNOLOGY2019

KEYNOTE SPEECHES

Wednesday, 9th October 20199:35 am - 11:55 am
6th Floor, UGSAS Building, Gifu University

ORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE, GIFU UNIVERSITY

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Keynote Speech: 01

Construction of a Metabolome Database for Uncovering the Chemical Worldin Foods
Nozomu SAKURAI1 *1

Bioinformation and DDBJ Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 4118540, Japan* Corresponding Author: sakurai@nig.ac.jp
SUMMARY
Food analysis is required for safety assessments, quality controls, traceability, evaluations of nutrition and functionsof foods. Metabolomics–a technology for comprehensive detection of small molecules in the samples–is prospective forfood analysis. However, a long-standing unsolved issue, namely, a lot of unidentified metabolite peaks remain in the dataobtained by mass spectrometry (MS)-based untargeted metabolome analyses, makes it difficult to understand the results.
A lot of computational tools for the prediction of chemical structure by MS data have been reported, and the prediction
accuracy has been improved rapidly. However, the substantial issue of the poor annotation of the compound peaks is, alack of information for prioritizing the candidate peaks for further detailed investigation, rather than the accuracy of theprediction tools. We constructed a database, Food Metabolome Repository (http://metabolites.in/foods), by which asample specificity of the queried peak can be obtained, and this information should be a key for the prioritization.
Untargeted metabolome data obtained from 222 foods using liquid chromatography (LC)-high resolution MS are available
at the repository. The repository can also be a good resource for annotation of unknown peaks detected in human biofluid.
By assuming the modified molecules such as glucuronidates and sulfur conjugates, the mass value of the original molecule
without the attached moiety (in this case, glucuronic acid and sulfate) can be calculated, and the existence of the original
molecule in foods can be searched. These molecules in human biofluid might be used as biomarkers for intake of a specificfood. A wide diversity of chemicals is expected in the food criteria because a wide range of organisms and various
compound modification processes such as cooking and fermentation are involved there. Therefore, Food Metabolome
Repository should be useful not only for the food science but also for the general metabolome analyses. In DNA Data
Bank of Japan (DDBJ), we are now constructing a new public repository for the metabolome data, named “MetaboBank”,
for primarily Japanese and Asian researchers. Enhancement of a variety of foods and analytical methods for foodmetabolome data should contribute to elucidate the chemical world in foods and further use of the chemical information
for food science and human health.
Keyword: food metabolomics, LC-MS, database, compound identification

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Keynote Speech: 02

Philippine Black Garlic (PhBG): Its Physico-Chemical Properties and Potential
for Food and Pharmaceutical Application
Shirley C. AGRUPIS1*, Milen Fileza M. INOCENCIO1, Aira Cassandra S. Castro1,
Dionisio S. BUCAO2*
1

Mariano Marcos State University, Brgy. 16 Quiling Sur, City of Batac 2906

2

University Innovation and Technology Services Office, Research Directorate, RL Building, MMSU, Cityof Batac 2906, Ilocos Norte, Philippines* Corresponding Author: mmsuop@yahoo.com
SUMMARY
Garlic is the flagship commodity crop in the entire province of Ilocos Norte. The province is considered a champion
garlic supplier in the Philippines (Ilocosnorte.gov.ph, 2016) for contributing 67 to 70 percent of the total garlic productionof the country annually. According to the records of the office of the Provincial Agriculturist of Ilocos Norte as reported
by Lazaro of balita.ph, the whole province produced at least 6,855 metric tons of garlic during the harvest season in thefirst quarter of 2014. It contributes to forty percent (40%) of the province’s gross domestic product and an effective tool
for poverty alleviation of the farmers. Garlic is known for its many health benefits. Garlic is widely used for several
conditions linked to the blood system and heart, including atherosclerosis (hardening of the arteries), high cholesterol,
heart attack, coronary heart disease and hypertension. It is also used today by some people for the prevention of lungcancer, prostate cancer, breast cancer, stomach cancer, rectal cancer, and colon cancer (Nordqvist, 2015). What makes raw
garlic such a healthful and potent vegetable is the compound allicin, which accounts for its pungent odor. However,according to Mercola (2014), allicin is a very unstable compound which is decomposed into simpler molecules in lessthan an hour when garlic is cooked, crushed, aged or processed.
Recently, a processed garlic called black garlic is becoming popular for its unique appearance, taste and most
specially, its health benefits. Black garlic is produced by processing ordinary garlic under constant high temperature andhumidity up to 30 days. During processing, the garlic changes its color from white to dark brown. This is due to Maillard
Reaction, a non-enzymatic browning reaction that also causes the change in flavor, texture, smell and nutrient content(Bae, et al., 2014). Studies have also shown that the compound s-allyl cysteine is higher in concentration in black garlicthan raw garlic. This compound, compared to allicin is absorbed more easily by the body (Hobson, 2012). Other articles
have claimed that antioxidants of black garlic increased (Choi, et al., 2014 and Sato, et al., 2006) which may also increase
the medicinal benefits of black garlic which include anticancer.
In partnership with Takara Inc. in Takko-machi, Aomori, Japan, black garlic was produced at MMSU, using low
cost available processing. At present, MMSU-Takara Inc. is currently implementing joint R&D project under the Japan
International Cooperation Agency (JICA). The PhBG produced was subjected to initial characterization and test toevaluate the physico-chemical properties and their potential medical application specifically in cancer treatment. To
evaluate these, two in vitro assays were performed- DPPH or antioxidant assay and the MTT cytotoxicity assay. The PhBG
was found as effective as Fluorouracil in destroying A549 cancer cell lines and better than raw garlic. On the other hand,
PhBG, raw garlic and Fluorouracil showed the same level of potency against HCT116 colon cancer cells. Chemically,
PhBG mineral contents’ such as sodium calcium, iron, potassium and zinc increased. It contains phytochemicals such asalkaloids, flavonoids, phenol, tannins, steroids and terpenoids. Furthermore, PhBG also has higher phenolic and flavonoid
content than raw garlic. The results of this study would provide information on the potential of Philippine black garlic asa food product and open possibilities in a cost-effective and non-invasive treatment for cancer.
Keywords: Black garlic, physico-chemical characteristics, phytochemicals, Ilocos Garlic, cytotoxicity, colon cancer, lungcancerProceedings of International Symposium on a New Era in Food Science and Technology 2019
Keynote Speech: 03

Sustainable Food Security: Importance of Policy and Technology
Utpal Bora*
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Kamrup
781039, Assam, India* Corresponding Author: ubora@iitg.ac.in
SUMMARY
Today the world is less undernourished then two decades ago. Although the proportion of undernourished people
declined from 15 per cent in 2000-2002 to 11 per cent in 2014-2016, more than 790 million people still lack regular accessto adequate food. Goal 2 of SDG focus on “Zero Hunger” by 2030 and aims at ensuring the global population with “enoughgood-quality food to lead a healthy life”. It seeks sustainable solutions to end hunger in all its forms and achieving foodsecurity.
Sustainable solutions lie at the level of production, processing, consumption of food and reduction of loss at every
step. Availability of land and water as well as technology and markets, increased investments through international
cooperation to bolster the agricultural productivity and food processing would be critical given the pressure due tourbanization and climate change. In my lecture I would be discussing about the interdependencies of various SDG goals
that could ensure us food security and how technology, policy and knowledge discovery both traditional and modern
would play critical roles in achieving Zero hunger.
Keyword: Sustainable Development Goals, Food security, Zero Hunger, Climate changeProceedings of International Symposium on a New Era in Food Science and Technology 2019
Keynote Speech: 04

Deodourization of Noni Extract: A Food Processing Approach
Mohamad Yusof Maskat, Haslaniza Hashim, Nur Hafiza Zairuh*
Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia*

Corresponding Author: yusofm@ukm.edu.my
SUMMARY

Noni (Morinda citrifolia) is a tropical fruit that has been traditionally used for various therapies. The use of noni is
not localized only to Malaysia but also in several other countries such as Indonesia, Polynesia, other Pacific Islands andother places. Traditionally, noni has been used for medicinal purposes by the Polynesians since 2000 years ago. Many
researchers have also studied the beneficial properties of noni which include antimicrobial properties, anti cancer
properties, antioxidant properties, anti inflammatory properties and others.
Although noni contained several beneficial properties, its usage has been limited due to its undesirable odour. Whennoni fruits ripen, it emits an undesirable odour which has been described as butyric acid-like rancid smell. Fifty one
volatile compoundshas been determined in noni fruit which include organic acids, alcohol (3-methyl-3-butane-1-ol), esther(methyl octanoate, methyl decanoate), ketone (2-heptanone), dan lactone. Organic acid content of noni was high especiallyoctanoic, hexanoic and decanoic acid which can reach 50-70%. From the volatile compounds that was identified, the main
volatile compound was octanoic acid (72%) and hexanoic acid (8%) which consisted of medium chain fatty acids of 6 and
8 carbons, respectively. Olfactory gas chromatography data also supported the finding that the undesirable odour of noni
was contributed mainly by octanoic acid.
Traditional production of noni extract involves fermenting the noni fruits. It has been reported that fermented noni
extract has less undesirable odour compared to fresh extract. It has been reported that Saccharomyces cerevisiae has an
acyl-coA synthetase that is able to degrade octanoic acid. Results showed that inoculum size of S. cerevisiae has negative
effect on octanoic acid content and undesirable odour intensity among trained panellists. Fermented noni extract also
showed reduced octanoic acid content and undesirable odour intensity compared with unfermented samples. To further
increase the capacity of octanoic acid degradation by S. cerevisiae, glucose at a concentration of 0-30 g/L was added
during fermentation. Addition of 6 g/L of glucose produced the lowest octanoic acid content and lowest intensity ofundesirable odour after 48 hours of fermentation. Thus, it can be concluded that the addition of glucose at limited amountfacilitated the metabolism of octanoic acid by S. cerevisiae in noni extract.
Due to octanoic acid being the contributing compound to the undesirable odour of noni extract, deacidification
using ion exchange resin was studied. Three types of weak base anion exchange resins (Amberlite IRA 67, Duolite A7
and Amberlite IRA 96) of different resin weight (0, 5, 10%, w/v) were used. Deacidification of M. citrifolia juice using
the resins significantly decreased the octanoic acid content compared to fresh juice where Amberlite IRA 67 gave thehighest reduction of octanoic acid followed by Duolite A7 and Amberlite IRA 96. The results indicated that deacidificationof the juice using 10% of resin weight (w/v) gave higher percentage of antioxidant activity and TPC. Results also showeda similar trend when different weight of resin was used where Amberlite IRA 67 > Duolite A7 > Amberlite IRA 96 for
DPPH, FRAP, TPC and antioxidant compounds.
Keyword: noni, Morinda citrifolia, deodourization, fermentation, ion exchange resin

-PART 1INTERNATIONAL SYMPOSIUM ON A NEW ERA
IN FOOD SCIENCE AND TECHNOLOGY

2019

SPECIAL LECTURE

Wednesday, 9th October 201913:00 pm - 14:00 pm
Room 100, Engineering Building, Gifu University

ORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE, GIFU UNIVERSITY

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Special Lecture

The Ethics of Scientific Writing: How to Write and How Not to Write a Paper
Gary D. Christian
Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700
christian@chem.washington.edu
SUMMARY
Scientific writing for peer-reviewed journals is how scientists communicate their work to the world.

It is importantto tell a clear and compelling story, beginning with justification for the work, placing it in the context of prior work, andits significance in advancing the field, i.e., what problem is being addressed? Manuscripts are submitted to peer-review
by experts, selected by the editor.

Only a select number will be published, depending on novelty, significance to the field,

demonstrated applicability, appropriateness for the journal, and so forth.

Peer-review is for the benefit of the author aswell as for the editor, and helps improve the quality and impact of the paper.

Ethics in publication is of paramountimportance, and has become more of an issue for editors in recent years, particularly with the advent of the electronic age.
I will relay my experiences as an Editor-in-Chief for Talanta over thirty years, providing guidance on how to structure
and present a paper so editors, reviewers and readers will have a good understanding of your accomplishments, and pitfalls
to avoid.

Real-world examples of manuscripts that do not follow established and ethical guidelines will be given, alongwith cases of outright scientific fraud in the chemical literature.
review to their advantage.

And hints will be given of how authors can use peer

-PART 1INTERNATIONAL SYMPOSIUM ON A NEW ERA
IN FOOD SCIENCE AND TECHNOLOGY2019

SCIENTIFIC SESSION

Wednesday, 9th October 201914:30 pm - 16:45 pm
Thursday, 10th October 20199:30 am - 12:15 pm
6th Floor, UGSAS Building, Gifu University
ORGANIZER:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE, GIFU UNIVERSITY

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 01

Green Tea Departs Baleful Effects on Health in Swiss Albino Mice
Das SHONKOR KUMAR*, Kundu SWARUP KUMAR
Bioresearch laboratory (Cancer and Herbal Research Center), Department of Anatomy and Histology,
Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202. Bangladesh*

Corresponding Author: skdas76@yahoo.com
SUMMARY

A myriad of health claims are being made in favor of the consumption of green tea due to its easy availability andgreater popularity in spite of its certain health risk have started to emerge. This study was aimed to determine the balefuleffects on health due to excessive green tea intake in model mice. Total ten (10) experimental Swiss albino mice (age of
5 weeks and avg. weight 18-20gm) were taken and divided into two (2) groups such as Control (C) and Treated (T) groups,
each having 5 mice. The control group (1 male+4 female) was fed with the normal mice pellet and water but the treatedgroup (1 male + 4 female) was given green tea instead of water (3ml/mice two times per day) and mice pellet for 21 days.
After the experimental tenure, the mice (both the control and treated groups) were sacrificed ethically and the samples(Blood, liver, kidney, lungs, brain and testes) were collected for investigation. The results of the study revealed that thetreated group became motionless than the control group. Grossly, the liver surface of the treated mice became dark red incolor with considerable hepatomegaly found; hemorrhage found in lungs whether the brain, testes and kidney were foundnormal although kidney glomerulus has a minor change. Histologically, mild central vein dilation with severe congestion,sinusoidal dilation and severe venous congestion were found in the portal vein of the liver of treated mice.
Hematologically, it was found that hemoglobin level of the treated female mice became lower than that of the control
group mice. On the other hand, within this experimental period female mice of both groups gave births (18 pups) whichwere devoid of any abnormality. Therefore, it can be concluded that excess green tea intake in a day might have balefuleffects on health and people need to be conscious about this. It also needs further in-depth investigation to understand thepathway of causing such baleful effects on health both at gross and cellular level.
Keywords: green tea, baleful effects, Swiss albino mice, health impacts
properties (Cooper et al., 2005). Green tea is also used as

Introduction

protective agent in exercise enhancement, diabetes,
Tea is one of the most popular beverages, which aremost commonly used in China and Japan (Nawab andinflammatory bowel disease, skin disorders, hair loss,
weight loss and iron overload (Sinija and Mishra, 2008).

Farooq, 2015). Green tea is an evergreen shrub derived

High doses of green tea consumption (i.e. 5–6 litersfrom the plant Camellia sinensis (Theaceae). The principal

per day) may cause nausea, vomiting, abdominal

active ingredients of green tea include polyphenolic

bloating/pain, dyspepsia, flatulence and diarrhea (Laurie etcompounds such as epicatechin (EC), epicatechin-3-

al., 2005) as well as excessive consumption of caffeine

gallatefrom green tea may also cause central nervous system(ECG),

epigallocatechin

(EGC)

and

epigallocatechin-3-gallate (EGCG) along with the otherstimulation

such

as

dizziness,

insomnia,

polyphenols in green tea include flavanols and theirrestlessness, confusion, diuresis, heart rate irregularities

glycosides and depsides such as cholorogenic acid, quinic

and psychomotor agitation (Tomasulo, 2004). Regularacids, carotenoids, trigalloylglucose, lignin, protein,

consumption of green tea is thought to reduce the risk ofchlorophyll, minerals, caffeine and a small amount ofcardiovascular disease (CVD) but has also been associatedmethylxanthines (Liu et al., 2008).

with liver toxicity (Frank et al., 2008).

Green tea offers many attractive & beneficial effects.
It

possesses

antiproliferative,

antioxidant,
antitumor

andanti-angiogenesis,anti-carcinogenic

Materials and Methods

tremors,

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 01

Total ten (10) experimental Swiss albino mice (at the

became dark red in color with considerable hepatomegaly

age of 5 weeks and avg. weight 18-20gm) were purchasedin the treated group of mice. Hemorrhage found in lungs

from the Department of Pharmacy, Jahangirnagar

whether the brain, testes and kidney were found normal.

University, Dhaka, Bangladesh. The mice were adapted at
Animal Care Room, for the period of 7 days before being
used for the experiment. Then the mice were divided into
two groups such as Control (C) and Treated (T) groups,
each having 5 mice. The control group (1 male+4 female)
was fed with the normal mice pellet and water whether thetreated group (1 male + 4 female) was given green tea
instead of water (3ml/mice two times per day) and micepellet for 21 days. After the experimental tenure, the mice
(both the control and treated groups) were sacrificed
ethically and the samples (Blood, liver, kidney, lungs, brain
and testes) were collected in order to investigate the gross,histologicalandhematologicalchanges.

Finally

Haematoxylin & Eosin staining was done for histological

From the histological study, we found that, mild
central vein dilation with severe congestion, sinusoidal

study

dilation and severe venous congestion in the portal vein of
the liver of treated group of mice.

Results and Discussion
The results of the study revealed that the treatedgroup mice became motionless than the control group mice.
On the other hand, within this experimental period female
mice of both groups gave births (18 pups) which were
devoid of any abnormality.

From the hematological study, it was found that
hemoglobin level of the treated female mice significantly(** p<0.01) reduced than the control group mice.
From the gross study, we found that liver surface[Results are as Mean ± SE (standard error) of 5 micein each group. One-way ANOVA was performed as the testProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 01of significance. There was a significant variation in theamong the health conscious people but the exact balefulhemoglobin level. The difference was considered to beeffects rendered by it on health is unclear until today. The

significant when ** p<0.01, * p<0.05 compared to normal

present study indicates that green tea has detrimental

control group].

effects on health specially hepatomegaly, decreasing
hemoglobin concentration and massive congestion in both
central and portal vein. For future it needs further indeed
investigation to understand the pathway of causing suchbaleful effect on the body both in gross and cellular level.
Acknowledgements
Sincere thanks to the Department of Anatomy andHistology, Faculty of Veterinary Science, Bangladesh
Agricultural University, Mymensingh-2202 for technical
help

in

histologicalslide

preparation. A special

acknowledgement to MoST, Bangladesh for providing the
NST fellowship as a financial support to conduct the
research smoothly.
Green tea is a natural antioxidant that has been usedin the most enduring of food cultures-Chinese andJapanese tea. The aim of this research was achieved in
Swiss albino mice in where liver became dark red in color
with considerable hepatomegaly as well as severe venous
congestion in the portal vein (PV) and mild central vein
dilation with severe congestion found due to excessive
green tea intake. Similar finding was found by El Daly,(2011). Mazzanti et al., (2009) stated that hepatotoxicity isprobably occurs due to epigallocatechin gallate or its
metabolites. From our research we found, the principal
active ingredients of green tea is polyphenolic compounds
such as epicatechin (EC), epicatechin-3-gallate (ECG),
epigallocatechin (EGC) and epigallocatechin-3-gallate(EGCG). So green tea may cause hepatotoxicity.
According to Fan, (2016) iron deficiency anemia occurs
due to excessive green tea drinking. In our study we found,hemoglobin level of the treated female mice significantly(**p<0.01) reduced than the control group mice. Green tea
is safe to drink in moderate amounts during pregnancy andepigallocatechin is an active ingredient and a powerfulantioxidant in green tea which is important during
pregnancy stated by Navarro-Perán, (2005). In our study,
we found that mice of both (control and treated) groups
gave births (18 pups) which were devoid of any
abnormality.

References
Cooper, R., Morré, D. J., & Morré, D. M. (2005).
Medicinal benefits of green tea: Part I. Review of
noncancer health benefits. Journal of Alternative &Complementary Medicine, 11(3), 521-528.
El Daly, A. A. (2011). Effect of Green Tea Extract on the
Rat Liver; Histo-architectural, Histochemical andUltrastructural Studies. Journal of American Science,
7(5), 65-73.
Fan, F. S. (2016). Iron deficiency anemia due to excessive
green tea drinking. Clinical case reports, 4(11), 1053.
Frank, J., George, T. W., Lodge, J. K., Rodriguez-Mateos,
A. M., Spencer, J. P., Minihane, A. M., & Rimbach, G.
(2008). Daily consumption of an aqueous green tea
extract supplement does not impair liver function oralter cardiovascular disease risk biomarkers in healthy
men. The Journal of Nutrition, 139(1), 58-62.
Laurie, S. A., Miller, V. A., Grant, S. C., Kris, M. G., &Ng, K. K. (2005). Phase I study of green tea extract inpatients

withadvancedlungcancer.

Cancerchemotherapy and pharmacology, 55(1), 33-38.
Liu, J., Xing, J., & Fei, Y. (2008). Green tea (Camelliasinensis) and cancer prevention: a systematic review of
randomized trials and epidemiological studies. Chinese
medicine, 3(1), 12.
Mazzanti, G., Menniti-Ippolito, F., Moro, P. A., Cassetti,

Conclusion
Although green tea is found to be popular recently

F., Raschetti, R., Santuccio, C., & Mastrangelo, S.
(2009). Hepatotoxicity from green tea: a review of theProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 01

literature and two unpublished cases. European journalof clinical pharmacology, 65(4), 331-341.
Navarro-Perán, E., Cabezas-Herrera, J., García-Cánovas,
F., Durrant, M. C., Thorneley, R. N., & RodríguezLópez, J. N. (2005). The antifolate activity of tea
catechins. Cancer research, 65(6), 2059-2064.
Nawab, A., & Farooq, N. (2015). Review on green tea
constituents

and

its

negative

effects. J

Pharm

Innov, 4(1), 21-24.
Sinija, V. R., & Mishra, H. N. (2008). Green tea: Healthbenefits. Journal of Nutritional & Environmental
Medicine, 17(4), 232-242.
Tomasulo, P. (2004). Natural Medicines Database
http://www.

naturaldatabase.

com.

Journalconsumer health on the internet, 8(2), 75-85.

ofProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 02

Gut Microbiota Alterations from Different Lactobacillus Probiotic-Fermented
Yoghurt Treatments in Slow-transit Constipation
Chen-Jian Liu1, Xiao-Ran Li1*, Xiao-Dan Tang2, Jie Yu3, Hai-Yan Zhang1
1Faculty of Life Science and Technology, Kunming University of Science and Technology, Chenggong,

2

Department of Gastroenterology, First People Hospital of Yunnan Province, Kunming 650032, Yunnan,

Kunming 650500, Yunnan, China
China
3

Yunnan University of Traditional Chinese Medicine, Kunming 650500, Yunnan, China*

Corresponding author: starkeyran@163.com
SUMMARY

Constipation is a frequent complaint, and probiotics could have a potentially synergistic effect on intestinal transit.
In the present study, yoghurt intake improved the symptoms of slow-transit constipation, which was confirmed in 144
mice (nine groups, n=16) with loperamide-induced constipation. Yoghurt fermented with different probiotics wasadministered orally. Loperamide treatment caused a marked increase in first defecation time and a decrease in the charcoal
transit ratio (P<0.05), while loperamide treatment after intake of a new formulation of yoghurt could significantly improvedefecation time and intestinal health. Significant (𝑃𝑃 < 0.001) decreases in butanoic acid content were observed in groups
given three different strains of yoghurt. Yoghurt intake could also change the intestinal bacterial community composition,
which was supported by operational taxonomic unit-related analysis as well as principal coordinates analysis. Our results
showed conclusive evidence indicating that yogurt is an excellent functional food that improves the symptoms of slowtransit constipation.
show favourable effects on constipation with fewer toxic

Introduction

effects (Choi et al. 2014).
Constipation can be divided into three broad
categories:

normal-transit

constipation,

To evaluate the effects of different probiotic strains ofslow-transit

fermented yoghurt on changes in faecal parameters, theconstipation, and disorders of defecatory or rectal

present study examined the intestinal charcoal transit ratio,

evacuation (obstructive defecation) (Lembo and Camilleri

short-chain fatty acid content, bacterial community2003). There is a clear relationship between increased

composition in the faeces, and intestinal contents aftercolonic transit time (CTT) and increased symptom severity

dissection in loperamide-induced constipated mice afterin patients with chronic constipation (Park et al. 2015).

administration of yoghurt. Two formulations of strains

Probiotics are nonpathogenic microorganisms that,when ingested in adequate amounts, exert health benefits

were used: Lactobacillus bulgaricus and Streptococcus
thermophiles (Fig. 1).

on the host. There has been growing evidence suggestingthat probiotics may have a beneficial role in alleviating

Materials and Methods

constipation symptoms, at least with certain probiotic
strains (Turan et al. 2014). Functional foods such as

1. Yoghurt product preparation

yoghurt could be promising in alleviating motility

The commercial formulation used in this study

problems of the gastrointestinal tract that relate toconsists of two species: Lactobacillus bulgaricus andconstipation. Yoghurt fermented by probiotic is considered

Streptococcus thermophilus. The novel formulation used ina probiotic source with anti-bacterial, anti-mycotic, anti-

this study consisted of three species: Lactococcus lactis,

neoplastic, and immunomodulatory properties. Yoghurt-

Lactobacillus

derived probiotics have been raised as alternative

Bacterial concentrations in the yogurt were 106 colonies

therapeutics for various digestive disorders, and they also

forming units (CFU) per millilitre.

plantarumandLactobacillus

casei.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 02

2. Experimental design
A total of 144 male Kunming mice (eight weeks oldupon receipt; Dashuo, China) were used following

Guidelines for Care and Use of Laboratory Animals of
Kunming University of Science and Technology, Kunming,
Yunnan, China.

acclimatization for 7 days in this study (Fig. 1). Samples

Eight mice randomly selected from each group werecollected before the experiment were identified as being

euthanized by cervical dislocation 25 min later. Mesentery

collected on day 0. The mice were randomly assigned to

was isolated from the intestinal cavity and intestinal

nine groups (n=16 in each group, eight mice per cage) in asamples were collected at the upper end of the clipping

room with controlled temperature (25±2°C) and humidityfrom the pylorus, bottom to the ileocecal bowel. The length(50–70%). The mice were kept under a 12 h: 12 h light:of the total small intestine and the pylorus to the forefront

dark cycle. Eight groups were: control mice (C),of ink were measured. The intestinal charcoal transit ratio

constipated mice without yoghurt (Y0), constipated micewas calculated as follows: charcoal transit ratio (%) =receiving two probiotic strains of fermented yoghurt with((total small intestinal length − charcoal meal transit

different doses per day (4 mL, Y2H; 2 mL Y2M; 1 mL,

distance)/total small intestinal length) × 100.

Y2L), and constipated mice receiving three probiotic
strains of fermented yoghurt with different doses per day

3. Determination of SCFAs(4 mL, Y3H; 2 mL Y3M; 1 mL, Y3L). The positive control

The standards of acetic acid (A116165), propionicgroup (PC) was given Live Combined Bifidobacterium andacid (D110443), butanoic acid (B110439) and hexanoic

Lactobacillus tablets at 650 mg/kg (Jinshuangqi, China)acid (H103630) were chromatographically pure. Hexanoic(Fig. 1). Constipation was induced in the animals by oralacid was used as an internal standard. Oxalic acidadministration of 6 mg/kg loperamide hydrochloride

dehydrate, sodium azide and ultrapure water were also

(Sigma, MO, USA) in a volume of 6 mg/kg (dissolved inused in this study. The content of short chain fatty acid was

ddH2O), whereas the control mice (C group) were

calculated according to the standard curve. The analysis ofadministered normal water only. Half an hour after taking

short chain fatty acid was conducted with a gas

loperamide or water, all mice were given ink gastricchromatography system (GC7890, Agilent, USA) coupled

perfusion (10% activated carbon powder and 10% gum

to a ﬂame ionization detector (FID).

arabic). All animals were treated in accordance with theFig. 1 Experimental design. Nine groups of mice were used as recipients of different treatments: (C) controls;(Y0) constipated mice without yoghurt; constipated mice receiving yoghurt fermented with two probiotic
strains at a different dose per day (4 mL, Y2H; 2 mL Y2M; and 1 mL, Y2L); constipated mice receiving
fermented yoghurt with three probiotic strains at a different dose per day (4 mL, Y3H; 2 mL Y3M; and 1 mL,
Y3L); (PC) positive control.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 02

4. Bacterial analysis procedure and statistics analysis

were detected with all three concentrations of the three

The following four groups were selected for analysis

yoghurt strains, compared with the control group and non-of gut bacterial community: C, Y0, Y2M and Y3M groups.

yoghurt group. The highest intestinal charcoal transit ratio

Faecal and small intestine content samples were stored at -was detected in the groups receiving the yoghurt with three

20°C prior to DNA extraction. The bacterial 16S rRNA

bacterial strains. A significant (𝑃𝑃 <0.05) increase in thegene was amplified with the following barcoded primers:

intestinal charcoal transit ratio was detected in the group

343F and 798R. Purified PCR product was mixed forreceiving a high dose of the three-strain yoghurt comparedsequencing using the Illumina MiSeq

system (Illumina,

to the control group and non-yoghurt group. More

USA). Sequence processing was performed by combining

favourable increases in intestinal charcoal transit wereTM

features of Mothur v1.35.1 (Schloss et al. 2009). In

detected in all three-strain yoghurt concentration groups

addition,compared to the control group and non-yoghurt groupsequenceswereassignedtooperationaltaxonomic units with 97% sequence homology.
All results are presented as the mean±s.d. Differences(Table 1). A normal intestinal charcoal transit ratio is an
obvious sign of improvement in clinical symptoms and an

between groups were investigated using two-way analysis

increase in intestinal charcoal transit ratio in yoghurt intake

of variance (ANOVA). Differences were considered asgroups was in agreement with other studies (Park, Chae etsignificant at P < 0.05. Differences in bacterial taxa andal. 2015). Chronic constipation is a common clinicalhost phenotypes among treatments were determined bycondition, which is associated with marked decreases in

one-way analysis. RDAs of the C, Y0, Y2M, and Y3M

the quality of life. A marked increase in colonic transit timegroup based on faecal parameters, intestinal charcoal

as well as a decrease in intestinal charcoal transit ratio, thetransit ratio and levels of SCFAs were performed using

basic

Canoco 5 software.

significantly improved after yoghurt intake especially withsymptomsofslow-transit

constipation,arethe three-strain yoghurt.
5. Nucleotide sequences
The sequences were deposited in the Short Read
Archive under the accession number SRP093954.

Table 1 also summarizes the results of major SCFA
concentrations in intestinal content samples from eachgroup of mice. Among the three major SCFAs, the content
of acetic acid was significantly lower only in the Y2L

Results and Discussion

group. Significant (𝑃𝑃 < 0.001) decreases in butanoic acidcontent were observed in three Y3 groups. SCFAs in PC

Mice were observed daily during the experimentalgroup didn’t show significant difference to the control

period, and no meaningful changes in body weight gaingroup and non-yoghurt group. SCFAs producing gut

were detected in the mice in all groups during the wholebacteria may affect constipation (Kang et al. 2015). In

experiment. Favourable effects on first defecation timeprevious studies, SCFAs being administered into the gut

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 02

demonstrated conflicting findings; some studies showed

study. The top 100 abundant OTUs were defined as coreaccelerated colonic transit (Mitsui et al. 2005), while others

OTUs which represented 79.1% of the total sequences (Fig.

slowed colonic transit (Dass et al. 2007). The reason may

2). The names of OTU were based on the number ofbe that SCFAs enhance colonic fluid and sodium

sequences. The most abundant OTU0001 showed 100%

absorption and thus may aggravate the symptoms ofidentity

constipation (Binder and Mehta 1989).

HFDE2698FD10 from dietary-fat-induced taurocholic

to

the

uncultured

bacteriumclone

The Silva database v123 was used to assign taxonomy

acid promoting pathobiont expansion and colitis in Il10(-

to sequences from phylum to genus level. At the phylum/-) mice (Devkota et al. 2012). This OTU could be aligned

level, a total of 28 different bacterial phyla and Candidatus

with Bacteroidales, which was caused by saturated (milk-

phyla were identified in each sample. The whole bacterial

derived) fat diets. OTU0002 was similar to uncultured

community was similar in each of the four groups. The

Bilophila sp. Clone GS16, which was also isolated frommajority

Bacteroidetes

the mouse gut (Berry et al. 2015). This bacterium belonged(55.5±3.4%), Firmicutes (26.5±2.7%) and Proteobacteria

to the family Desulfovibrionaceae, which might be related(16.9±1.3%), which encompassed more than 98% of all

to protein digestion. Both OTU0001 and OTU0002 did not

sequences. The two groups receiving yoghurt showed ashow any difference between samples of the four groups

higher percentage of the phylum Bacteroidetes but lower

on day 8.

ofsequencesbelongedtopercentage of the phylum Firmicutes. The sequences could

Comparing the sequence percentages of day 8

be definitively assigned into 170 different genera, with ansamples between Y3M and C groups, OTU0003,

average of 56.2% of sequences being unclassified at the

OTU0008, OTU0012, OTU0019, OTU0029, OTU0032,

genus level. The nine bacterial genera Parasporobacterium,

OTU0037, OTU0046, OTU0047, OTU0049, OTU0069,

Desulfobaculum, Natronoflexus, Pseudoflavonifractor,

OTU0093 and OTU0094 showed significant differences

Isobaculum, Alkalitalea, Sporotomaculum, and Cosenzaea.

(P<0.05). The percentage of OTU0003 was higher in Y3M

A total of more than 3000 OTUs were observed in thisthan in the C group. However, the percentage of OTU0003

Fig. 2 The abundance of the top 100 OTUs in four groups during the eight-day experimental procedure dependingon OTUs identified with a 3% cut-off. The red letters after OTU names indicate OTU percentages of the day 8
samples that are significantly different between different groups: a, C compared to the Y2M group; b, Y0 comparedto the Y2M group, c, C compared to the Y3M group; and d, Y0 compared to the Y3M group.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 02

was much higher in the Y0 group The OTU0003 wassimilar to the OTU0019 mentioned above, and both OTUs
showed a similar trend, especially low abundance, in the
Y3M group. OTU0008, OTU0012, OTU0029 andOTU0069 showed less than 90% similarity to any known
bacterium but were 100% similar to uncultured bacterium
clones isolated from mouse guts (Devkota, Wang et al.
2012; Lawley et al. 2012).
Previous studies have found that mammalian gut
microbial composition can be associated with probiotic
intake(Quigley2007).

An

exploratory

principal

coordinates analysis (PCA) on weighted UniFrac
dissimilarities was performed to evaluate variation ofdifferent groups in bacterial composition. Overall, 33.3%of the total variation was explained by the first threeprincipal coordinates (PC1 = 13.5%, PC2 = 11.7%, PC3 =8.1%) (Fig. 3). In faecal and intestinal content samples,Y3M and Y2M groups were slightly different from othergroups, and were not exactly similar to each other. Hence,yoghurt intake seems to play dominant roles in determining
variation in gut microbial composition. Further, samples

as those determined by one-way analysis. Y2M and Y3Mfrom the same group were not exactly clustered, whichgroups were significantly related to length of charcoal meal

meant even the same diet could result in different intestinal

transferred and intestinal charcoal transit ratio indicating

microbes.

yoghurt intake indeed changed the intestinal microbial

The correlations between mouse physiologicalcommunity structure, and these changes can significantly

properties and day 8 samples of the four groups weredecrease intestinal charcoal transit ratio. The Y0 group has

analysed using Redundancy Analysis (RDA) (Fig. 4). The

a great influence on the first defecation time, which was

results of RDA did not show exactly similar relationships

confirmed by first defecation time results (Table 1). C andProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 02

Y0 groups showed a relationship with acetic acid andconstipation. Evid Based Complement Alternat Med

butanoic acid content, which was not similar with one-way

2014: 878503-878503.
Dass, N. B., A. K. John, et al. (2007). The relationshipanalysis.

between the effects of short-chain fatty acids on
intestinal motility in vitro and GPR43 receptor

Conclusion

activation. Neurogastroenterology and Motility 19(1):Constipation is a common problem and probiotics
may contribute to ameliorating the symptoms of functional
constipation. This study suggested that the use of yoghurt
decreased the defecation time and led to a significant
improvement in stool consistency. Yoghurt, especially thethree-strain yoghurt used in this study, was also associatedwith improvements in SCFA concentrations in faeces andincreased the intestinal charcoal transit ratio. Further, nosignificant changes in mouse body weight after yoghurt
intake were found, indicating that neither two-strain or
three-strain yoghurt will affect mouse health. Althoughyogurt intake as a functional food could not significantly
increaseprobiotic

numbers,

intestinal

microbial

community structures were changed after yoghurt intake,which was confirmed by PCA and RDA analyses. In the
top 100 abundant OTUs, a total of 18 OTUs showedsignificant differences between yoghurt intake and C
groups, and 10 OTUs showed significant differences
between yoghurt intake and Y0 groups. These OTUs weremost often detected in studies of intestinal inflammation,
indicating that these OTUs might be correlated with an
unhealthy gut. Our results suggest that yoghurt improved
gut health, making the development of new yoghurts
fermented by different strains necessary.
Acknowledgements
This work was supported by the Discipline Direction
Team foundation of Kunming University of Science andTechnology (grant number 14078326).
References
Berry, D., E. Mader, et al. (2015). Tracking heavy water(D2O) incorporation for identifying and sorting active
microbial cells. Proc Natl Acad Sci U S A 112(2):E194-E203.
Binder, H. J. and P. Mehta (1989). Short-chain fatty acids
stimulate active sodium and chloride absorption in vitro
in the rat distal colon. Gastroenterology 96(4): 989-996.
Choi, J.-S., J. W. Kim, et al. (2014). Synergistic effect offermented rice extracts on the probiotic and laxative
properties of yoghurt in rats with loperamide-induced

66-74.
Devkota, S., Y. W. Wang, et al. (2012). Dietary-fat-induced
taurocholic acid promotes pathobiont expansion andcolitis in Il10(-/-) mice. Nature 487(7405): 104-108.
Kang, D.-W., J. K. DiBaise, et al. (2015). Gut microbial
and short-chain fatty acid profiles in adults withchronic constipation before and after treatment withlubiprostone. Anaerobe 33: 33-41.
Lawley, T. D., S. Clare, et al. (2012). Targeted restorationof the intestinal microbiota with a simple, definedbacteriotherapy resolves relapsing clostridium difficile
disease in mice. PLoS Pathog 8(10): e1002995.
Lembo, A. and M. Camilleri (2003). Current concepts:
Chronic constipation. N Engl J Med 349(14): 13601368.
Mitsui, R., S. Ono, et al. (2005). Neural and non-neural
mediation of propionate-induced contractile responses
in the rat distal colon. Neurogastroenterology andMotility 17(4): 585-594.
Park, H. J., M. H. Chae, et al. (2015). Colon transit time
may predict inadequate bowel preparation in patientswith chronic constipation. Intest Res 13(4): 339-345.
Quigley, E. M. M. (2007). Bacteria: A new player ingastrointestinal motility disorders-infections, bacterial
overgrowth, and Probiotics. Gastroenterol Clin North
Am 36(3): 735-748.
Schloss, P. D., S. L. Westcott, et al. (2009). Introducing
mothur:open-source,

platform-independent,

community-supported software for describing andcomparing microbial communities. Appl Environ
Microbiol 75(23): 7537-7541.
Turan, I., O. Dedeli, et al. (2014). Effects of a kefir
supplement on symptoms, colonic transit, and bowel
satisfaction score in patients with chronic constipation:
A pilot study. Turkish Journal of Gastroenterology
25(6): 650-656.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 03

Effects of Food Polysaccharides on Host’s Health Associated with the
Modulation of Gut Microbiota
Dan Chen, Guijie Chen, Yu Ding, Hong Ye, Xiaoxiong Zeng*
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095,
People’s Republic of China*

Corresponding Author: zengxx@njau.edu.cn
SUMMARY

Natural polysaccharides show a variety of effects on host’s health. In our recent works, polysaccharides from
Fuzhuan brick tea (FBTPS), flower of tea plant (Camellia sinensis) (TFPS), fruits of Lycium barbarum (LBPS), bee pollenof Chinese wolfberry (WBPPS), Gracilaria rubra (GRPS), leaves of mulberry (Morus alba) (MALPS), Brassica rapa
(BRPS), mycelium of Pholiota dinghuensis Bi (PDPS), Paecilomyces hepiali (PHPS) and Stachys floridana (SFPS) wereprepared for the evaluation of their bioactivities including antioxidation, hypoglycemic, hypolipidemic andimmunomodulating effects. FBTPS, TFPS, LBPS, GRPS, MALPS, BRPS and SFPS were typical acidic
heteropolysaccharides. FBTPS was mainly composed of Man, Rha, GalA, Glc, Gal and Ara with little molar contents of
Rib and GlcA, TFPS composed of GalA, Gal, and Rha, GRPS consisted of Gal and Fuc, and LBPS consisted of GlcA,
GalA, Glc, Gal and Ara. All of the polysaccharides exhibited antioxidative capacity in vitro or in vivo. FBTPS could
significantly impair obesity induced by high-fat diet. TFPS, LBPS, MALPS, BRPS, PDPS, PHPS and SFPS intervention
remarkably alleviated the immunocompromise in cyclophosphamide-induced mice. It was revealed that thepolysaccharides mentioned above could reach the large intestine safely and were broken down and gradually utilized byhuman gut microbiota, so the distal gut is the key location where the polysaccharides played its role in. On the contrary,
the polysaccharides could significantly modulate the composition and abundance of gut microbiota, and regulate their
metabolites, such as short-chain fatty acids (SCFAs) to affect host’s health. It was found that the effects of FBTPS on
impairing high-fat-induced obesity and TFPS and LBPS on alleviating cyclophosphamide-induced immunocompromise
were related to their modulation of gut microbiota.
compounds in the cytoplasm (such as starch) and structural

Introduction

components of the biofilm or cell wall of organisms (suchPolysaccharides are a class of structurally diverse

as cellulose). The separation, purification and utilization ofbypolysaccharides are essentially dependent on theirmonosaccharide residues via glycosidic bonds. It is worth

structural characteristics. The main structures of natural

macromolecules

that

are

linkedtogethernoting that polysaccharides have the greatest structuralpolysaccharides are extremely complex and diverse, butvariability compared with other natural biopolymers suchthe basic structure of the main chain is often dextran, xylan,

as proteins and nucleic acids and therefore have the highest

fructan, mannan, etc., or a polymer of two or morecapacity to carry biological information. The nucleotides

monosaccharides (such as galactomannan and pectin).

in the nucleic acid and the amino acids in the protein can

Their branched structures are diverse and show great

only be linked to each other in a manner, and thediversity (Chen & Seviour, 2007; Shi, 2016; Shi, Dong, &monosaccharide units in the polysaccharide can be

Ding, 2014; Shi, Fu, & Chen, 2007; Thombare, Jha, Mishra,connected to each other at a plurality of points to form a& Siddiqui, 2016).

plurality of branches or linear structures (Shi, 2016).
The sources of polysaccharides are very diverse. The

Emerging studies have shown that polysaccharides
have complex biological activities and multiple functions,higherespecially influence on the immune function of the host.

photosynthetic plants, fungi, algae, bacteria, etc. At the

Most of the biological activities and functions ofcellular level, polysaccharides represent both reserve

polysaccharides, as it were, related to the immune system

polysaccharides

may

be

derivedfromProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 03(Wang, Petrella, Nicosia, Bellomi, & Rizzo, 2016).

polysaccharides and host health. Thus, a number of

Although the research of polysaccharides started later thanpotential strategies or candidates based on modulation of

the other three types of life macromolecules (proteins,gut microbiota by dietary polysaccharides, have been

nucleic acids and lipids), due to its important physiologicalpresented and evaluated for prevention of diseases andbeing

improvement of our health. In recent years, we have been

continuously advanced in the life process, causing

specialized in systematic study on the preparation andincreasing people’s interests. For now, polysaccharides

valuation of health-promoting functions of dietary

have become a crucial part of the development of naturalpolysaccharides. So far, a series of polysaccharides frommedicines and healthcare products. On the basis of

more than 10 kinds of food source materials with greatincomplete statistics, at least 30 kinds of polysaccharides

potential for exploitation were investigated. Recently andin the world are currently undergoing standard clinicalmore strikingly, our recent works showed that dietary

trials in anti-tumor, anti-viral, and diabetes treatments,

polysaccharides can generally pass through digestive

respectively (Villares, Mateo-Vivaracho, & Guillamón,

system untouched by human enzymes and transporters,2012). In 2002, global sales of carbohydrate drugs andand survive to encounter gut microbiota in large intestine,

healthcare products surpassed $19.3 billion (Shi & Wang,

which act as an important source of energy for gut2011). Obviously, the sleeping carbohydrate giant ismicrobiota thereby shape the composition and physiology

waking up. Allegedly, studies of the structure and functionof gut microbiota. Thus, we report here the researchof polysaccharides have become the third landmark in the

progress of dietary polysaccharides in our team.

functionsandextensive

applications

areexploration of the mysteries of life after the research ofproteins and nucleic acids. At present, many countries
attempt

touseadvancedbiochemical

tools

Materials and Methods

andbiotechnology to find more productive polysaccharides

1. Materials

and their derivatives from traditional medicine, microbes

The polysaccharides in our team were mainly fromand marine organisms to overcome diseases such as tumors.

Fuzhuan brick tea, flower of tea plant (Camellia sinensis,

Besides, the structure-activity relationship and mechanism

Longjing 43), the fruits and bee pollens of L. barbarum L.,of action of polysaccharides were studied. So far, pectin

seaweed G. rubra, mulberry leaves, Brassica rapa L.,from some plants, β-glucans from higher fungi especiallymushrooms, and some sulfate polysaccharides from algae

Pholiota dinghuensis Bi, Paecilomyces hepiali HN1 andStachys floridana Schuttl. ex Benth, respectively.

have been intensively studied, among polysaccharides
from natural products.

2. Preparation of polysaccharides

Complex carbohydrates are an important part of theGenerally, the polysaccharides were extracted by hot

human diet and are ingested in the form of soluble starchwater at about 90 ℃ for several times. Each infusion wasand non-starch polysaccharides. Analysis of the humanfiltered and centrifuged at 4000 rpm for 20 min, and the

genome revealed that the lack of genes was founded inresulting supernatants were collected, mixed with four

encoding non-starch polysaccharide degrading enzymes,

times volume of ethanol and kept overnight. After

recognizing that the composition of the human gut

centrifuged again, the precipitation was collected,

microbiota is of considerable worth to human nutrition

dissolved with water, deproteinized using Sevag method,(Kaoutari, Armougom, Gordon, Raoult, & Henrissat, 2013;

dialyzed against water and lyophilized, affording crudeTasse et al., 2010). In recent years, with the discovery ofpolysaccharides. The crude products were purified bylytic polysaccharide monooxygenases (LPMOs) andcolumn chromatography of DEAE cellulose, Sephadex G-

polysaccharide utilization loci (PULs), the classical view

50/100, affording purified fractions of polysaccharides forof microbial polysaccharide degradation has been further

structural and biological evaluation.

developed. Usually the number of enzymes contained in an
enzymatic system of a microorganism is directly related to

3. Structural characterization

the complexity of the polysaccharide target (Hemsworth,

The structure of polysaccharides was investigated by

Déjean, Davies, & Brumer, 2016). A growing amount ofhigh-performance liquid chromatography (HPLC), Fourier

evidence suggests that the gut microbiota may serve as antransform-infrared spectroscopy (FTIR), methylation

important modulator of the crosstalk between dietary

analysis, gas chromatography-mass (GC-MS), and nuclear

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 03

magnetic resonance (NMR) spectroscopy.

of mannan with the units of →2,6)-α-D-Manp-(1→ andside chain →6)-α-D-Manp-(1→O-2 including →2)-α-D-

4. Assays of bioactivities and digestibility

Manp-(1→ and non-reducing end α-D-Manp, β-D-Galp.

The antioxidant activities in vitro of polysaccharides

The main chain of MALPS-3a was composed of α-(1→3)-

were evaluated by chemical methods, such as Fe

chelating power and scavenging activities on 2,2-azinobis-

Ara and β-(1→4)-D-Gal, and the side chain of 1,3,5-Ara,
1,3,6-Glc, 1,2-Rha, 1,4-GalA and the side chain ends were

(3-ethyl-benzothiazolin-6-sulfonic acid) (ABTS), 1,1-

Glc and Ara (Wu, 2014). The main chain of MALPS-3b

diphenyl-2-picrylhydrazyl (DPPH) free radicals andwas composed of a smooth region formed by repeatedhydroxyl superoxide, and cell methods such as H2O2-

ligation of α-1,4-GalA and a hair region formed by the

2+

The

alternate connection of α-1,4-GalA (methylation andhepatoprotective activity of polysaccharides was studied

inducedoxidative

injury

in

PC12

cell.

acetylation of a partial carboxyl group of GalA) and α-1,2Rha (Rha has a branch at the 4-position with the side chain

using carbon tetrachloride-induced liver injury mice. A
RAW 264.3 cell model was used to investigate theimmunomodulation capacity in vitro (Di et al., 2017), theincludes β-1,4-Gal, α-1,5-Ara, and α-1,3-Ara) (Yuan,2016).

cyclophosphamide-induced mice were used to investigate

As abundant evidences have demonstrated thatthe in vivo antioxidative and immunomodulation effectsoxidative stress is related to a wide range of disease.

(Chen et al., 2019; Di et al., 2017; Ding etal., 2019). The

Thereinto, antioxidant enzymes in liver, which contribute

hypolipidemic activities were evaluated by high-fat dietto the major defense against reactive oxygen species (ROS),

induced mice (Chen et al., 2018). The digestibility ofplay an important role in controlling lipid peroxidation.

polysaccharides in vitro was investigated by simulate

Our results showed that all of these polysaccharides

digestive system (saliva, simulated gastric and small

exhibited antioxidative capacity in vitro in cell or in vivo in

intestinal conditions). The fermentation in vitro by gut

mice (Chen et al., 2018; Di et al., 2017; Yuan et al., 2015).

microbiota was carried out to investigate the effect ofFor example, GRPS exhibited strong scavenging activities

polysaccharides on the gut microbiota.

against ABTS and superoxide radicals and lipid
peroxidation inhibition. At a cellular level, GRPS-3-2

Results and Discussion

showed the strongest protective effect on H2O2-induced
oxidative injury in PC12 cells. FBTPS showed significant

10 kinds of representative polysaccharides fromamelioration of high-fat diet-induced oxidative injury invarious natural materials were obtained and themice (Chen et al., 2018). LBPS could reverse themonosaccharide compositions are shown in Table 1.

cyclophosphamide (Cy)-induced liver injury in mice, by

Overall, all of these polysaccharides were typicalsignificantly decreasing the levels of ALT and MDA, andheteropolysaccharides. FBTPS was mainly composed ofincreasing the levels of GSH, SOD and CAT. TFPS, LBPS,

Man, Rha, GalA, Glc, Gal and Ara with little molar

MALPS, BRPS, PDPS, PHPS and SFPS intervention

contents of Rib and GlcA, TFPS composed of foremost

remarkably alleviated the immunocompromise in Cy-

GalA, Gal and Rha, GRPS consisted of Gal and Fuc, andinduced mice (Chen et al., 2019; Ding, Yan, Chen, et al.,

LBPS consisted of GlcA, GalA, Glc, Gal and Ara. WBPPS

2019).

was mainly made up of Ara, Gal and GalA. MALPS

Digestion is the necessary way for food to play aconsisted of Man, Rha, GlcA, Glc, Gal, and Ara. BRPS was

nutritional function, so it is necessary to evaluate themainly composed of GlaA, Ara and Gal, PDPS was mainly

digestibility of dietary polysaccharides. We have foundmade up of Glc. PHPS was mainly composed of Man, Glc,that the molecular weights of these polysaccharides were

Gal with little molar contents of Rha, Rib, Ara, Xyl, GalA,

not changed under the human saliva and simulated

and GlcA. SFPS consisted of Rha, GlcA, GlaA, Glc, Gal

gastrointestinal

and Ara.

polysaccharides were not degraded in the alimentary tract.

conditions,indicating

that

these

Structure of polysaccharide is very complicated, so it

However, the molecular weights of FBTPS, TFPS, LBPS,

is quite difficult to obtain homogeneous polysaccharide

WBPPS and GRPS were reduced after 24 h of fecal

fractions. This is one of the main factors to impede

microbiota

polysaccharide research development. It was found that

decomposition

PHP-1, PHP-2 and PHP-3 were consisted of the backbone

fermentation
capacity

revealinginvitro,

ofmicrobiota

on

a

these

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 03

polysaccharides. Thus, it was considered that distal gut was

health, has been presented. Recently, we have revealed thatthe most important place for these polysaccharides to exert

the immunomodulate effects and the protection of liver

their bioactivities.

against oxidative damage of TFPS and LBPS was

Trillions of bacteria colonized in the gut forming a

associated with the modulation of gut microbiota and theirmutually beneficial symbiotic relationship with the hostmetabolites such as SCFAs (Chen et al., 2019; Ding, Yan,

during long-term co-evolution. Gut microbes and theirChen, et al., 2019). Additionally, FBTPS-induced

metabolites play an important role in maintaining hostmodulation in the gut microbiota was highly associatedhealth (Nicholson et al., 2012). It was found that FBTPS,

with metabolic syndrome in obese mice, and these changes

TFPS, LBPS, WBPPS and GRPS had the significant effectmight be an important mechanism for FBTPS mediating its

on regulating gut microbiota and promoting the SCFAs

beneficial metabolic effects. Therefore, the possible

production (Chen et al., 2017; Chen et al., 2017; Di et al.,mechanism of metabolic syndrome prevention by FBTPS

2018; Ding, Yan, Peng, et al., 2019; Zhou et al., 2018).

is that indigestible FBTPS reach the large intestine andSpecifically, either FBTPS TFPS, LBPS or GRPS could

then modulate the gut microbiota (Chen et al., 2018).

to

To normal mice, polysaccharides also exhibited

Bacteroidetes in phylum level, which was associated withpositive effects on maintain host’s health. The abundanceenergy metabolism and the risk of obesity (Ley, Turnbaugh,of Lactobacillus in the gut of mice that were administrated

Klein, & Gordon, 2006). Besides, FBTPS significantlywith TFPS or LBPS at the dosage of 200 mg/kg/d for 3

increased the levels of Prevotella and Bacteroides, TFPS

months was significantly increased, while the abundanceenriched Prevotella. The relative abundances of generaof Akkermansia was significantly reduced. Besides, theBacteroides,

content of short-chain fatty acids in the gut after ingestion

significantly

reducetheratio

Bifidobacterium,ofFirmicutes

Phascolarctobacterium,

Clostridium XlVb, Prevotella and Collinsella wereof TFPS and LBPS was significantly higher than that in theenhanced after LBPS fermentation. WBPPS could

normal diet group. TFPS and LBPS intervention showedsignificantly increase the relative abundances of genera

no significant effect on intestinal morphology, but the

Prevotella, Dialister, Megamonas, Faecalibacterium, andexpression of claudin1 and claudin5 was increased. These

Alloprevotella and decrease the numbers of genera

results indicated that TFPS and LBPS had a positive effectBacteroides,

Parabacteroides,

on improving intestinal homeostasis (unpublished data).

Escherichia/Shigella,

Overall, a summary of our recent work is shown in Fig. 1.

Parasutterella,

Clostridium

XlVa,

Phascolarctobacterium, Clostridium sensu stricto andFusobacterium.
Thus, a potentially viable concept, modulating the gut
microbiota by dietary polysaccharides as new therapeutictarget for the prevention of diseases and improvement ofProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 1 – Bioactives and Health / Prebiotics and Probiotics –: 03

Development of Jiangsu Higher Education Institutions.
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Acknowledgements

intestinal juices and fermentation by human fecal

The studies were supported by the National Key
ResearchandDevelopment(2017YFD0400200,

Program

2017YFD0400800

of

China
and

2018YFC1604404), Postgraduate Research & Practice
Innovation Program of Jiangsu Province (KYCX18_0725
and

KYCX17_0632),

National

NaturalScienceFoundation of China (31201454 and 31972025) and a
project funded by the Priority Academic Program

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characterization and antioxidant activity in vitro ofGibson, G., Jia, W., & Pettersson, S. (2012) Host-gut

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 01

Effect of Micellar κ-Casein Dissociation on the Formation of Soluble Protein
Complexes and Acid Gel Properties
Md. Sultan MUHOMUD1,2, Nakako KATSUNO1, Takahisa NISHIZU1*
1

Department of Applied Life Science, Gifu University, Yanagido1-1, Gifu 501-1193, Japan2

Department of Food Engineering and Technology, Hajee Mohammad Danesh Science and Technology
University, Dinajpur-5200, Bangladesh.

*

Corresponding author: nishizu@gifu-u.ac.jp
SUMMARY

This study investigated the role of the dissociation of micellar κ-casein by using cross-linking agent(glutaraldehyde) on the formation of soluble protein complexes and texture of resulting gels. Reconstituted skim milkwith different levels of added glutaraldehyde (SM-GTA) and skim milk without glutaraldehyde (SM) were processed withor without heating followed by study of structural properties of prepared acid gel. Acid gels made from heated SM(without GTA) had higher firmness, water holding capacity, storage modulus and dense microstructure than that fromheated SM-GTA. The electrophoretic analysis demonstrated that the levels of κ-casein in the serum phase was decreased
with increasing GTA concentration in SM attributed to the GTA depreciated the dissociation of micellar κ-casein andsubsequent decline in the formation of the soluble protein complexes. Such reduction of soluble protein complexes in
SM-GTA resulted in weaker acid gels in comparison to the gel made from SM without GTA.
Keywords: casein micelles, κ-casein, glutaraldehyde, soluble protein complexes, acid gel
resulting in severely inhibited dissociation of micellar κ-

Introduction

casein as well as the formation of soluble protein
Textural characteristic of acid milk gel is one of thecomplexes.

most important attribute which play a crucial role in

Therefore, the aim of this study was to investigate thesensory evaluation and in consumer acceptability. Heat

effect of the dissociation of micellar κ-casein by using

treatment of milk causes the denaturation of whey proteins,cross-linking agent (GTA) on the formation soluble protein

which can in turn interact with the κ-casein via thiol-

complexes and on the properties of acid gels. By adding

disulphide exchanges reactions to form whey protein/κ-the small amount of GTA to SM, it is possible to protect

casein aggregates (Vasbinder et al., 2003; Ozcan et al.,casein micelles from dissociation of micellar κ-casein and2015). Theses aggregates strengthen the firmness of acid

reduced the formation of soluble protein complexes.

gels. Moreover, it is demonstrated in our previous study(Mahomud et al., 2017) that the formation of the soluble

Materials and Methods

protein complexes as a new significant complex in theheated milk as well as whey proteins enriched milk may

1. Preparation of milk samples

increase the network connectivity and promote the number

Skim milk (SM) was prepared by adding 12 g skimand strength of contact points which cause the firmer gels.

milk powder (Difco™ Skim milk, Wako, Osaka, Japan)

However,formation of such soluble

proteincomplexes is dependent on dissociation of κ-caseinwith 100 g deionized water to give a milk solution of
10.7% total solids (w/w).

followed by interaction with denatured β-lactoglobulin
which can be increased continuously with increasing the

2. Addition of a cross-linking agent to milk samples

temperature (Anema et al., 2008). Another approach to

The cross-linking agent GTA (25% in water, Nacalaialter casein micellar structure is to add cross-linking agent,

Tesque, Kyoto, Japan) was added to unheated SM to givesuch as glutaraldehyde (GTA) or genipin into milk

GTA concentrations of 0.1, 0.3, and 0.5mM, respectively.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 01

11. Statistical analysis

3. Heat treatment and centrifugation of milk samples
The treated milk samples were subjected to heat

Any significant difference between sample means

treatment at 85 °C for 30 min. After heating, the milk

was determined with Tukey’s honest significant difference

samples were rapidly cooled in an ice bath and put 1.5 mL

test at a significance level of P < 0.05.

plastic tubes for centrifugation at 20,100 × g and 25 °C for
60 min.

Results and Discussion
1. Effect of adding glutaraldehyde to skim milk on the

4. Polyacrylamide gel electrophoresis
Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE) was performed using a BioRad mini-gel slab electrophoresis system.

protein interactions and distribution
Reducing and non-reducing SDS-PAGE of milk
samples were carried out to observe the covalentaggregation of denatured whey proteins with micellar κ-

5. Measurement of casein micelle size

casein (Fig. 1A & B). The dissociating micellar κ-casein

The size of the casein micelles in heated and unheated

was involved with denatured β-lactoglobulin in themilk samples were analyzed by dynamic light scatteringformation of the heat-induced aggregation in the serum(DLS) at 20 °C using a Malvern Zetasizer Nano-ZS

phases of heated samples which was treated as soluble(Malvern Instruments Ltd., Malvern, UK).

protein complexes (Singh & Creamer, 1991).

6. Preparation of acid gels

was observed in the heated SM (Fig. 1 B, lane 2) compared

It has been shown that high intense band of κ-casein
The milk samples were acidified using GDL at ato the heated SM-GTA (Fig. 1 B, lane 4, 6, 8). As theconcentration of 2.0% (w/w) at a temperature of 30 °C until

concentration of added GTA to SM was increased, the level

the pH of the milk reached 4.6.

of dissociating κ-casein was decreased in serum phases.
The dissociation of micellar κ-casein from casein micelles

7. Water holding capacity of the acid gels

was affected adversely by the addition of GTA in SM.

A sample of about 25 g of acid gels (AG) was

The reason behind the formation of soluble protein

centrifuged (KN-70, Kubota, Tokyo, Japan) at 1250 × g forcomplexes is due to the dissociation of micellar κ-casein

25 min. The whey expelled (WE) was carefully separated

from the casein micelles to the serum phase in heated SM

and weighed. WHC was calculated as follows:(Guyomarc’h et al., 2009). The heat induced aggregation

WHC (%) =8. Firmness of the acid gels

A − WE
× 100
A

The firmness of the acid gels was determined by atexture analyzer (Sun Rheometer CR-200D, Sun ScientificCo., Tokyo, Japan) using a single-compression cycle test.
9. Rheological measurement of the acid gels
The rheological properties of the acid gels during andafter acidification were measured using a controlled stressrheometer (AR2000, G2KG, TA Instruments, Newcastle,
DE, USA) with a cup and bob geometry consisting of two
coaxial cylinders (diameters 37.03 and 35.01 mm).
10. Confocal laser scanning microscopy
The microstructure of the acid gels was monitored
using confocal laser scanning microscopy (LSM710, Carl
Zeiss microscopy GmbH, Jena, Germany).

Fig. 1 SDS-PAGE of Gels A (non-reducing) and gels B(reducing) where Lanes 1, 3, 5 and 7 are unheateddispersions and lanes 2, 4, 6 and 8 are heated dispersionof SM and SM-GTA, respectively.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 01

between β-lactoglobulin and κ-casein was remarkablestructure, into which free water was immobilized andhigher in serum of SM compared to SM-GTA. This is due

entrapped.

to the preferential heat-induced aggregation of the β-

The effect of addition of GTA to SM on the firmness

lactoglobulin and dissociating κ-casein in the serum phasesof set acid gels is presented in Fig. 2B. The acid gel madewhich was also reported by Rodriguez and Dalgleish

from heated SM was showed significantly (P < 0.05)(2006).

higher firmness compared with those made from heated
SM-GTA. Addition of GTA to SM caused a significant

2. Effect of adding glutaraldehyde to skim milk on thelower firmness in set acid gels compared with no addition

particle size of the casein micellesof GTA to SM. This result is in agreement with that of

The particle size of the casein micelles of milk

Donato et al. (2007) who reported that soluble protein

samples before and after heat treatment is presented in thecomplexes may be important to increase stiffness of theTable 1. For the heated milk samples, the particle size ofacid gels.

the SM-GTA was lower than that of the SM samples.
Casanova et al. (2017) demonstrated that the decrease in
the casein micelle size could take place when casein
micelles were treated with cross-linking agents. The
decrease in particle size of SM-GTA with increasing theconcentration of GTA could be explained by the smoothing
surface of the casein micelles.
Table 1 Z-average particle diameter of unheated andheated milk samples treated with 0, 0.1, 0.3, 0.5 mM
glutaradehyde (GTA), respectively.
SampleZ-average particle diameter inmilk samples (nm)Unheated

HeatedSM

205.33 ± 1.94a

192.78 ± 3.27a

SM+0.1 mM GTA

199.80 ± 4.38ab

188.14 ± 2.33ab

SM+0.3 mM GTA

196.41 ± 1.64ab

187.68 ± 1.57ab

SM+0.5 mM GTA

192.85 ± 4.86b

185.81 ± 2.23b

Results are expressed as means of three trials.
Different lowercase
superscripts in the same column are significantly different (P < 0.05).

Fig. 2 Water holding capacity (A) and firmness (B)of acid gels made from unheated dispersions (□)
and heated dispersions (■) of SM and SM-GTA.

a, b

4. Rheological properties of acid gels prepared from skim
milk with added glutaraldehyde

3. Water holding capacity and firmness of acid gels
prepared from skim milk with added glutaraldehyde
The water holding capacity (WHC) of acid gels madefrom SM and SM-GTA is shown in Fig. 2A. A significant(P < 0.05) increase in WHC was observed in the acid gels
prepared from heated SM compared with those preparedfrom heated SM-GTA. Increased the WHC of acid gelsmade from heated milk samples was indicated that heat
treatment had a great impact on the WHC of acid gels dueto the denaturation of whey proteins and their interaction
with micellar κ-casein. According to Lee and Lucey (2003),
the denaturation of whey proteins and formation of soluble
protein complexes creating a homogeneous porous

The changes in storage modulus (G′) of the SM and
SM-GTA samples as the reduction of pH from 6.7 to 4.6
using GDL are presented in Fig. 3A. The effect of theaddition of small concentration GTA to SM on therheological properties of acid gels was monitored todetermine G′ every 3 min during acidification. Acid gels
prepared from heated SM without addition of GTA showed
higher final G′ values than those made heated SM withaddition of GTA (Fig. 3A). Heating milk at a higher
temperature accelerates the denaturation of β-lactoglubolin
which forms complex with κ-casein either in the serum
phase or in casein micelles (Singh & Creamer, 1991). The
active participation of denatured whey proteins in the gelProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 01

network by the formation of heat-induced aggregation of

On the other hand, acid gels prepared from heated SM with

β-lactoglubolin with κ-casein stimulates to build theor without added GTA had a more branched and compact

number and strength of bonds between protein complexes,

protein network with less porous space (Fig. 4 E, F, G &resulting in the increase of storage modulus of acid gels

H). However, there was more compact microstructure(Lucey et al., 1998).

observed in the acid gels made from heated SM compared

Other studies (Guyomarc’h et al., 2009) reported thatwith those of heated SM with the addition of different

the increased level of soluble protein complexes in theconcentration of GTA. The possible reasons for increasing

heated SM enhanced to increase the G′ values of the acidcompact networks is the presence of more aggregatinggels. Therefore, it may be concluded that dissociation ofprotein complexes between denatured β-lactoglubolin andmicellar κ-casein stimulates to form a soluble protein

κ-caseins in the serum phase (Schorsch et al., 2001). In

complexes which is responsible for increasing the storage

heated SM without GTA, as a high number of denatured β-

modulus (G′) of acid gels.

lactoglubolin was aggregated with dissociating κ-casein toform soluble protein complexes and some of them wasaggregated with micellar κ-casein to form micelle-bondcomplexes (Lucey et al., 1998). Consequently, the total
number of bonds and amount of protein involvement in theprotein networks would be higher in acid gel made fromheated SM than those made from SM-GTA.

Fig. 4 Confocal laser scanning microscopy images of acidgels made from unheated dispersions of SM (A),Fig. 3 Storage modulus (G′; A) of gel and storage

SM+0.1mM GTA (B), SM+0.3mM GTA (C), andmodulus (G′; B) as a function of frequency sweep

SM+0.5mM GTA (D), respectively and heated dispersions

made from unheated milk (dotted line) of SM (□),
SM+0.1mM GTA (◇), SM+0.3mM GTA (△), andSM+0.5mM GTA (), respectively and heated milk(solid line) of SM (■), SM+0.1mM GTA (◆),
SM+0.3mM GTA (▲), and SM+0.5mM GTA (),respectively.

of SM (E), SM+0.1mM GTA (F), SM+0.3mM GTA (G),and SM+0.5mM GTA (H), respectively.
Conclusion
The dissociation of micellar κ-casein from casein
micelles after heat treatment is reduced remarkably with

5. Microstructure of acid gels prepared from skim milk withincreasing the concentration of GTA in SM. Formation ofadded glutaraldehyde

soluble protein complexes are hence a result of dissociation

The effect of the addition of cross-linking reagents toof micellar κ-casein, which is limited after addition of GTA

SM on the microstructure of acid gels analyzed by CLSM

to SM. The gelation properties of the resultant acid gels areis shown in Fig. 4. Acid gels prepared from unheated SM

influenced by the application of the small concentration ofwith or without added GTA appeared to have a coarse

GTA to SM. The acid gels made from heated SM were

network with little interconnectivity and more open

showed a high WHC, high firmness, and dense

networks with larger porous space (Fig. 4 A, B, C & D).

microstructure compared those of made from heated SM-

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 01

made from milk heated at different pH values. Journal

GTA.

of Dairy Science. 98: 1–10.
Acknowledgements

Rodriguez, C., and Dalgleish, D. G. (2006). Structures and

The authors are grateful to the Japanese Government
for granting the fund through the MEXT scholarship to the
PhD scholar.

Research International. 39: 472–479.
T. (2001). Gelation of casein-whey mixtures: effects ofAnema, S. G. (2008). On heating milk, the dissociation of
kappa-casein from the casein micelles can precedeinteractions with the denatured whey proteins. Journalof Dairy Research, 75(4): 415–421.
Casanova, F., Silva, N. F. N., Gaucheron, F., Nogueira, M.
H., Teixeira, A. V. N. C., Perrone, I. T., Alves, M. P.,
Fidelis, P. C., and Carvalho, A. F. D. (2017) Stability ofcrosslinked

withphysicochemical

study

functionInternational

Dairy

micellesasagenipin:of

Journal.

a
pH,
doi:

10.1016/j.idairyj.2016.12.006
Donato, L., Alexander, M., and Dalgleish, D. G. (2007).
Acid gelation in heated and unheated milks:interactions between serum protein complexes and thesurfaces of casein micelles. Journal of Agricultural andFood Chemistry. 55: 4160–4168.
Guyomarc’h, F., Jemin, M., Le Tilly, V., Madec, M. N., andFamelart, M. H. (2009). Role of the heat-induced whey
protein/κ-casein complexes in the formation of acidmilk gels: a kinetic study using rheology and confocal
microscopy. Journal of Agricultural and Food
Chemistry. 57: 5910–5917.
Lee, W. J., and Lucey, J. A. (2003). Rheological properties,
whey separation, and microstructure in set-styleyogurt: effects of heating temperature and incubation
temperature. Journal of Texture Studies. 34(5-6): 515536.
Lucey, J. A., Tamehana, M., Singh, H., and Munro, P. A.
(1998). Effect of interactions between denatured whey
proteins and casein micelles on the formation andrheological properties of acid skim milk gels. Journalof Dairy Research. 65: 555–567.
Mahomud, M. S., Katsuno, N., Zhang, L., and Nishizu, T.
(2017). Physical, rheological and microstructuralproperties of whey protein enriched yogurt influenced
by the heating milk at different pH values. Journal of
Foodby the heating of reconstituted skim milk powder. Food
Schorsch, C., Wilkins, D. K., Jonest, M. G., and Norton, I.

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andPreservation.

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10.1111/jfpp.13236.
Ozcan, T., Horne, D. S., and Lucey, J. A. (2015). Yogurt

heating whey proteins alone or in the presence of casein
micelles. Journal of Dairy Research. 68(3): 471–481.
Singh. H., and Creamer, L. K. (1991). Aggregation anddissociation of milk protein complexes in heatedreconstituted concentrated skim milk. Journal of Food
Science. 56: 238–246.
Vasbinder, A. J., Alting, A. C., Visschers, R. W. and deKruif, C. G. (2003). Texture of acid milk gels:formation of disulfide cross-links during acidification.
International Dairy Journal. 13: 29–38.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 02

Impacts of Anti-browning Agents and Blanching Against Browning andFungal Growth in Trimmed Aromatic Coconut Fruit
Kittiya PAYUHAMAYATAKUL1, Apiradee UTHAIRATANAKIJ1,2, Varit SRILAONG1,2,
Panida RENUMARN3, Kanlaya SRIPONG, Pongphen JITAREERAT1,2*
1

Postharvest Technology Program, School of Bioresources and Technology, King Mongkut’s University ofTechnology Thonburi, Bangkok 10150, Thailand

2

Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand

3

Department of Innovation and Product Development Technology, Faculty of Agro-Industry, King
Mongkut’s University of Technology North Bangkok, Prachinburi Campus, Prachinburi province,
Thailand.

*Corresponding Author: pongphen.jit@kmutt.ac.th
SUMMARY

Major problem of exporting trimmed aromatic coconuts are fungal infection and browning of coconut mesocarp.
Thus, the effectiveness of sodium chlorite )SC( and acidified sodium chlorite )ASC( for controlling browning and fungal
infection on trimmed coconut fruit were studied in compared with filtrated water and 3% sodium metabisulfite ) SMS;
commercial treatment( . Dipping pieces of coconut mesocarp in 250 mg/ l SC and ASC for 5 min showed the great
inhibition of browning but did not inhibit fungal growth. Comparative effectiveness of SC and ASC was investigated bydipping trimmed fruit in filtrated water, 3% SMS and 250 mg/ l of SC and ASC for 5 min, then kept at 4 °C for 20 days.
The SC treated fruit showed lower browning, brown pigment, and the activity of polyphenol oxidase )PPO, key browning
enzyme( in compared with filtrated water and ASC treated fruit. However, the effectiveness of SC to inhibit browningand fungal growth still was lower than SMS treatment. Effectiveness of SC was enhanced when incorporated with
blanching and PCV film wrapping. Effects of blanching with hot vapor ) HV( and PVC film on browning and fungal
contamination of trimmed fruit were studied. The fruit were treated with filtrated water, SMS and HV for 90 sec followed
by 250 mg/ l SC (HV + SC). After that, each treated fruit were separated into two groups to wrap with PVC film and unwrapped before kept at 4°C for 25 days. Fruit treated with HV+SC+PVC showed lower color changes (ΔE) of mesocarp,
brown pigment and o-quinone than filtrated water )both wrapped and unwrapped) and HV + SC )un-wrapped) treatments.
Application of HV+SC+PVC also inhibited PPO and peroxidase activities, but did not affect on TA, TSS, percentage oftransmittance and thiobarbituric acid ) TBA) value of coconut juice. Moreover, overall acceptance of coconut had thehighest score compared with other treatments, except SMS treatment. This result suggested that HV + SC + PVC could
suppress browning and control fungal contamination without any negative effects on the quality of coconut fruit, eventhough its effectiveness was not equal with SMS treatment.
Keywords: aromatic coconut, blanching, browning, hot vapor, sodium chlorite
Introduction

such as glucose, fructose, sucrose, potassium, sodium,
vitamins and antioxidants (Kailaku et al., 2015). For theThailand is a great source of agricultural production

coconut trading, the green shell of coconut fruit is peeledthat serve to the world population. Thailand produces

and trimmed for being the convenience to consumers andmany tropical fruits that are used for consuming in the

reducing the transportation costs (Paull and Ketsa, 2015).

country and exporting to aboard. Aromatic coconut (Cocos

Peeling and trimming the coconut is a cause of rapid

nucifera L.) is an important economical fruit that has beenbrowning and easy to infect by various fungi on coconut

promoted as the sport drink by The Food and Agriculture

mesocarp leading to short storage life. It has been known

Organization of the United Nations (FAO, 2000). Becausethat browning of fruit or vegetables happens after

coconut water contains a lot of health benefit components

wounding. The browning is a result of the formation ofProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 02

brown compounds from the oxidation of phenolic

Each treatment had four replicates. The inhibition ofcompounds by polyphenol oxidase (PPO) (He and Luo,browning and fungal growth on mesocarp pieces were2007). Sodium metabisulfite (SMS) at 3% is commerciallyevaluated every 2 days.

used to prevent browning and fungal infection of trimmed
coconut but it can be a cause of asthma for consumers (Luet al., 2006). Thus, this research was looking for thealternative methods that be safe to the consumer andenvironment.
Sodium chlorite (SC, NaClO2) and acidified sodium
chlorite (ASC) are strong oxidizing agents, which havebeen accepted by the FDA for use in foods. SC and ASC
have dual effects as a anti-microbial agent and antibrowning inhibitor (Lu et al., 2006; He et al., 2008).
Recommended concentration of SC to use for fresh
produce ranges 500-1200 mg/l (Rao, 2007). In case of ASC,
it is produced by the addition of citric acid to SC solution
and adjust the pH to be 2.3-2.9. The USDA Food Safety
and Inspection Service (FSIS) allows to use 500-1,200
mg/l ASC dip or spray in fruit and vegetables (Rao, 2007).
Heat treatments have been commercially used tomaintain the quality to harvested products by minimizing
disease infection and insect infestation during storage.
Moreover, heat treatment can inhibit the enzyme activities
of microorganisms and plant tissue (Corcure et al., 2004).
Luckanatinvong et al. (2011) reported that hot steaming thearomatic coconut fruit at 60, 80 and 100 ˚C for 90, 180 and
270 sec followed by dipped in 0.9% SMS for 3-5 min, afterthat wrapped up with polyvinyl chloride (PVC) film
reduced mesocarp browning and decay during cold storage(2±1°C). Especially coconut steamed with hot vapor at
100˚C for 90 sec showed the best quality in term ofaromatic and sweet water.
Thus, this research was to study the combined effectsof SC, ASC, blanching with hot vapor and PVC film onbrowning, fungal growth and quality of trimmed aromatic
coconut.
Materials and Methods
1. Effects of SC and ASC at different concentrations onbrowning and fungal growth of coconut mesocarp pieces
Aromatic coconut fruit 6 months after anthesis were
peeled the green shell. Pieces of coconut mesocarp wereobtained by vertical shearing and cutting them into small
pieces, approximately 4 x 4.5 x 0.7 cm in size, andimmediately dipped in filtrated water )control(, 3% SMS,
250-1000 mg/l SC or ASC for 5 min. All samples weredrained and placed into plastic baskets, then covered witha polyethylene plastic )PE( bag and kept at 4 °C for 10 days.

2. Effects of SC and ASC on browning and fungal growthof trimmed coconut fruit
Green shelled coconut fruit were peeled and
immediately dipped in filtrated water )control(, 3% SMS,
250 mg/l SC or ASC, each for 5 min. All fruit samples were
packed as described above and kept at 4 ºC for 20 days.
Each treatment had four replicates (fruit). Fungal growth
inhibition, browning pigment and PPO activity of coconut
mesocarp were investigated 5 days interval.
3. Combined effects of SC, hot vapor and PVC film onbrowning and quality of trimmed coconut fruit
Green shelled coconut fruit were peeled and
immediately dipped in filtrated water )control(, 3% SMS,
hot vapor for 90 sec followed by 250 mg/l SC (HV + SC).
After that, each set of treated fruit were separated into twogroups for wrapping with PVC film and un-wrapping with
PVC film. All fruit were kept at 4°C for 25 days. Eachtreatment had four replicates (fruit). Fungal growth
inhibition, color difference (∆E), browning pigment, theactivity of PPO and POD of coconut mesocarp were
investigated 5 days interval. Titratable acid (TA), totalsoluble solids (TSS) content, percentage of transmittanceand thiobarbituric acid )TBA) value of coconut juice werealso determined 5 days intervals.
4. Quality and biochemical analysis
Color changes of coconut mesocarp were determinedusing a Minolta DP-301 colorimeter )Konica Minolta;
Tokyo, Japan(. L*, a* and b* values were recorded for
calculating the difference color of mesocarp )∆Ε( andpercentage of browning inhibition as described by Maskan)2001( and Sapers and Douglas )2007( respectively. The
total soluble solid content, titratable acid and percent oftransmittance of the coconut water was determined using
hand refractometer )ATAGO PAL-1(, the method of
A.O.A.C. (1990) and Campos et al. )1996( respectively. Oquinone

content,

polyphenol

oxidase

)PPO(

and

peroxidase activity in mesocarp were measured according
to the method of Degl’ Innocenti et al. (2007), Duan et al.
)2007( and Zhang and Shao (2005), respectively.
5. Microbiological analysis
Coconut mesocarp were cut into the small pieces (1.0

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 02x 1.0 cm). Five pieces randomly selected and placed on(A)potato dextrose agar )PDA( contained antibiotic to inhibit
bacterial growth. PDA plates were incubated at 27±2 °C
for 3 days. Numbers of coconut pieces showed the fungal
mycelium were counted and calculated as the percent offungal growth.
6. Statistical analysis
Data were analyzed using the ANOVA procedure in(B)

the general linear models procedure of SAS 9.0 )SAS
Institute; Cary, NC, USA( for completely randomized
design experiments. Data were presented as mean ± SE.
Each treatment contained four replicates. Significance wastested at p ≤ 0.05 using Duncans multiple range test.
Results and Discussion
Browning and fungal infection are the major problem
for trimmed aromatic coconut trade. The present work
showed the effectiveness of SC and ASC on browning andfungal infection of the pieces of coconut mesocarp. Result

Fig. 1 Browning pigment (A) and polyphenol oxidase
activity (B) of the mesocarp of trimmed coconut treatedwith filtered water (control), 3 % SMS and 250 mg/l
SC or ASC solution for 5 min. (A)Treated samples werestored at 4 °C for 20 days.

found that dipping pieces of coconut mesocarp in 2501,000 mg/l SC or ASC for 5 min showed the great
inhibition of browning in compared with the control(filtrated water) but there were not effects to inhibit fungal
growth (data not shown). Effect of SC and ASC on fungal
growth in this experiment differed from the previous
research. Microbial contamination on fresh cut pear and(B)

fresh cut potato was reduced by treating with 300 mg/l SC(Xiao et al., 2011) and 500 mg/g ASC (Sun et al., 2012).
250 mg/l SC and ASC were selected to compare theireffectiveness on browning inhibition of trimmed coconut
fruit. Trimmed fruit were dipped in filtrated water, 3%
SMS and 250 mg/l of SC and ASC for 5 min before storage.
The SC treated fruit showed lower brown pigment and(C)

PPO activity in compared with filtrated water and ASC
treated fruit (Fig. 1). This result implied that SC is a greater
strong oxidizing agent than ASC although the effectivenessof SC and ASC to inhibit browning still was lower than
SMS treatment. Several researches demonstrated thatapplication of SC could reduce browning by inactivating
the browning enzyme such as PPO activity in fresh-cut
apple (Luo et al., 2011) and rose apple (Mola et al., 2016).
He et al. (2008) stated that SC might oxidize the co-factor(Cu ) of PPO resulting to change the enzyme structure to2+

be inactivate form.
Enhancing the effectiveness of SC solution on theinhibition of browning and fungal growth was found when

Fig. 2 Color difference (∆E, A), browning pigment (B) andO-quinone content (C) of the mesocarp of trimmed coconut
treated with hot vapor for 90 sec, then dipping in 250 mg/l
SC for 5 min followed by wrapping or unwrapping with
PVC film .Coconut treated with filtered water or 3% MSwith and without PVC film were used as the controls. All
samples were stored at 4 °C for 25 days.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 02

incorporated with HV for 90 sec with PCV film. Trimmed(A)

coconut fruit treated with HV+SC with and without PVC
wrapping showed greatest effects to inhibit browning as
indicated by lower values of color difference (∆E), brown
pigment and o-quinone content (Fig. 2). Treatment of
HV+SC+PVC also inhibited PPO and peroxidase activities(Fig. 3), but was not effects on TA, TSS, percentage of(B)

transmittance and thiobarbituric acid )TBA) value of
coconut juice (data not shown). Moreover, it was foundthat HV+SC+PVC treatments could suppress the fungal
growth when compared with the filtrated water treatment(Fig. 4) These results could explain that the reduction of
microbial growth and browning might be caused by heat
treatment inactivated the enzyme activities of microbial
and plant tissue (Corcure et al., 2004) such as browning

Fig. 3 Polyphenol oxidase (A) and peroxidase (B) of the

enzymes (PPO and POD) (Klein and Lurie, 1992; Luo etmesocarp of trimmed coconut treated with hot vapor foral., 2015). Oday Husham Kamil et al. (2011) stated that

90 sec, then dipping in 250 mg/l SC for 5 min followed

heat treatment affected on the chemical reactions of fungal

by wrapping or unwrapping with PVC film .Coconut

cells. Fungal growth become slow down if the temperature
is higher than the optimal temperature due to lessefficiently chemical reaction. However, if the temperature

treated with filtered water or 3% SMS with and without
PVC film were used as the controls. All samples werestored at 4 °C for 25 days.

is over than the critical point, the fungal growth will stop,
the lipid and protein structure of fungi are changed, andcell components begin to be damaged resulting to fungal death. The other reason of fungal death may be involved
with antimicrobial property of SC. SC is a strong oxidizing
agent that can provide active chlorine dioxide )ClO2(. ClO2
can interfere the microbial protein synthesis resulted tolimit microbial growth )Trinetta et al., 2012(. However,this result showed that trimmed coconut fruit treated with
HV+SC with PVC wrapping showed the better result to

Fig. 4 Fungal growth on the mesocarp of trimmed coconut
treated with hot vapor for 90 sec, then dipping in 250 mg/l

control browning than HV+SC without PVC wrapping.

SC for 5 min followed by wrapping or unwrapping withThis can be explained that PVC wrapping prevented

PVC film .Coconut treated with filtered water or 3 %SMS

oxygen transmission from the outside through inside of thewith and without PVC film were used as the controls. Allfilm. Therefore, under the low level of oxygen condition,

samples were stored at 4 °C for 25 days.

PPO activity will be slow down due to low oxidation incoconut mesocarp (He and Luo, 2007).
Therefore, this result suggested that HV+SC+PVC
could reduce the fungal infection and browning of coconut
without negative effect on the coconut quality even throughits effectiveness was not equal with SMS treatment.

Conclusion
Sodium chlorite (SC) at 250 mg/l showed the greater
anti-browning effect than acidified sodium chlorite (ASC)at 250 mg/l but at this concentration were less effect tocontrol fungal growth on trimmed coconut. The efficiency
of SC against browning and fungal infection of trimmed
coconut was enhanced when cooperated with hot vapor forProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 02

90 sec followed by wrapping with PVC film although theircharacteristics of coconut water from different varieties

effectiveness was not equal with SMS treatment.

and its potential as natural isotonic drink. International

Furthermore, HV + SC + PVC treatment did not have any

Journal on Advanced Science Engineering Information

negative effects on the quality of trimmed coconut fruit

Technology 5(3), 174–177.

such as TA, TSS, percentage of transmittance andthiobarbituric acid (TBA) value of coconut juice.

Klein, J.D. and Lurie, S. (1992) Reviews: Heat treatments
for improved postharvest quality of horticultural crops.
Hort Technology 2(3), 316–320.
Lu, S., Luo, Y. and Feng, H. (2006). Inhibition of apple

Acknowledgements

polyphenol oxidase activity by sodium chlorite.
This research was supported by The National
Research Council of Thailand )NRCT( of the fiscal year
2017. The authors thank to The United Graduate School of
Agricultural Science (UGSAS), Gifu University, Japan forsupporting some equipment in this study.

Campos, C.F., Souza, P.E.A., Coelho, J.V., and Gloria,
M.B.A. (1996). Chemical composition, enzyme
activity and effect of enzyme inactivation of flavor
quality of green coconut water. Journal of Food
Processing and Preservation 20, 487–500.
Corcuera, J.I.R.D., Cavalieri, R.P. and Powers, J.R. (2004).
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b5ce8bef8cf3863542aa8ed.pdf [9 August 2019].
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(2007). Physiological basis of sensitivity to enzymaticbrowning in “lettuce”, “escarole” and “rocket salad”
when stored as fresh-cut products. Food Chemistry,
104(1), 209–215.
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(2007). Effect of nitric oxide on pericarp browning of
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and athletes-First patent granted to un-food agency.
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control in fresh-cut produce. Stewart Postharvest 6, 1–
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Kailaku, S.I., Syah, A.N.A., Risfaheri, Setiawan, B. andSulaeman,A.

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(2011). Quality of blanched aromatic coconut for

Luo, Y., Lu, S., Zhou, B. and Feng, H.

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and Jitareerat, P. (2016). Impacts of sodium chlorite
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Kamil,

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Paull, R.E. and Ketsa, S. (2015). Coconut: postharvest
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Session 2 – Physical Properties of Food –: 02Sun, S.H., Kim, S.J., Kwak, S.J. and Yoon, K.S. (2012).
Efficacy of sodium hypochlorite and acidified sodium
chlorite in preventing browning and microbial growthon fresh-cut produce. Preventive Nutrition and Food
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dioxide for microbial decontamination of food.
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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 03

Effect of Edible Laccase on Chinese Bread Texture
Maharani Pertiwi KOENTJORO1*, Desy Lailatul RACHMAH2,
Endry NUGROHO PRASETYO2
12

Department of Medical Laboratory Technology, Faculty of Health, Nahdlatul Ulama University ofSurabaya, Jl. Jemursari 51–57, Surabaya, 60237, Indonesia

2

Department of Biology, Faculty of Science, Institut Teknologi Sepuluh Nopember, Gedung H Kampus*

Corresponding Author: maharani@unusa.ac.id

ITS Keputih Sukolilo, Surabaya, 60111, Indonesia

SUMMARY
Laccase application in the food industry is an alternative and innovative solution for natural ingredients since it isable to improve the food quality especially for baking industry. Laccase is a natural catalyst which could improve thestructure of gluten in dough by increasing phenol oxidation resulting a better bread texture, taste, volume, and freshness.
The aim of this study was to evaluate Trametes versicolor laccase oxidation on physicochemical quality of Chinese bread.
Laccase were pasteurized to obtain edible laccase with the final activity as 774.7 U/ml. Laccase treatment managed toreduce bread hardness until 9.55 N. The softening phenomenon was related to the laccase–mediated depolymerization of
the cross–linked network. Chemical parameters of laccase treatment were 0.065 ppm lower than control. The functionalgroup in the polymer pretreatment mixture of edible laccase observed by Fourier Transform Infrared Spectrometer (FTIR)
showed the formation of disulfide group at a wavelength of 450–500 cm–1. The Scanning Electron Microscope analysis
revealed formation of gluten protein which has smaller and uniform pores compared to the control group. Based on the
results obtained in the present study, laccase from Trametes versicolor can be considered a promising application to
improve quality of Chinese bread at industrial level.
Keywords: laccase, Trametes versicolor, physicochemical quality, Chinese bread
Introduction

Khatkar, 2015).
Generally, the baking industry uses artificial food

Laccase (EC 1.10.3.2) is a type of enzyme containing

additives such as baking soda (sodium bicarbonate) to

polyphenol oxidase, which can oxidize polyphenols,

increase volume and delighting texture. Long–term of

methoxy–substituted phenols, and diamine (Minnusi et al.

chemicals consumption could result negative impact of2002). Among plants, fungi, and bacteria, white–rot fungi

human health thereby reducing serum iron and calcium

are the major laccase producer. Laccase by Trametes

levels in the body which lead to specific doses toxicity

versicolor has been produced using lignin containing(Fakhri et al. 2016). It is well known that the addition ofmaterials (Shraddha et al. 2011; Imran et al. 2012; Offia–

certain enzymes to the dough have unwanted side effects.

Olua, 2014).

Since the enzyme are denaturated and inactivated by heat

Laccase in the food industry has been implemented induring cooking process (Gallagher et al. 2009). Therefore,a dough as bread quality improver to increase volume or

the application of laccase as an enhancer of the texture of

crumb structure and softness in the baked products

the bread is less health risks.

(Minnusi et al. 2002; Imran et al. 2012; Osma et al. 2010).

The objective of this study was to determine theLaccase from T. versicolor have been evaluated to theireffects of edible laccase from T. versicolor on the texture

ability to increase the strength of the gluten structure priorof Chinese bread by measuring phenol content, functional

oxidizing lignocellulose in flour (Kaur et al. 2014; Minnusi

groups, and micro visual characters.

et al. 2002). The elastic properties of low–protein flour
used in making Chinese bread has a weak gluten index
therefore it needs to be improved using laccase (Gulia andMaterials and Methods

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 03

Laccase activity was determined as the increase in

1. Laccase production

absorbance at 468 nm due to the oxidation of 2,2 –azino–

1.1 Organism and cultivation conditions
The T. versicolor fungi are a culture collection ofbis (3 ethylbenzothiazoline–6–sulfonate) (ABTS) at 30°C

Microbiology and Biotechnology Laboratory, Department(Koentjoro et al, 2016). The reaction mixture containedof Biology, Institut Teknologi Sepuluh Nopember (ITS)

200 µL 10 mM ABTS, 300 µL enzyme solution in 500 µL

Surabaya, Indonesia. The fungi cultures were maintained

100 mM citrate buffer, pH 4.5.

on Potato Dextrose Agar (PDA) medium and incubated at

Oxidation

of

ABTS

was

observed

28°C, for 6 days before using as seeding. The procedures

spectrophotometrically at 436 nm for 5 minutes. The

of maintaining fungi culture is briefly explained as follows.

laccase activity is calculated based on the ABTS standard

Sporulated PDA medium were dislodged and washed withcurve. One unit (U) of laccase activity was defined as the

PBS sterile. As much as 10 mL of 108 CFU fungi spores’

amount of enzyme required to oxidize 1 μmol ABTS per

suspension were inoculated into 100 mL of Potato

minute and considered equivalent to 1 unit of enzyme

Dextrose Broth (PDB) (pH 6.0) and incubated for 6 days

activity (ε420 = 36000 M−1 cm−1 and path length l = 1 cm)

at room temperature (25°C) with a rotary shaker at 130 rpm(Wolfenden and Wilson, 1982).

(Jo et al. 2010; Qin et al. 2017).

2.2 Isoelectric point (pI)

1.2 Partially edible laccase production

To investigate the isoelectric point of supernatant, as

All experiments were carried out in 300–mLmuch as one mL of laccase was incubated in each tube

Erlenmeyer flasks containing 100 mL culture medium. The

containing a phosphate buffer with variable pH level (3.0,

medium contained 4.5 g/L rice husk, 1.5 g/L yeast extract,

4.0, 5.0, 6.0, 7.0 and 8.0) at room temperature for overnight.

1 g/L C6H12O6, 0.5 g/L NH4Cl and 100 mL of salt solution.

The occurring of precipitation was monitored and justified

The salt solution contained 2 g/L KH2PO4, 0.5 g/L

as the isoelectric point of laccase in particular pH level.

MgSO4.7H2O, 0.1 g/L CaCl2.2H2O and 0.5 g/L KCl and

2.3 Total Protein determination in enzyme solution

maintained pH 5.0, (Koentjoro et al. 2016). CuSO4 was

Total Protein concentration in supernatant wasadded by dissolving 100 mg in 1 L of dH2O as laccase

determined by Bradford method using Bovine Serum

inducer. The medium was autoclaved at 121°C for 15 min.

Albumin (BSA) as standard. Reaction was started by

Each flask was inoculated using an agar piece 1 cm cut

addition of 0.1 mL of supernatant and 5 mL of Bradfordfrom an actively growing fungal culture. The flasks werereagent (10 mg of Coomassie Brilliant Blue G–250 in 5 mL

incubated at 30°C on a rotary shaker (130 rpm). Theof 95% ethanol, 10 mL of 85% phosphoric acid, and 85 mL

culture broth was filtered, clarified by centrifugation atof dH2O). The changes in absorbance due to laccase

10,000 rpm for 15 min, frozen, defrosted and then filteredcontent

to remove the precipitated polysaccharides. The resulting

spectrophotometer in absorbance at 595 nm.

2insupernatant

wererecordedbyclear filtrate was used for the further experiment. Partiallyedible laccase was sterilized by pasteurizing (or High

3. Preparation of laccase modified Chinese breadTemperature Short Time) (HTST) is as follows, a total ofDough was made manually by mixing flour with

50 mL of laccase solution was placed in a water bath heatedenzymes, yeast, water and sugar. The experimental

at 72°C for 25 seconds (Ranieri et al. 2009).

formulations set are shown in Table 1. The average of
duplicate data was used in analysis. The dough in each

2. Characterization of laccase

experimental formulation was divided into 6 similar

2.1 Laccase activity

weight. The dough was placed in a bowl covered with a

Table 1 Composition of bread with different levels of edible laccase
SampleSample 1
Sample 2Treatment
Negative control
Non–Edible Laccase
Flour wheat low protein (gr) 100
100
Non–Edible Laccase
0
1.5
Edible Laccase
00Baking soda (gr)00Yeast (gr)2.2
2.2
Water (mL)50
50
Sugar (gr)20
20

Sample 3
Edible Laccase
100
0
1.5
0
2.2
50
20

Sample 4
Baking soda
100
0
0
400
2.2
50
20

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 03

plastic foil and allowed to stand at room temperature(28°C) for 45 minutes. The dough was then steamed for 7

5. Statistical analysis

minutes (Laohaprasit and Sricharoenpong, 2018).

The data obtained were then analyzed using of one–way
variance (ANOVA) Duncan test using Minitab 14.0

4. Laccase modified Chinese bread analysis(Minitab 14 Statistical software, 2014). Meanwhile, a

4.1 Total phenol content

qualitative descriptive design was conducted for texture

Total phenolic content was determined according toand functional group analysis.

Folin–Ciocalteu method with slightly modified. Every
extraction solution of bread (10 mL) was mixed with 10

Results and Discussion

mL of 96% ethanol and 5 mL of 0.1% HCl for 10 min. The
reaction mixtures were allowed shaking in a rotary shaker

1. Production and characterization of edible laccasefor 30 minutes at room temperature. The mixtures were

The activity of laccase before and after pasteurization

then centrifuged for 10 minutes at 4,000 rpm and thewere calculated as 799.4 and 774.7 U/mL, respectively

supernatant was collected. Every extraction of supernatant(Table 2). These results demonstrated the pasteurization(1 mL) was mixed with 100 µL of Folin–Ciocalteu reagent

processes has no effect on laccase activity.

and 1 mL of sodium carbonate 15% (w/v). After incubationfor 1 hour at room temperature, the absorbance wasmeasured at 710 nm using a spectrophotometer. Total
phenol was calculated from the calibration curve of tannic
acid and expressed as milligram of tannic acid equivalent
per gram of dough (mg TAE/g).

Table 2 Activity and quantitative estimation of laccase
production
LaccaseActivity
Protein Contain(U/mL)(mg/mL)
Before pasteurization
799.4
0.02
After pasteurization
774.7
0.11

4.2 Texture profile analysis
Evaluation of dough properties (hardness) was carried

The pI of the non–and edible laccase was determinedout by texture profile analysis. The test consists ofon pH 3.0. This information is helpful to developing

compressing a bite–piece of bread two times in apurification scheme as a crucial factor in enzyme

reciprocating motion that imitates the action of the jaw andpurification, the precipitation occurs is avoided (Xia, 2007).

extracting from the resulting force–time curve a hardness

This result is consistent with the study of Afreen et alparameters. The test was applied on bread by using(2017) which stated that generally, laccase has anfollowing procedures. Each sample was placed on theisoelectric point between at pH 3.0–3.1.

Texture Analyzer (SMS) mod.TA.HDi 500 (Stable Micro

The pasteurization did not change the total protein in

Systems, Surrey, UK) preparation table followed bylaccase solution as shown in Table 2. It is indicated that

applying a pressure using a piston diameter of 75 mm

laccase can be pasteurized using heating with a certain(Ayala–Soto et al. 2017).

temperature and time that serves to kill pathogenic

4.3 Scanning Electron Microscope (SEM)

microorganisms while maintaining physical properties

The micro structural of starch granules in bread withdifferent formulation were studied using Scanning(Verhoeckx et al. 2015).
2. Texture Analysis (Test Hardness)

Electron Microscope. Samples were prepared by knife

Bread treated with edible laccase was demonstrated

cutting then placed on a carbon tip followed by gold andin deformation in cavity characters (Fig. 1). The

palladium coating. The SEM images of each samples werepretreatment–control had a greater cavity compared totaken at magnification of 3,000 (Wojciechowicz–Budzisz

non–and edible laccase pretreatment. Moreover, between

et al. 2015).

non–and edible laccase Chinese bread has a small anduniform cavity. The result confirms the findings of

4.4 Fourier Transform Infrared Spectroscopy (FTIR)

Salinheimo et al (2007) and Tsegaye et al. 2018.that theAnalysisbread treated with laccase has a small and uniform cavity.

The FTIR analysis in this study has been carried out in

The small and uniform cavities, due to the ability of theorder to identify functional groups shifting in every

laccase to form crosslinking in arabinoxilan through ferulic

treatment at a wavelength between 800–3600 cm (Amiracid side groups allowing strengthen of gluten and more

et al. 2013).

ability to retain gas (Meybodi et al. 2015). Therefore, the–1Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 03

pasteurization has no effect on the laccase protein
component due to glycosylation. The edible laccase has agreater hardness value (9.55N) compared to non–edibleone (9.15 N), this is most probably because of the different
laccase activity shown in Table 2.

pasteurization does not affect the protein component ofTable 4 A comparison of the physicochemical
characteristics of bread
Pretreatment
Hardness (N)Control
21.9 ± 0.269b
Baking soda
17.97 ± 3.514b
Non–Edible Laccase
9.15 ± 0.869a
Edible Laccase
9.55 ± 0.262a
Notes: Values with different superscript letters within acolumn are significantly different (p≤0.05. Data areexpresses as means values of two samples ± standard
deviationlaccase.

4. Scanning Electron Microscope (SEM) Analysis

Fig. 1 Cross Section of Chinese Bread with a Variety ofPretreatment a) Control b) baking soda c) non–ediblelaccase d) edible laccase.

3. Total Phenol Analysis
Analysis of total phenols in various treatments fordough are reported in Table 3. The non–pretreatment andedible laccase experimental group both of them have alower total phenol concentration of 0.096 and 0.065 ppm
respectively compared to a control of 0.168 ppm. This isbecause of ability of laccase to oxidize phenolic
compounds such as ferulic acid that act as laccase mediator

SEM analysis was taken to investigate the effect oflaccase on microstructure of whole bread. This techniqueallowed a qualitative description of the structural cell andtheir distribution.
As shown in Fig. 2, that the microstructure of gluten
bread was more uniform and more number of pores after
adding of laccase due to higher volume of gas was released
during dough fermentation.

in a laccase–mediator system (LMS) reaction mode (Imranet al. 2012; Everette et al. 2014). The higher amount ofoxidized phenol, it will increase the formation of disulfide
groups (Linares–García et al. 2019). The ferulic acid isoxidized and formed radicals and continues to oxidize thesulfidryl (SH) groups. The oxidation can affect the natural
disulfide formation (S–S) in gluten polymers and increase
the rate of protein depolymerization lead to increase gluten
networks (Linares–García et al. 2019).
Texture analysis on various treatments are shown in
Table 4. Pretreatment of edible laccase and controls had asignificantly different hardness value of 9.55 and 21.9 N
respectively. These results indicated that the addition of
laccase to the dough was able to reduce the hardness of theChinese bread. This is consistent with the statement (Osmaet al. 2010; Brijwani et al. 2010) that the laccase shows anoxidizing effect to improve the structure of gluten in thedough and can reduce stiffness, increase volume, improve
crumb structure, and increase the softness of bread. Indeed,
one good parameter of bread quality is a soft texture andlow hardness (Borla et al. 2014). In addition, in Table 4 thenon–and edible laccase pretreatment showed non–significantly different of hardness level which shows that

Fig. 2 SEM of a) control; b) baking soda; c) non–ediblelaccase; d) edible laccase at 1000X magnification Arrows
show pores on gluten tissue.
The pore structure can be caused by the presence ofan elastic gluten protein matrix which is able to withstandthe development of gas while the cell walls was not easily
broken (Ortolan and Steel, 2017).
5. FTIR analysis

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 03

FTIR spectroscopy of bread functional groups at
various pretreatments was done in the range of 400–4000

decolorization of anthraquinonic dye. PLOS ONE,https://doi.org/10.1371/journal. pone.0175144.

cm (Fig. 4). Characters of spectral bands were similar for

Amir, R. M, Anjum, F. M., Khan, M. I., Khan, M. R., Pasha,

control and laccase containing bread. Major peaks wereI., Nadeem, M. (2011) Application of Fourier transform

indicated absorption of OH groups at a wavelength range

infrared (FTIR) spectroscopy for the identification ofof 3000–3,700 cm–1.

wheat varieties. Journal Food Science and Technology–1

In the range of 450–500cm there is a change in the–1

50, 1018–1023.

peak (peak) spectra at the pretreatment of non–edible

Ayala–Soto, F. E., Serna–Saldívar, S. O., Welti–Chanes, J.

laccase and edible laccase. At this wavelength range is the(2017) Effect of arabinoxylans and laccase on batter

absorption of disulfide groups (S–S) (Amir et al. 2013).

rheology and quality of yeast–leavened gluten–free

Disulfide groups are formed due to the oxidation process

breads. Journal of Cereal Science 73, 10–17.

in sulfidryl groups which can affect the natural disulfide
formation (S–S) (Linares–García et al. 2019).

Bertrand B, Martínez–Morales F, and Trejo–Hernándes
MR. 2013. Fungal laccases: Induction and production.
Revista Mexicana de Ingeníería Química, 12 (3): 473–
488.
Borla OP, Motta EL, Saiz AI, Fritz R. 2004. Quality
parameters and baking perfoemance of commercial
gluten flours. Swiss Society of Food Science andTechnology, 37: 723–729.
Brijwani, K., Rigdon, A., Vadlani, P. V. (2010) Fungal
laccase: prduction, function and application in foodprocessing. Enzyme Research 2010, 1–11.
Everette, D. Jace., Q. M. Bryant., A. M. Green., Y. A.

Fig. 3 FTIR spectra of bread in variety treatment

Abbey., G. W. Wangila., R. B. Walker. A ThoroughStudy of Reactivity of Various Compound Classes
Toward the Folin–Ciocalteu Reagent. J Agric Food

Conclusion

Chem 58(14) (2014) 8139–8144.
Fakhri, Y., Amanidaz. N., Zandsalimi, Y., Dadar. M.,

Laccase can be pasteurized excellently without

Moradi. A., Moradi, B., Amirhajeloo, L. R., Keramati,

decrease their activity. Pretreatment of edible laccase was

H., Rafieepour, A.

able to reduce the value of bread hardness up to 9.55 N.

sodium bicarbonate consumption and human health: A

The functional group in the polymer pretreatment mixture

systematic review. International Journal of Medical

edible laccase formed a disulfide group formation at a(2016) Association between

Research and Health Sciences 5, 22–29.

wavelength of 450–500cm . The structure of gluten was

Koentjoro, M. P, Fitriana, M., Isdiantoni, Prasetyo, E. N.

formed in uniform pores compared to controls. Therefore,(2016) Enzymatic modification of cotton fiber for

applying laccase as natural food additives is able topromising smart medical based material. Proceedings

improve bread texture quality.

– 2018 1st International Conference on Bioinformatics,–1

Biotechnology, and Biomedical Engineering, BioMIC
Acknowledgements
The authors would like to thank the anonymous
reviewers for their insightful suggestions.
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Imran, M., Asad, M. J., Hadri, S. H., Mehmood. S. 2012.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 03

Production and Industrial Applications of Laccase

Boor. K. J. (2009) High temperature, short time

Enzyme. Journal of Cell and Molecular Biology 10, 1–

pasteurization temperatures inversely affect bacterial

11.

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Jo W–S, kang M–J, Choi S–Y, Yoo Y–B, Seok S–J, Jung
H–Y. 2010. Culture Conditions for Mycelial Growth of

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Kaur, S., Nigam V. (2014) Production and application ofin wheat bread making. Journal of Agriculture andlaccaseenzyme

in

pulp

and

paper

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A. (2011) Laccase: Microbial sources, production,and Social Sciences 2, 153–158.
Laohaprasit N and Sricharoenpong K. 2018. Development

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 04

Physical, Chemical and Sensory Properties of the White Breadfrom Acetylated Jack Bean Flour
Achmad Ridwan ARIYANTORO1*, Bambang Sigit AMANTO1, Rizki NOVITASARI2
1

Department of Food Science and Technology, Faculty of Agriculture, Sebelas Maret University, Jalan Ir.

2Alumni of Department of Food Science and Technology, Faculty of Agriculture, Sebelas Maret

Sutami 36 A, Kentingan, Surakarta, 577, Indonesia
University, Jalan Ir. Sutami 36 A, Kentingan, Surakarta, 577, Indonesia*

Corresponding Author: ridwan030586@gmail.com
SUMMARY

White bread is the preferred type of bread and it can be consumed by a variety of ages. Production of white bread inIndonesia is increasing every year. The main ingredients of white bread are wheat flour. Unfortunately, wheat could not
grow very well in Indonesia. It will affect the increment of the number of imports in wheat flour. Therefore, it is necessary
to substitute wheat flour with the others. The alternative is jack bean flour, which has been made from local commodities
and it has high carbohydrate and protein content. Native jack bean flours still have weaknesses such as unstable viscosity
on high temperature and shear stress, high retrogradation and syneresis. The modification can overcome these problems,
one of the methods is acetylation process.
The aim of this research was to determine the selected formula of white bread, which has good sensory properties.
Physical properties (baking expansion) and chemical properties (moisture, ash, fat, protein, carbohydrates and saltcontent) of white bread using acetylated jack bean flour (AJBF) also were investigated. The results show white bread withgood sensory is F2. It has baking expansion (126.35 %), moisture (37.86 %wb), ash (1.93 %db), fat (2.70 %db), protein(5.68 %db), carbohydrate (52.26 %db) and salt content (1.88 %db). The results suggest that 10% of AJBF can be used inbread making and it has higher protein content than wheat bread (control).
Introduction

and taste. Therefore, it need to improve the functional
properties with modification.

The production of white bread in Indonesia is

Starch modification is a method for improving theincreasing every year. According to Setyawan (2006), thefunctional properties of native starch. There are many

production of white bread increased from 2000 to 2003

modification methods have been used to increase starchreached 25.106.495 kg. The main material of white bread

properties, which can be divided into physical, chemical,

is wheat flour. Unfortunately, wheat cannot be cultivated inand enzymatic treatment (Zavarese and Dias, 2011). One

Indonesia, so Indonesia have imported from abroad. Wheatof chemical modification method is acetylation. Starchflour imports in 2014 reached 756,241 tons. Therefore, it

acetate is widely used in the food industry because it

needs a substitute flour with other flour in making bread toincreased viscosity, solubility compared with native starch.

reduce wheat imports One of flour which it is can be used

The objective of the research is to investigate the effect ofas substitute is jack bean flour.

acetylated jack bean flour on the properties of the white

Jack bean has high potency because it has highbread.

nutrition such as protein, carbohydrates, amino acid nonessentials and mineral. The carbohydrates content on jackMaterials and Methods

bean is very high (50-60%) (Olalekan, and Bosede, 2010).
It has also contained protein content approximately 29-

1. Material

32% (Doss, 2011). Also, it has high starch content, so it can

The main material in this research is jack bean flour,be changed to flour. The native jack bean flour has somewhich is obtained from the local market. The other material

weaknesses, such as low ability to swell and the unpleasant

for the bread making and the chemical material for theProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 04

aroma, taste and overall. The sensory properties of white

analysis.

bread were shown in Table 1. The results show that themore AJBF lead to decrease in sensory properties (colour,

2. Acetylated Jack Bean Flour (AJBF)Jack bean seeds were soaked in 1% baking soda

aroma, taste, texture and overall). The AJBF has darker

solution for 8 hours, and it was boiled for 5 minutes. It wascolour than wheat flour, so the use more AJBF cause theremoved the skin to obtain jack bean seed without skin. It

colour of white bread darker compare with white bread

was soaked again in 1% baking soda solution for 16 hours

from wheat flour. The dark color of AJBF of white bread(with water changes every 8 hours). It was cut and it was

make the panellist give low score (2.93) compare withdried in a cabinet dryer at 60 C for 7 hours. Then, it wascontrol (4.20).

o

The AJBF has unpleasant odour compare with wheat

grounded and sieved to obtain jack bean flour.
Jack bean flour was soaked with ratio of 1: 3 withflour. The increment of more AJBF in bread making leads

solution acetic acid (0.15% acetic acid in 450 ml of

to decrease the aroma score by panellist. The aroma score

distilled water). It was heated in a water bath at aof F1, F2, and F3 were 3.13, 2.63 and 2.50, respectively.

temperature of 45 C for 90 minutes. The water was

The other sensory property in this research is taste. The

separated from the solution. Wet flour was dried using a

AJBF has special taste with beany taste. White breadcabinet dryer at 60 C for 7 hours. It was grounded andusing more the AJBF could decrease panellist score.

oosieved to obtain AJBF.
Table 1 The effects of AJBF on sensory properties of whitebread

3. Bread making
All ingredients (wheat flour (WF), AJBF, milk

SampleColour

Aroma

Taste

TextureOverallpowder, yeast, bread improver, water, sugar and salt) was

F0

4.20a

3.93a

4.23a

3.90a

4.23a

mixed

are

F1

3.83ab

3.13b

3.20b

3.70b

3.37b

F1:100%WF;F2:90%WF:10%AJBF;F3:80%WF:20%AJ

F2

3.47b

2.63c

3.00b

3.27bc3.20bc

BF;and F4:70%WF:30%AJBF). It was kneaded until

F3

2.93c

2.50c

2.77b

3.00c

2.90c

with

mixer.

The

formulaofbreadssmooth, fermented and baked at 180 C for 20 minutes.
oValues represent the mean of triplicate measurements ± SD
(Standard Deviation). Means within columns with

4. Sensory test
The sensory test was done using scoring method withpanellist. Panellists give score on white bread parameter:
colour, aroma, taste, texture and overall. They give scorewith range 1 to 5, which is 1 is disliked a lot and 5 is liked
a lot.
5. Baking expansion test
The baking expansion of white bread was calculated
by measuring the volume before and after baking process.
The volume of white bread was performed by measuring
the surface area of the sliced bread.
6. Statistical analysis
Data were analyzed statistically using the SPSS
Software (Version 12, IBM, USA). Analysis of varianceand DMRT tests with a significance threshold of p < 0.05
were used to compare the differences among samples.
Results and Discussion
1. Sensory properties
Sensory analysis was carried out in colour, texture,different letters are significant.
Score : 1 = disliked a lot, 2 = disliked, 3 = neutral, 4 = liked,
5 = liked a lot.
Sample :F0 : 100% wheat flour (WF)
F1 : 90% WF : 10 % AJBF
F2 : 80% WF : 20 % AJBF
F3 : 70% WF : 30 % AJBF
The texture of white bread from AJBF has harder thanwhite bread. The AJBF has not gluten content comparewith white bread. It cannot hold the gas from fermentation
stage in the bread making. Therefore, the texture of the
AJBF white bread has denser and harder compare withwheat flour. Affandi (2016) reported that brownies withmodified jack bean flour lead to decrease the texture. The
decrease in texture due to wheat flour has high water
absorption capacity. In other hand, another flour has low
water absorption capacity, so in the baking process
resulted in more water evaporation lead to denser texture(Dahlia, 2014).
The panellist score in overall parameters shows theProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 04

same trend with the other parameter of sensory properties.

part of wheat protein which when hydrated cannot be

The more AJBF lead to decrease overall score compare

dissolved in water. In addition, gluten is a wheat proteinwith white bread. But, the F2 has better sensory properties

which is very important role in the final results of thecompare with F2 and F3 based on the results of sensory

dough.

properties.
3. Chemical properties
From the sensory and physical properties, F2 has

2. Physical Properties
Baking expansion test was used in this research forchosen for chemical analysis. The result of chemical

physical properties. The baking expansion of white bread

properties of white bread was presented in Table 3. The

was calculated by measuring the volume before and after

moisture, ash, fat, protein, carbohydrate and NaCl content

baking process. The baking expansion of white bread waswere investigated. The results suggest that moisture, ash,presented in Table 2.

fat and NaCl were not significantly differenced with F1.

The baking expansion is closely related to the abilityof the dough to form and hold the gas produced during

Table 3 The effects of AJBF on chemical properties offermentation. The most important component of wheat

white breadflour is gluten, which affects the elasticity of the dough andSampleMoist.

Ash

Fat

Protein

Carbo.

NaCl

support viscoelastic properties. The substitution of AJBF

F032.29a

2,32a

3,57a

4,55a

57,10a

2,03a

F1

37,86

1,93a

2,70a

5,68b

52,26b1,88a

has significantly different effect on the baking expansionof white bread.

a

Values represent the mean of triplicate measurements ± SD

Table 2 The effects of AJBF on baking expansion ofwhite bread
SampleBaking expansion (%)

F0157,78aF1

126,35bF2

112,41bF391,50cValues represent the mean of triplicate measurements ± SD
(Standard Deviation). Means within columns with different
letters are significant.
Sample :F0 : 100% wheat flour (WF)
F1 : 90% WF : 10 % AJBF
F2 : 80% WF : 20 % AJBF
F3 : 70% WF : 30 % AJBF

(Standard Deviation). Means within columns with different
letters are significant.
Sample :F0 : 100% wheat flour (WF)
F1 : 90% WF : 10 % AJBF
F2 : 80% WF : 20 % AJBF
F3 : 70% WF : 30 % AJBF

F2 has significantly different in protein content(5.68%) higher than F1 (4.55 because the AJBF has higher
protein compared with wheat flour. The result in agreementwith Ariyantoro (2014), the protein content of modified
jack bean flour has higher than wheat flour. If the AJBF
was used in the white bread-making, so white bread has
higher protein content compare with wheat bread.
Conclusion

It can be seen that the greater of AJBF, the baking
expansion of white bread will be decreased from 157.78%
to 126.35%. This is caused by a decrease in the gluten
content in the white bread dough. If the gluten content in
the dough was reduced, the development of the dough is
also getting smaller. Gluten was only obtained from wheat
flour because the AJBF does not have gluten. Gluten
functions to keep the dough steady and can hold CO2 gas
during the fermentation process lead to the dough can
expand. that the reaction of gliadin and glutenin with water(hydration) will form an elastic mass called gluten which
is a gas-binding (Bennion and Hughes 1970). Gluten is a

The making of white bread using AJBF were
investigated. More concentration of AJBF in the breadmaking lead to decrease sensory and baking expansion
properties. 10% AJBF of white bread has not significantly
different with wheat bread in moisture, ash, fat and NaCl.
However, the use 10% AJBF in the white bread making has
successfully increased protein content compare with wheat
bread.
References

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 2 – Physical Properties of Food –: 04

Affandi, D.R., Ariyantoro, A. R., Khairini, R. S. (2016)
The Effects Of Adding Modified Jack Bean Flour(Canavalia Ensiformis) As Wheat Flour Substitution
On The Chemical, Physical, And Sensory Charactreristics of Baked Brownies. Jurnal Teknosains Pangan,
5.
Ariyantoro, A. R., Rachmawanti, D., Imroah, I. (2016)
Physicochemical characteristics of modified jack beanflour (Canavalia ensiformis) with various lactic acidconcentration and soaking time. Agritech, 36, 1–6.
Bennion, M.,

Hughes, O. (1970). Introductory Foods, 6thedition. Collier Macmillan Publisher. London.
Dahlia, Lies. 2014. Hidup Sehat Tanpa Gluten. Alex Media
Komputindo. Jakarta.
Doss, A., Pugalenthi, M., Vadivel, V. (2011) Nutritional
evaluation of wild jack bean (Canavalia ensiformis
DC) seeds in Different locations of south India. World
Apllied Sciences Journal 13, 1606–1612.
Olalekan, A. J., Bosede, B. F., (2010) Comparative Studyon Chemical Composition and Functional Properties ofThree Nigerian Legumes (Jack Beans, Pigeon Pea and
Cowpea). Journal of Emerging Trends in Engineeringand Applied Sciences (JETEAS) 1, 89–95.
Setyawan, H. (2006) Analisis Sikap dan Preferensi
Konsumendalam

Pembelian

ProdukBakeryTradisional Kartika Sari Bandung. Bachelor Thesis.
Bogor Agricultural Institute.
Zavareze, E.D.R., Dias, A.R.G. (2011) Impact of heatmoisture treatment and annealing in starches: A review.
Carbohydr. Polym. 2011, 83, 317–328.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 01

Metabolomics and Bioactivity Profiles of Torbangun (Plectranthus amboinicus)
Grown in Japan and in Indonesia
Nancy Dewi YULIANA1*, Muhammad Anwari SUGIHARTO1, Hanifah Nuryani LIOE1,
Masao GOTO2, Yuko Takano ISHIKAWA2
1

Department of Food Science and Technology, Bogor Agricultural University, IPB Dramaga Campus,
Jalan Raya Dramaga, PO BOX 220, Bogor, Indonesia

2

Functionality Evaluation Unit, Food Function Division, Food Research Institute, NARO, 305-8642 Kannondai 2-1-12, Tsukuba Ibaraki Japan*

Corresponding Author: nancy_dewi@ipb.ac.id; juliana.luthfia@gmail.com
SUMMARY

The leaves of torbangun (Plectranthus amboinicus, PA) are commonly consumed as vegetable in North Sumatra. It
is traditionally used to stimulate breastmilk in breastfeeding women. Recent study showed that PA have antidiabetic andantioxidant activities. NMR-metabolomics was used in this study to show phytochemicals variation between PA grownin Japan and in Indonesia since environmental factors might influence the plant’s secondary metabolite profile and theirhealth beneficial properties. The results showed that flavonoids and triterpenes were among discriminating factors of thevariation between the two groups. Targeted comparative analysis of PA specific flavonoids concentration using a validated
HPLC-MWD method showed that Japanese samples contained higher concentration of total flavonoids than that ofIndonesian samples. The Japanese samples contained higher amount of luteolin and apigenin than Indonesian samples.
Eriodyctiol was detected only in Indonesian samples. On the contrary, more intensive DPPH reduction and α-glucosidase
inhibition activities were found in the Indonesian samples. Thus, the flavonoids might not the only group of compounds
related to the bioactivity, but it should be confirmed by further research targeting other groups of compounds such astriterpenes.
Introduction

Materials and Methods

Plant metabolites has provided as an incomparable

1. Single solvent plant extraction method

source for bioactive compounds. Plectranthus amboinicus

All plant are freeze dried, powdered and stored inLour., or being famous with its local name, Torbangun, isfreezer prior analysis. About 100 g of each are sonicatedone of Plectranthus species commonly used as vegetableusing 80%and condiment in North Sumatra Indonesia. In this region,

rotavapor to remove the solvent. This step will be

it is also traditionally used to stimulate breastmilk

conducted in Indonesia.

MeOH for 30 minutes, filtered and put inproduction of breastfeeding women (Damanik, 2006). This
species was also reported to show) anti-inflammatory andantitumor activities (Gurgel et al. 2009).

However,

2. α-glucosidase-inhibition assay in-vitro
α-glucosidase

inhibition

assay

is

conducted

different climate in the place where the plants are grownaccording to method described by Sancheti et al. (2009)

might affect the active compounds responsible for theusing 96 wells plate and Elisa Reader.

abovementioned Torbangun functional properties.
In this research, NMR metabolome profile ofTorbangun grown in Japanese and Indonesia werecompared. Their antioxidant and α-glucosidase inhibition
activities were also measured to see whether the different
in the metabolome lead to activities variations.

3. Total flavonoids content as described by Ahn et al.
(2007)
To 0.5 ml of sample extract was added 0.5 ml of 2%
AlCl3–ethanol solution. After 1 h at room temperature, theabsorbance was measured at 420 nm. EEP samples were
evaluated at a final concentration of 20 lg/ml. Total

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 01

flavonoid contents were calculated as milligrams per gram

Torbangun had better antioxidant and anti-diabetic

quercetin equivalents (mg/g QE) from a calibration curve.

properties than its Japanese counterpart.

Analysis was performed in 5 replicates, each in duplo.

both samples is presented in Table 1. The difference could

4. Comprehensive extraction for metabolomics study
Extraction was performed in a stainless-steel column(5.0 cm length, 2.0 cm diameter) according to a method
described by Yuliana et al. (2011). The mixture of 1.0 g of

IC50 value forbe attributed to the differences of phytochemicals due tothe different in the climate where the plants grown. The
OPLS-DA analysis of NMR data showed that Japanese andIndonesian samples are well separated (Fig. 1), whichmeans that their metabolome profiles indeed significantly
different.

dried powdered sample and 5 g of methanol washed 20-35

Flavonoids profiling for these two samples were thenmesh sea sand were packed into the column. Sea sand was

conducted using validated HPLC – MWD method and thethen again added until it reached the top of the column. The

results were presented in Table 2. The table shows thatcolumn was closed at the lower end with cotton andconnected to Jasco PU-980 Gulliver HPLC pump (Jasco,Tokyo, Japan). The combination of solvents used for the
extraction was n-hexane, acetone, and milliQ-water. The
solvents were delivered by gradient started from n-hexane,

Indonesian Torbangun had lower total flavonoids content
that Japanese Torbangun. The result of specific flavonoids
measurement indicated that Indonesian Torbangun had
lower luteolin but higher eriodictyol than Japanese
Torbangun, while the amount of apigenin of both samples
was almost similar.

acetone, then water. Extraction was performed for 75 min

From these results (Table 1 and Table 2), we mightwith solvent flow rate of 3 ml/min. The fractions wereassume that the compounds responsible for antioxidant andcollected in 10 ml tubes every 5 min with an automatic
fraction collector. A total of 15 fractions were obtained at
the end of the extraction. Three replicates were made witheach extraction. Every fraction was then dried using

α-glucosidase inhibition activity could be specificflavonoids which is only present in Indonesian sample,
such as eriodictyol, or probably non-flavonoids compound,which should be confirmed further in the next experiment.

vaporizer (Eyela Centrifugal Vaporizer CVE-200D, Tokyo
Rikakikai Co., Ltd., Japan).
5. NMR measurement
Dried methanolic extracts of each plants, aredissolved in methanol-d6 subjected to 1H NMR
measurement. NMR spectra were recorded according tomethod described previously by Kim et al. (2010) which issuitable for metabolomics-based experiment. The 1H NMR
spectra were automatically reduced to ASCII files.
Bucketing was performed by AMIX software (Bruker,
Karlsruhe, Germany). Spectral intensities were scaled tototal intensity and reduced to integrated regions of equal
width (0.04) corresponding to the region of δ -0.5 – 10.0
ppm. Chemical shift of NMR solvent and water are

Fig. 1 OPLS-DA Score plot of 1H NMR profile ofTorbangon grown in Japan (1) and Indonesia (2)Table 1 Comparison of antioxidant and α -glucosidase
inhibition activity of Indonesian and Japanese
Torbangun
SampleIC50 (µg/mL)
DPPH
AGI*
14.4 ± 1.2b
1181.9 ± 113.5b

excluded. The data was analyzed with OPLS-DA

Indonesia(Orthogonal projection to the least square-discriminantJapan

24.0 ± 0.3a

4451.4 ± 290.0a

analysis) using SIMCA ver. 13 (Umetrics, Umea Sweden).

Vitamin C

1.6 ± 0.3c-

Results and Discussion
The results of this study showed that Indonesian

Acarbose
0.02 ± 0.001c*AGI = α -glucosidase inhibition activity All value aremeans from 3 replications. Different letter in samerow shows significant differences (p = 0.05)

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 01

Journal of Pharmacology and Toxicology 4(1):8-11.

Table 2 Flavonoid profile of Indonesian andJapanese Torbangun (Api=apigenin, Lut = luteolin,
Er = eriodictyol, TF = total flavonoid)
SampleIndonesia

Flavonoid Concentration (µg/g)
Api
Lut
Er
TF*
571a
584b
4547
4146b

Japan

532b

1100.6a

Yuliana, ND., Khatib, A., Verpoorte, R., Choi, YH (2011)
Comprehensive extraction method integrated with
NMR metabolomics. A new bioactivity screening
method for plants: Adenosine A1 receptor binding
compounds in orthosiphon stamineus benth. Analitycal
Chemistry 83(17), 6902-6906.

nd

7934 a* in µg quercetin/g. All value are means from 3
replications. Different letter in same row shows
significant differences (p = 0.05)
Conclusion
NMR based metabolomics and targeted HPLC
analysis revealed that bioactive compounds content ofTorbangun leaves grown in Japan and Indonesia aresignificantly different, at least for several flavonoids
measured in this study. The differences lead to different
level of DPPH antioxidant and α -glucosidase inhibition
activity. Further research on the detail profiling ofcompounds other than flavonoids would be interested to
conduct.
Acknowledgements
The authors would like to thank the Ministry of
Research Technology and Higher Education of the
Republic of Indonesia for funding this research underInternational

Collaboration Competitive

Grant

No.

1572/IT3.11/PN/2018. We would also like to acknowledge
the UNU-KIRIN Fellowship Follow-up Grant for 20162018 for partial financial contribution to this study.
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Zhu F, Nakayama T. (2007) Antioxidant activity andconstituents of propolis collected in various areas ofChina. Food Chemistry 101 1383–1392.
Gurgel APAD., da Silva, JG., Grangeiro, ARS., Oliveira,DC., Lima, CMP., da Silva, ACP., Oliveira, RAG.,
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(2009)

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antiinflammatory and antitumor activities of the leaves
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amboinicus (Lour) Spreng(Lamiaceae). Journal of Ethnopharmacology 125: 361363.
Sancheti, S., Seo, SY. (2009) Chaenomeles sinensis: apotent α- and β-glukosidase inhibitor. AmericanProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 02

Evolving Potential Functional Foods to Meet Modern Needs
in Commercial Product
Irmanida BATUBARA1, 2*, Susi INDARIANI1, C. Hanny WIJAYA1,3, Katrin ROOSITA1,4
1

Tropical Biopharmaca Research Center, Bogor Agricultural University, Bogor, Indonesia

2

Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor, Indonesia

3

Faculty of Agricultural Technology, Bogor Agricultural University, Bogor, Indonesia

4

Faculty of Human Ecology, Bogor Agricultural University, Bogor, Indonesia*

Corresponding Author: ime@apps.ipb.ac.id
SUMMARY

Over the last decade, demand for “healthy” foods and beverages has increased in many parts of the world. Thisphenomenon shows that consumers nowadays have the tendency to go back to nature, including the use of functional
foods for disease prevention and treatment. Indonesia is known for its local wisdom with functional food potential, suchas ethnobotanically valuable plants, traditional foods and traditional drinks or ‘jamu’. Functional foods believed topromote general health and reduce the risk of diseases are becoming increasingly popular. The development of theseproducts is a key research priority for functional foods development and a challenge for both industry and science sectors.
The paper will highlight the findings obtained during the product development starting from the idea, invention and into
the ready to market product. Several studies were carried out in the scientific investigation contexts of functional fooddevelopment, including Java Tea based functional drinks, cajuputs candy, galohgor cookies and powder drinks, and red
fruit.
physiological functions, proven to be harmless andIntroduction

beneficial to health (BPOM, 2005). The development ofOver the last decade, demand for “healthy” foodsthese products is a key research priority for functionaland beverages has increased in many parts of the world

foods development and a challenge for both industry and(Ozen et al., 2012) and the diffusion of functional foods

science sectors. The paper will highlight the findings

throughout the market has blurred the distinction between

obtained during the product development starting from thepharma and nutrition (Eussen et al., 2011). Thisidea, invention and into the ready to market product.

phenomenon shows that consumers nowadays have theSeveral studies were carried out in the scientifictendency to go back to nature, including the use ofinvestigation contexts of functional food development,functional foods for disease prevention and treatment.

including Java Tea based functional drinks, cajuputs candy,

Indonesia is known for its local wisdom withgalohgor cookies and powder drinks, and red fruit.

functional food potential, such as ethnobotanicallyvaluable plants, traditional foods and traditional drinks or‘jamu’. These foods have health benefits, beyond thebasic nutritional functions. In general, functional food is

Java Tea Based Functional Drinks
Many of various traditional drinks, we called herbal

food that not only provides essential nutrients to the body,

drinks or ‘jamu’, are produced from traditional medicinal

but also promote general health, provides a protective effectingredients such as Java tea. Research on Java tea-basedon the body (or even healing) for some diseases and reduce

functional drinks has shown promising health benefits suchthe risk of diseases. The Indonesia National Agency of Drugas antioxidant (Wijaya et al., 2007). A Java tea-based

and Food Control define functional foods is processed

functional drink prepared using fresh ingredients was

foods containing one or more functional components

reported to possess 532.307–726.818 ppm ascorbic acidwhich

equivalent antioxidant capacity (AEAC) (Wijaya et al.,

based

on

scientificstudieshavecertainProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 022007). Furthermore, this drink has antihyperglycemic

microencapsulated beverages in suppressing the reduction

activity (Wijaya et al., 2011; Indariani et al., 2014). Thisof feed intake and body weight, decreasing blood glucose

functional drink was able to restrain the increase of bloodlevel (7.98 %). Nano encapsulated beverage was better inglucose level in diabetic mice, while it didn’t cause aprotecting the viability of Langerhans (49.09 %) and β celldecrease of blood glucose level in the normal mice.

(32.50 %) than microencapsulated and ready-to-drink

Beverage formulas with the addition of ginger extract had

beverages. This encapsulated Java tea-based beverages cana steadier hyperglycemic ability because it was able tointervention attenuated the formation of malondialdehyde

lower the blood glucose level for 20 days during the study

in diabetic rats from 36.63% to 51.95%. In addition, micro-

period. The beverage provision to diabetic mice was able

and nano-encapsulated drink suppressed the fluctuation of

to restrain further damage on islet of Langerhans.

blood glucose level and body weight. In vitro assessment

Langerhans in diabetic mice, which were given beverage

showedwith or without the addition of ginger extract or insulin,

suppressed the formation of MDA at 5.25% and 72.16%,

has a larger amount and larger size of Langerhans whenrespectively (Naibaho et al., 2019).

that

micro-

andnano-encapsulated

drink

compared to positive control mice (diabetic), although the

However, utilization of fresh ingredients is often

amount is still less than the amount of Langerhans fromlimited by availability and short shelf-life. To produce thisnormal mice group. In the diabetic mice group treated withtea on an industrial scale, extracts of Java tea, sappan wood,

insulin, an improvement of the Langerhans were observed

ginger, and curcuma were modified using simplicia instead(Indariani et al., 2014). Wijaya (2010), also reported thatof fresh ingredients. Modification of extraction processes

ginger extract can increase the glucose uptake by isolate

and ingredients may impact the functional efficacy of thehemidiaphragm cells. It is postulated that gingerol fromproduct as well as its sensory properties. Formula

ginger extract could increase the insulin sensitivity upon

optimization has been done by Wijaya et al. (2018) for the

glucose, and improve the hyperglycemia condition (Sekiya

modified Java tea-based functional drink in terms of bothet al., 2004).

maximum antioxidant properties (efficacy) and sensory

Java Tea-based beverage in ready-to-drink (RTD)

properties (color, taste, and aroma). Formula optimization

type has a limitation in distribution. Converting the ready-was performed by response surface methodology (RSM)

to-drink beverage into effervescent, which is simple andusing Design Expert® 7 software. Java tea-based

easy carrying, using nanoencapsulation technology alsofunctional drink was successfully formulated from

improve the shelf life (Chusen et al., 2012). Encapsulation

simplicia extracts with optimum sensory and antioxidant

protects bioactive compounds from degradation byproperties. The antioxidant activity of the optimum

hindering their direct exposure to adverse environments,

formula was 335.69 ± 48.30 ppm ascorbic acid equivalentsuch as the light, oxygen, chemicals, etc. A study has

antioxidant capacity and the sensory acceptance scores (onproved that encapsulation can increase bioavailability ofa scale of 1–7) were 5.6 for color, 5.8 for taste, 5.2 forbioactive compounds (Fathi et al., 2014). Another study

aroma, and 5.2 for overall attributes (corresponding to ‘likehas proved that encapsulation of active ingredients withinslightly’ to ‘like moderately’). Simplicia extracts inpolysaccharide is a powerful means of protecting themappropriate combinations can be used instead of freshfrom degradation and for enhancing their bioactivity

ingredients for the large-scale production of a Java tea-

(Putheti, 2015). Chitosan is one of the polysaccharides thatbased functional drink while maintaining efficacy andis used in nanoparticles-based playing a vital role inpalatability (Wijaya et al., 2018).

sustained and targeted drug delivery, nowadays.
Effervescent Java Tea-based functional beverage

Cajuputs Candy

using nanoencapsulation technology has been reported by
Wijaya et al. (2013). It showed that nanoencapsulation

Cajuputs candy includes medicated sweets in the

could reduce the size of particle (223.4±23.8 nm), with theform of "hard candy" with mixed flavor formulations

polydispersity index (0.747), and suppressed the bitter

which consist of primary flavor (cajuput oil) and secondary

taste intensity (2.74) of the Java tea-based beverage. It had

flavor (crystalline menthol, cinnamon oil and peppermint

preferred taste than that the non-nano encapsulated one.

oil). Hard candy is the name for candy that is processed at

Rekasih et al. (2018) has been reported nano encapsulated

a temperature of 140-150 °C, hard textured and has a clear

beverage was more effective than the ready-to-drink andappearance or like a crystal. Cajuputs candy can relieve theProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 02

throat and maintain homeostasis of the mouth flora. Theof 0.37 g/kg body weight of rats that consumed Galohgor

main composition and process of making candy original

decreases the lower levels of T3 and T4 and increases the

cajuputs refers to patent that was granted in 2000 (ID 0 000

body antioxidant levels (Roosita 2003; Leatemia 2010). It

385 S) with slight modifications, where the composition

can also lower blood glucose levels, increases serum adi-consisting of sucrose (granulated sugar), glucose (DE 42)

ponectin and reserves the liver glycogen level in theand water, additional flavor of pure cajuput oil, peppermint,

streptozotocin-induced mice (Firdaus 2016). This effect ismenthol, and maltol (Wijaya et al., 2000).

thought to be caused by the activity of various bioactive

The formula candy has a balance between thesubstances contained in the galohgor.

superiority of taste (sensory reception) and active

The galohgor is traditionally made by roasting andphysiological ability with its ability to inhibit S. mutans

pounding method to produce a coarse powder consumed

biofilm formation which can cause dental caries anddirectly (Roosita & Wientarsih 2013). The crude herbal

suppresses viability of C. albicans which can infectmixtures are generally difficult to consume in the long termwounds in the oral cavity (Wijaya et al., 2012). The

because not everyone is accustomed to consume herbs andprovision of flavor components of cajuput and peppermint

to accept the taste in its original form (Goel & Kaur 2013).

oil could produce synergistic effects compared to single

Therefore, the galohgor cookies and powder drinks are

flavor component. The addition of cajuput oil at 0.6% has

made to improve the acceptance and flavor of the productbeen able to inhibit the viability of C. albicans. The

so that it can serve as an alternative choice for functional

activities of the cajuput oil showed positive correlation

food products. Cookie form is chosen because it is one ofwith its concentration (Wijaya et al., 2014).

the most popular snacks in Indonesia. While powder drink

While cajuputs candy non sucrose with the main

has a longer shelf life than any other drink. Setyaningsih etcomposition is isomaltose, acesulfame-K, water, cajuputal. (2017), reported that galohgor products in the form ofoil, peppermint and honeydew flavor (Wijaya et al., 2014).

cookies and powdered drinks significantly decreased

Because it uses the main ingredients isomaltose and water,oxidative stress in type 2 diabetes mellitus. This researchthen in addition to low-calorie cajuputs candy non sucrose

showed that adjusted mean changes of antioxidant activity

also contains ingredients that do not cause dental caries, sowith DPPH method for intervention group compared withthat it is potentially used as an oral health care product.

control group were +4,07% and +1,78 % (p>0,05), andCurrently the two variants of Cajuputs Candy are being

MDA were -0,56 nmol/ml and +7,05 nmol/ml(p<0,05).

investigated for their ability to maintain balance

Damayati et al., (2018) also reported that theadministration of cookies and galohgor powder drinks(homeostasis) of the mouth flora.

tends to lower total cholesterol, triglyceride levels, LDL
levels, and maintain HDL levels better than the control

Cookies Galohgor and Powder Drinks

group. However, the differences were not statisticallyGalohgor is one of the traditional herbal medicine insignificant between the groups. Thus, intervention package

West Java, Indonesia. The traditional herb was prepared

containing cookies and galohgor cookies and powder

from 56 kinds of material consist of 38 medicinal plants, 5drinks as much as 2 g of Galohgor extract per day for 38

herbs and spices, 7 nuts and 6 rhizomes in composition.

days is suggested for type 2 DM patients because it has

This traditional herb commonly consumed by postpartum

health benefits specially to decrease VAT to help prevent

mothers in the Sundanese tribe in West Java. Roosita et al.

DM complications.

(2003) reported that galohgor has efficacious for increasemilk breast production and health maintain. It contains

Red Fruit (Pandanus conoideus Lamk)

various important minerals, such as Fe, Zn, Cu, and Mn. In
addition, it also contains many other functional compounds
such

as

antioxidants,

alkaloids,

triterpenoids

Buah Merah or red fruit (Pandanus conoideus

andLamk) is an indigenous plant from Papua Island thatglycosides as well as steroids, flavonoids, and saponins

belongs to genus Pandanus. This plant spread in Papua,(Roosita et al. 2014).

New Guinea, and began planted in some areas in Indonesia

Besides, it was also reported that galohgor have

(Hadad et al., 2006). Papuan people usually utilize redlong been studied as therapeutic agents in DM manage-

fruits as food and consume directly, as sauce or use the oilment (Ghorbani 2014; Roosita et al. 2006). Administration

for so much purpose. Red fruit oils used as natural products

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 02

for medicine (Arumsari et al., 2013; Wijaya and Pohan,(Pandanus conoideus Lamk) from Hexane and2009).

Chloroform fraction. J. Food Pharm. Sci 1, 30-34.
Local people in Papua extracted the oil from theBPOM. The Indonesia National Agency of Drug and Foodfruits by heating method and use for some purpose likeControl. 2005. PerKa BPOM No. HK 00.0s.52.06

food and medicine (Hening et al., 2008). Red fruit oils has

Tahun 2014 Tentang Ketentuan Pokok Pengawasan

high level of monounsaturated fatty acid (oleic acid), β-Pangan Fungsional (Main Provisions for Functional

carotene,

Food Monitoring). Jakarta, Indonesia: BPOM.

β-cryptoxanthin,compound andα-tocopherol,

phenolic

flavonoid that potentially become

Cushen, M, Kerry, J, Morris, M, Cruz-Romero, M,

functional food and medicine. Red fruit oils empiricallyCummins, E. (2012) Nanotechnologies in the foodused by local people as natural medicine for many diseases

industry: Recent developments, risks and regulation.

such as cancer, rheumatoid arthritis and stroke, HIV Aids

Trends in Food Sci & Tech 24, 30-46.

(Kie et al., 2009; Rohman et al., 2012; Mun’im et al., 2006).

Damayati, RP, Roosita, K, Sulaeman, A. (2018) Effect of

The potential plant as natural products has to be

Galohgor Cookies and Powder Drinks on Visceralfollowed by the data of safety level of the product. The

Adipose Tissue and Lipid Profile in Patients with Typesubstance and chemical compound of plant products may

2 Diabetes Mellitus. J. Gizi Pangan 13(3), 137-144.

result in chronic toxicity or acute toxicity. Therefore, theEussen, SR, Verhagen, H, Klungel, OH, Garssen, J, vaninformation about the toxicity of red fruit oils is very

Loveren, H, van Kranen, HJ, Rompelberg, CJ. (2011)

important. Wismandanu et al. (2016), reported that red

Functional foods and dietary supplements: products at

fruit oils can be categorized as Category 5 GHS (Globallythe interface between pharma and nutrition. Eur J

HarmonizedPharmacol 668, S2–9.

Systemfor

ChemicalClassification

Substances and Mixtures) as practically non-toxic

Fathi, M, Mart, AI, McClements, DJ. (2014) Review

materials, based on the 2001 OECD acute toxicity. No

Nanoencapsulation

animals showed toxic symptoms in 300 and 2000 mg/kg

carbohydrate based delivery systems. Trends in Fooddosing group. One animal in 5000 mg/kg BW dosing group

of

food

ingredientsusing

Sci & Tech 39, 18-39.

had diarrhea one hour after administration. No animal deadFirdaus. (2016) Peran ekstrak nutrasetika galohgor untukin this experiment after 14 days observation. AST and ALT

mengatasi resistensi insulin pada tikus diabetes yangmean value for rats on 300 mg/kg BW, 2000 mg/kg BW,

diinduksi Streptozotocin (STZ) [tesis]. Bogor: Institut

and 5000 mg/kg BW dosing groups are 22.70±1:05 IU/L,

Pertanian Bogor.

24.15±8.89 IU/L and 24.54 ± 6.26 IU/L and 18.04±0.77

Ghorbani, A. (2014) Clinical and experimental studies on

IU/L, 19.69±3.08 IU/L, 16.78±1.60 IU/L, respectively. No

polyherbal formulations for diabetes: Current status

statistically significant difference of the value of AST andand future prospective. J Integr Med 12(4), 336-345.

ALT levels in each treatment group (p>0.05). Therefore, in

Goel, S, Kaur, T. (2013) Impact of hypoglycemic herbal

this research, red fruit oils no cause liver injury.

mixture based biscuits intervention on blood glucose
level and lipid profile of type 2 diabetic subjects.
IJFANS 2(2), 84-90.

Conclusion

Hadad, Atekan, M., Malik, A., Wamaer, D. (2006)
There is a lot of potential natural resources and local

Karakteristik dan potensial tanaman buah merah

wisdom that are potential as functional food. The(Pandanus conoideus Lamk.) di Papua, Prosiding

development of ethnobotanically valuable plants and

Seminar Nasional BPTP Papua, Jayapura 24-25 Juli

traditional products is a key research priority for functional

2006. Balai Besar Pengkajiandan Pengembangan

foods development. It’s a challenge for both industry andTeknologi Pertanian, Bogor, 243-255.

science sectors to study it scientifically, from the idea,

Minyak Buah Merah (Pandanus conoideus Lamk)

invention and into the ready to market product.

Terhadap Pertumbuhan In Vivo Tumor Kelenjar Susu

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based on Java tea (Orthosiphon aristatus) in

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pada tikus putih (Rattus sp.) [tesis]. Bogor: Program

ProductnAntioxidant

Activityand310-318.
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consumption of functional foods: a systematic review.

Pasca Sarjana, Institut Pertanian Bogor.
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Nutr Rev 70, 472–81.

hyperglycemic activity of encapsulated Java tea-based

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nutraceuticals and pharmaceuticals. e-Journal of Sci& Tech.

9(04), 088–095.
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Wijaya, CH, Halimah, Taqi, FM. (2000) Cajuputs candy

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 03

Evaluation of Whole Mitochondrial DNA Sequence in Healthy and Type 2
Diabetic Individuals with Reference to Bangladeshi Population
Sajoy Kanti Saha, Md Hasib, Ashish Das, Nafiul Huda, Tahirah Yasmin,
Md Ismail Hosen, A.H.M. Nurun Nabi*
Laboratory of Population Genetics, Department of Biochemistry and Molecular Biology, University of
Dhaka, Dhaka-1000, Bangladesh.
*

Correspondence to nabi@du.ac.bd
SUMMARY

Type 2 diabetes (T2D) is one of the global health burden and food is one of the modifiable risk factors of T2D. The
genes of the mitochondria are important to the energy production, both to ATP production and heat generation.
Mitochondrial DNA (mtDNA) variation is likely to contribute to an imbalance in cellular energy homeostasis, insulin
secretion and beta cell functions which in turn is linked to the risk of developing diabetes. We aim to explore the whole
mtDNA sequence in Bangladeshi healthy individuals and thus, compare mtDNA variation with that of diabetic patients.
A total of 26 pairs of primers were designed. mtDNA was extracted from a total of 29 unrelated Bangladeshi (thirteen
healthy and sixteen type 2 diabetic patients) individuals and sequences were obtained by Sanger sequencing platform.
Contigs were assembled and analyzed using Geneious software by comparing with the revised Cambridge reference
sequence of mtDNA (NC_012920.1). Haplogrep2 and Phylotree17 tools were employed for determining haplogroups.
MITOMASTER facilitates determination of total variants of each participant. Total 25 diverse haplogroups wereidentified. Haplogroup M and its subgroups were the most frequent (65.5%) followed by haplogroup R (24.2%), U (6.9%)
and C (3.4%). It was found that healthy individuals had a total of 879 variants (varied from 43-183). Of them, 11 werepredicted to be the most pathogenic while 18 were possibly pathogenic. On the other hand, out of 1139 variants (varied
from 45-117) found in diabetic patients, 3 were the most pathogenic and 9 were possibly pathogenic. We found 47 variants
unique to healthy individuals while 74 were unique to diabetic patients. Among these, frequencies of 8 unique variants(30%-54%) and 6 unique variants (31%-50%) were found to be the highest respectively in healthy individuals and diabetic
patients. We believe that our baseline data of whole mtDNA sequence of our population would facilitate further research
to validate frequencies of new variations unique to diabetes in diagnosing and investigating genetic linkages of noncommunicable diseases in Bangladesh.
Introduction

life

qualitycases.
According to International Diabetes Federation (IDF),

or

leads

to

Mitochondrion ismetabolism through glucose

death
involvedinsome

in energy

metabolism and insulin

425 million people world-wide are suffering from diabetes

secretion. Electron-transport chain within mitochondria

and among them, 82 million people resides in South East

synthesizes ATP that mediates insulin secretion from

Asian region. By the year 2045, the prevalence will be 151

pancreatic

million. Bangladesh is also one of the worst affected region

disturbances in insulin secretion are critical in thewhere in 2017, 6.9% of the Bangladeshi adults were

pathogenesis of T2D [6].

β-cells [5]. Beta

cell destruction andsuffering from diabetes [1]. It influences functions ofMany strains of mice and rats were used to study

different organs that either reduces life expectancy or leads

diabetes at genetic levels. For example, BHE/Cdb rat

to death [2.3]. Diabetes is a polygenic diseases and it is a

mimics the human phenotype with a mutation inphenotypes of >150 genotypes, which are characterized by

mitochondrial DNA. Studies of twins have shown that if

impaired glucose tolerance and impaired control of

one twin develops type 1 diabetes, the other twin has a 60%

intermediary metabolism [4]. The functions of different

chance of developing the disease. In contrast, with type 2

organs are influenced by the disease, which either reduces

diabetes, if one twin develops diabetes, the other has aProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 03100% chance of developing the disease [7,8]. A decrease

Materials and Methods

in mitochondrial function or mitochondrial DNA (mtDNA)
copy number has been correlated with insulin resistance

A total of 29 unrelated Bangladeshi individuals wereand dysregulated lipid metabolism [9-11]. Alterations ofenrolled. Among them, 13 were healthy individuals and 16

mitochondrial DNA has been found to be associated with

were individuals with type 2 diabetes. This study was

the risk of developing type 2 diabetes in different

approved by the ethical review committee of the Faculty of

populations with incompatible findings. A mutation,

Biological Sciences, University of Dhaka, Bangladesh.

3243A→G, within mtDNA has been found to be associatedUsing the levels of plasma fasting glucose (≥7.0 mmol/L)with maternally linked diabetes [12]. mtDNA G10398A

and levels of HbA1c (≥6.5%) set by the World Healthvariant in ND3 gene has shown association with type 2

Organization, patients with type 2 diabetic patients werediabetesthroughselected. Each individual was informed about the study andinteracting with other candidate genes [15,16]. A weakafter getting their consent, 5 mL of venous blood was taken

association of T2D with mtDNA variants in Tunisianin EDTA containing vacutainer tubes. Anthropometric andpopulation was reported but such association was missing

demographic data were obtained from the data available inwhen values were adjusted with age, gender and BMI. Also,

the structured questionnaire obtained during interview.

association of certain mtDNA genetic backgrounds

Mitochondrial DNA was extracted from each sample(haplotypes, haplogroups) with T2D was reported inaccording to the method described by Sajoy et al (2019)

Asians [17], but not in Caucasians [18,19]. Studies showed[33].

that mitochondrial C5178A contributes to human longevity(http://bioinfo.ut.ee/primer3-0.4.0/), 26 pairs of primers[20], has protective effects against oxidative damage [21],wereanti-atherosclerotic effects in diabetic patients [22], andReference Sequence (rCRS) of the Human Mitochondrial

decreases the possibility of lung cancer [23]. It has also

DNA (NC_012920.1). For each pair of primer, reaction

been reported that mtDNA polymorphisms are related toconditions were set and amplicons (sizes ranges frominterindividual functional variability in human cognition

542bp-891bp) were validated in 1.5% agarose gel[24], personality [25], athletic performance [26], andelectrophoresis. PCR products were cleaned directly or

longevity [27]. Mutations in mitochondrial DNA are also

from gel using the Wizard® SV Gel and PCR Clean-Up

associated with mitochondrial myopathy, encephalopathy,

System (Promega, USA) where necessary followed bylactic acidosis, and stroke-like episodes (MELAS) [28,29],

single pass Sanger Sequencing. Chromatograms werevariety of malignancies, including breast, colorectal,

analyzed using DNA Baser (v 4.36.0.2) and Geneious

ovarian, gastric, hepatocellular, kidney, pancreatic, thyroid,

software (v R11). MEGA (v 10.0.4) was used for aligning.

and prostate cancer [30]. Using complete mitochondrial

Haplogrep2 and Phylotree17 tools were employed forgenome sequencing, US Caucasian, Hispanic, and African

determining haplogroups. MITOMASTER available atAmericans were differentiated [31]. Thus, mtDNA has

https://www.mitomap.org/foswiki/bin/view/MITOMAST

enormous contribution in forensic purposes. Considering

ER/WebHome facilitates determination of total variants ofthe variation among different ethnic groups, theeach participant. Human mtDNA Genome Polymorphism

understanding of genetic diversity for specific populations

Database was used for the comparative analyses of theis mandatory, not only for research but for practicalvariants present in ancestral haplogroups and Asianapplications such as human identification [32]. However,

population.

[13,14]eitherindependently

or

Using

Primer3

designedby(v

0.4.0)

deployingweb

Revised

based

tool

Cambridge

information related to whole mtDNA sequence andvariants

arecompletely

missingfor

Bangladeshi

Results

population.
Thus, this study aims to i) identify all mtDNA

1. Haplogroup analyses

variants and probable potential mutations, ii) identify

One major M haplogroups and three sub-haplogroups

major haplogroups in Bangladeshi healthy individuals andR, U, C were respectively identified in 46.14%, 30.76%,

T2D diabetic patients, iii) compare mtDNA variants from

15.38%, 7.69% of the total healthy participants (Table 1).

type 2 Bangladeshi diabetic individuals with that of Asian

More analyses revealed that healthy individuals belonged

populations. In this report, we provide the results ofto the M64, M53b, M30c, M18, M5b, M2a and C7b

mtDNA analysis of 29 Bangladeshi individuals.

subgroups were originated from M haplogroup while restProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 03of them fit in R5a2b, R6a2, R6b, U2c’d and U2b2

respectively were found in 100% Asian people with T2D

subgroups originated from major N haplogroup. On thethat completely matched with the variants identified inother hand, 78.57% and 21.43% of diabetic individuals

Bangladeshi population with T2D. While SNP at position

belonged to M haplogroup and R sub-haplogroup. Of them6680 within NADH-dehydrogenase subunit 4 (ND4) was

subgroups M3a, M5a, M18, M34a, M35a, M38, M42, M45,

100% prevalent in Asian diabetic individuals, inM49c, M65 were from M haplogroup and subgroups R0,

Bangladeshi individuals with T2D, this SNP was missing.

R5a1, R6a were from R haplogroup.

Other SNPs were identified at varying frequencies.
However, though prevalence of SNPs at positions 4883,

2. Comparison of SNPs between Asian and Bangladeshi

5178, 8414, and 14668 in Asian diabetic population varieddiabetic population with respect to rRNAs

from 35%-40%, such variants in Bangladeshi population

A total of 41 single nucleotide polymorphisms were
found within the mtDNA sequences that encode for rRNAs

were not identified. Table 4 demonstrated the variants
between the two groups.

in T2D Asian population. For comparison, SNPs reported
more than >10% in the database were considered. A total

5. Comparison of SNPs between Asian and Bangladeshi

of 22 and 19 SNPs within mtDNA sequence encoding

diabetic population with respect to tRNAs

respectively 12s rRNA and 16s rRNA are reported among

According to database, 29 variants were found in type

Asians. While our sequence data revealed 40 SNPs in

2 diabetic Asian individuals within the mtDNA sequences

Bangladeshi population with T2D. According to databasethat encode 16 tRNAs at varying frequencies. In our(www.http://mtsnp.tmig.or.jp),

ofpopulation, 22 variants were identified within the mtDNA

variations were observed at positions 2706 (transition,

sequences that encode tRNAs for Thr, Arg, Met, Trp andA/G), 3106 (Deletion, C/-), 3010 (transition, G/A) within

Glu as shown in Table 5. Among them, the most variants

16s rRNA and 1438 (transition, A/G), 709 (transition, G/A)

were observed within the sequence that encode Arg inwithin 12s rRNA. In our population, all frequencies

Bangladeshi individuals with T2D compare to Asianmatched with that of the SNPs reported in the databasecounterparts.

highestfrequency

except the deletion mutation found at position 3106. The
data have been presented in Table 2.

Discussion

3. Comparison of SNPs between Asian and Bangladeshi

Though exact causative gene responsible fordiabetic population with respect to non-coding sequencesdeveloping or commencing diabetes is not identified yet

A total of 127 single nucleotide polymorphisms werebut multiple genes associated directly or indirectly with thereported in the database while in Bangladeshi population

development or with the risk of diabetes have been

67 SNPs have been identified (Table 3). For therecognized. Mitochondria, the power house of a cell andcomparative analysis, we considered the most prevalent

one of the central organs in energy metabolism in human

SNPs reported in the database from 47%-100%. Highest

body [34], became part of the cell through endosymbiosis,

frequency of variations were observed at positions 73, 263,

contains 16569 nucleotides containing circular DNA that

489, 16223, 16362, 16519 and all of them were transition

encodes for 13 proteins, 22 transfer RNAs (tRNAs) and 2

mutations.

ribosomal RNAs [35] , which is inherited maternally [36].
Thus, any mutation within mtDNA sequence related to

4. Comparison of SNPs between Asian and Bangladeshi

disease only follow maternal lineage not paternal [37].

diabetic population with respect to protein coding

Sanger sequencing is one of the most comprehensive, costsequences

effective and relatively faster methods of determining

According to database, Asian individuals with type 2

variants/mutations within a DNA sequence [38,39] .

diabetes had total of 351 SNPs within the protein coding

Capillarybased

Sangerregions of mitochondrial DNA. For the comparative

determining whole mitochondrial DNA sequence inanalysis, we considered the most prevalent SNPs reported

Bangladeshi

in the database from 35%-100%. SNPs at positions 4769,

associated single nucleotide polymorphisms or variants

7028, 11719, 14766, 15326 within the protein coding

indeed play pivotal role in understanding the pathogenesis

regions of ND2, COI, CytT71, CytbT194A and ND5,

of diabetes. In this regard, nuclear DNA has attained

individuals.

sequencetechnique

Identification

of

for

diseaseProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 03

more attention than mtDNA. However, due to lack ofof deletion mutation (C/-) at position 3106 within mtDNA

intronic sequences and protection of histones, mtDNA has

sequence that encodes 16s rRNA, transition mutations

got possibility of 6-17 fold higher rate of mutation than(A/G, C/T, C/A, C/T, C/T) respectively at positions 8860,

nuclear DNA [40] that may put mtDNA more vulnerable

4883, 5178, 14668, 8414 within non-coding regions of

towards its association with diseases. Thus, analyses of

mtDNA sequence are absent in Bangladeshi population

mtDNA to find out disease association should get similar

with T2D which were reported in Asian diabetic

priorities as it was achieved by nuclear DNA. We, for the

population. According to database, SNPs have been

first time, are comparing the whole mtDNA sequence ofidentified in 16 out of 22 regions within mtDNA that

individuals with T2D and healthy controls with reference

encode tRNAs for 20 amino acids while in our population

to Bangladeshi population using capillary based Sanger

SNPs were identified in regions that encode 5 tRNAs.

sequencing platform. We deployed primer walking

This study attempted to analyze whole mitochondrial

approach by designing 24 pairs of primers that generated

DNA sequence in Bangladeshi population using Sanger

overlapping regions between each of the amplicon. The

sequencing

primers were designed in such a way that each ampliconthroughput sequencing technologies may reveal more

overlapped at least 60 bases. As a result, contigs werevariants with lower frequency.

platform.

Furtherstudies

with

high

assembled with high confidence by removing first 15-40
Acknowledgement

nucleotides with poor quality.
mtDNA sequence

analysis

revealedvarying

frequencies of variants within the regions that encoderRNA, tRNA, coding sequence, non-coding sequenceboth in healthy individuals and patients with T2D. A totalof 4 and 2 different haplogroups were identified
respectively in healthy and diabetic individuals. It was
found that individuals with M haplogroup and its

This study was supported by the research grant
provided by the Ministry of Education, Government of thePeoples Republic of Banglade sh. Sajoy Kanti Saha is a
PhD student and recipient of PhD fellowship from
the Ministry of Science and Technology, Government of
the Peoples Republic of Bangladesh.

associated subhaplogroups are more prevalent in diabetic

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 04

Rice Quality Under Ratooning Farming System
KOMARIAH1*, Rahajeng Putu Widiani PRISWITA2
1

Faculty of Agriculture, Sebelas Maret University, Jl. Ir. Sutami No. 36A, Kentingan, Surakarta, 57126,
Indonesia

2

Master Program of Environmental Science, Graduate School, Sebelas Maret University, Jl. Ir. Sutami
No. 36A, Kentingan, Surakarta, 57126, Indonesia*

Corresponding Author: komariah@staff.uns.ac.id
SUMMARY

The increasing rice demand to meet the growing population need should pay attention to the natural resources
availability.

Producing rice with ratooning farming system could save time, water, seeds and labor resources, therefore

this system must be introduced and implemented worldwide with improved practices according to local condition. Veryfew studies reported that the quality of rice produced from ratooning system was not lower than the main crop. However,more related studies is urgent to support the policy decision on rice ratooning farming system.
Table 1 Rice (paddy production) over 3 decades at some

Introduction

continents and the world in tones
Rice is the staple food for most of the Asian people,

Region

1996

2006

2016

hence the rice production must be improved in the term ofAfrica15.944.498

21.989.568

32.497.773

quality and quantity. The world rice production in 2018

Americas27.219.877

33.684.603

36.029.484

was approx. 728 million tones, where 63% (approx. 459

Asia

521.318.379

580.605.576

667.932.238

Europe3.204.652

3.406.389

4.219.382

World

568.659.290

640.705.682

740.961.445

million tones) were produced in Eastern and Southeastern
Asia (IRRI (International Rice Research Institute), 2019).
The rice (paddy) production during 3 decades at eachcontinent (except Australia) and the world is presented inTable 1, showing the rice production increased year by year.
This is in contrast with the land conversion issues whichlead to the shrinking farm land areas. Green revolution,
rice breeding technology andthe genetic engineering tocreate high yielding potential rice seeds seemed to meet thechallenges on rice demand (Khush, 2005).
However, rice demand increase should be able tomeet the less land, less water, and less chemical or other
inputs due to competition with the growing population.
Less water availability and water scarcity has been thecritical issue of crop production under the climate change
condition across the world. Rice ratooning offers

thehigh efficiency of water, seeds and chemical inputs hence
lower the production cost (Negalur, Yadahalli, Chittapur,
Guruprasad, & Narappa, 2017).
Ratoon is the practice of farming to obtain a secondor more crop from tillers originating from the stubble ofthe harvested crop (Chauhan, Vergara, & Lopez, 1985),
with the shorter vegetative period after the first harvested

Source: (IRRI, 2019)(Busyra, Adri, 2014). Ratooning is also defined as thebassal sucker for the propagation, such as in banana,sugarcane, pineapple, sorghum, cotton, pearl millet andrice. The first harvest crop is called as main crop and eachsucceeding harvest after is determined as first ratoon,
second ratoon, and so on (Santos, Fageria, & Prabhu, 2003).
Rice ratooning farming system had been practiced atlimited areas in some countries, including Philippines,
Indonesia, India, Thailand, United States, Brazil, Japan,
Ethiopia (Bahar & De Datta, 1977). Twice harvesting canbe performed in southwestern Japan and warmer regions(Nakano & Morita, 2007). Ratooning could save time(nursery and field preparation, transplanting, etc.),
resources (labour, seeds, etc.) and gave higher productivity(Munda, Das, & Patel, 2009).
Crop Productivity Under Ratooning
Ratoon is a very special local-wisdom farmingProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 04

system, which can be performed up to more than 3(Samson & Zandstra, 1980).

Although starch wasregenerations on sorghum (House, 1995). The rice yield oftranslocated from the stem to the panicle in the second crop

the first ratoon was found to be approximately 50% lower

when the first crop was cut at a height of 15 cm, insufficientthan the main crop (Calendacion, Garrity, & Ingram, 1992;

starch was translocated to the panicle when the first crop

Pasaribu, Triadiati, & Anas, 2018). In the contrary, (Itabiyi,

was cut at 5 and 0 cm because of the low temperatures after

Adebowale, Shittu, Adigbo, & Sanni, 2016) reported thatheading, hence it is effective to cut stubble lower to reduce

the ratoon agronomic properties of the NERICA rice

the overall loss of nutrition from a crop (Nakano et al.,variety were higher than the main crop.

2010).

However, proper

land management practices such as proper fertilizer

Not many information found regarding the rice

application, earlier planting season and optimal variety

quality produced from the ratooning system, but it was

selection (Dou, Tarpley, Chen, Wright, & Mohammed,

found that the genetic characteristics positively and2016).

strongly correlated with the sucrose concentration of

Ratoon grain yield of Bellemont and Lemont ricesugarcane’s ratoon (Milligan, Gravois, & Martin, 1996).

cultivars were not influenced by row spacing or seeding

On the other hand, the main crop of sorghum contained

rate, while the filled grain number accounted for over 85%

much higher mean concentrations of the cyanogenic

than the main crop (Jones & Snyder, 1987). The yield of

glycoside, and dhurrin, than the ratoon (Vinutha, Anil

ratoon season mainly depended on more ratoon spikes,

Kumar, Blümmel, & Srinivasa Rao, 2017). So far, the

35%-40% of dry matter accumulation in ratoon spikes

article reported by (Alizadeh & Habibi, 2016) is found to

derived from the transportation of main crop stubble andbe the only information on rice quality regarding with

60%-65% from the photosynthetic production of the leaves

ratooning farming system. In general, rice ratoon resultingof ratoon rice itself (ShangShou, ChuanYing, WeiZhao,

in bigger grain width and thickness, and higher Amylose

YiZhen, & CongHua, 2003). Improved practices of rice

Content and Alkali Spreading Value (APV) Value, butratooning using IR-64 cultivar gave 202% higher

lower grain length and whiteness degree (Table 2).

productivity over the local practices (Munda et al., 2009).
Conclusion
Rice Quality
Rice ratoon is a promising efficient farming
The capacity of rice plants to produce a ratoon crop

technology that supports environment and meet the global

used to be noticeably influenced by means of theirchallenge. In general, the quality of rice resulted fromcarbohydrate content and the phytohormones that continue

ratooning was, somehow, not lower than the main crop.

to be in the intercalary meristem tissues of stubble after

Therefore, more studies regarding rice quality under

harvest. (Alizadeh & Habibi, 2016) reported that the

ratooning farming system is urgent to support the policy

amylase content in the ratoon crop were higher than thefor rice ratooning implementation.

main crop (22.93 and 21.97%, respectively), and the alkali
spreading also showed similar results (5.5 and 4.27,
respectively (Table 2).

Proximates, Amino acids, fatty

acids, minerals and vitamins were not significantly
different between the main crop and ratoon crop, except thefiber and Vitamin B2 were higher under the main crop
(Nam et al., 2013).
The total carbohydrate concentration was positively
correlated with growth duration, while higher nitrogenconcentration was found in the shorter growth duration
period, 117 and 137 days (Garcia, Mabbayad, & Vergara,1980). The sugar and starch concentrations in the main
crop promoted the grain yield of the ratoon crop (Manjappa& Prabhakar, 2003), while the carbohydrate in the stubble
and the root of the main crop determined the ratoonability

Acknowledgements
The authors acknowledge The United Graduate
School of Agriculture Science, Gifu University , japan forprovide funding to present this study in the International
Symposium of a New Era in Food Science and Technology
in Gifu University 9-10 October 2019.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 04

https://doi.org/10.2134/agronj1977.0002196200690
0040003x
Busyra, Adri,and E. (2014). No Title. In Seminar

Nasional Lahan Suboptimal 2014 (pp. 578–584).
Palembang.
Calendacion, A. N., Garrity, D. P., & Ingram, K. T. (1992).
Lock lodging: a new technology for ratoon rice
cropping. Philippine Journal of Crop Science.
Chauhan, J. S., Vergara, B. S., & Lopez, F. S. S. (1985).
Rice ratooning. IRRI Research Paper Series.
Dou, F., Tarpley, L., Chen, K., Wright, A. L., &Mohammed, A. R. (2016). Planting Date and Variety
Effects on Rice Main and Ratoon Crop Production inSouth Texas. Communications in Soil Science andPlant

Analysis.

https://doi.org/10.1080/00103624.2016.1243705
Garcia, R. N., Mabbayad, B. B., & Vergara, B. S. (1980).
Effects of growth duration and different levels oflight intensity on the ratooning ability of rice.
Philippine Journal of Crop Science.
House, L. R. (1995). Sorghum and Millets : History,
Taxonomy, and Distribution. In Sorghum andMillets: Chemistry and Technology.
IRRI (International Rice Research Institute). (2019). No
Title.

Retrieved

August

25,

2019,

from

http://ricestat.irri.org:8080/wrsv3/entrypoint.htm.
Itabiyi, O. V. I., Adebowale, A. A., Shittu, T. A., Adigbo, S.
O., & Sanni, L. O. (2016). Effect of ratooning
process on the engineering properties of NERICA
rice varieties. Quality Assurance and Safety of Crops
and Foods. https://doi.org/10.3920/QAS2014.0449
Jones, D. B., & Snyder, G. H. (1987). Seeding Rate andRow

Spacing

Effects

on

Yield

and

Yield

Components of Ratoon Rice1. Agronomy Journal.
https://doi.org/10.2134/agronj1987.0002196200790
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Khush, G. S. (2005). What it will take to Feed 5.0 Billion
Rice consumers in 2030. Plant Molecular Biology,59(1),

1–6.

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2159-5

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Milligan, S. B., Gravois, K. A., & Martin, F. A. (1996).
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Srinivasa Rao, P. (2017). Evaluation of yield and

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Munda, G. C., Das, A., & Patel, D. P. (2009). Evaluation of

forage quality in main and ratoon crops of different

transplanted and ratoon crop for double cropping ofsorghum

rice (Oryza sativa L.) under organic input

Tropicales. https://doi.org/10.17138/TGFT(5)40-49

management in mid altitude sub-tropical Meghalaya.
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Nakano, H., Hattori, I., Sato, K., Morita, S., Kitagawa, H.,& Takahashi, M. (2010). Effects of cutting height of
the first crop on estimated total digestible nutrient
concentration and yield in double-harvested rice.
Agronomy

Journal.

https://doi.org/10.2134/agronj2009.0347
Nakano, H., & Morita, S. (2007). Effects of twice
harvesting on total dry matter yield of rice. Field
Crops

Research.

https://doi.org/10.1016/j.fcr.2006.12.001
Nam, K. H., Nam, K. J., An, J. H., Jeong, S. C., Park, K.
W., Kim, H. B., & Kim, C. G. (2013). Comparative
analysis of key nutrient composition between
drought-tolerant transgenic rice and its nontransgenic

counterpart.

Food

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Negalur, R. B., Yadahalli, G. S., Chittapur, B. M.,
Guruprasad, G. S., & Narappa, G. (2017). Ratoon
Rice: A Climate and Resource Smart Technology.
International Journal of Current Microbiology andAppliedSciences.

https://doi.org/10.20546/ijcmas.2017.605.179
Pasaribu, P. O., Triadiati, & Anas, I. (2018). Rice ratooning
using the salibu system and the system of riceintensification method influenced by physiologicaltraits. Pertanika Journal of Tropical Agricultural
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Samson, B. T., & Zandstra, H. G. (1980). The effect of two
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Santos, A. B., Fageria, N. K., & Prabhu, A. S. (2003). Riceratooning management practices for higher yields.
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ShangShou, Z., ChuanYing, Z., WeiZhao, J., YiZhen, L., &CongHua, G. (2003). Yield formation of andcultivation techniques for super high-yielding
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TropicalGrasslands-Forrajes

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 05

Elucidating the Molecular Mechanism of β-Citraurin Accumulationin Citrus Fruit
Gang MA, Lancui ZHANG, Masaya KATO*
Department of Bioresource Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga,
Shizuoka 422-8529, Japan*

Corresponding Author: kato.masaya@shizuoka.ac.jp
SUMMARY

β-Citraurin, a C30 apocarotenoid, is a color-imparting pigment responsible for the reddish color of citrus fruits. In
citrus fruits, the accumulation of β-citraurin is not a common event, it is only observed in the flavedos of some varieties
during the fruit maturation. Although more than seventy years have passed since β-citraurin was first identified, thebiosynthesis pathway is still unknown. In this study, to elucidate the molecular mechanism of β-citraurin accumulation,
carotenoids contents and expression of genes related to carotenoid metabolism were investigated in two citrus varietiesof Satsuma mandarin (Citrus unshiu Marc.), ‘Yamashitabeni-wase’, which accumulates β-citraurin predominantly, and‘Miyagawa-wase’, which does not accumulate β-citraurin. The results suggest that CitCCD4 was the key gene
contributing to the biosynthesis of β-citraurin. In the flavedo of ‘Yamashitabeni-wase’, the expression of CitCCD4
increased rapidly from October, which was consistent with the accumulation of β-citraurin. In the flavedo of ‘Miyagawawase’, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent
with the absence of β-citraurin. Functional analysis showed that the cleavage of β-cryptoxanthin and zeaxanthin at 7,8 or
7’,8’ position by CitCCD4 enzyme led to the formation of β-citraurin in citrus fruits. These results presented herein might
contribute to elucidate the mechanism of β-citraurin accumulation in citrus fruits, which might facilitate the improvementin the citrus nutritional and commercial qualities.
Introduction

heterogeneous family (CCD1, CCD4, CCD7, CCD8 and
NCEDs). The similarity among the different members is

Carotenoids, a diverse group of pigments widely

very low apart from four strictly conserved histidine

distributed in nature, fulfill a variety of important functions

residues and a few glutamate residues (Kloer and Schulz,

in plants and play a critical role in human nutrition and2006; Walter et al., 2010). In Arabidopsis, the CCD family

health (Krinsky et al., 2003; Ledford and Niyogi, 2005). As

contains nine members (CCD1, NCED2, NCED3, CCD4,

carotenoids contain a series of conjugated double bonds in

NCED5, NCED6, CCD7, CCD8 and NCED9), andthe central chain, they can be oxidatively cleaved in a site-orthologues in other plant species are typically named

specific manner (Mein et al., 2011). The oxidative cleavage

according to their homology with an Arabidopsis CCD

of carotenoids not only regulates their accumulation, but(Huang et al., 2009). In plants, NCEDs locate in plastids

also produces a range of apocarotenoids (Walter et al.,and catalyze the cleavage of 9-cis-violaxanthin and 9’-cis-2010). In higher plants, many different apocarotenoids

neoxanthin to form C25 epoxy-apocarotenal and xanthoxin,

derive from the cleavage of carotenoids, and havea precursor of ABA. This reaction is a rate-limiting step inimportant metabolic functions, such as plant hormones,

ABA biosynthesis (Chernys and Zeevaart, 2000; Kato etpigments, aroma and scent compounds, as well as signalingal., 2006). CCD1 symmetrically cleaves the 9,10 and 9’,10’compounds (Fig.1).

double bonds of multiple carotenoid substrates to form C14

Carotenoid cleavage dioxygenases (CCDs) are a

dialdehyde and two C13 products, which vary depending ongroup of enzymes that catalyze the oxidative cleavage ofthe carotenoid substrate (Schwartz et al., 2001). When the

carotenoids (Ryle and Hausinger, 2002). CCDs aresubstrate of CCD1 was β-carotene, the volatile apo-

nonheme iron enzymes present in plants, bacteria andcarotenoid β-ionone, which contributes to the fragrance ofanimals. In plants, CCDs belong to an ancient and highly

flowers and flavor of fruits, was generated (Kato et al.,

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 05

2006). CCD7 and CCD8 were identified from shoot

3. Isolation and Identification of β-Citraurinbranching mutants (Booker et al., 2004; Sorefan et al.,The carotenoids extracted from the flavedo of2003). Alder et al. (2012) reported that CCD7 and CCD8‘Yamashitabeni-wase’ were loaded on a column of silica

together with D27 were involved in the biosynthesis of

gel using a hexane:ethyl ether: isopropyl alcohol (7:3:1

strigolactone, a shoot branching inhibitor hormone, using[v/v]) solution as eluent. The red pigment was collected

all-trans-β-carotene as a substrate. Compared with otherand evaporated to dryness. The collection was subjected to

CCDs, the function of CCD4 is poorly understood. In

HPLC and identified by spectrophotometry and mass

Chrysanthemum morifolium, CmCCD4a contributed to thespectrometry. The UV-visible spectra were taken with awhite color formation by cleaving carotenoids into

spectrophotometer.

colorless compounds (Ohmiya et al., 2006). Recently, it

spectrometry (FAB-MS) analysis was performed with thehas been reported that CsCCD4, CmCCD4a and MdCCD4

API 2000 triple stage quadrupole mass spectrometer.

Fast

atom

bombardment

mass

could cleave β-carotene to yield β-ionone (Rubio et al.,2008; Huang et al., 2009).
β-Citraurin, a C30 apocarotenoid, is a color-imparting

4. Total RNA Extraction and Real-time Quantitative RTPCR

pigment responsible for the reddish color of citrus fruits

Total RNA was extracted from the flavedos and juice(Farin et al.,1983). In 1936, it was first discovered insacs of ‘Yamashitabeni-wase’ and ‘Miyagawa-wase’ atSicilian oranges (Cual, 1965). In citrus fruits, thedifferent stages according to the method described byaccumulation of β-citraurin is not a common event, it isIkoma et al. (1996). The cDNA was synthesized and theonly observed in the flavedos of some varieties during thegene expression was confirmed by real-time PCR.

fruits maturation. The citrus varieties accumulating βcitraurin are considered more attractive because of their

5. Functional Analysis of CitCCD4 Enzyme in vitro

red-orange color (Ríos et al., 2010). Although more than

The CitCCD4 cDNA from ‘Yamashitabeni-wase’ wasseventy years have passed since β-citraurin was firstcloned into the pCold I vector. The recombinant plasmid

identified, the pathway of its biosynthesis is still unknown.

was transformed into XL1-Blue cells. The transformants

In the present study, we found that CitCCD4 gene waswereinvolved in the synthesis of β-citraurin in citrus. To

carbenicillin (50 μg ml ), and incubated at 37 ˚C for 20 h.

confirm the role of the CitCCD4 gene further, functional

The colonies were incubated in 100 ml of 2×YT medium

analyses of the CitCCD4 enzyme were performed in vitro.

with carbenicillin (50 μg ml-1) at 37 ˚C for 16 h. Then, 2

The present study is the first to investigate the biosynthesis

ml of culture solution was inoculated into 200 ml of 2×YTof β-citraurin in citrus fruits. The results might provide new

medium with carbenicillin (50 μg ml-1). Cultures werestrategies to enhance the nutritional and commercial

grown at 37 ˚C until an OD600 of 0.7 was reached. The

qualities of citrus fruits.

culture solution was quickly refrigerated at 15 ˚C and left

platedinLBmediumsupplemented

with

-1

to stand for 30 minutes. The expression of proteins wasMaterials and Methods

induced by the addition of IPTG, and the cultures weregrown at 15 ˚C for an additional 24 h. The cells were

1. Plant Material

harvested by centrifugation, frozen in liquid nitrogen, andTwo varieties of Satsuma mandarin (Citrus unshiu

then resuspended in 2.5 ml of extraction buffer (0.1 M Tris-

Marc.), ‘Yamashitabeni-wase’ and ‘Miyagawa-wase’,HCl, pH 7.2, 30 mM Na-ascorbate, 5 mM dithiothreitol

cultivated at the Fujieda Farm of Shizuoka University(DTT), 10% (v/v) glycerol, 0.05% (v/v) Triton X-100).

(Shizuoka, Japan) were used as materials.

After sonication and centrifugation, the complex was
subjected to gel filtration using a PD-10

2. Extraction and Determination of Carotenoids

column.

The enzymatic activities of the recombinant CitCCD4

The identification and quantification of carotenoids

protein were assayed in a reaction mixture consisting of 0.1

were conducted according to the methods described by

M Tris-HCl, pH 7.2, 30 mM Na-ascorbate, 50 μM FeSO4,

Kato et al. (2004). Total carotenoid is the sum of the

20 μg catalase, 0.05% (v/v) Triton X-100, 20% (v/v)

content of various carotenoids detected in this study. The

glycerol, 1 mM carotenoid substrate (β-cryptoxanthin,carotenoid concentration was estimated by the standard

zeaxanthin, all-trans-violaxanthin and 9-cis-violaxanthin)curves and expressed as micrograms per gram fresh weight.

and 5 μg of the recombinant protein in a total volume ofProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 05

200 μl at 27 ˚C for 3 h. After the incubation, 1 ml of water

Octoberin‘Yamashitabeni-wase’,

while

it

was

was added to the reaction mixture. The reaction products

undetectable in ‘Miyagawa-wase’ throughout the ripening

were partitioned three times into 1.2 ml of ethyl acetate,process (Fig. 2). This difference led to different peel colors

evaporated to dryness, and dissolved in methanol. An

between the two varieties. The accumulation of β-citraurin

aliquot (20 μl) was analyzed by HPLC.

contributed to the reddish peel in ‘Yamashitabeni-wase’,
while the complete absence of β-citraurin led to theyellowish peel in ‘Miyagawa-wase’.

6. Treatment with Ethylene and Red LED light
Fruits of ‘Yamashitabeni-wase’ harvested in October

Although more than seventy years have passed sincewere used as materials. For the ethylene treatment, fruits

β-citraurin was first identified in citrus fruits, the pathway

were treated with 50 μl L ethylene for three days at 20 ˚C.

of β-citraurin biosynthesis has yet to be elucidated (Cual,

For the red LED light treatment, fruits were irradiated with1965). In the present study, the contents of β-cryptoxanthin,-2 -1

red LED lights (660 nm) at an intensity of 150 μmol m s

zeaxanthin and β-citraurin rapidly increased from October

for three days at 20 ˚C. Fruits stored at 20 ˚C (RH 75%) in

in ‘Yamashitabeni-wase’. The concomitant increase of β-

the dark were used as the control.

cryptoxanthin, zeaxanthin and β-citraurin indicated that β--1

A

Results and Discussion
1. Accumulation of β-Citraurin in the Flavedos of Citrus
Fruitsβ-Citraurin, a C30 apocarotenoid, is a red pigment
responsible for the attractively reddish peel color of citrus(Farin et al., 1983). However, the accumulation of βcitraurin is not a common event in citrus; it has been

B
433.6

detected in only a few varieties (Xu et al., 2011). In thepresent study, we isolated β-citraurin from the flavedo of‘Yamashitabeni-wase’ using a silica gel column and HPLC.
The mass spectrum and absorption maximum of the βcitraurin were consistent with those reported by Farin andAgócs (Farin et al., 1983; Agócs et al., 2007; Fig. 1).
Additionally, changes in the content of β-citraurin
were examined in the flavedos of two varieties of Satsuma
mandarin, ‘Yamashitabeni-wase’ and ‘Miyagawa-wase’,during the fruit maturation process. The results showed
150-1Carotenoid content ( µg gFW )

120

20

Phy

15

90

10

60

5

30

0
S O N D
100
C-vio
80

J

60
40
20
0

S O N D

J

ζ-Car

25
20

β-Car

15
10
5

0
0
S O N D J
S O N D
7
45
Lut
α-Car
6
36
5
27
4
3
18
2
9
1
0
0
S O N DS O N D J

Fig. 1. Isolation and identification of β-citraurin fromthe flavedo of ‘Yamashitabeni-wase’. (A) HPLC
analysis and UV-visible light spectra of β-citraurin. (B)
FAB-MS spectra of β-citraurin.
30
16
0.5
Zea
β-Cry
T-vio
25
0.4
12
20
0.3
15
8
0.2
10
4
0.1
5
0
0
0.0
S O N D J
S O N D J
S O N D J
J
140
300
β-Cit
Total
120
250
'Yamashitabeni-wase'
100
200
80
'Miyagawa-wase'
150
60
100
40
50
20
0
0
J
S O N D J
S O N D J
MonthsFig. 2. Carotenoids accumulation in flavedos of ‘Yamashitabeni-wase’ and ‘Miyagawa -wase’ during the ripening process. S, September; O,
October; N, November; D, December; J, January. Phy, phytoene；ζ-Car, ζ-carotene; β-Car, β-carotene; β-Cry, β-cryptoxanthin; Zea,
zeaxanthin; T-vio, all-trans-violaxanthin; C-vio, 9-cis-violaxanthin; α-Car, α-carotene; Lut, lutein; β-Cit, β-citraurin; Total, total carotenoids.

that the content of β-citraurin increased significantly from

citraurin might be a breakdown product of β-cryptoxanthin

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 05

and zeaxanthin. Additionally, in ‘Miyagawa-wase’, β-the white color formation by cleaving carotenoids into

citraurin was not accumulated, zeaxanthin was further

colorless compounds (Ohmiya et al., 2006). A common

converted

9-cis-

feature of CCD4 identified in several recent studies is aviolaxanthin, and as a result, the contents of all-trans-

9,10 or 9’,10’ cleavage activity to yield β-ionone (Huang

violaxanthin and 9-cis-violaxanthin were higher than thoseet al., 2009; Rubio et al., 2008). However, the 7,8 or 7’,8’in ‘Yamashitabeni-wase’ (Fig. 2).

cleavage activity of CCD4 remains to be confirmed.

into

all-trans-violaxanthin

andBouvier et al. (2003) reported that CsZCD specifically
2. Isolation and Sequence Analysis of CitCCD4
To find the enzyme responsible for β-citraurin
biosynthesis, we compared the expression of genes related

catalyzed the cleavage of zeaxanthin at the 7,8 and 7’,8’positions of the chromophore and initiated the formation ofsaffron secondary metabolites.

to carotenoid metabolism between the two varieties ofIn the present study, we found that CitCCD4 enzyme

Satsuma mandarin, ‘Yamashitabeni-wase’ and ‘Miyagawa-exhibited substrate specificity. It cleaved β-cryptoxanthin

wase’. In the flavedo, a significant difference in theand zeaxanthin at the 7,8 or 7’,8’ position (Fig. 4). But

expression of CitCCD4 was observed between the twoother carotenoids tested in the present study (lycopene, α-varieties. In ‘Yamashitabeni-wase’, the expression of

carotene, β-carotene, all-trans-violaxanthin and 9-cis-

CitCCD4 increased rapidly with a peak in December,violaxanthin) were not cleaved by CitCCD4 enzyme.

which was consistent with the accumulation of β-citraurin

Moreover, the cleavage of β-cryptoxanthin and zeaxanthin(Fig. 3). In ‘Miyagawa-wase’, the expression of CitCCD4

by CitCCD4 enzyme led to the formation of β-citraurin.

remained at a low level during the ripening process, which

The specific cleavage reaction of CitCCD4 enzyme

was consistent with the absence of β-citraurin in thepresented

flavedo (Fig. 3). Moreover, in the juice sacs, where β-

speculations that β-citraurin was a breakdown product of

citraurin was not accumulated, the expression of CitCCD4β-cryptoxanthin and zeaxanthin. In addition, when β-

was extremely low in both ‘Yamashitabeni-wase’ andcryptoxanthin was used as the substrate for the cleavage‘Miyagawa-wase’ (data not shown). These results
suggested that CitCCD4 was a key factor for regulating β-

reaction of the recombinant CitCCD4 enzyme, trans-βapo-8’-carotenal, which eluted at 43 min, was also detected

citraurin biosynthesis.

except for β-citraurin (Fig. 4). However, we have not been

3. Functional Analyses of Recombinant CitCCD4 Enzyme

hereinfurtherconfirmed

the

A

I
HO

Compared with other CCDs, the information about

previous

I
CHO

III

the functions of CCD4 is limited. In potato, downregulation of CCD4 gene expression using RNAi resulted

I

ControlIIin increased violaxanthin content (Campbell et al., 2010).

III

mRNA levels (arbitrary units)

CitCCD4

6
CitCCD1
5
4
3
2'Miyagawa-wase''Yamashitabeni-wase'
10
S O N D J
8CitNCED3

6
4
2

5
4

10

CitNCED2

3
2
1
0
S O N D J
6
CitCCD4
5
4
3
2
1
0
S O N D J

20

30

Minutes

40

50

60

B
IV
HO

IV

HO

II

CHO

II
IV

Control
CitCCD41020

30

Minutes

40

50

60

Fig. 3. Changes in the expression of genes related tocarotenoid metabolism in flavedos of ‘Yamashitabeniwase’ and ‘Miyagawa-wase’ during the ripening process.

Fig. 4. Functional analysis of CitCCD4 enzyme in vivo.
(A) HPLC analysis of the cleavage products from the
incubation of β-cryptoxanthin with recombinant
CitCCD4. (B) HPLC analysis of the cleavage products
from the incubation of zeaxanthin with recombinant
CitCCD4. Peak I, β-cryptoxanthin; Peak II, β-citraurin;
Peak III, trans-β-apo-8’-carotenal; Peak IV, zeaxanthin.

In Chrysanthemum morifolium, CmCCD4a contributed toable to detect trans-β-apo-8’-carotenal in the flavedos or0

S O N D J

MonthsProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 3 – Comprehensive Food Science and Others –: 05

Conclusion

juice sacs of citrus fruits. It is possible that trans-β-apo-8’carotenal might be further cleaved by other CCDs in the

In the present study, the biosynthetic pathway of β-

citrus fruits.

citraurin was investigated using two citrus varieties of
4. Effects of Ethylene and Red LED Light on β-Citraurin

SatsumaContent and CitCCD4 Expression

accumulates β-citraurin predominantly, and ‘Miyagawa-mandarin,‘Yamashitabeni-wase’,which

It has been reported that ethylene treatment increased

wase’, which does not accumulate β-citraurin. The results

the contents of carotenoids; as a result, the degreening

suggested that CitCCD4 was a key gene regulating theprocess of citrus fruits was accelerated (Rodrigo andbiosynthesis

Zacarías, 2007). We previously found that red LED light

cryptoxanthin and zeaxanthin by CitCCD4 enzyme led towas effective to enhance carotenoids contents, especiallythe formation of β-citraurin (Fig. 6). In addition, thethe content of β-cryptoxanthin, while blue LED light had

ethylene and red light treatments were effective to enhanceno significant effect on the carotenoid content in thethe content of β-citraurin by up-regulating the expression

flavedo of Satsuma mandarin (Ma et al., 2012). In theof CitCCD4 in the flavedo of ‘Yamashitabeni-wase’. These

present study, to investigate the regulatory effects ofresults presented herein might contribute to elucidate theethylene and red LED light on β-citraurin accumulation,mechanism of β-citraurin accumulation in citrus fruits,

fruits harvested in October were used as materials, as thewhich might facilitate the improvement in the citruschanges in β-citraurin content were most significant at thisnutritional and commercial qualities.

stage. As shown in Fig. 5, the content of β-citraurin was

ethylene and red light treatments was attributed to anincrease in the expression of CitCCD4. The resultspresented herein provide more insights into the regulatory
mechanism of β-citraurin metabolism in citrus fruits.

β-citraurin.

The

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 01

Metabolite Fingerprinting of Assam Tea (Camellia sinensis L.(O) Kuntze)
Under Alumnium Stress
Sanjenbam Sanjibia Devi1, Sanjib Kumar Panda1,2*
1Plant Molecular Biotechnology laboratory, Department of Life Science & Bioinformatics, Assam

2

Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Bandarsindri,

University Silchar
305817, Ajmer Rajastha*

Corresponding author: profskpanda73@gmail.com, sanjib.panda@curaj.ac.in
SUMMARY

Tea, Camellia sinesis (L.) Kuntze in the genus of Camellia, belongs to the family of Theaceae, is an economicallyimportant horticultural crop and most popular beverages and an important financial source for those tea producing
countries in the world. Assam, a north eastern state of India is famous as the largest tea producing state, forming one of
the major tea producing area globally. From the structural difference the tea catechins can be divided into dihydoxylated(ECG, EC and C) and trihydroxylated (EGCG, EGC and GC) with 5’ position in the B ring . The presence of hydroxyl
group at the 5’ position in the B ring is shown by structure activity relationship and is the most potent scavenging
antioxidant. Flavonoid 3’-hydroxylase (F3’H) and flavonoid 3’,5’-hydroxylase (F3’5’) are the soul enzymes which
control the hydroxylation of narigenin and dihydrokaempferol at either 3’ position or in both 3’ and 5’ position in B ring.
With the flow of their downstream processing enzymes the intermediate resultant is to form dihydroxylated andtrihydroxylated flavonoids. NMR is one of the popularly used multivariate method which offers a broad range of
secondary metabolites evaluation to differentiate the quality of tea. Metabolomic study exemplifies the consolidation of
the genetic background information that changes by the environmental stresses in plants. In our study, metabolite
fingerprinting of tea leaves was

performed to examine the response to alumnium stress in two tea varieties S3A3 andS3A1 (Assamica) by 1H NMR. Certain secondary metabolites were found from NMR peak of tea leaves. Among themcarbohydrates were found less in stress conditions and in case of phenolic compounds (catechins) the number of peaks
and intensity were slightly increased at stressed tea leaves. And other metabolites amino acids, caffeine, gallic acids, myo
inositol and some organic acids were detected. Flavonoid 3’-hydroxylase (F3’H) and flavonoid 3’,5’-hydroxylase (F3’5’)
are the enzymes affecting the ratio of dihydoxylated to trihydroxylated catechins in Camellia sinensis. Expression of thesegenes were analyzed by real time RT-PCR. Slight Changes were observed in ratio of dihydroxylated to trihydroxylated(RDTC) catechins

under alumnium stress showing that alumnium stress affects the tea composition and could change

the metabolic pathways.
Introduction

sweetness and an earthy flavor.

Due to the extensive

secondary metabolites in tea leaves yields many healthTea, Camellia sinesis (L.) Kuntze in the genus ofbenefits to human beings which includes polyphenols,

Camellia, belongs to the family of Theaceae, is antheanine and volatile oils (Yamamoto et al. 1999, Rogers

economically important horticultural crop and model

et al. 2008). India, beside being the second largest tea

system for studying self-incompatibility as well as anproducer, is the highest consumer of tea. 82% of the total

evergreen flowering plant. Tea is one of the most popular

production is consumed inside and the remaining 18% isbeverages and an important financial source for those tea

exported (Annual report 2016-17, Dept of Commerce,

producing countries in the world. Assam contributes

Govt. of India; www.commerce.gov.in). Assam, a northaround 50 percent of total tea produced in India. Barakeastern state of India is famous as the largest tea producing

valley and upper Assam has the maximum numbers of tea

state, forming one of the major tea producing area globally.

plantation. The climate of the state is such that it gives

The crops, growing in the soil of North East India is

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 01

inflicted with alumnium (Al) toxicity due to soil acidity inchanges of chemical constituents after exposure to

this region (Kumar et al. 2013). Excessive rainfall inalumnium stress in tea leaves.

culmination with deforestation is the main reason behindthe flushing of essential cations from the soil rendering theMaterials and Methods

soil acidic (Saha et al. 2012). However the tea plant(Camellia sinensis) being a typical alumnium (Al) hyper-

1. Sample Preparation for NMR

accumulator grow relatively well on acidic soil polluted

Two to three leaves of tea (soft and tender) werewith high levels of soluble ionic Al. To understand the fullcollected and were ground with the help of precooledmechanisms that are used by the Al; tolerant plant andmortar and pestle with liquid nitrogen. The ground samples

species to fight with Al toxicity is important and to find out

were transferred into a plastic bag by a sterilized spatula.

the specific genotype with most suitable and tolerant inAllow it for free drying and then to deep freezer for further

acidic environment mainly to enhance the productivity,

analysis. In the freeze dryer the samples were kept for 1-2

yield. One of fundamental economic solution for making

days and after this lyophilization process done weight thethe food production increased new cultivars development

sample with Eppendorf tube for the analysis. Re-

may be from the Al rich region is necessary (Silva 2012).

suspension of lyophilized samples in D2O containing 0.3

The raw materials of tea making is one most important

mM TSP as a concentration standard and chemical shift

factor contributed to the quality of Northeast India Tea, thisreference (δ= 0 ppm). Centrifuge the sample at 14000g at

is mainly given by their constituents of polyphenol. The

40C for 15 min to remove any particle. After thequality of black tea is contributed mainly by flavanol

centrifugation, samples cannot be stored further and should

precisely catechins. Among the antioxidant catechins,

go for NMR analysis immediately. Desired amount (0.7ml)

epigallocatechin gallate (EGCG) is the most potent one andof the sample is transferred into an NMR tube and takeused as biochemical marker for NorthEast Indian tea

spectra (Bruker, H1 NMR).

(Gupta et al. 2002). The tea from Northeast India are

For the annotation of unknown compounds, themainly processed from Camellia Assamica leaf and it haschanges of chemical shift were taken from the previous

the uniqueness due to the presence of wide range ofreports through literature survey and name of the authors

catechins (Sabhapondit et al. 2012). The tea plant can be

and their related details were summarized in the table form

distinguished into three different verities according to theiralong with compounds which were found in our samples.

phenotypic characteristics, Assam, China and Cambod

With the help of MestReNova x64 software was used totype. This classification of variation is followed only in

identify the metabolites in tea leaf samples.

Indian sun continent due to presence of huge population
tea plant (Wight 1962). The constituents of tea leaves can

2. Validation of gene expression by Quantitative RT-PCR

easily differentiate between hybrids, an interrelationshipanalysis of F3’H and F3’5’H

between taxa can be set up with the presence and absence

To validate the co relation of expression between theof different polyphenols (Roberts et al. 1958). NMR is one

key genes F3’H and F3’5’H and the ratio dihydroxylatedof the popularly used multivariate method which offers ato trihydroxylated catechin (RDTC) in tea plant underbroad range of secondary metabolites evaluation toalumnium stress according to the Wei et al. 2015. To see

differentiate the quality of tea. All these information al

the impact of alumnium stress in their ratio this analysis

together with metabolomics has the possibility to furnish

was performed. CsF3’5’H1 and CsF3’H (CsF3’H1,

an authentic and conclusive envision or scenario of

CsF3’H2 and CsF3’H3) were picked for quantitative RT-

composition of tea related to their quality. 1H NMR

PCR (qPCR) examination. The RNA was isolated as per

metabolic profiling and fingerprinting are a treasure of

method Muoki et al, 2011 from control and stressed

information in wide range of secondary metabolites(500µM) of leaf; whereas cDNA was prepared according(Tarachiwin et al. 2007). For the tea quality evaluation

to manufacturer’s instructions (PrimeScript 1st Strand

NMR technique are preferred than the other analytical

cDNA synthesis kit, Takara, Japan). The synthesizedtechniques due to giving more structural information. It

cDNA was normalized using GAPDH as housekeeping andhelps to obtain the information about the molecular

the transcript analysis. Real time expression was calculated

structure of tea constituents from NMR spectra.

according to Wei et al. 2014.

The aim of this investigation is to evaluate theProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 01

Results

ECGC, ECG, EGC, EC, C, some unknown catechins,theogallin, quinic acid,

NMR is widely used for metabolic profiling in

digallate,theaflavintheaflavins, thearubigin 3,3’3,3’-digallatehydroxycinnamic

medicinal plants. It provides a fast detailed analysis. The

compound ; organic acids,

γ-Amino- butyrate, citric acid,effects of Al stress in tea leaf were studied for the

malic acid , fumaric acid, succunic acid, proline, theanine,

metabolites study. Five different varieties were used in thistryptophan, glutamic acid, arginine, threonine, leucine,study. 1H-NMR spectra was recorded at 23°C on Brucker.

lysine,anomeric protons of carbohydrates were shown by thepresence of metabolites were signicantly different. The

region of β- glucose, α-glucose, fructose and sucrose. We

phenolic region was depicted at area. 5-8.5 ppm.

caffeine other fatty acids were detected. The

summarized the information based on the chemical shift

Epicattechins derivatives and caffeine were detected in thefrom the previously reported and described authors. Showntea leaves. For the identification of the compounds we took

the H NMR spectral signal assignments of two different

the help from the software MestReNova x64 and identified

varieties of tea leaves and arranged in terms of PPM,

the compounds from tea leaf samples. The detected

intensity, width and area of the annotated compounds. The

compounds or seconadry metatloites, chararcteristics of 1H

samples shown a characteristic changes between control

NMR signals were assigned with respected compoundand stress one as well as variety wise variation were clearly

name chemical shift (ppm) and authors. In S3A1 variety

observed. The identification of compounds from tea plants

the control and stressed there were a drastic different

control and stress of five varieties were performed andbetween control and stressed one, almost half of themetabolites or compounds were assigned and comparison

number of peaks were lessly detected in stressed one. This

with published data (Yuan et al. 2014; Lee et al. 2010, 2011variety shown that the alumnium stress in variety werea, b; Wahyuni et al. 2013; Tarachiwin et al. 2007; Le Gall

highly impacts especially on carbohydrates, significantly

et al. 2004; Kumar et al. 2016; Ohno et al. 2011; Kim et al.

decreased in stressed and slightly increased in phenolic2010). Enhancement of phenolic content and decrease ofcompounds as well as the flavonoid components. Slightly

sucrose were mainly observed. Phenolic contents likeincreased in inositol compound but sudden dropping down

Fig.1 The Representative of 1H-NMR spectra of tea

Fig. 2 The Representative of 1H-NMR spectra of tea

leaf (a) spectrum of S3A1 control leaf; (b) spectrum ofleaf (c) spectrum of S3A3 control leaf; (d) spectrum of

S3A1 stressed leaf.

S3A3 stressed leaf.

1Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 01of sucrose level and could dected glucose at stressed one.

Discussion

Four key genes F3’H (CsF3’H1, CsF3’H2 CsF3’H3)and F3’5’H (CsF3’5’H1) were selected and went for their

Carbohydrates played an important role in stressreal time RT-PCR expression analysis. These aretolerance mechanism and it is straightly connected withresponsible for the formation of dihydroxylated andperformance of photosynthesis. Plants utilized both starchtrihydroxylated catechins. RNA were isolated according toand fructans as a source of energy during stress in placed

the protocol (Mouki et al. 2012) from leaf only. The coof glucose. In our experiment the sucrose level in 1H MNR

relation and relationship between key genes and the ratio

peak as well from total carbohydrate quantification wereof dihydoxylated to trihydroxylated catechins (RDTC)

decreased from control to stress in all five varieties.

were performed. (Fig; 3) . Only one concentration 500 µM

Sucrose is mainly decreased in stress condition andwere selected for the real time analysis. The ratios of their

this compound may be trans-located to root tips to promote

expressions of key genes (CsF3’H1+ CsF3’H2 +growth. The decrease of total sugar concentration were

CsF3’H3) to CsF3’5’H1 were calculated by the following

reported in tea plant (Chen et al. 2010; Mukhophadya et al,

protocol Wei et al. 2015. From the co relation analysis2012) and in tobacco (Abdel –Basset et al. 2010). In our

shown both two genes were highly co related each other instudy also, carbohydrates content was decrease fromboth stress and unstressed condition of tea plant bycontrol stress and significant increase were at 500 µM (Fig.

pointing towards the expression ratios F3’H to F3’5’H

1&2). From investigation of total phenolic content, total

genes has the crucial role in determining the differentiation

flavonoid content, allocation of 1H MNR peaks ofof RDTC under Al stress of tea plant.

Different changes

phenolic compounds, real time PCR of four genes

were observed in all the five varieties of tea plant and their

CsF3’H1+, CsF3’H2, CsF3’H3 and CsF3’5’H1. Similar

relative expressions shown in Fig. 3, shows the CsF3’H1,reports were found by (Chen et al. 2011 and Hajiboland etofal. 2013a) in tea plant. By the effect of certain enzymes

CsF3’H1+CsF3’H2+CsF3’H3 i.e F3’H showing thephenolic compounds of tea plants occur in polymerization

relative expression of CsF3’5’H1 gene underCsF3’H2andCsF3’H3,

thesumaluminium

reaction with the variation of growth condition likestress and last were the ratio of dihydoxylated totemperature, location, drainage, light and weather cantrihydroxylated catechins (RDTC). We observed thecause the variation in the types of polyphenols (Chen et al.

highest ratio were found at S3A3 than S3A1. By our study2013). In our investigation in hydroponic condition acidic

revealed, aluminium treatment has the impact on the ratio

medium, the changes in the catechins profile were foundof catechin composition on tea plant.

along with the phenolic related compounds under Al stressin tea plant. Tea plant content varieties of polyphenol
compounds formed a complex along with Al. this makes toaccelerate the absorption of Al without affecting otherfunctions translocate into various parts of tea plants. The
influence of catechins status in tea under aluminium stresswere reported by Chen et al. 2011and found statisticallysignificant co relation were found along with Al and ECG
as well with total phenolic content. Presence of catechincomplexes with Al in leaf were detected through Al NMR
spectroscopy, there the chemical form of Al wascomplexed with catechin and uptake by the root cells,
translocated to xylem cell and finally complex reached toleaf cells (Chen et al. 2011). Metabolomics study
exemplifies the consolidation of the genetic backgroundFig.3 Co relation between F3’H and F3’5’H expression
pattern ; Relative expression of F3’H1, F3’H2, F3’H3;
relative expression of (F3’H1+F3H+,F3’H3); relative
expression of F3’5;H1; ratio of F3’H and F3’5’H.

information changes by the environmental stresses inplants. And thus, can distinguish accurately the phenotypeof plant species. To avoid the stress by the plant’s
responses certain stimuli of adverse effects is solely
depend on particular stress tolerance. To do such things

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 01

plants make various alterations of their metabolism atConclusion

certain conditions and mainly associated with modificationof metabolism. The metabolites include sugars, amino

In summary, our investigation furnishes an important

acids, sugars alcohols, TCA intermediates. Although theinformation regarding the fingerprinting of metabolomics

alterations in the secondary metabolites are specific to eachof tea plant. Our finding may help to insight the secondary

given species as well as highly specific to the type of stress.

metabolites accumulation of North East India (Assam) tea

Substantial enhancement of catechins in tea plantand selection of good quality based on biochemical

under Al stress might be the presence of potential binding

constituents which confers the characteristics of the liquor.

site of Al ortho-dihydroxyl group of catechin.

Inoue et al.

Not even this the existence of pure archetypes is become

2002; Chen et al. 2006 (b) reported Al binds to catechinvery doubtful due to extreme hybridization. This

with help of C-3 and C-4 hydroxyl groups of ring B. The

biochemical biomarkers could be provided as a tool inbrowning of catechin was significantly enhanced by Al

chemotoxonomic

ions was shown through visible absorption spectroscopy

resources of tea. Therefore, an authentic clarification of

investigation (Chen et al. 2006, b). Not only the catechinvarieties of Assam, Cambod and China could be built using

profile alterations there were the changes in the nutritional

catechin as a biomarker.

markers

mineral parameters also observed. From 1H NMR peak

Derivative of polyphenolics are synthesized throughshikimatepathwayleadingtophenylalanine

andphenylalanine ammonia lyase (PAL) an important enzyme
in the biosynthesis of phenolic compound. Under theadverse and stressful conditions, the enzyme activity of
PAL along with other enzymes which is responsible forphenylpropanoid pathway was increased (Wahid A et al.
2007). From the structural difference the tea catechins canbe divided into dihydoxylated (ECG, EC and C) andtrihydroxylated (EGCG, EGC and GC) with 5’ position in
the B ring (Wei et al. 2015). The presence of hydroxyl
grouped at the 5’ position in the B ring is shown bystructure activity relationship and is the most potent
scavenging antioxidant. Flavonoid 3’-hydroxylase (F3’H)
and flavonoid 3’,5’-hydroxylase (F3’5’) are the only soul
enzymes which controls the hydroxylation of narigenin
and dihydrokaempferol at either at 3’ position or in both 3’
and 5’ position in B ring (Toda et al. 2002). With the flowof their downstream processing enzymes the intermediate
resultant is to form dihydroxylated and trihydroxylated
flavonoids. By this way the dihydroxylated andtrihydroxylated flavonoids that F3’Hs and F3’5’Hs haveplayed an important role to affect the composition of thesetwo. Calletarrin et al. 2006 studied the expression of both
F3’H and F3’5’H genes and showed this affects directly totheaccumulationofcyaniding(dihydroxylated)/dephinidin- (trihydroxylated) in berry skin of grave vines
based on anthocyanins that ascertains the variation of color
among grape varieties, Callisterin et al. 2006.

differentiate

various

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 02

Improving Postharvest Handling of ‘Merah Delima’ Papaya to Reduce Quality
Loss During Transportation and Storage
Khandra FAHMY*
Faculty of Agricultural Technology, Andalas University, Kampus Unand Limau Manis, Padang, 25163,
Indonesia*

Corresponding Author: khandrafahmy@ae.unand.ac.id
SUMMARY

This study aimed to determine the favorable condition for maintaining quality of ‘Merah Delima’ papaya duringtransportation and storage. Papaya was transported by using cardboard and cardboard with Modified Atmosphere
Packaging (MAP) using Low Density Polyethylene (LDPE) under ambient temperature. After arrival in the market, fruit
were stored at 10oC and 25oC for 21 days to check the shelf-life. The results showed that fruit packaged in cardboard with
MAP reduced the mechanical injury of papaya after transportation. Combination with low-temperature storage at 10°C
maintained the quality and extended the shelf-life of papaya after transportation. Moreover, cardboard with MAP also
alleviated chilling injury in papaya fruit during storage at 10°C.
Introduction

Nakano, 2014a).
In order to determine the favourable condition for‘Merah Delima’ papaya is a superior variety being

preventing quality loss during transportation and extended

developed in West Sumatra province, Indonesia. The

the shelf-life after transportation, the effect packing stylecharacteristics of this papaya are orange-red flesh, sweetand temperature storage were investigated. In thistaste and chewy flesh. Usually, this papaya is distributed inexperiment, a practical distribution of papaya chain fromlocal market and other provinces in Sumatra island.

farm to table, papaya fruits were transported cardboard box

However, papaya is perishable commodity. Therefore, isand cardboard with MAP, and stored at 10oC and 25oC afterneeded the proper technology for maintaining qualitytransportation.

particularly during transportation and storage.
In actual distribution chain, this papaya transported

Materials and Methods

via land transportation using truck under ambient
temperature. Fruits were put on the floor of the truck“Merah Delima” papaya (Carica papaya L.) atwithout any packaging. This condition is injury the fruit,commercial maturity were harvested from the garden atresulting shorten the shelf-life and increases the amount of

Solok Regency, West Sumatra Province, Indonesia. Fruits

losses.

were sorted and selected on the basis of uniform size and

Low temperature storage is primary method forabsence of visual defects. Fruits were packed in cardboard

maintaining quality of perishable product. However,box (40.5 x 21 x 21.5 cm), and cardboard with MAP using

papaya is chilling-sensitive product (Kader, 2002), prolong

LDPE. Both sides of cardboard were made four holes withstorage at chilling-temperature increases susceptible toa diameter 2 cm, respectively. After packaging, fruit weredecay. MAP has been subjected in many studies fortransported to the retailer market (Padang City) by using a

alleviating CI in chilling-sensitive product. Packaged

mini-truck

persimmon in MAP prevent CI during transportation andtransportation. During transportation, the vibration wasstorage at low temperature (Fahmy and Nakano, 2016).

recorded by using vibration recorder (data not shown).

The ability of MAP to reduce CI is thought as reduction in

After arrival in the market, the mechanical injury wasO2 and elevation of CO2 inside the package. In previous

checked, and then stored at 10°C and 25°C in both of

study, the individual and combined effect of low O2 andpackaging for 21 days to check the shelf-life. Quality

high CO2 has been investigated to reduce CI (Fahmy andparameters such as weight loss, CI index, total soluble

which

is

commonvehicle

using

for

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 02solid (TSS) and malondialdehyde (MDA) content were
investigated every 3 days. MDA content was measured as
describe in Fahmy and Nakano (2014a), CI index was
calculated according to Pesis et al. (2000), and TSS was
measured by using refractometer (Atago, Japan).
The research was completely randomised with 3
replications (3 fruits per test). Statistical significance wasdetermined by subjecting the mean values to analysis ofvariance and means were compared by Tukey’s test at the
5% level of significance using R 3.5.1 (R Foundation).
Fig. 2 Weight loss of ‘Merah Delima’ Papaya after
Results and Discussion

transportation followed by 10°C and 25°C for 21 days.
Fruits were packaged with cardboard and cardboard with

Figure 1 showed that the percentage of mechanicalinjury after transportation from the farm to the market

MAP. Vertical bars represent standard error of the means(SE) for triplicate samples.

which observed by visual. The percentage of mechanicalinjury was higher on fruit packaged in cardboard than fruit

CI index of ‘Merah Delima’ papaya appeared on day

packed in cardboard with MAP. It has been reported that

15 for both of packaging during storage at 10°C (Fig. 3).

application of MAP reduces the softening of persimmon

However, the increase of CI index on the fruit packaged in

fruit on the day of delivery and the day after delivery

cardboard with MAP was supressed until day 18, and then(Fahmy and Nakano, 2016). Packaged in MAP also

increased slightly until the end of storage. While, fruit

maintains the quality during long term transportation of

packaged in cardboard without MAP, the index of CI

persimmon (Liamnimitr et al., 2018).

increased rapidly, and on the end of storage was higher
significantly compared with fruit packaged in cardboard
with MAP. Pesis et al. (2000) also reported that MAP
suppressed the development of CI symptoms in mango
fruit.
TSS of papaya increased for both of packaging duringstorage at 10 ° C and 25 ° C, and then decreased after
achieving the maximum value (Fig. 4). Fruit stored at 25°C,
the maximum values of TSS were occurred on day 3 and
6 for fruit packed in cardboard and cardboard with MAP,
respectively. While, for fruit stored at 10 °C, showed

Fig. 1 Percentage of mechanical injury of ‘Merah Delima’
papaya after transportation. Fruits were packaged withcardboard and cardboard with MAP. Vertical bars
represent standard error of the means (SE) for triplicate
samples.
The weight loss of fruit increased significantly on the
fruit packaged in cardboard without MAP, and rapid
increased was shown on the fruit stored at 25°C (Fig. 2).
While, the increased of weight loss was suppressed on the
fruit packaged in cardboard with MAP, and it was mostFig. 3 CI index of ‘Merah Delima’ Papaya after

suppressed stored at 10 C. The result also shows that fruits

transportation followed by 10°C for 21 days. Fruits werestored at 25°C had shelf-life 9 days and 12 days for

packaged with cardboard and cardboard with MAP.

cardboard and cardboard with MAP, respectively. Fruit

Vertical bars represent standard error of the means (SE)stored at 10°C could be kept until the end of storage.

for triplicate samples.

oProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 02trend among both of packaging, which the maximum(Fahmy and Nakano, 2014b). Stored of papaya fruit undervalue of TSS occurred on day 15, and then decreased

high CO2 suppressed the increases of MDA content

gradually until the end of storage.

(Fahmy et al., 2019)Conclusion
Packaged of ‘Merah Delima’ papaya in cardboard
with MAP reduced quality loss during transportation andstorage. Combination treatment with low temperaturestorage showed best performance for maintaining quality
and extending the shelf-life of papaya.
Acknowledgements

Fig. 4 TSS of ‘Merah Delima’ Papaya after transportation
followed by 10°C and 25oC for 21 days. Fruits were
packaged with cardboard and cardboard with MAP.
Vertical bars represent standard error of the means (SE)for triplicate samples.
MDA content increased for both of packaging duringstorage at 10°C and 25°C (Fig. 5). Rapid increased in MDA
content was showed on fruit packaged in cardboard at
25oC, and it was suppressed on fruit packed in with MAP
until day 3, and then increased quickly until the end ofstorage. While, for fruit stored at 10 C, the MDA content°

showed similar trend until day 15 on both of packaging,and then it increased rapidly for fruit packaged in
cardboard without MAP. Fahmy and Nakano (2014a) also
found that the increases of MDA content of cucumber fruit
were suppressed on the fruit stored under low O2. MAP
with LDPE film supressed the increases of MDA content

We would like to thank to Faculty of Agricultural
Technology for financial support. We are grateful to The
United Graduate School of Agricultural Science, Gifu
University, Japan for supporting laboratory instrument at
Lab Station in Postharvest Technology.
References
Fahmy, K., Nakano, K. (2014a) The individual andcombined influences of low oxygen and high carbon
dioxide on chilling-injury suppression in cucumber
fruit Environ. Control Biol. 52 3 149–153.
Fahmy, K., Nakano, K. (2014b) Optimal design ofmodified atmosphere packaging for alleviating chilling
injury in cucumber fruit Environ. Control Biol. 52(4),233–240.
Fahmy, K., Nakano K., Violalita, F. (2015) Investigation
on quantitative index of chilling injury in cucumber
fruit

based

on

the

electrolyte

leakageandmalondialdehyde Int. J. Adv. Sci. Eng Inform. Technol.
5 71-74.
Fahmy, K., Nakano, K. (2016) Effective transport andstorage condition for preserving the quality of Jiro
persimmon in export market Agric. Agric. Sci.
Procedia 9 279-290.
Fahmy, K., Violalita, F., Chatib, O.C., Yulia, R., Nakano,
K. (2019). The individual influences of high CO2 on
chilling injury suppression of ‘Merah Delima’ papaya
fruit.

IOP

Conferences

Series:

Earth

and

Environmental Science, 230 (1), 012016.
Fig. 5 MDA content of ‘Merah Delima’ Papaya after
transportation followed by 10 C and 25 C for 21 days.
°°

Fruits were packaged with cardboard and cardboard with
MAP. Vertical bars represent standard error of the means(SE) for triplicate samples.

Kader, A. A. (2002). Postharvest biology and technology:Anoverview.

In“Postharvest

technologyofhorticultural crops” (ed. by Kader, A. A.), Ed. 3. Univ.
California, Div. Agric. Nat. Resour., Oakland, CA, p
39–47.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 02

Liamnimitr, N., Thammawong, M., Techavuthiporn, C.,
Fahmy,K.,

Suzuki,T.,

Nakano,K.

(2018).

Optimization of bulk modified atmosphere packaging
for long-term storage of “Fuyu” persimmon frui.
Potsharvest Biol. Technol., 135, 1-7.
Pesis, E., Aharoni, D., Aharon, Z., Ben-Arie, R., Aharoni,N., Fuchs, Y. (2000). Modified atmosphere andmodified humidity packaging alleviates chilling injury
symptoms in mango fruit. Postharvest Biol. Technol.
19: 93–101.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 03

Effects of Maturity Stages and Organic Treatments to Control Postharvest
Fungal Infection, Shelf life Extension and Quality Retention of Papaya for
Nutritional Food Safety
Md. Harun Ar RASHID1*, Md. Ahsan HABIB1
1

Department of Horticulture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh*

Corresponding Author: harun_hort@bau.edu.bd
SUMMARY

An experiment was conducted to study the effects of maturity stages and organic treatments to control postharvest
fungal infection, shelf life extension and quality retention of papaya at the Laboratories of the Departments of Horticultureand Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh during the period from August to
November 2018. The two-factor experiment consisted of two maturity stages viz. M1: mature green colour and M2: 010% yellowing on the fruit; and six organic postharvest treatments viz. control (T0), hot water @ 50°C for 10 minutes(T1), aloe vera extract @ 1% (T2), garlic extract @ 1:1 (T3), hot water + aloe vera (T4), and hot water + garlic (T5). The
experiment was laid out in a completely randomized design with 3 replications. At the end of the storage period, it was
found that combined application of maturity stages and organic treatments were significant on almost all the parametersstudied except titratable acidity. Peel colour was light greenish yellow in hot water plus aloe vera and hot water plus garlic
extract fruits, while yellow in rest of the treatment combinations under both maturity stages. The maximum weight loss
(18.82%), TSS (14.68%), disease incidence (100%) and severity (80.67%) were recorded from control fruits, whereas theminimum values (18.82, 10.30, 58.33 and 15.17%, respectively) were found from hot water plus aloe vera followed by
hot water plus garlic treated fruits under both maturity stages. However, the highest pulp to peel ratio (4.33), pulp pH(6.32), vitamin C (53.96 mg/100g) and shelf life (15.08 days) were obtained from the combined treatment of hot waterplus aloe vera extract followed by hot water plus garlic extract, while the lowest values (3.10%, 5.02%, 39.77 mg/100g
and 8.27 days, respectively) were observed in control under maturity stage 2 fruits. Therefore, it can be concluded thatcombined application of hot water plus aloe vera extract, followed by hot water plus garlic extract under both maturity
stages found to be better in respect of reducing fungal infection, extension of shelf life and quality retention of papaya
fruits without affecting the nutritive value.
Introduction

flavors by partial hydrolysis of protein, production of
dehydrated pulses and beans, and improvement of protein

Papaya (Carica papaya L.) belonging to the family
Caricaceae, is an evergreen herbaceous commercial fruit

dispersibility index of soya flour (El Moussaoui et al.
2001).

plant of tropical and subtropical region (Srinu et al. 2017).

Global papaya production in recent years is about

Papaya is consumed as fresh, as a vegetable or used for13.02 M tons (FAOSTAT, 2019), which is quite satisfactory,

processed products such as drinks, jams, jellies, ice cream,

although approximately 40-70% of the produce is damaged

pies and as dried and crystallized fruits world-wide (Jaime

due to postharvest fungal decay, pest disinfestations,

et al. 2007). Papaya is a nutritious table fruit of highphysiological disorders, mechanical and chilling injuries

digestive value and an excellent source of natural vitamins(Rashid et al. 2015). After harvesting, the fruits start to live

and minerals. Biochemically, papaya contains several

by using their own reserves and fruits mature progressively

proteins, alkaloids and proteolytic enzymes, which are

and during the storage, conditions are favourable tosuccessfully used in the pharmaceutical, medicinal andmicroorganisms, which cause fruit damage. Because of its

other industries such as preparation of chewing-gum, chill-

naturally fragile and thin skin, papaya is damaged very

proofing beer, tendering meats, preparation of fish protein

easily by handling and infected by the main postharvest

concentrates, animal feed, developing roast beef-like

disease anthracnose (Colletotrichum gloeosporioides),Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 03

which is a major problem for papaya producers, traders and2. Treatments and design of the experimentconsumers in the tropics (Palhano et al. 2004).

The two-factor experiment consisted of two maturity

Sensitiveness to low temperature increases difficulties ofstages viz. M1: mature green and M2: 0-10% yellowing on

papaya during. Harvesting at proper maturity stage is a

the fruit; and six organic treatments viz. control (T0), hotvery important determinant for storage-life and final fruit

water @ 50°C for 10 minutes (T1), aloe vera extract @ 1%

quality, while at improper maturity can lead to uneven(T2), garlic extract @ 1:1 (T3), hot water + aloe vera extract

ripening and over ripe fruits. Postharvest losses can be(T4), and hot water + garlic extract (T5). Trials were set up

reduced by appropriate wrapping materials, heat treatment,

in a completely randomized design (CRD) with three

low-dose gamma irradiation, chitosan and wax coating,replications and 5 different days of storage (0, 3, 6, 9, 12)

plant extracts (aloe vera, onion, garlic, neem, etc.).

for a total of 360 samples and each sample represented by

Postharvest research related to fruit ripening and handling

one papaya.

became more important during the last decade because ofincreasing consumer awareness, expansion in production

3. Application of postharvest treatments

and exports. The most important research issues are on

Postharvest treatments were sequentially applied to

keeping quality and postharvest storage life since extreme

the fruits in order to study physico-chemical characteristics.

or fluctuating temperature treatments and mechanicalFor control treatment (T0), ten fruits under each maturity

damage combined with improper harvesting and handlingstages were taken randomly from a papaya fruit lot. Hot

can result in fruit with poor appearance, flavour andwater treatment (T1) was done by individually dipping thenutritional value (Proulx et al. 2005). However, a limited

fruits into the hot water at 50 °C for 10 minutes in an

work has been done on the combined application of hotelectric hot water bath. For aloe vera coating (T2), thewater and plant extracts under different maturity stages.

selected fruits were dipped in 1% aloe vera solution for 5

Therefore, the present study was undertaken to investigate

minutes. Aloe era gel was extracted from fresh aloe vera

the effects of maturity stages and organic treatments toleaves and gel solution was prepared as described bycontrol postharvest fungal infection, shelf life extension

Sharmin et al. (2015). For garlic treatment (T3), initiallyand quality retention of papaya without affecting thestock garlic extract (1 kg garlic cloves and 1 L water) was

nutritive value.

prepared by crushing the fresh cloves in distilled waterusing a blender through straining and then cheesed. The
stock extract was then used to prepare treatment of 1:1

Materials and Methods

concentration. To improve wettability of the solution 0.1
1. Experimental location and material
themL Tween 20 was added to the solution. The selected fruits

The experiment was conducted at the Laboratories ofunder both maturity stages were dipped into the treatment

Departments

solutions for 5 minutes. For combined treatment, hot water

Chemistry,of

Horticulture

Bangladeshand Agricultural
University,

dipping was done first followed by aloe vera extract (T4)

Mymensingh during the period from August to November

Agriculturaland garlic extract coating (T5), respectively. After2018. Papaya fruits (Carica papaya var. Shahi) of two

application of all the treatments, fruits were air-dried,maturity stages (mature green and beginning of yellow

wrapped in non-absorbent paper and stored at 25 ± 1°C forcolour up to 10% of yellow surface) were collected from anormal ripening and data were monitored at 0, 3, 6, 9 andfarmer’s field near the Bangladesh Fisheries Research

12 days of storage.

Institute, Mymensingh in perfect quality conditions. On
arrival at the laboratory, the fruits were washed with

4. Parameters measured

distilled water, cleaned properly and air-dried. The selected

Changes in external colour of papaya fruits of two

fruits were uniform in shape, size (20-25 cm in length) andmaturity stages were observed carefully, visually examinedweight (700-800 g). After grading, the fruits wereand recorded at four stages during storage through scoring

individually wrapped in non-absorbent paper and thenusing a colour chart. Weight loss of papaya was measured

placed in a commercial packaging material under ambient

by weighing the individual fruits using a top pan electric

temperature (25 ± 1°C) prior to further treatments. Freshbalance (Rashid et al. 2019). Pulp to peel ratio wasaloe vera leaves and garlic cloves were bought from thedetermined by separately weighing pulps and peels ofSupermarket in Mymensingh.

fruits using electrical balance and measured by the ratio ofProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 03

weight of fruit pulp to weight of peel. The pH of fruit juice

stages. In the control, protoplast was changed into

was recorded using an electric pH meter, which waschromoplast normally, while in treated samples, thisstandardized with buffer solution as described byprocess was suppressed by the treatment effect (Rashid et

Ranganna (1994). The remaining juice from pH

al. 2019). The changes of these components affected thedetermination was used to measure TSS of fruit pulp andfruit colour from green into yellow in the skin. The results

was determined by using a hand refractometer (Model N-1

showed that combined treatment had better externalα, Atago, Japan). Titratable acidity and ascorbic acidappearance than the others of both maturity stages.

content of papaya fruit pulp was determined by Ranganna
method (1994). For calculating disease incidence and2. Weight loss

severity, ten fruits were taken for each treatment and stored

Combined application of maturity stages and organicat ambient temperature (25 ± 1°C) for normal ripening.

treatments had significant influence on weight loss ofFruits were critically examined every day for thepapaya, which was gradually increased with the storage

appearance of the disease symptoms already published (Alperiod (Table 1). At 12 DAS, the maximum weight loss

Eryani-Raqeeb et al. 2009), and data were recorded every

was observed in M2T0 (18.82%) followed by M1T0

3 days starting from the 3rd day of storage and used to(17.85%), while the minimum weight loss was found from

calculate the percentage of visibly infected fruits in the

M1T4 (9.20) followed by M2T5 (9.78). This might be due toentire population (Rashid et al. 2015). Disease severity was

the coating effects of aloe vera and garlic extracts alongmeasured as described in terms of the proportion of thewith hot water, which could reduce transpiration andfruit surface occupied by lesions with <5% being

respiration by the papaya fruits.

acceptable for trading, 5–25% being viewed as tolerable
for trade, 25–50% seen as having little possibility of

3. Pulp to peel ratio

commercial use and >50% as unusable (Azevedo, 1998).

Pulp to peel ratio was significantly influenced by the

Shelf life was calculated from daily estimates of diseasecombined effects of maturity stages and organic

severity on the same 10 fruits from each replicate andpostharvest treatments (Table 2). It showed a decreasing

considered as ended when the fruits had little or no

trend from harvest to ripening. At 12 DAS, the maximum

commercial viability and eating quality (disease severity

pulp to peel ratio was observed in M1T4 (4.33) followed by>25%).

M2T4 (4.19), while the minimum pulp to peel ratio was

5. Data analysis

decrease in pulp to peel ratio might be due to the change inrecorded in M2T0 (3.10) followed by M1T0 (3.20). The
The collected data were statistically analyzed usingsugar concentration in the peel compared to the pulp, whichanalysis of variance (ANOVA) where the significance ofcontributed to different change in osmotic pressure (Rashid,difference between pair of means was tested by least

et al. 2019).

significant difference (LSD) at 1 and 5% levels ofprobability using MSTAT Computer programme.

4. Pulp pH
Combined effects of maturity stages andResults and Discussion

organic postharvest treatments exhibited significant
variation in pulp to peel ratio of papaya during

1. External colour

storage (Table 2). Results revealed that pulp pH was

The change in external colour was noticiable due toincreased with the storage period. At 12 DAS, the highest

maturity stages and organic postharvest treatments (datapulp pH was recorded from M1T4 (6.32) followed by M2T4

not shown). The results showed that the external colour(6.19), while the lowest was observed in M2T0 (5.02)

changes in fruits of control and single treatments were

followed by M1T0 (5.05). This could be due to theobserved earlier than the combined treatments under bothincreasing fashion of papaya pH during storage.

maturity stages. At the end of the storage period (12 DAS),
peel colour was light greenish yellow in combined

5. Total soluble solids (TSS) content

treatment of hot water plus aloe vera extract (M1T4) and

The combined effect of maturity stages and organic

hot water plus garlic extract (M1T5) fruits, while yellow in

postharvest treatments has significant influence on TSS ofrest of the treatment combinations under both maturity

papaya (Table 2). Ghanta (1994) reported that TSS content

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 03

Table 1 Combined effects of maturity stages and organic

Table 2 Combined effects of maturity stages and organic

treatments on the weight loss of papaya fruits at 12 days

treatments on weight loss of papaya fruits at 12 days after

after storage (DAS)

storage (DAS)

Treatment

WeightPulp to

Pulp

TSS

Treatment

Titratabl

Vitamin

Disease

Diseasecombination

loss

peel

pH

(%)

ratio

content

combinatione acidity

C (mg/

incidenseverit

(%)

100g)

ce (%)

y (%)

M1T0

17.85

3.20

5.05

14.12

M1T0

2.40

39.77

100.0

73.33

M1T1

14.68

3.77

5.47

13.77

M1T1

2.30

45.83

93.33

46.67

M1T2

12.58

4.13

5.67

12.85

M1T2

1.67

48.02

81.67

31.67

M1T3

13.56

4.09

5.55

13.00

M1T3

1.95

46.68

83.33

39.00

M1T4

9.20

4.33

6.32

10.30

M1T4

1.05

53.96

58.33

15.17

M1T5

10.53

4.15

6.10

10.67

M1T5

1.19

51.29

71.67

17.67

M2T0

18.82

3.10

5.02

14.68

M2T0

2.55

41.40

100.0

80.67

M2T1

15.20

3.69

5.43

14.12

M2T1

2.36

42.81

96.67

47.67

M2T2

13.25

4.01

5.34

13.09

M2T2

1.74

47.11

86.67

33.33

M2T3

13.66

3.82

5.88

12.72

M2T3

2.09

45.31

88.33

41.00

M2T4

9.78

4.19

6.19

10.82

M2T4

1.29

49.63

61.67

16.50

M2T5

10.95

4.09

6.04

11.30

M2T5

1.45

47.74

76.67

19.33

LSD0.01

0.324

0.075

0.092

0.266

LSD0.01

0.107

0.881

2.14

2.08

**

**

**

**

NS

**

**

**

Level ofsignificance(%brix)Level ofsignificance**= Significant at 1% level of probability, M1=Maturity stage
1 (Mature green colour), M2=Maturity stage 2 (0-10%
yellowing on the fruit surface), T0=Control, T1=Hot
water@50°C for 10 minutes, T2=Aloe vera extract@1%,
T3=Garlic extract@1:1, T4=Hot water+aloe vera extract,
T5=Hot water+garlic extract.

**= Significant at 1% level of probability, NS=Not significant,
M1=Maturity stage 1 (Mature green colour), M2=Maturity
stage 2 (0-10% yellowing on the fruit surface), T0=Control,
T1=Hot water@50°C for 10 minutes, T2=Aloe vera
extract@1%, T3=Garlic extract@1:1, T4=Hot water+aloe vera
extract, T5=Hot water+garlic extract.

in pulp gradually increased during the storage period

C (53.96 mg/100g) was observed in combined treatment ofirrespective of postharvest treatments. At 12 DAS, thehighest TSS was observed in M2T0 (14.68%) followed by
M1T0 (14.12%), while the lowest in M1T4 (10.30%)
followed by M1T4 (10.67%). This might be due to thehydrolysis of starch into sugar (Pimentel and Walder, 2004).
Sugar content is one of the main components of quality of
papaya, so it is important that papaya should be harvested
more mature and, if possible, in the maturation stage thatelicits higher sugar concentration.
6. Titratable acidity (TA)
Combined effects of maturity stages and organic
treatments exhibited no significant influence on TA
content of papaya fruits (Table 2).
7. Vitamin C content
The combined treatment of maturity stages andorganic treatments showed significant influence on
vitamin C content of papaya (Table 2). The highest vitamin

hot water plus aloe vera extract under maturity stage 1
fruits (M1T4) followed by 51.29 mg/100g in M1T5, while
the lowest vitamin C was recorded in M1T0 (39.77
mg/100g) followed by M2T0 (41.40 mg/100g). Vitamin C
levels rose immediately following each treatment and thendeclined in the expected way over the storage period.
8. Disease incidence
Three fungal diseases viz. anthracnose, stem end rot
and Rhizopus rot were identified during storage byobserving the typical symptoms caused by Colletotrichum
gloeosporioides, Botryodiplodia theobromae and Rhizopus
stolonifer, respectively (Fig. 1).
8.1 Assesment of percent disease incidence
Combined effect of maturity stages and organic treatments
had significant influence on disease incidence of papaya(Table 2). Highest disease incidence (100%) was found in
M1T0 and M2T0 at the end of the storage period and theProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 03

2011; Nur Fatima et al. 2018).
10. Shelf life
Shelf life is the period from harvesting up to the last
edible stage. This is the basic quality of fruits, which helps
long marketing time, and it is the most important aspect inloss reduction technology of fruits. The extension of shelf
Fig.1

Anthracnose

life of fruit has been one of the prime concerns of(Colletotrichum gloesporioides), (b) Stem end rot

marketing throughout the record of history. Statistically(Botryodiplodia theobromae) and (c) Rhizopus fruit rot

there was significant difference between the two maturity(Rhizopus stolonifer) of papaya in control treatment.

stages and organic postharvest treatments with respect to

Photographshowing(a)shelf life of papaya (Figure 2). Results revealed that thelowest was observed in M1T4 (15.17%) followed by M1T5

longest shelf life of papaya fruits was observed in M1T4

(17.67%). Combined treatments such as heating and plant(15.08 day) followed by M2T4 (14.05 day), while theextracts may give synergistic effect and could give lessshortest shelf life (8.27 day) was recorded in M2T0

detrimental effect to quality attributes of the papaya fruits.

followed by 8.48 day in (M1T0). The delay in ripening on

Miller and McDonald (1999) also reported that decay was

hot water and aloe vera coated fruits can occur due to the

manifested more in 1/3 yellow coloured fruits than mature

lower capacity of these fruits in producing ethylene, sincegreen stage, probably because of the higher incidence of

this hormone has a stimulation role in the generalpeel injuries during handling; decay organisms are known

metabolism, and seems to be implicated in the activation

to enter into fruit at sites of injury. Hot water dipping forand regulation of some enzymes involved in ripening

10 min at 55 °C can be used to reduce postharvest fungal(Mondal et al. 2011). The increase in shelf life was

infection (Rashid et al. 2015). Though the hot waterprobably due to the changes in the concentrations oftreatment reduce the incidence of anthracnose by reducing

various gases like the increase of O2, the reduction of CO2

the available inoculum on the fruit surface, this treatment

and ethylene as well as the slowing down of the processes

affects the polygalacturonase activity, ascorbic acidleading to delayed ripening and reducing decay by aloe

content and titratable acidity levels (Ferguson et al. 2000).

vear and garlic extracts and hot water treatments.

9. Disease severity
Combined effect of maturity stages and organic
postharvest treatments had significant influence on disease18

severity of papaya (Table 2). The highest disease severity(16.50%). Recently, aloe vera gel has been used as an
edible coating materials, which have various favourable
effects on fruits and vegetables such as imparting a glossy
appearance and better colour, retarding weight loss, or
prolonging shelf-life by preventing microbial spoilage andbiologically safe for different types of foods because of its

14.13

14.05

13.12

Shelf life (days)

and the lowest in M1T4 (12.36%) followed by M2T4

15.08

15

was found at M2T0 (80.67%) followed by M1T0 (73.33%

12.20

12

9

12.12

10.57

13.82

11.83

10.12

8.48

8.27

6

3

0

0
1T
M

1
1T
M

2
1T
M

3
1T
M

4
1T
M

5
1T
M

0
2T
M

1
2T
M

2
2T
M

3
2T
M

4
2T
M

5
2T
M

Treatment combination

film-forming properties, biodegradability and biochemical
properties (Sharmin et al. 2015). It is composed mainly ofFig. 2 Combined effect of maturity stages and organic treatments

polysaccharides and acts as a natural barrier to moisture

on shelf life of papaya fruits. The vertical bar represents LSD atand oxygen, which are the main agents of deterioration of

1% level of probability. (M1=Maturity stage 1 (Mature green

fruits and vegetables (Misir et al. 2014). The ethanoliccolour), M2=Maturity stage 2 (0-10% yellowing), T0=Control,

extract and fresh garlic cloves contain biologically natural

T1=Hot water@50°C for 10 minutes, T2=Aloe vera extract@1%,

fungicide substances (allicin), which are potentially used

T3=Garlic extract@1:1, T4=Hot water+aloe vera extract, T5=Hot

for control of many fungal diseases of fruits (Mondal et al.

water+garlic extract.)Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 03Conclusion

as a novel edible coating for fresh fruits: a review.
American Journal of Food Science and Technology

From the experimental results and discussion, it can2(3), 93-97.

be concluded that combined application of hot water plus

Mondal, M. F., Islam, M. S. and Rashid, M. H. A. (2011)

aloe vera extract, followed by hot water plus garlic extract

Effects of plant extracts on shelf life and postharvest

under both maturity stages found to be better in respect of

diseases of papaya. Journal of the Bangladesh Society

reducing fungal infection, extension of shelf life andfor Agricultural Science and Technology 8 (3&4), 93-

quality retention of papaya fruits without affecting the96.
Nur Fatimma, A., Munirah, M. S., Sharifah Siti Maryam,

nutritive value.

S. A. R., Najihah, A., Nur Ain Izzati, M. Z. (2018)
Acknowledgements

Efficacy of Allium sativum extract as post-harvest
treatment of fruit rots of mango. Plant Pathology &The authors are pleased to thank the Bangladesh
Agricultural University Research System (BAURES) for

Quarantine 8(2), 144–152.
Palhano, F. L., Vilches, T. T. B., Santos, R. B., Orlando, M.
T. D., Ventura, J. A., Fernandes, P. M. B. 2004.

funding this work.

Inactivation of Colletotrichum gloeosporioides spores
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the plant defence mechanism. Cell and Molecular Life

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Rome(http://faostat3.fao.org/faostatgateway/go/to/download/Q/*/E)

under two different maturity stages. Journal of
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T. M. M. (2015) Low-dose gamma irradiation

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following hot water immersion of papaya (Carica

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papaya Linn.) fruits provides additional control of

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postharvest fungal infection to extend shelf life.

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Sharmin, M. R., Islam, M. N., Alim, M.A. (2015) Shelf-S., Abed G., Manoel, T. S. J., Paula, F. T. (2007) Papaya

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Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 04

Extraction of Chitosan from Shrimp and Crab Shell and Its Effect on the
Quality of Pineapple Juice
Sharmin SULTANA1, Md. Sajjad HOSSAIN2, Abdullah IQBAL1*
1

Department of Food Technology & Rural Industries, Bangladesh Agricultural University, Mymensingh-

2

Department of Chemical and Food Process Engineering, Rajshahi University of Engineering &2202, BangladeshTechnology, Rajshahi-6204, Bangladesh*

Corresponding Author: iqbal21155@bau.edu.bd
SUMMARY

The present study was undertaken to extract chitosan from shrimp and crab shell by chemical method and to assess
its effects on the qualities of pineapple juice. Chitosan was derived from shrimp and crab shell by the process of chemicaltreatment demineralization (2%HCl, 16hr), deproteinization (4% NaOH, 20hr) and deacetylation (60% NaOH, 650C,
20hr). Extracted chitosan’s physicochemical and functional properties like fat binding capacity (FBC), water binding
capacity (WBC), solubility, average molecular weight, ash content, moisture content and degree of deacetylation werestudied. The degree of deacetylation and solubility of chitosan extracted from shells of shrimp and crab were found to be
76 and 65% and 91 and 70%, respectively. In a similar way, the WBC of shrimp and crab shell chitosan were found to be
445 and 260 % and the and FBC was found to be 360 and 210%, respectively. The average molecular weight of shrimp
and crab chitosan was found 6273 and 6267 Dalton, respectively. Chitosan was added to pineapple juice at 0.2, 0.4 and0.6 g/L concentration as a clarifying agent. The results showed that addition of the shrimp chitosan as a clarifying agent@ 0.6g/L at 25±2ºC for 90 min was more effective than the crab chitosan. Statistical analyses showed significant effecton sensory characteristics when added chitosan at 0.2-0.6 g/L in pineapple juice. This study showed that shrimp shell was
better than crab shell for the preparation of chitosan and shrimp chitosan was more effective than crab chitosan as apreservative and clarifier on the quality of pineapple juice.
Introductionused as an important source for production of value-added
product such as chitin and its deacetylated derivative

Chitosan is deacetylated derivative of chitin, whichchitosan.

is one of the most abundant polysaccharides occurring in

Chitosan have been used as additive in the processing

nature next to cellulose (Ramasamy and Shanmugam,of fruit juices, for reducing the turbidity (Soto-Peralta et al.,1885) and found on the outer surface of the arthropod body,1989), reduction of acidity (Imeri and Knorr, 1988),

in the cell wall of mushrooms and in cell structures of algae

reductionand yeast (Kaya et al., 2015). Crustacean waste from crab

demonstrated antimicrobial activity against bacteria,

shells is composed of chitin which forms a chitin-protein

yeasts and molds, with low toxicity in mammalian cells

complex with proteins. Fish scales, shrimp and crab shells(Martín-Diana et al., 2009), as antioxidant agent (Chien et

are composed of different components such as proteinsal., 2007) as well as shelf life extender for selected fruit(15–50%), minerals (30–50%) and chitin (15–30%) (Hajji

and fruit juice (Martín-Diana et al., 2009, Hossain and

et al., 2015, Hossain and Iqbal, 2014). The growing

Iqbal, 2014).

quantities of shell wastes from processing of crustaceansofbrowning(Sapers,1992),

and

also

Pineapple juice is very popular and abundantly

have become a major concern for seafood processing

consumed in many countries.

plants since their biodegradation is very slow. Global

large amount of pineapple every year. Its popularity isannual production of shell wastes from crustaceans isbased on attractive aroma and flavor characteristics andestimated at 1.41 million metric tons (Pérez Roda et al.,beneficial components that play a primary role in avoiding2019). These processing byproducts, however, may be

the risk of chronic diseases. Pineapple juice is one of the

Bangladesh produces aProceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 04

fruits juices that contain high contents of antioxidant andthe quality of chitosan (Toan, 2009). The isolation ofphenolic compounds (Wen and Wrolstad, 2002). To

chitosan was carried out following 3 (three) steps namely-

prolong the juices shelf life, chemical additives are used.

demineralization, deproteinization and deacetylation as

However, some countries limit their use because of

described by Toan (2009) and Hossain and Iqbal (2014).

possible health damage. These facts, combined with

The resulting chitosan was then dried at cabinet dryer for 4

growing consumer demand for natural foods, encourages

hours at 65±50C and stored in polyethylene bag until they

the study of the area of food science to gain new

are used as a preservative in pineapple juice.

perspectives preservatives that meet market demands
without neglecting the quality of products (Raybaudi-

2. Physicochemical and functional properties of chitosan

Massilia et al., 2009). In this context, chitosan has gained

Moisture and ash content, Solubility, Bulk density,

popularity to be applied widely in the pharmaceutical andintrinsic viscosity, Molecular weight, Color, Water binding

food industries due to its unique features such as

capacity, Fat binding capacity, degree of deacetylation etc.

non-of chitosan were measured as per the standard methods

toxicity and biodegradability etc.
Processing of clarified fruit juices commonly
involves the use of clarifying aids, including gelatins,

described by several researchers (AOAC, 2005, Wang etal., 2007, Knorr, 1982, Hossain and Iqbal, 2014).

bentonite, tannins or combinations of these compounds(Soto-Peralta et al., 1989). Chitosan with a partial positive

3. Application of chitosan to Pineapple juice

charge has been shown to possess acid binding properties

Pineapple juice was prepared from mature and fresh(Imeri and Knorr, 1988) and to be effective in aiding thepineapples. Clarification of pineapple juice is performed

separation of colloidal and dispersed particles from foodby addition of three different concentrations of shrimp andprocessing wastes (No and Meyers, 2000), while thermal

crab chitosan S1, C1 (0.2%), S2, C2 (0.4%), S3, C3 (0.6%)

pasteurization of juice significantly reduces the risk of foodincluding a control sample P (0), where S and C indicatesborn disease and can also reduce the nutrient content. For

chitosan prepared from Shrimp and Crab, respectively. The

these reasons, the scientific community and food industry

clear juice was obtained after centrifugation at 5000 rpm

focus their attention on non-thermal emerging technologies

for 10 min. Unclarified juice was used as a control forand alternative novel treatments to control undesirable

comparison among clarification treatments. The clarified

microorganisms with less adverse effects on quality.

juice was stored at 40C until further analysis (Oszminanski

Therefore, in this research work, it is attempted to
extract and characterize chitosan

and wojdylo, 2007).

from shrimp and crab

shell and subsequently to study the feasibility of applying
chitosan to improve the quality of pineapple fruit juice and4. Quality evaluation of Pineapple juice
Total soluble solids (TSS), pH, potential browning etc
were measured as per approved and standard methods.

to enhance the shelf life.

Results and Discussion

Materials and Methods

1. Physiochemical and functional Properties of shrimp and

1. Extraction of Chitosan
The experiment has been conducted in the laboratoryof the Department of Food Technology and Rural

crab chitosan
Properties of shrimp and crab chitosan are

University,

summarized at Table 1. Chitosan’s moisture content,

Mymensingh. Fresh shrimp and crabs were collected fromobtained from shrimp shells and crab was found as 4.23%

local market. Shell of shrimp and crab were separated fromand 3.78%, respectively which indicates shrimp chitosan

the whole body using a sharp knife. The collected wastes

has more moisture than the crab counterparts.

Industries,

Bangladesh

Agricultural

were then washed with tap water and crushed with mortar-

Ash content indicates that crab chitosan has more ash

pastel. Other essential reagents and utensils were used

content compared to shrimp chitosan.

from the laboratory stock. Clean and crushed shrimp andet al. (2014) reported that commercial chitosan consists ash

crab wastes were kept in a polyethylene bags at ambient

content around 2.28% which is very similar to the result

temperature (28±2 C) for 24 hours for partial autolysis toobtained in this study, particularly for crab shell.

0

facilitate chemical extraction of chitosan and to improve

Mohanasrinivasan

Shrimp chitosan was more soluble than crab

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 04

chitosan. Higher temperatures during the deacetylation

with increasing chitosan concentration. At 90 min., theprocess lead to a reduction in solubility. The quality of

optical density of shrimp chitosan added juice (@0.6 g/L)

chitosan depends on the degree of deacetylation which

was found less than that of crab chitosan added juice.

in-turn affects its solubility (Madhavan and Nair, 1974).

Domingues et al. (2012) reported that treatments

The degree of deacetylation of shrimp and crab was

employing chitosan ensures a turbidity reduction of almost

found to be 76% and 65% respectively. Because of low

100% regarding the raw juice or even to the juice

deacetylation of crab chitosan, solubility was found to be

centrifuged at 4000 rpm, similar behavior is observed

low whereas shrimp chitosan’s solubility was more.

regarding color removal.
The effect of chitosan on the TSS showed that

Hossain and Iqbal (2014) reported that several criticalincludingchitosan reduced the TSS value of the pineapple juice andtemperature and deacetylation reaction time, alkali

significant changes over storage time were observed. This

concentration, the ratio of chitin to alkali solution, andresult showed that the TSS reducing effect of shrimp

particle size.

chitosan is higher than Crab chitosan.

Table 1 Physiochemical and functional properties ofchitosanParameter/properties
Chitosan from
ShrimpCrabMoisture content (%)
4.23
3.78
Ash content (%)
0.29
2.3
Yield (%)
16.0
20.2
Solubility (%)
91
70
Degree of deacetylation (%)
76
65
Bulk density (g/ml)
0.09
0.20
Intrinsic viscosity (dl/g)
11.5
8.20
Ave. molecular weight (Da)
6273
6267
Water binding capacity (%)
445
260
Fat binding capacity (%)
360
210

chitosan concentration and storage time. All the samples

factors

affectingchitosansolubility

The bulk density found for chitosan from shrimp

It is seen that pH was significantly affected byshowed a similar pH increasing trend (from 3.7 to 4.18)
over storage time and no interaction effect resulted fromstorage time and chitosan concentration.
An interactive effect between chitosan concentrationand storage time on the browning potential (BP) was also
observed. In samples with lower or no chitosan
concentration, the BP increased rapidly over storage time,
while it remained almost constant in samples with higher
chitosan concentration.
3. Sensory evaluationand crab indicates that shrimp chitosan is more porous

Results of the sensory (hedonic) evaluation ofthan crab chitosan. The intrinsic viscosity of chitosan

pineapple juice using chitosan as preservative are

obtained from shrimp and crab in this study was found topresented in Table 2. In all the treatments, the averagebe 11.5 dl/g and 8.20dl/g. The average molecular weightscore ranged between 7-9 for color, taste and overallof chitosan derived from shrimp and crab was 6273

acceptability on a 9-point hedonic scale, the data are theDalton and 6267 Dalton, respectively. Crab chitosan hasmean scores of 10 panelists selected randomly (Table 2).

less average molecular compared to that of shrimp. Water

These data showed that there is significant increase in thebinding capacity of chitosan derived from shrimp anddegree of acceptance of each score with the increase of

crab was found to be 445% and 260%, respectively whichchitosan concentration (i.e., scores for color, taste andindicated that water binding capacity of shrimp chitosan

overall acceptability of the chitosan added samples were

was better than that of crab chitosan. The fat-binding

higher than the control sample). This result agrees withcapacity of shrimp and crab chitosan was found to be
360% and 210% respectively. No et al., (1989) reported
that fat binding capacity values of chitosan ranged from
314% to 535%, which indicates that the value obtained inTable 2 Sensory evaluation scores of pineapple juice
enriched with chitosan

this research is within the range.
2. Effect of chitosan on pineapple juice quality
The effect of chitosan at different concentration (0.2,
0.4, 0.6 g/L) on the clarification of pineapple juice has been
investigated. The juices showed increased in luminosity

S for samples treated with chitosan from Shrimp shell and
C for samples treated with chitosan from crab shell;
P=control, S1=(0.2g/L), S2=(0.4g/L), S3=C1=(0.6g/L);
C1=(0.2g/L), C2=(0.4g/L), C3=(0.6g/L);**

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 04

Hossain,
Chatterjee et al. (2004) who reported improvement of
acceptability and appearance when treated with chitosan.
The result showed that Shrimp chitosan added juice ismore acceptable than crab chitosan added juice.

M.S., Iqbal, A.

(2014) Production andcharacterization of chitosan from shrimp waste.
Journal of the Bangladesh Agricultural University,
12(1), 153–160.
Imeri, A.G., Knorr, D. (1988) Effect of chitosan on yieldand compositional data of carrot and apple juice.
Journal of Food Science, 53(3), 1707–1709.

Conclusion

Kaya, M., Baran, T., Asan-Ozusaglam, M., Cakmak, Y.,

Comparative studies on chitosan extracted fromshrimp and crab waste showed that shrimp shell is better
choiceforextractionofchitosan

based

on

all

physicochemical properties. Chitosan application on
pineapple juice showed increased in luminosity withincreasing chitosan concentration. Chitosan reduced
the Brix value of the pineapple juice. The pH
increased significantly with increasing chitosan
concentration and shrimp chitosan added juice
showed greater pH than crab chitosan added juice.
Chitosan retarded potential browning of pineapple
juice during storage. As the outcome of this research
is promising and optimistic, it will not be confined
only to pineapple, but also may be used for otherfruits like apple, orange and litchi etc.

Official

Analytical

Chemists.

Washington, D.C.
Chatterjee, S., Chatterjee, S., Chatterjee, B.P., Guha, A.K.
(2004) Clarification of fruit juice with chitosan.
Process Biochemistry, 39(22), 2229–2232.
Chien, P., Sheu, F., Huang, W.T., Su, M.S. (2007) Effectmolecular weight of chitosans on their antioxidative
activities in apple juice. Food Chemistry 102, 1192–
1198.
Domingues, R.C.C., Junior, S.B.F., Silva, R.B., Cardoso,
V.L., Reis, M.H.M. (2012) Clarification of passion fruit
juice with chitosan: Effects of coagulation process
variables and comparison with centrifugation andenzymatic treatments. Process Biochemistry, 47, 467–
471.
Hajji, S., Ghorbel-Bellaaj, O., Younes, I., Jellouli, K., Nasri,
M. (2015) Chitin extraction from crab shells by
Bacillus bacteria. Biological activities of fermented
crab supernatants. International journal of biologicalmacromolecules, 79, 167–173.

with antimicrobial and antioxidant activities fromcosmopolitan

Orthopteranspecies(Insecta).

Biotechnology and Bioprocess Engineering, 20(1),168–179.
Knorr, D. (1982) Functional properties of chitin andchitosan. Journal of Food Science, 47, 593-595.
Madhavan, P., Nair, K.G.R., (1974) Utilization of prawn
waste-Isolation of chitin and its conversion to chitosan.
Fishery Technology, 1, 50–53.
Martín-Diana, A.B.M., Rico, D., Barat, J.M., Barry- Ryan,
C. (2009) Orange juices enriched with chitosan:
Optimisation for extending the shelf life. Innovative
Food Science and Emerging Technologies, 10(4), 590–
600.
S.K., Selvarajan, E., Suganthi, V., Subathra Devi C.

AOAC (2005) Official Methods of Analysis. 15th Edition.
ofExtraction and characterization of chitin and chitosan

Mohanasrinivasan, V., Mishra, M., Paliwal, J.S., Singh,

References

Association

Tozak, K., Mol, A., Mentes, A., Sezen, G. (2015)(2014) Studies on heavy metal removal efficiency andantibacterial activity of chitosan prepared from shrimp
shell waste, Biotechnology, 4 (2), 167–175.
No, H.K., Meyers, S.P., Lee, K.S. (1989) Isolation andcharacterization of chitin from crawfish shell waste.
Journal of Agriculture and Food Chemistry, 37, 575–
579.
No, H.K., Meyers, S.P. (2000) Application of Chitosan for
Treatment of Wastewaters. In: Ware G.W. (eds)
ReviewsofEnvironmental

Contamination

andToxicology. Reviews of Environmental Contamination
and Toxicology, vol 163. Springer, New York, NY.
Oszmianski, J., Wojdylo, A. (2007) Effects of various
clarification treatments on phenolic compounds andcolor of apple juice. Eur. Food Res. Technol., 224, 755–
762.
Pérez Roda, M.A. (ed.), Gilman, E., Huntington, T.,
Kennelly, S.J., Suuronen, P., Chaloupka, M. andMedley, P. (2019). A third assessment of global marine
fisheries discards. FAO Fisheries and Aquaculture
Technical Paper No. 633. Rome, FAO. 78 pp.
Ramasamy, P., Shanmugam, A. (1885) Characterization

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 04

and wound healing property of collagen–chitosan film
from Sepia kobiensis. International journal of
biological macromolecules, 74, 93–102.
Raybaudi-Massilia, R.M., Mosqueda-Melgar. J., SolivaFortuny, R., Martih-Belloso, O. (2009) Control ofpathogenic and spoilage microorganisms in fresh-cut
fruits and fruit juices by traditional and alternative
natural antimicrobials. Comprehensive Reviews inFood Science and Food Safety, 8, 157–180.
Sapers, G.M. (1992) Chitosan enhances control of
enzymatic browning in apple and pear juice byfiltration. Journal of Food Science, 57(5), 1192–1193.
Soto-Peralta, N.V., Mller, H., Knorr, D. (1989) Effects ofchitosan treatments on the clarity and color of apple
juice. Journal of Food Science, 54(2), 495–496.
Toan, N.V. (2009) Production of Chitin and Chitosan from
Partially Autolyzed Shrimp Shell Materials. The OpenBiomaterials Journal, 1, 21-24.
Wen, L., Wrolstad, R.E. (2002) Phenolic composition ofauthentic pineapple juice. Journal of Food Science, 67,
155–161.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 05

Electrical Impedance Analysis for Evaluating Tissue Conditionsof Processed Vegetables
Teppei IMAIZUMI1*, Haruki ANDO1, Satoshi IWAMOTO1
1*

Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu city, 501-1112, Japan
Corresponding Author: imaizmi@gifu-u.ac.jp
SUMMARY

Tissue conditions are strongly related to quality of processed vegetables. To establish a technique which enables toevaluate the tissue conditions, we applied electrical impedance analysis to heated vegetables such as carrot. Carrot
samples were heated at 50 °C. Electrical impedance spectroscopy was performed over a frequency range of 50 Hz to 5
M Hz. The obtained impedance values were analyzed using an equivalent circuit model, then cell membrane capacitance,
extracellular resistance and intracellular resistance were determined. The model was successfully fitted to the measured
values. As expected, cell membrane capacitance decreased by long-time heating. By using a confocal laser scanningmicroscope, we observed cell membrane structure which stained with 0.05 % DiI. The images also indicated that cell
damage appeared when the sample was heated. Our research confirmed that the changes of electrical properties were
occurred by cell membrane damage.
Introduction

The parameters are considered to be directly related to the

Recently, electrical impedance spectroscopy (EIS) isoften used to know cell membrane soundness of vegetables.
The advantage of this technique is that the measurement iscompleted easily and quickly, and there is almost no
adverse effect on vegetables. Cell membrane of plant tissue,
which is formed by lipid bilayer, has a dielectric function.
When AC current is applied to healthy cellular tissue, atissue structure of vegetables. Because the tissue structureis strongly related to quality aspects including texture ofvegetables, the equivalent circuit parameters of processed
vegetables can be one of quality indexes. However,knowledge about changes in the parameters during
processing and the relevance to actual tissue structures is
not enough. The purpose of this study was to clarify theapplicability of equivalent circuit analysis to heatedfor obtaining detailed tissue state information from
impedance measurement results. In this method, models inwhich the tissue structure is regarded as electric circuits areused and the parameters are determined by complex
nonlinear least square method. An equivalent circuitdescribed in Fig.1a is popularly used in past studies (Wu,Ogawa, & Tagawa, 2008; Zhang & Willison, 1992).
Recently, another circuit which substitutes constant phaseelement (CPE) for capacitance (Fig.1b) is also used formore accurate analysis (Imaizumi et al., 2015).

vegetables and relationships between the parameters andcell membrane structure.

Cm
Ri

Re

(a) Hayden’s model

CPE

dielectric relaxation occurs due to the cell membrane
structure in a specific frequency band. While, it is assumed
that deteriorated or damaged cell membrane decrease thefunction. To date, researchers reported that the impedancevalue of fruit and vegetables changed under various
situations such as drop shock (Watanabe et al., 2018) anddecay (Euring, Russ, Wilke, & Grupa, 2011).
Equivalent circuit analysis is an effective technique

RiRe(b) CPE modelFig. 1 Equivalent circuit models for biological tissues.
Materials and Methods
1. Sample preparation
Carrot roots were cut into cylinders having 16 mm ofdiameter and 10 mm of height. The carrot cylinders were
immersed into 150 mL of distilled water which kept at

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 05

50 °C, then immediately cooled in iced water after theheating.

part of the cylindrical sample. Each section was stainedwith 0.05 % 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) (Wako, Japan) and2. Electrical impedance measurement
Two needle electrodes (diameter: 0.25 mm)
connected to a LCR tester (3532-50，HIOKI, Japan) wereinserted into the sample. The impedance |Z|, resistance R
and reactance X of the sample were measured at 100 points
over the frequency range from 50 Hz to 5 MHz at ameasuring voltage of 1 V, and automatically recorded by acomputer for analysis.

gently washed with distilled water. All confocal images
were obtained and digitized using a confocal laser scanningmicroscope. A HeNe laser (543 nm) was used as theexcitation source for the observation of DiI.

3. Electrical impedance measurement
In this study, equivalent circuit analysis using CPE
model (Fig.1b) was conducted according to the past study(Imaizumi et al., 2015). The impedance of CPE wasdefined as following equations:1(𝑗𝜔)+ 𝑇ππ
sin 7 2 𝑝8cos(2 𝑝)
=
−𝑗
𝜔+𝑇
𝜔+𝑇

𝑍"#$ =(1)(2)

Results and Discussion
We measured impedance values from the heatedsamples with different heating time. The CPE model was
successfully fitted to the measured values (R2 > 0.99), andresistance Re and intracellular resistance Ri. Figure 2A weobtained cell membrane capacitance Cm, extracellular
shows that the Cm values of each heated sample. Cell
membrane of plant tissue is formed by a lipid bilayer,
which is thought to function as a capacitor. Since this value
was lowered by heating for 40 min or more, it is consideredthat the membrane structure was damaged. Figure 2B
shows the ratio of extra and intra cellular resistance Re/Ri.
Biological tissues maintain electrolyte concentration insideand outside the cell. It is assumed that R e and Ri arewhere j is the imaginary unit, ω is angular frequency and T
and p are constants (0 ≤ 𝑝 ≤ 1). Thus, the impedance of
CPE model is expressed as follows:π
𝑅< =1 + 𝜔 + 𝑇 ?(2𝑅@ + 𝑅< ) cos 7 2 𝑝8 + 𝜔 +𝑇𝑅@ (𝑅< + 𝑅@ )AB𝑍=π{𝜔 + 𝑇(𝑅< + 𝑅@ )}E + 2𝜔 + 𝑇(𝑅< + 𝑅@ )cos 7 𝑝8 + 1
2−𝑗π𝜔 + 𝑇𝑅<E sin 7 2 𝑝8

(3)π{𝜔 + 𝑇(𝑅< + 𝑅@ )}E + 2𝜔 + 𝑇(𝑅< + 𝑅@ )cos 7 𝑝8 + 1
2

where Re is extracellular resistance, Ri is intracellularresistance. The equivalent circuit parameters (Re, Ri, T, p)
were estimated using complex nonlinear least squares
curve fitting (Macdonald, 1992). In addition, the cell
membrane capacitance Cm was defined using the following
equation (4) (Ando et al., 2014):
𝐶H = 𝑇I/+ (𝑅< + 𝑅@ )(IK+)/+(4)

4. Confocal laser scanning microscopy
The cell membrane structure of potato tissue was
evaluated by confocal laser scanning microscopy (CLSM).
Sections for observation were obtained from the central

Fig.2 The values of cell membrane capacitance Cm (A) andratio of extra and intra cellular resistance Re/Ri (B) obtained
from fresh and heated carrot samples. Bar: S.E. Different
letters indicate significant difference (p < 0.05).

Proceedings of International Symposium on a New Era in Food Science and Technology 2019
Session 4 – Quality Control of Food –: 05

https://doi.org/10.1016/J.PROFOO.2011.09.177
Imaizumi, T., Tanaka, F., Hamanaka, D., Sato, Y., &Uchino, T. (2015). Effects of hot water treatment on

Fig. 3 CLSM images observed from fresh carrot sample(A) and sample heated at 50 °C for 40 min (B). Bar: 200µm
influenced by the electrolyte concentrations. Our result
suggests that the function of adjusting electrolyte
concentration was impaired after 40 min of heating. Thus,
the Re/Ri result also supported occurrence of cell
membrane damage.
In our research, in order to confirm the cell membrane
damage which was assumed from these electrical changes,
we observed the structure with a confocal laser scanningmicroscope. Figure 3 shows that CLSM images of freshand heated (40 min) samples. While cell membrane
structure was clearly observed in the fresh tissue, theboundary formed by cell membrane lost its continuity. In
this way, destruction of cell membrane structure was also
indicated by CLSM observation.
Conclusion
Impedance measurement helps us to obtain somebiological information without complicated procedure.
In this study, it was indicated that electrical properties ofcarrot, which obtained by impedance analysis, greatlychanged by heating treatment. Additionally, we also
investigated that the relationships between the electricalproperties and cell membrane structure. By CLSM
observations, it was supported that the electrical properties
changed due to cell membrane damage.
References
Ando, Y., Mizutani, K., & Wakatsuki, N. (2014). Electrical
impedance analysis of potato tissues during drying.
Journal of Food Engineering, 121(121), 24–31.
https://doi.org/10.1016/j.jfoodeng.2013.08.008
Euring, F., Russ, W., Wilke, W., & Grupa, U. (2011).
Development of an impedance measurement system
for the detection of decay of apples. Procedia Food
Science, 1, 1188–1194.

electrical properties, cell membrane structure andtexture of potato tubers. Journal of Food Engineering,
162, 56–62.
https://doi.org/10.1016/j.jfoodeng.2015.04.003
Macdonald, J. R. (1992). Impedance spectroscopy. Annalsof Biomedical Engineering, 20(3), 289–305.
https://doi.org/10.1007/BF02368532
Watanabe, T., Nakamura, N., Ando, Y., Kaneta, T.,
Kitazawa, H., & Shiina, T. (2018). Application and
Simplification of Cell-Based Equivalent Circuit Model
Analysis of Electrical Impedance for Assessment ofDrop Shock Bruising in Japanese Pear Tissues. Foodand Bioprocess Technology, 11(11), 2125–2129.
https://doi.org/10.1007/s11947-018-2173-7
Wu, L., Ogawa, Y., & Tagawa, A. (2008). Electrical
impedance spectroscopy analysis of eggplant pulp andeffects of drying and freezing–thawing treatments on
its impedance characteristics. Journal of FoodEngineering, 87(2), 274–280.
https://doi.org/10.1016/j.jfoodeng.2007.12.003
Zhang, M. I. N., & Willison, J. H. M. (1992). Electrical
impedance analysis in plant tissues: The effect offreeze-thaw injury on the electrical properties of potato
tuber and carrot root tissues. Canadian Journal of Plant
Science, 72(2), 545–553.
https://doi.org/10.4141/cjps92-068

-PART 2UGSAS-GU & BWEL JOINT POSTER SESSIONON AGRICULTURAL
AND BASIN WATER ENVIRONMENTAL SCIENCES

2019

POSTER PRESENTATIONS
Thursday, 10th October 201913:00 pm - 15:00 pm
6th Floor, UGSAS Building, Gifu University

ORGANIZERS:
THE UNITED GRADUATE SCHOOL OF AGRICULTURAL SCIENCE,
GIFU UNIVERSITY&
GIFU UNIVERSITY REARING PROGRAM
FOR BASIN WATER ENVIRONMENTAL LEADERS

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P01

The effect of Japanese persimmon (Diospyros kaki) extract on theprevention of sarcopenia
Nayla Majeda Alfarafisa1, Kohji Kitaguchi1,2 and Tomio Yabe1,2,3
1. The United Graduate School of Agricultural Science, Gifu University
2. Faculty of Applied Biological Sciences, Gifu University
3. G-CHAIN, Gifu University

INTRODUCTION
Sarcopenia is an age-related syndrome which characterized byprogressive loss of mass, strength, and function of skeletal muscle(Budui, et al., 2015). People with sarcopenia syndrome usuallyexperienced a progressive and irreversible loss of body weight,
followed by muscular atrophy, decreased muscle strength, and evendeath. Sarcopenia is a multifactorial syndrome. The exact underlying
mechanisms and pathophysiology of sarcopenia remain unclear. As ofnow, there is no effective clinical strategies to overcome sarcopenia
Nowadays, the use of natural bioactive compound from plant extract
as therapeutic agent had gained much attention. Various botanicalcompound proved to be effective in maintaining muscle health.
Compared with conventional medicine, natural compound possessed
lower physiological and psychological addiction among patients withrelatively lower side effect. Japanese persimmon (kaki fruit
persimmon), Diospyros kaki, is one of a well known and highly
distributed fruits around the world. This fruit enriches with many
bioactive compounds that have beneficial effect on human health(Yaqub, et al., 2016). It is suggested that bioactive compound inJapanese persimmon also had an effect to pathological condition of
sarcopenia.
This study aims to investigate the effect of Japanese persimmon
extract on skeletal muscle health. For mimicking real-life condition,
co-culture system using a human Caco-2 intestinal cell as intestinal
barrier model and a murine C2C12 skeletal muscle cell will beestablished for evaluating its possible effect on muscle cellproliferation, differentiation, and atrophic prevention. Therefore, eachfraction was added into co-culture system, follow by the collection ofculture media from the basolateral components and then C2C12 cellproliferation, cell differentiation, and its role on muscle protein
turnover will be evaluated. Currently in this research progress, theextraction and fractionation steps have been done. Evaluation of eachfractions in the form of antioxidant activity and total phenolic content
showed different potentiality with hydrophobic fractions tend to show
stronger antioxidant activity

MATERIALS AND METHODS

Materials
Diospyros kaki Nishimura cultivar was provided by Gifu Prefectural
Agricultural Technology Center (Gifu, Japan) and used as a sample.
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid andTrolox (Sigma-Aldrich, St. Lois, USA) were used as a standard formeasuring antioxidant activity, and 1,1-diphenyl-2-picrylhydrazyl(DPPH; Wako Pure Chemical Industries, Osaka, Japan) was used as astable free radical which turns yellow when scavenged. 2,2-azobis-2methyl-propanimidamide dihydrochloride (AAPH) was obtained from
Wako Pure Chemical Industries. Cell Counting Kit-8 (CCK-8) werepurchased from Dojindo Molecular Technologies Inc. (Kumamoto,Japan).
Japanese persimmon extraction
After washed and properly handled, the raw material of Japanesepersimmons was freezed at –30ºC until it was ready to process. The
samples were completely dried by lyophylization methods. Afterexclude the calyx and seeds, the remaining parts of the samples
weighed and were recorded as dry weight. The samples then crushed
using mortar in order to improve the efficiency of extraction byincreasing their surface area. Powdered samples (42.5 g) wereextracted in 420 mL of 98% aqueous ethanol for 30 min in boiled
condition. After three cycles of extractions, the extract was mixed
combined and filtered using a Buchner funnel. The solvents werevaporized using rotary evaporator and dried up using lyophylization
methods. All crude extract was kept on –30ºC until further analysis.

Japanese persimmon fractionation
Japanese persimmon crude extract was fractionated using different
concentrations of ethanol (25, 50, and 75%). The extracts (5.00 g)
were dissolved in each solvent (50 mL) and homogenized using
magnetic stirrer for 1 h at room temperature. When the crude extract
completely dissolved in ethanol solvent, the samples were fractionatedagain using butanol (1:1) solvent until two different phases wereobtained. Solvent were vaporized using rotary evaporator and dried up
using lyophylization methods. All samples were kept on –30ºC until
further analysis.
DPPH radical scavenging activity
The free radical scavenging activity of the extract was measured interms of hydrogen donating or radical scavenging ability using thestable free radical DPPH. Gradient concentration (3.00-11.00 mg/mL)of Japanese persimmon fraction solutions in water was prepared. On
the other hand, trolox solution was prepared by dissolving trolox inethanol. Gradient concentration of trolox were used as a standard
curve. 30 µL of Japanese persimmon fraction and trolox solutions
were added to 96-well plate, followed by 240 µL of methanol. Water
was used as a blank. 1 mM solution of DPPH in ethanol was prepared
and added to each well of the samples. After 30 min of incubation indark condition, the absorbance was measured at 540 nm. The assay
was performed in triplicate. Percentage of inhibition of free radical
DPPH was calculated by using equation bellows:
DPPH Scavenging Activity (%) =Ab = Absorbance of the blank
As = Absorbance of the sample(Ab − As)
x 100
AbOxygen Radical Absorbance Capacity (ORAC) Assay
Different concentration of Japanese persimmon fraction solutions inwater, blank, and trolox standard solutions (50 µL) were added to 96well plate. After that, 50 µL of fluorescein was added. Incubated theplate in dark condition at 37oC. After 15 minutes, 25 µL of AAPH wasadded to each well. The fluorescence intensity was monitored every 5
min for 90 min by a micro plate reader in excitation 485 nm andemission 535 nm with 37oC incubation temperature. The assay was
performed in triplicate. ORAC value is the result of net area under the
curve of the fluorescence decay (AUC) of standard or sample minus
AUC of blank.
Determination of Total Phenolic Content
The total phenolic content of the extracts was determined by the Folin–
Denis method. Briefly, 1 mL of the diluted sample extract wastransferred to reaction tubes containing 1 mL of Folin-Denis reagent.
After 3 minutes, 1 mL of sodium carbonate solution was added to the
mixture. The tubes were then allowed to stand at room temperature for
1 hour before measuring absorbance at 760 nm. The total phenoliccontent of the extracts was calculated as catechin equivalents.
Cell Culture and Treatments
C2C12 cell lines were maintained in growth medium consisting ofDulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and L-glutamine. On the other hand,
Caco-2 cells were also maintained in DMEM supplemented with 10%
fetal bovine serum and Non-Essential Amino Acid (NEAA). Cells aremaintained under 70% confluency. After exceeds 70%, the cells were
harvested with trypsin/EDTA and place to the new vessels. Both cells
were incubated at 37 °C and 5% CO2.
Cell Viability Assay
WST-8 that utilized in CCK-8 kit (2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4 disulfophenyl)-2H tetrazolium, monosodium salt)
was used to evaluate cell viability and cytotoxicity. WST-8 solution(3%) in complete medium was added to the culture in 96 well plate.
After 2 h incubation, the absorbance was measured at 450nm.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P01

RESULTS AND DISCUSSION

Japanese persimmon extraction and fractionation
Table 1 showed an extraction yield of hot ethanol extraction fromJapanese persimmon. Theoretically, the quantity of extractive yield
was directly affected by the type of extraction methods/solvents andtheir extraction efficiency (Sharma & Cannoo, 2016). In this research,
we conducted three cycles for each extraction step. Relatively highnumber of yield percentage from extraction step indicated the methodof extraction which was chosen can be said sufficiently effective toisolate water soluble bioactive compound from Japanese persimmon.
Table 1. Japanese persimmon extraction yield
Dry Sample (g)Crude Extract (g)Yield (%)

42.5

27.8

65.3

Japanese persimmon fractionation
After underwent an extraction step, Japanese persimmon crude extract
was fractionated. Table 2 showed total yield of fractionation from 5 g
Japanese persimmon crude extract. There are six different fractions
obtained from Japanese persimmon crude extract: ethanol (A) andbutanol (B) phase from 25% ethanol fractionation, ethanol (C) andbutanol (D) phase from 50% ethanol fractionation, ethanol (E) andfiltrate (F) phase from 75% ethanol fractionation.
Table 2. Total yield fractions
Fraction

A

B

C

D

E

F

Yield (g)

1.75

2.85

3.53

0.92

4.30

0.64

Figure 2: ORAC value (µmol/100 g TE) of persimmon fractions.
Values are means ± SD, n=4/group. Statistically significant
difference of each group was determined by Tukey test.
Total Phenolic Content of Persimmon Fractions
In line with ORAC value, fractions with higher antioxidant activity
from ORAC analysis also possessed higher phenolic content. In Figure
3, F fraction had the highest phenolic content followed by D, B or E,
and then A or C fractions. However, total phenolic content of F
fraction was way too high compare to others. Otherwise, according to
ORAC analysis, antioxidant activity from F fraction was not
significantly different with fraction B, D, or E. It was suggested thatthe major antioxidant components in several fractions might not be
phenolics compound. Therefore, among Japanese persimmon
fractions with relatively high antioxidant activity only F fraction thatshowed high phenolic content.

To evaluate the extraction and fractionation methods of plantcompound, several analytical procedures were carried out. Below are
the results of qualitative and quantitative chemical analysis ofJapanese Persimmon.
DPPH IC50 values of Japanese persimmon fractions
Figure 1 showed IC50 values of each fraction of Japanese persimmon.
DPPH IC50 value is the capability of an antioxidant and necessary
concentration to reach 50% of scavenging DPPH free radical activity.
Therefore, this means that the fraction with lowest IC50 concentration
have strongest antioxidant activity among others. In Figure 1, D
fraction possessed the strongest antioxidant activity, followed by B or
E, and then C or F. Meanwhile A fraction showed the lowest radical
scavenging activity with the IC50 values was more than 11 mg/mL.
These findings are almost in line with Jang, et al. (2011) which statedthat Nishimura-wase persimmon hydrophobic extract has better DPPH
IC50 values in compare with hydrophilic extract, except the fact that E
sample as hydrophilic fraction gave a comparable value among othershydrophobic fractions.

Figure 1: IC50 (mg/mL) values of persimmon fractions for DPPH
radical scavenging activity. Values are means ± SD, n=7/group.
Statistically significant difference of each group was determinedby Tukey test.
ORAC Values of Persimmon Fractions
ORAC values of Japanese persimmon fractions can be seen in Figure
2. Compared with DPPH, ORAC measures the degree of inhibition ofperoxy-radical-induced oxidation by the compounds of interest in achemical milieu. The results showed slightly different pattern incompare with DPPH results. Among fraction B, D, E, and F, there
were no significantly different results. On the other hand, fraction C
and A are significantly lower among others. In general, hydrophobic
fraction have better ORAC value compare to hydrophilic fraction,
except for E fraction that again give a comparable value among othershydrophobic fractions.

Figure 3: Total Phenolic Content (g/100 g CE) of persimmon
fractions. Values are means ± SD, n=6/group. Statisticallysignificant difference of each group was determined by Tukey test.
In general, hydrophobic fractions (B, D, F) from Japanese persimmon
tend to have stronger antioxidant activity compare to hydrophilic
fractions (A and C). On the other hand, E fraction always showed thehighest antioxidant activity and phenolic content among otherhydrophilic fractions, which even almost comparable withhydrophobic fractions. Several papers stated that there was a positive
relationship between antioxidant activity and anti-atrophy potential formuscle cell in vitro (Hirasaka, et al., 2013). Therefore, we choose
fractions B, D, E, and F for further analysis. As future plan,cytotoxicity of each fractions for C2C12 and Caco-2 cells will beanalyzed to decided fractions ideal range of concentration.
Furthermore, we will study the effect of each fractions on thephysiological factor of myoblast, which are cell proliferation,differentiation, and finally its potential on preventing inducedmuscular atrophy.

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Budui, S. L., Rossi, A. P. & Zamboni, M. (2015) The pathogenetic bases ofsarcopenia. Clin. Cases Mater. Bone Metab., 12, 22-26.
Hirasaka, K. et al. (2013) Isoflavones Derived from Soy Beans Prevent MuRF1Mediated Muscle Atrophy in C2C12 Myotubes through SIRT1 Activation.
J. Nutr. Sci. Vitaminol., 59, 317-324.
Jang, I.-C.et al. (2011) Antioxidant Activity of 4 Cultivars of Persimmon Fruit.
Food Sci. Biotechnol., 20, 71-77.
Sharma, A. & Cannoo, D. S. (2016) Comparative evaluation of extraction
solvents/techniques for antioxidant potential and phytochemicalcomposition from roots of Nepeta leucophylla and quantification ofpolyphenolic constituents by RP-HPLC-DAD. Food Measure, 10, 658669.
Yaqub, S. et al. (2016) Chemistry and Functionality of Bioactive Compounds
Present in Persimmon. J. Chem. 2016, 3424025.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P02

Lipidomic profiling of cabbage stored at low temperature by liquid
chromatography-tandem mass spectroscopy
Putri Wulandari Zainal1, Daimon Syukri2, Teppei Imaizumi1, Masayasu Nagata3 and Kohei Nakano1
1. The United Graduate School of Agricultural Science, Gifu University, 1-1 yanagido, Gifu, 501-1193, Japan
2. Faculty of Agricultural Technology, Andalas University, Limau Manis, Padang, 20362, Indonesia
3. National Food Research Institute, NARO, 2-1-2 Kannondai, Tsukuba, Ibaragi, 305-8642, Japan
LC-MS/MS condition
Lipids were separated by liquid chromatography in binary
INTRODUCTION
gradientmode with a C18 reversed-phase column and wereFresh produce continues the biological activity after
ionized by electrospray ionization (ESI) using Turbo V ion
harvesting, and it is higher than the legume and grain because
source, then detected using multiple reaction monitoring
the fresh produce has a high moisture content around 95 %(MRM) by a triple-quadrupole mass spectrometer (Q-TRAP
which relatively have high metabolic activity (Seymour, Taylor,4500 AB-Sciex, Framingham, MA, USA). In detecting lipid& Tucker, 1993). The high metabolic causes rapid maturity andspecies, total 900 channels were set in MRM transitions.
senescence, which lead to deterioration of chemical
Data processing
composition, texture, flavor, aroma, appearance, and color. This
The peak area of lipid from MRM chromatogram was
metabolic process is strongly influenced by temperature during
processed using MultiQuant software (AB-Sciex, Framingham,storage (Lyons and Breidenbach, 1987), and low temperature is
MA, USA). Each peak area was normalized using that of theone of a method to reduce them. Give that, it has been used ininternal standard and the weight of the sample. Hierarchicalpreserving quality of fresh produce in market or transportation
clustering analysis (HCA) was conducted based on Pearson’s
since long ago (Wismer, 2003).
correlation using Multiple experiment viewer (MeV 4.9.0).
The process of metabolism can be slowed down by the
Moreover, principal component analysis (PCA) was applied toutilization of the low temperature, even the deterioration
the auto-scaled data for finding out the most important lipid
process still continues. Senescence has related to membrane
species.
deterioration caused by membrane lipids and proteins
catabolism (Paliyath & Droillard, 1992). Lipid composition inplants is very diverse and have an essential function such as
signaling (Munnik, Irvine and Musgrave, 1998) and energy
storage (Meer, Voelker, and Feigenson (2008). Therefore, lipid
profiling needs to know for learning about the senescenceprocess, subsequently leads to the development of the novel
assessment method for freshness of the postharvest products.

MATERIALS AND METHODS

Plant material and storage condition
The cabbage ‘cv. Kinkei 201’ was harvested at Kanagawa
Prefecture Agricultural Technology Center on 25th April 2019and stored at 5 °C for 0 and 16 days. The leaf was cut into small
pieces using φ3-mm of cork-borer, and approx. 100 mg ofsample was put into 2 ml cryotube with a piece of φ5-mm ofzirconia ball. Then, it was rapidly frozen by liquid nitrogen andstored at -80 °C until lipid analysis.
Lipid extraction
Lipid was extracted using a methyl-tert-butyl ether (MTBE)
method as described by Vitali et al. (2008) with somemodifications. Each sample was homogenized with methanoland 0.01% Butylated Hydroxytoluene (BHT) for 1 minute by abeads crusher. The homogenates were centrifuged at 12,000rpm for 10 minutes at 10°C, and 400 µl of supernatant wascollected in 10 ml testing tube. Then, 50 µl of internal standard(a mixture of phosphatidylcholine; PC 17:0 andphosphatidylethanolamine; PE 17:0) were added and put 3ml of
MTBE and was shaken in a water bath for 1 hour. After that,
400 µl of 1% potassium chloride was added and mixed
thoroughly by a vortex mixer.
The suspension was centrifuged for 3 minutes at 2,000 rpm,
and the supernatant was collected into a different testing tube.
Then, the lipid extraction was evaporated by a centrifugal
evaporator at 30°C for approx. 2 hours, and dissolve again in
600 µl of the solvent used for the mobile phase in HPLC
separation (acetonitrile/20 mM ammonium acetate/isopropanol(27.3:9.1:63.6 v/v) with 0.01% acetic acid). The concentrate
was filtrated using a membrane filter (pore size; 0.2 µm), andused for subsequent LC-MS/MS analysis. Lipids were extracted
from each 5 cabbage sample.

RESULTS AND DISCUSSION

Around 555 kinds of lipid species were detected in the stored
cabbage. The number of species in each lipid class was shownin Table 1. PC, PE and 3 kinds of glycolipids, namely MGDG,
DGDG, and SQDG class have rich diversity compared to otherones. In membrane, lipid has 3 major class which classified into
glycerophospholipids, sphingolipids, and sterols (Harayama
and Riezman, 2018) and glycerophospholipids are the main
constituent in membrane bilayer, for example, PC and PE areabundant content in plant membrane (Nakamura, 2017). On theother hand, especially in leaf, glycolipids are the main lipids
which account for about 40% of total lipids, and MGDG is the
major single component (Kates, 1970).
Figure 1 shows the difference of the lipid class distribution
between non-stored and 16 days stored cabbage at 5 °C. HCA
could separate into two main clusters in which several classes
were increased, and the other ones were decreased duringstorage. Figure 2 (A) shows the PCA score plot between two
samples and each group could be successfully separated into
two on PC1 axis. Figure 2 (B) is box plots of top 6 important
lipid species in the senescence process which were chosen fromthe loading plot of PCA. 6 lipid species in LGPL class shown
here clearly increased in the cabbage stored at 5 °C for 16 days.
Several researchers have discussed the change of lipid
composition in the plant under stress and postharvest conditions
using LC-MS (Welti et al., 2002, Kong et al., 2017, Bustamante
et al., 2018) and detected about 200 kinds of lipid species.
However, in this study we successfully found 555 lipids species
in stored cabbage using LC-MS/MS. Lipid profiling with alarge number of species could give more accurate results fordiscussing the mechanism of bio-membrane deterioration
during senescence of fresh produces.
The lipid profile obtained in this study will also contribute to
the development of postharvest technology for assessing thedegree of freshness in fruits and vegetables.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P02
Table 1 Lipid profile in stored cabbage.
Lipid classes
Phosphatidylcholine (PC)
Phosphatidic Acid (PA)
Phosphatidylethanolamine (PE)
Phosphatidylglycerol (PG)
Phosphatidylinositol (PI)Phosphatidylserine (PS)
Lysoglycerophospholipids (LGPL)
Lysoglyceroglycolipids (LGGL)
Monogalactosyldiacylglycerol (MGDG)
Digalactosyldiacylglycerol (DGDG)
Sulfoquinovosyl Diacylglycerols (SQDG)
Diacylglycerol (DAG)
Triacylglycerol (TAG)
Fatty Acid
Ceramide
Acylated Sterylglucoside (ASG)
Steryl Glycoside (SG)Steryl Ester (SE)
Total

Number of detected
lipid species
0d
16 d
63
54
8
10
75
69
40
34
26
21
28
22
15
16
19
19
66
63
34
24
58
52
26
26
51
34
6
6
42
36
13
10
3
3
12
11
592
517

Fig. 1 Hierarchical clustering analysis (HCA) showing thedifferential of lipid classes between non-stored and stored
cabbage at 5°C for 16 days.
(A)

(B)

Fig. 2 Score-scatter plot of PCA (A) and six important
lipid species selected by loading plot of PCA in stored
cabbage for 0 and 16 days (B).

REFERENCES

Bustamante, C. A., Brotman, Y., Monti, L. L., Gabilondo, J.,
Budde, C. O., Lara, M. V., and Drincovich, M. F. (2018).
Differential lipidome remodeling during postharvest ofpeach varieties with different susceptibility to chilling injury.
Physiologia plantarum, 163(1), 2-17.
Harayama, T., & Riezman, H. (2018) Understanding thediversity of membrane lipid composition. Nature Reviews
Molecular Cell Biology, 19(5), 281.
Kates, M. (1970). Plant phospholipids and glycolipids. In
Advances in lipid research (Vol. 8, pp. 225-265). Elsevier.
Kong, X., Wei, B., Gao, Z., Zhou, Y., Shi, F., Zhou, X., & Ji, S.
(2017) Changes in membrane lipid composition and function
accompanying chilling injury in bell peppers. Plant and Cell
Physiology, 59(1), 167-178.
Lyons, J.M., and R.W., Breidenbach. 1987. Chilling injury. In
J. Weichmann (Ed.), Postharvest physiology of vegetables.
Newyork: Marcel Dekker, Inc.
Nakamura, Y. (2017) Plant phospholipid diversity: emerging
functions in metabolism and protein–lipid interactions.
Trends in plant science, 22(12), 1027-1040.
Munnik, T., Irvine, R. F., & Musgrave, A. (1998) Phospholipid
signaling in plants. Biochimica et Biophysica Acta (BBA)Lipids and Lipid Metabolism, 1389(3): 222-272.
Matyash, V., Liebisch, G., Kurzchalia, T. V., Shevchenko, A.,& Schwudke, D. (2008) Lipid extraction by methyl-tert-butyl
ether for high-throughput lipidomics. Journal of lipid
research, 49(5): 1137-1146.
Paliyath, G., & Droillard, M. J. (1992) The mechanisms ofmembrane deterioration and disassembly during senescence.
Plant Physiology and Biochemistry, 30; 789–812.
Seymour, G. B., Taylor, J. E., & Tucker, G. A. (Eds.). (2012).
Biochemistry of fruit ripening. Springer Science & BusinessMedia, 454.
Van Meer, G., Voelker, D. R., & Feigenson, G. W. (2008)
Membrane lipids: where they are and how they behave.
Nature reviews Molecular cell biology, 9(2): 112.
Welti, R., Li, W., Li, M., Sang, Y., Biesiada, H., Zhou, H. E.,& Wang, X. (2002) Profiling membrane lipids in plant stressresponses role of phospholipase Dα in freezing-induced lipidchanges in Arabidopsis. Journal of Biological Chemistry,
277(35), 31994-32002.
Wismer, W.V. (2003) Low temperature as a causative agent ofoxidative stress in postharvest crops. In Hodges, M.D,
Postharvest oxidative stress in horticultural crops.
Binghamton: The Haworth Press, Inc.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P03

First record of wolbachia infection in camellia spiny whitefly,
Aleurocanthus camelliae (Hemiptera: Aleyrodidae), during their invasion
stages in JapanEko Andrianto and Atsushi Kasai
1. The United Graduate School of Agricultural Science (UGSAS)-Gifu University
2. Laboratory of Applied Entomology, Faculty of Agriculture, Shizuoka University
MIE
KYO

INTRODUCTION

Mie
Kyoto

2009
2009-2010

4
2

The camellia spiny whitefly (Aleurocanthus camelliae
Kanmiya & Kasai) was decribed as a new species that closely
related to the citrus spiny whitefly (A. spiniferus), the mostserious pest of citrus plantation in Japan in 1920s (Kanmiya et
al. 2011). The occurance of this whitefly firstly reported in Uji,
Kyoto prefecture in 2004 and had rapidly spreads all around
Japan (Kasai et al. 2010). This pest became serious by causing
reduction of tree vigour, sap loss and sooty mould (Yamashita& Hayashida, 2006).
A huge number of sap feeding herbivore e.g. whiteflies
harbour the bacterial symbionts to provide the essential
nutrients. The one of most widely distributed endosymbiont inarthropods is Wolbachia, a group of rickettsial endocelluar
bacteria, belonging to the α-proteobacteria. Wolbachia
endosymbiont was suggested can affect the evolution of closely
related insect host species by affecting the genetic structure anddiversity of mitochondria of insect host (Narita et al. 2006).
Recently, the study that compare haplotype of the Rhagoletis
cerasi,theEuropeancherry
fruit
fly
and
its Wolbachia infection status revealed a strong association
between the endosymbiont and the mitochondrial haplotype—individuals of haplotype 1 (HT1) are Wolbachia-uninfected,
whereas Wolbachia-infected flies are associated with haplotype
2 (HT2) (Schuler et al. 2016). Similar with R. cerasi, In Japan,
A. camelliae has a low genetic diversity, with only a single
haplotype in mtCOI (haplotype B1) (Uesugi et al. 2016). We
hypothesized that if this low genetic diversity caused by
Wolbachia infection status, the population of A. camelliae inJapan would be either positively infected or uninfected by
Wolbachia. The infection of Wolbachia in the A. camelliae are
not yet reported. This study is trying to measure the frequencyof Wolbachia infection and its diversity and assessing themitochondrial genotypes of the host under natural conditions
during the invasion stages of A. camelliae.

DNA extraction and PCR The DNA both of Wolbachia andtheir hosts were extracted using modified STE method. Ten ofnymphs whiteflies were crushed in eppendorf tube contains 100
μl of STE buffer (100mM Nacl, 10mM tris-HCl, ImM
EDTA,pH 8.0) using Biomasher. About 30μl of aliquot wastransfered into PCR tube (0.2μL) and proteinase K (10mg/ml,2μl) was added. And then it placed in the thermocycler at 95oC
for 15 and then temperature was reduced to 4oC.
The polymerase chain reaction were performed in a total
volume of 20μl GoTaq® Green Master Mix. PCR profile
used was: Pre-denaturation (98oC for 10 sec). Then followed by
35 cycles 98oC for 10 sec, Annealing temperature (Table 2) for
50 sec and 72 oC for 1 min. Extension period at 72 oC for 4 min.
PCR products were visualized after subjected to an agarose gelelectrophoresis. The positive results of Wolbachia detection
were sequenced. And to avoid false negative results of PCR wealso performed the nested PCR using the primer WacF/WacR(Table 2) to confirm the results. This a set of primer were
designed based on the sequence data of Wolbachia in A.
camelliae from Shizuoka (2010).

MATERIALS AND METHODS

Data analysis. The all sequences including the partial mtCOI
sequences and wsp genes of 14 samples, which wererepresentative ofthe genetics groups in each locality, were
determined. Sequence similarity was analysed using BLAST on
nucleotide sequences deposited in NCBI/GenBank databases.
Sequences were aligned using MUSCLE using MEGA version
X (Kumar et al. 2018). Phylogenetic analyses were performed
using both the maximum likelihood (ML) and Neighbor Joining(NJ) methods, In both analysis, 1000 bootstrap replicates were
performed.

Sample collection. Samples were collected from tea field inseveral cities in Japan during 2009-2011 and 2017-2018. In
total, there are 14 specimen from 6 cities. Mostly samples arecollection of Laboratory of Applied Entomology in Shizuoka
University from previous study and reanalyzed for the infectionof Wolbachia.
Table 1. Sampling locations including code, city, year andnumber of population samples
Code
KKG
SZK1
SZK2
KNY
SHG

City
Kikugawa
Shizuoka
Shizuoka
Kanaya
Shiga

Year
2011
2010
2018
2017
2010

n
3
1
1
1
2

Table 2. The set of primers in this study
Gene/
Primername
COI
HCO2198
LCO1490
wsp
81F
691R
WacF
WacR

Sequence (5’→3’)

Annealing
temperature/
product size

Referencetaaacttcagggtgaccaaaaaatca
ggtcaacaaatcataaagatattgg

52 oC/710bp

Folmer etal. 1994

tggtccaataagtgatgaagaaac
aaaaattaaacgctactcca
atagctggtggtggtgcatt
aaccgaagtaacgagctcca

55 oC/600bp

Braig etal. 1998
This
study

55 oC/358bp

RESULTS AND DISCUSSION
Infection of Wolbachia. The detection of Wolbachia
infection are common using wsp gene as a target PCR. A
standard PCR using 81F/691R as wsp gene primer in this study
revealed the negative results of 6 samples or in other words
there are more 40% samples were not infected by Wolbachia.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P03
Considering the false negative results that also often found in
Wolbachia detection, hence the nested PCR were performed toreconfirm the negative result samples using the second set ofwsp primers. The target size of the second primer is shorter thanthe previous one. The results of nested PCR shows the positive
infection in all samples of this study. This results indicate that
the A. camelliae haplotype B1 might be infected by Wolbachia.
However this results is not sufficient to proof the Wolbachia
effect to the genetic diversity of A. camelliae since there are twoother haplotypes of A. camelliae (B2 and B3) that exclude inthis study. Haplotype B2 only detected in the populations ofChongqing (China) that might be native to that region.While
haplotype B3 only detected on Theaceae plants in Japanesequarantine (China) (Uesugi et al. 2016).
Table 3. The infection rate of Wolbachia in A. camelliae
Code

n

KKG
SZK1
SZK2
KNY
SHG
MIE
KYO

3
1
1
1
2
4
2

Standard
PCR
+
─
3
0
1
0
1
0
1
0
0
2
1
3
1
1

%
100
100
100
100
0
25
50

Nested
PCR
+
─

%

2
3
1

100
100
100

0
0
0

Total
(%)
100
100
100
100
100
100
100

Wolbachia diversity in the A. camelliae populations.
Based on the wsp sequences data, at least there are seven cladesof Wolbachia in the A. camelliae population samples (I-VII).
Those clades indicates the Wolbachia group which wereseparated into two supergroup (A and B). A supergroup is
generally defined as a cluster of phylogenetically related
strains, though type and number of genes used for phylogenetic
inference are not set.

Fig.1. Neighbour-joining phylogenetic tree of Wolbachia
infected A. camelliae. Taxa are labeled as the host from which
Wolbachia isolated.
The supergroup B consisting the populations of A. camelliae
that infected by Wolbachia group (I-IV), the rest groups (VVII) clustered into supergroup A. This study is the first reveal
the positive infection status of Wolbachia in all A. camelliae
population samples. A modified nested PCR is an apporiate
method to measure the infection status of Wolbachia using wsp
gene target. This results supports the previous study that used amodified nested PCR to measure infection rates of Wolbachia
in the Bemisia tabaci populations (Ji et al. 2015). However toconclude that Wolbachia infection affects the genetic diversityof A. camelliae, more samples data are needed. At this point,
we noted that the populations of A. camelliae in Japan were
infected by Wolbachia during the early invasion of this whiteflyin Japan.

ACKNOWLEDGMENTS
We thank to Prof. Tsutomu Saito for the permission to use A.
camelliae sample collections.

REFERENCES
Braig, H. R., Zhou, W., Dobson, S. L., and O'Neill, S. L. (1998).
Cloning and characterization of a gene encoding the major
surface protein of the bacterial endosymbiont Wolbachia
pipientis. Journal of Bacteriology 180 :2373-2378.
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994).
DNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebrates.
Molecular Marine Biology and Biotechnology 3(5):294-299.
Ji, H.L., Qi, L.D., Hong, X.Y., Xie, H.F. & Li, Y.X. (2015).
Effects of Host Sex, Plant Species, and Putative Host Specieson the Prevalence of Wolbachia in Natural Populations ofBemisia tabaci (Hemiptera: Aleyrodidae): A Modified
Nested PCR Study. Journal of Economic Entomology 108(1):210-218
Kanmiya K., Ueda, S., Kasai, A., Yamashita, K., Sato, Y., andYoshiyasu, Y. (2011) Proposal of new specific status for teainfesting populations of the nominal spiny whitefly
Aleurocanthus spiniferus (Homoptera: Aleyrodidae).
Zootaxa 2979: 25-44
Kasai, A., K. Yamashita and Yoshiyasu, Y (2010) Tea infesting
population of Citrus Spiny Whitefly, Aleurocanthus
spiniferus (Homoptera: Aleyrodidae), Does not accept citrus
leaves as host plants. Japaese Journal of Applied Enomology
and Zoology 54: 140-143 (In Japanese)
Kumar S, Stecher G, Li M, Knyaz C, and Tamura K (2018)
MEGA X: Molecular Evolutionary Genetics Analysis acrosscomputingplatforms. Molecular
Biologyand
Evolution 35:1547-1549.
Narita, S., Nomura, M., Kato, Y., and Fukatsu, T. (2006)
Genetic structure of sibling butterfly species affectedby Wolbachia infection
sweep:
Evolutionary
andbiogeographical implications. Molecular Ecology 15: 1095–
1108.
Schuler, H., Kern, P., Arthofer, W., Vogt, H., Fischer, M.,
Stauffer, C., and Riegler, M. (2016) Wolbachia in parasitoids
attacking native European and introduced Eastern cherry fruit
flies in Europe. Environmental Entomology 45: 1424–1431.
Uesugi, R., Sato, Y., Han, B.-Y., Huang, Z.-D., Yara, K., &Furuhashi, K. (2016) Molecular evidence for multiple
phylogenetic groups within two species of invasive spiny
whiteflies and their parasitoid wasp. Bulletin of
Entomological Research. 106(3):328-340.
Yamashita, K. and Hayashida, Y. (2006) Occurance and controlof the citrus spiny whitefly, Aleurocanthus spiniferus(Quaintance), on tea tree in Kyoto Prefecture. Plant
Protection 60:378-380

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P04

Physicochemical properties of chitosan films contained curcumin
nanoemulsion
Fakfan Luangapai 1, Methavee Peanparkdee2,3 and Satoshi Iwamoto2,3
1. Graduate School of Natural Science and Technology, Gifu University
2. Faculty of Applied Biological Sciences, Gifu University
3. The United Graduate School of Agricultural Science, Gifu University

INTRODUCTION
Chitosan is a derivative of chitin, which can be found in
crustaceans, insects and fungi. Because of the effective
biodegradability and antimicrobial activities, chitosan has beencommonly applied as biopolymer in food, agricultural andpharmaceutical industries (Luangapai et al. 2019).
Furthermore, the chitosan films provide as good barrier whencompared to other polymers such as methylcellulose and corn
starch (García et al. 2009). Chitosan films easily to interact
with environmental moisture which changes physicochemical
properties such as tensile strength, sorption ability andpermeability of the films (Yoshida et al. 2009). Therefore, inorder to prevent the undesirable changes in chitosan films, anaddition of bioactive compounds into the films has been
investigated (Mujtaba et al. 2019).
One of the remarkable bioactive compounds, turmeric has
been used as food additive, food preservative and coloring
agent for ages in Asian countries. Indeed curcumin, one of themain components of turmeric, has an excellence anti-oxidant
and anti-microbial ability against E. coli, B. subtilis and S.
aureus (Maheshwari et al. 2006), it exhibits low water solubility
which leads to lower bioavailability (Manju and Sreenivasan2011). To improve the solubility, a number of methods havebeen studied, including nanoemulsion method. Since,
nanoemulsion technology has attracted wide attention in thefood and pharmaceutical industry for years. The nanoemulsion
can be to strengthen the solubility, stability and bioactivity ofvarious oil-soluble chemical substance due to its small droplet
size and high kinetic stability which this method widely
accepted many industries (Mazzarino et al. 2012).
In the present work, the study on combining of chitosan films
with nanoemulsion of bioactive compound such as curcumin in
physicochemical perspective will be determined. Nevertheless,for the first step, the evaluation to find optimal condition of
curcumin nanoemulsion on uniformity stability and size
distribution need to be achieved. Thus, the aim of the study isto evaluate appropriate condition for curcumin nanoemulsion inorder to be using for further study.

MATERIALS AND METHODS
Chitosan films preparation
Chitosan film-forming solution was prepared according to
the procedure of Siripatrawan and Harte (2010) with slightly
modification. Dissolved specific amount of chitosan powder
into 1% acetic acid solution and glycerol, as plasticizer, wasadded to the film-forming solution at the constant concentrationof 30% w/w of chitosan powder. The solution was heated at
60 °C in a water bath shaking incubator at 100
oscillation/minutes for 30 minutes. The solution was cooled toroom temperature and the turmeric nanoemulsion were added.
The film-forming solutions were homogenized for 2 minutes
using a homogenizer (NS-60, Microtec, Japan) and removed air
bubbles using ultrasonicator (ASU-10D, AS ONE, Osaka,

Japan). The amount of films forming solution, 100 ± 1 g, were
cast on a 12 × 28.5 cm on rubber plate and dried. The dried films
were adjusted to proper conditioned in an environmental
chamber at 25 °C and 50% relative humidity (RH) by using
saturated Mg (NO3)2·6H2O for 48 hours before measurement.
All property measurements were performed immediately after
removing film sample from the chamber to lessen moisturevariances of these natural films.
Preparation and characterization of curcumin nanoemulsion
About 30 mg of curcumin was dispersed into 10 mL Medium
Chain Triglyceride (MCT oil) as oil phase. The aqueous phase
was prepared by mixing the surfactant (Tween 20) and water.
The compositions of oil, surfactant, and water. The formulationof each curcumin nanoemulsion was varied by ultrasonication
time, amount of curcumin and ratio of oil phase and aqueous
phase. First, oil phase and aqueous phase were mixed andhomogenized by a homogenizer followed Table 1 (NS-60,
Microtec, Tokyo, Japan) at 13500 rpm for 15 minutes. Then,curcumin nanoemulsion was subjected to ultrasonicemulsification using 43 kHz ultrasonicator (ASU-10D, AS
ONE, Japan) with a maximum power output of 500 W durationvary by Table 1. Then, the formulated nanoemulsion wascharacterized and the uniformity stability of the emulsion wasinvestigated (Ghosh et al. 2013).
Table 1 formulation of curcumin nanoemulsion.
Curcuminnanoemulsion
SampleCurcumin(mg)

Ratio of oilphase andaqueous phase

Ultrasonictime(minute)

1
2
3
4
5

70
70
70
30
30

1:4
1:4
1:4
1:3
1:9

30
90
120
15
15

Determination of stability of nanoemulsion under processing
conditions
Curcumin nanoemulsion was evaluated particle size
distribution by dynamic light scattering method using Malvern
Zetasizer Nano ZS (Malvern Instruments, UK). Then, zeta
potential, an electrical charge on the oil droplets in theemulsions was evaluated at approximate temperature of 25°C
and electrical voltage 3.9 V.

RESULTS AND DISCUSSION
Chitosan films was prepared in good condition. The
appearance of the films shows smooth surface without air
bubbles, yellowish color and transparency. The color of thefilms depends on quality and sources of chitosan powder, in thiscase, chitosan powder obtained from red snow crab. However,Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P04
chitosan isolated from squid pens provide perfectly white andshrimp or lobster sources provide highly pink color.
From Table 1, size and zeta potential value are analyzed by
Dynamic Light Scattering (DLS) method. The Zetasizer shownthat the particle size in the nanoemulsion in each sample arevaried by amount of curcumin, ultrasonication time and ratio ofoil phase and aqueous phase. The sample 1,2,3,4 and 5 haveaverage size distribution, zeta potential and Polydisperse index(PdI) as shows in Table2. The sample 2 and 3 tend to have lower
size distribution compare to other sample, however,ultrasonication time of both samples are 90 and 120 minutes
which takes longtime for creating nanoemulsion. Perhaps, thelongtime of ultrasonication generate heat which can increase
temperature result in active substances such as phenolic
compound is lessen during the ultrasonication process.
On the other side, sample 5 has size distribution resemble tosample 2 and 3 but using less ultrasonication time. Furthermore,in part of PdI value, which is a ratio of standard deviation toaverage of droplet size of nanoemulsion, so it indicates theability of accumulation depended on droplet size distribution,
the lower of size distribution means the lower in droplet
accumulation. In addition, PdI value, the higher the PdI value
shown the lower the potential on uniformity of the droplet
whereas the lower values shown the higher. Therefore, fromsample 5 exhibit notable lowest PdI values. This means thatsample 5 illustrate higher stability on uniformity than othersamples. From this result, sample 5 tend to has potential
applying as optimal condition for further study according tosize distribution and PdI value.
Table 2 size, zeta potential and polydisperse index of
nanoemulsion samples.
Curcuminnanoemulsion
Sample
1
2
3
4
5

Size
distribution

Zeta potential
(mV)

PdI

624.66±15.40
360.66±2.29
378.2±5.59680.06± 1.24
410.67 ± 1.80

-0.025±0.12-0.54±0.49-1.54±0.48-0.64±0.34-2.45±0.087

0.482±0.032
0.447±0.013
0.509±0.012
0.46±0.029
0.23±0.027

FUTURE WORK
Following the results in this study, need to achieve for thefurther study. Firstly, the stability of nanoemulsion time will be
determine by monitor the change of size and stability of
nanoemulsion during longer storage time. Then, the optimal
results of stability together with size distribution will be used to
combine with chitosan film in order to create chitosan film
incorporated curcumin nanoemulsion. The incorporated films
will be examined the physicochemical properties to monitor
inner structure change of films and incorporated films, forexample, Thermal property by using Differential scanningcalorimetry (DSC) measurements, Mechanical properties
determined by tensile strength (TS), elongation at break (EAB)
and Young’s modulus, Water vapor permeability (WVP). In
addition, as curcumin itself provide potential of antioxidant
properties, in order to know the alteration of antioxidant
properties need to be examined. The examination will be
conduct by the technique of Total phenolic compound (TPC),
analyzing phenolic content, and DPPH radical scavenging
assay, analyzing antioxidant content.

REFERENCES
Aider, M. (2010) Chitosan application for active bio-based
films production and potential in the food industry. Review
LWT Food Science Technology 43 (6): 837–842.
Anton, N., Benoit, J., and Saulnier, P. (2008) Design andproduction of nanoparticles formulated from nano-emulsion
templates. A review Journal of Controlled Release 128: 185199.
Chattopadhyay, I., Biswas, K., Bandyopadhyay, U., andBanerjee, R. (2004) Turmeric and curcumin. Biologicalactions and medicinal applications 87: 44-53.
Ghosh, V., Mukherjee, A., and Chandrasekaran, N. (2013)
Ultrasonic emulsification of food-grade nanoemulsion
formulation and evaluation of its bactericidal activity.
Ultrasonics Sonochemistry 20: 338-344.
García, A., Pinotti, A., Martino, N., and Zaritzky, E. (2009)
Characterization of starch and composite edible films andcoatings. Edible films and coatings for food applications 169209.
López-Mata, A., Ruiz-Cruz, S., de Jesús Ornelas-Paz, J., Del
Toro-Sánchez, L., Márquez-Ríos, E., Silva-Beltrán, P.,
Burruel-Ibarra, E. (2018) Mechanical, Barrier andAntioxidant Properties of Chitosan Films Incorporating
Cinnamaldehyde. Journal of Polymers and the Environment
26: 452-461.
Luangapai, F., Peanparkdee, M., and Iwamoto, S. (2019)
Biopolymer films for food industries: properties,
applications, and future aspects based on chitosan. Reviewsin Agricultural Science 7: 59-67.
Maheshwari, K., Singh, K., Gaddipati, J., and Srimal, C. (2006)
Multiple biological activities of curcumin. Jounal of Life
Sciences 78: 2081-2087.
Manju, S., and Sreenivasan, K. (2011) Conjugation of curcuminonto hyaluronic acid enhances its aqueous solubility andstability. Journal of Colloid and Interface Science 359: 318325.
Martins, T., Cerqueira A., and Vicente, A. (2012) Influence of
α-tocopherol on physicochemical properties of chitosanbased films. Food Hydrocolloids 27: 220-227.
Mazzarino, L., Travelet, C., Ortega-Murillo, S., Otsuka, I.,
Pignot-Paintrand, I., Lemos-Senna, E., and Borsali, R.
(2012) Elaboration of chitosan-coated nanoparticles loadedwith curcumin for mucoadhesive applications. Journal of
Colloid and Interface Science 370: 58-66.
Mujtaba, M., Morsi, E., Kerch, G., Elsabee, Z., Kaya, M.,
Labidi, J., and Khawar, M. (2019) Current advancements inchitosan-based film production for food technology.
International Journal of Biological Macromolecules 121:
889-904.
Siripatrawan, U., and Harte R. (2010) Physical properties andantioxidant activity of an active film from chitosan
incorporated with green tea extract. Food Hydrocolloids 24:
770-775.
Yoshida, M., Oliveira Junior, N., and Franco, T. (2009)
Chitosan tailor made films: the effects of additives on barrier
and mechanical properties. Packaging Technology and
Science 22: 161-170.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P05

Identification of Plantaricin A on Lactobacillus plantarum IYO1511
Yolani Syaputri1, Masanori Horie2 and Hitoshi Iwahashi1
1 The United Graduate School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu City, 501-1193, Japan
2 Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-Cho,
Takamatsu, Kagawa, JapanINTRODUCTION
Lactobacillus plantarum is a gram-positive bacteria,
commonly found in many fermented food products. L.
plantarum has anti-microbial activity in vitro against
potentially pathogenic species such as Listeria monocytogenes,
Bacillus cereus, Escherichia coli, Yersinia enterocolitica,
Citrobacter freundii, Enterobacter cloacae, Enterococcus
faecalis, Salmonella enterica subsp. Enterica, and Candida
albicans (Todorov, 2009). L. plantarum produced antimicrobial
substances such as organics acids, fatty acid, and bacteriocins.
Bacteriocins are defined as proteinaceous antibiotics which killor inhibit the growth of bacterial species closely related to
producer bacteria (Malik, Sumayyah, Yeh, & Heng, 2016).
Bacteriocin that produced by L. plantarum is plantaricin.
plnA expressed by L. plantarum inserts in the cytoplasmic
membrane of the target cell, thereby promoting membrane
depolarization and cell death (Ahmad, et al., 2017). The genes
encoding bacteriocin are located on operon clusters, which may
be placed on the chromosome (such as PlantaricinST31), or
plasmids (such as Plantaricin423) or transposons (such as Nisin
A) (Wada, et al., 2009). L. plantarum IYO1511 was isolated
from fresh tea leaf. This research aims to determine theexistence of the bacteriocin biosynthetic cluster of plnA in L.
plantarum and show the related structural cluster gene ORFs.

MATERIALS AND METHODS
Plasmid Isolation from L. plantarum IYO1511
Plasmids were extracted with the protocol based on
Lactobacillus spp. oriented High-Quality methods (O'Sullivian& Klaenhammer, 1993) with modification. One hundred EDTA
1M was added into the solution.
Identification of Gene Encoding Bacteriocin Production from
Lactobacillus plantarum Using Specific Primers
The identification of gene encoding bacteriocin was
performed using PCR. The plnA specific primer F: (5`TAGAAATAATTCCTCCGTACTTC-3`) and R: (5`ATTAGCGATGTAGTGTCATCCA-3`). Polymerase chain
reaction (PCR) was carried out in a Fast reaction Tube (Applied
Biosystems, USA) in a total volume of 25 μl containing 12.5 μl
2 × Green Master Mix PCR (Promega, USA), 1.25 µL of eachprimer (concentration 0.05 pmol/µL), 9 µL nuclease freedeionized water, and 1 µL template DNA, and was run under
the following temperature program: initial denaturation of DNA
for 5 min at 95°C, 30 cycles of 30sec at 94°C, 1 min at 55°C,
and 1 min at 72°C; and final extension for 10 min at 72°C.
Then, 5 µl aliquots of the PCR product were analyzed byelectrophoresis using 1% (w/v) agarose gel in TAE 1X buffer
at 100 V for 30 min. The gel was then placed in an Electronic
U.V. Transilluminator to detect the presence a 1500 bp band.
The size of the DNA fragments was estimated using a FastGene
100 bp DNA Ladder (Nippon Genetics, Germany). Fast
Gene™ Gel/PCR Extraction kit (Nippon Genetics, Germany)
was used for purification before sending the extracted DNA for
sequencing, according to the manufacturer’s instructions. An
average of 500 bp nucleotides for each sequence from each side

was read and compared against the NCBI database using
BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

RESULTS
Identification Plantaricin A on L. plantarum using SpecificPrimers
Identification of gene encoding bacteriocin was done toknow the presence of plantaricin on plasmid. Specific primer ofplnA was amplified on plasmid of L. plantarum IYO1511 using
PCR method. The cell harvest at the early of stationary phase toreached maximum production of plantaricin (data not shown).
The presence of plnA gene encoded on plasmid were confirmed(fig. 1) based on the observation of the PCR product using
specific primers of plnA with size approximately ~550bp.
Plasmid of L. plantarum COY-2906 isolated from Virgin
Coconut Oil that had confirmed present plnA is used as positive
control.

M

1

2

600bp
500bp

Fig. 1: Detection of plnA sequence in L. plantarum IYO1511
plasmid M: Molecular Weight Marker 100bp DNA Ladder.
1: Positive control, Amplification of plantaricin from L.
plantarum COY2906 isolated from Virgin Coconut Oil; 2Amplification of plnA from L. plantarum IYO1511.
Plantaricin A of L. plantarum IYO1511 on plasmid

Fig. 2: Gene cluster plnA of L. plantarum IYO1511 on
plasmid
L. plantarum IYO1511 DNA sequence analysis has highDNA sequence identity 100% that correspond to plnA from
GenBank, the plantaricin gene was shown elsewhere. The
cluster structure of plantaricin encoding region was not shown
yet around plantaricin A gene region (fig. 2). Open Reading
Frame 1 showed plnA from L. plantarum IYO1511 start on 4152 bp and ORF2 showed GHLK domain-containing protein(L. plantarum) start on 268-519bp. Plantaricin A of L.
plantarum IYO1511 encoded a protein consisting of 48 aminoacid residues, followed by a TAA stop codon. The start codon
plnA followed by 4-152 bp, ATG start codon.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P05

DISCUSSION
L. plantarum IYO1511 is one of the potential lactic acidbacteria (LAB) isolated from fresh tea leaf. Plantaricins wereencoded on plasmid and chromosome. This study, we focus onplasmid of L. plantarum IYO1511. PlnA of L. plantarum
IYO1511 encoded a protein consisting of 48 amino acidresidues, followed by a TAA stop codon. The sequence analysis
showed 100% that correspond to plnA GenBank. Manybacteriocins are produced by Lactic Acid Bacteria (LAB) andthe bacteriocins are mostly active against Gram-positivebacteria and foodborne pathogens such as Listeria
monocytogenes (Azizi, Najafi, & Dovom, 2017) (Malik,
Sumayyah, Yeh, & Heng, 2016) (Reenen, Zyl, & Dicks, 2006)(Cho, Hanak, Huch, Holzapfel, & Franz, 2010) (Gautam,
Sharma, & Ahlawat, 2014). The bacterial pathogens included
Staphylococcus aureus, Clostridium botulinum, Campylobacter
jejuni and Escherichia coli 0157:H7 (Gautam, Sharma, &Ahlawat, 2014). This plnA has potential to kill or inhibit
pathogen bacteria.
In conclusion, plantaricin A has due role to their potential tokill or inhibit pathogen bacteria. The most attractive feature ofplantaricin A in this study is improved potential as role infermentation process. Although, the antimicrobial activity ofplantaricin is relatively well understood, the used of plnA as a
key role for fermentation food is still limited to study in theprocess of product.

REFERENCES
Ahmad, V., Khan, M. S., Jamal, Q. M., Alzohairy, M. A.,
Karaawi, M. A., & Siddiqui, M. U. (2017) Antimicrobial
potential of bacteriocins: in therapy, agriculture and foodpreservation. International Journal of Antimicrobial Agents,
49, 1-11.
Azizi, F., Najafi, M. B., & Dovom, M. R. (2017) The
biodiversity of Lactobacillus spp. from Iranian raw milk
Motalcheeseand antibacterial
evaluation
based
on bacteriocin-encoding genes . AMB Express, 1-10.
Cho, G.-S., Hanak, A., Huch, M., Holzapfel, W. H., & Franz,
C. M. (2010) Investigation into the Potential of
Bacteriocinogenic Lactobacillus plantarum BFE 5092 for
Biopreservation of Raw Turkey Meat. Probiotics &Antimicro. Prot, 241-249.
Gautam, N., Sharma, N., & Ahlawat, O. P. (2014) Puriﬁcation
and Characterization of Bacteriocin Produced by
Lactobacillus brevis UN Isolated from Dhulliachar: a
Traditional Food Product of North East India. Indian J
Microbiol, 185–189.
Horie, M., Sato, H., Tada, A., Nakamura, S., Sugino, S., Tabei,Y., . . . and Toyotome, T. (2019) Regional characteristics ofLactobacillus plantarum group strains isolated from two
kinds of Japanese post-fermented teas, Ishizuchi-kurocha
and Awa-bancha. Bioscience of Microbiota, Food and
Health, 11-22.
Malik, A., Sumayyah, S., Yeh, C.-W., & Heng, N. C. (2016)
Identification and sequence analysis of pWcMBF8-1, abacteriocin-encoding plasmid from the lactic acid bacterium
Weissella confusa . FEMS Microbiology Letters, 1-8.
Møller, S. A., Jensen, P. Ø., Høiby, N., Ciofu, O., Kragh, K. N.,
Bjarnsholt, T., & and Kolpen, M. (2019) Hyperbaric oxygen
treatment increases killing of aggregating Pseudomonas
aeruginosa isolates from cystic fibrosis patients. Journal of
Cystic Fibrosis , 1-9.
O'Sullivian, D., & Klaenhammer, a. T. (1993) Rapid Mini-Prep
Isolation of High-Quality Plasmid DNA from Lactococcus

and Lactobacillus spp. Applied and Environmental
Microbiology , 2730-2733.
Reenen, C. A., Zyl, W. H., & Dicks, L. M. (2006) Expressionof the Immunity Protein of Plantaricin 423, Produced by
Lactobacillus plantarum 423, and Analysis of the Plasmid
Encoding the Bacteriocin. Applied and Environmental
Microbiology, 7644–7651.
Renois, F., Jacques, J., Guillard, T., Moret, H., Pluot, M.,
Andreolettu, L., & and Champs, C. d. (2011) Preliminary
Investigation of a Mice Model of Klebsiella pneumoniae
subsp. ozaenae Induced Pneumonia. Microbes and Infection ,1045-1051.
Todorov, S. D. (2009) Bacteriocins from Lactobacillus
plantarum - Production, Genetic Organization and Mode of
Action. Brazilian Journal of Microbiology , 209-221.
Wada, T., Noda, M., Kashiwabara, F., Jeon, H. J., Shirakawa,A., Yabu, H., . . . Sugiyama, M. (2009) Characterization offour plasmids harboured in a Lactobacillus brevis strain
encoding a novel bacteriocin, brevicin 925A, andconstruction of a shuttle vector for lactic acid bacteria andEscherichia coli. Microbiology, 1726–1737.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P06

Genome-wide association study for understanding aluminum signaling
genes expression in the shoots of Arabidopsis thaliana
Raj Kishan Agrahari1, Yuriko Kobayashi1, 2, Ayan Sadhukhan2, Sanjib Kumar Panda3 and Hiroyuki Koyama1, 2

1The United Graduate School of Agricultural Science, Gifu University

2Faculty of Applied Biological Sciences, Gifu University, Gifu 501- 1193, Japan

3Department of Biochemistry, Central University of Rajasthan, Rajasthan 305817, India

INTRODUCTION
Aluminum (Al) toxicity in acid soils (pH < 5.5) is a severe
global problem that affects greater than 30% of the world’s
arable land. Physiologically Al toxicity includes severe root
growth inhibition coupled with reduction in shoot biomass andcrop yield (Kochian, 1995).
Accumulating evidence suggests that the cell wall plays
important roles in the perception and manifestation of Al
toxicity in plants (Horst et al., 2010; Kochian et al., 2015). The
cell wall is the first point of contact when plants are exposed toAl, and serves as a major pool for the metal. Al bound to thecell wall negatively affects wall structure and function byincreasing the rigidity and reducing cell expansion andmechanical extensibility, thus affect plant growth (Van et al.,
1994; Ma et al., 2004; Yang et al., 2010). The major Al bindingsite in cell walls is generally the pectin, because their negatively
charged carboxylic groups have a high affinity for Al (Chang etal., 1999; Schmohl and Horst, 2000).
It was reported that ALS3 ALUMINIUM-SENSITIVE 3(ALS3) & PGIP1 (POLYGALACTURONASE INHIBITINGPROTEIN 1), regulated by STOP1 (SENSITIVE TO PROTONRHIZOTOXICITY1) are Al biomarker genes induced in shoot(Sawaki et al., 2016), ALS3 encodes a half type ATPase isinvolved in the Al-translocation process (Larsen et al., 1996,1997) where as PGIP1 stabilizes the cell wall (Kobayashi et al.,2014), But the detailed regulatory mechanisms leading to the
induction of STOP1-regulated genes under Al stress in shootremain unclear. Recently we identified involvement ofphospholipids upstream to STOP1 in ALS3 signaling. Here we
present our work regarding the new signaling molecule
upstream to PGIP1.

MATERIALS AND METHODS

every 2 days, and grown under controlled environmental
conditions for 10 days (Sawaki et al., 2016).
Gene Expression Analysis
10 days old seedlings were then transferred to the treatment
solution containing 25 μM Al. after a 24-h stress treatment, theshoots were collected and freeze in liquid nitrogen, and crushed
using tissue homogenizer. Total RNA was extracted from theshoot and used for cDNA. PGIP1 expression level was
determined by real-time qPCR.
Genome-wide Association Study
The GWAS calculations were completed using the compressed
mixed linear model of the TASSEL program as previously
described (Sadhukhan et al. 2017). The genome-wide SNPs
used in this study were obtained from a public database.
Missing data or a minor allele frequency below 10% wasremoved. Genes nearest to the most significantly associated
SNPs were annotated according the TAIR 10 database.
PGIP1 expression analysis in KO line
Total RNA was extracted from a 24-h stress (25 μM Al) treatedshoot sample, and used for cDNA. PGIP1 expression level was
determined by real-time qPCR.

RESULTS & DISCUSSION
From a GWAS on PGIP1 expression we found 16 SNPs (P<103.5) to be strongly associated to PGIP1expression. Role of thegenes, linked to the most significant SNPs, in regulating PGIP1
was validated by reverse genetics.

Seeds of natural A.thaliana accessions and KO lines wereobtained from the Arabidopsis Biological Resource Center(Columbus, OH, USA), the Nottingham Arabidopsis Stock
Center (Nottingham, UK), and the RIKEN BioResource Center(Tsukuba, Japan), We treated 85 A.thaliana accessions with 25
μM Al for the GWAS. Progeny lines were obtained bycontrolled self-pollination method. The homozygocity of KO
lines was confirmed by PCR using SALK recommended
methods.
Plant Growth Condition
Germinated A.thaliana seedlings were grown on mesh andtransferred to to modified 2% MGRL (pH was 5.6) culture
solution (Fujiwara et al., 1992). The solution was changed

Number of A.thaliana
accessions

Plant Materials
30
25
20
15
10
5
0
Relative Fold Change of PGIP1
Fig 1. Histogram of PGIP1 expression in A. thaliana
accessions.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P06

A

B

Fig 2. (A) Expression of PGIP1 in T-DNA insertion mutants of
GWAS detected genes. (B) Expression of trx SF in WT and stop1

As PGIP1 encodes a POLYGALACTURONASE
INHIBITING PROTEIN 1 responsible for cell wall stability
under Al stress, though it’s signaling is unclear. We did
GWAS
to identify a signaling cascade regulating PGIP1
expression under Al stress. From the GWAS we identified
thioredoxin (Trx) superfamily protein downstream of STOP1
that involved in PGIP1 signaling.

REFERENCES
Chang, Y.C., Yamamoto, Y., Matsumoto, H. (1999).
Accumulation of aluminium in the cell wall pectin in
cultured tobacco (Nicotiana tabacumL.) cells treated with a
combination of aluminium and iron. Plant, Cell andEnvironment 22,1009– 1017.
Fujiwara, T., Hirai, M.Y., Chino, M., Komeda, Y., and Naito,
S.(1992). Effects of sulfur nutrition on expression of the
Soybean seed storage protein genes in transgenic petunia.
Plant Physiol. 99:263–268.
Horst ,W.J., Wang, Y., Eticha, D. (2010) The role of the root
apoplast in aluminium-induced inhibition of root elongationand in aluminium resistance of plants: a review. Annals of
Botany 106, 185–197.
Kochian, L.V. (1995) Cellular mechanisms of aluminum
Toxicity and resistance in plants. Annu. Rev. Plant Physiol.
Plant Mol.Biol. 46, 237–260.
Kochian, L.V., Pineros, M., Liu, J., Magalhaes, J. (2015)
Plant adaptation to acid soils: the molecular basis for crop
aluminum resistance. Annual Review of Plant Biology 66,
23.1–23.28.
Kobayashi, Y., Ohyama, Y., Kobayashi,Y., Ito,H., Iuchi,S.,
Fujita, M., Zhao, C.R., Tanveer, T., Ganesan, M., Kobayashi,
M. and Koyama,H. (2014) STOP2 activates transcription ofseveral genes for Al- and low pH-tolerance that are regulated
by STOP1 in Arabidopsis Mol. Plant, 7 (2014), pp. 311-322.
Larsen, P.B., Kochian, L.V. and Howell, S.H. (1997) Al inhibits
both shoot development and root growth in als3, an Alsensitive Arabidopsis mutant. Plant Physiol. 114, 1207–1214.
Ma, J.F., Shen, R., Nagao, S., Tanimoto, E. (2004) Aluminumtargets elongating cells by reducing cell wall extensibility inwheat roots. Plant and Cell Physiology 45, 583–589.
Schmohl, N., Horst, W. ( 2000). Cell wall pectin content
modulates aluminium sensitivity of Zea mays (L.) cells grownin suspension culture. Plant, Cell and Environment 23, 735–742.
Sadhukhan A., Kobayashi Y., Nakano Y., Iuchi S., Kobayashi M.,
Sahoo L. & Koyama H.(2017) Genome-wide Association Study
Reveals that the Aquaporin NIP1;1 Contributes to Variation in
Hydrogen Peroxide Sensitivity in Arabidopsis thaliana.
Molecular Plant 10, 1082–1094.

Sawaki,K.,Sawaki.Y.,Zhao,C.R.,Kobayashi,Y., andKoyama,H.(20 16) Specific transcriptomic response in theshootsof Arabidopsis thaliana after exposure to Al
rhizotoxicity: - potential gene expression biomarkers forevaluating Al toxicity in soils. Plant Soil. 409:131–142.
Van, H.L., Kuraishi, S., Sakurai, N. (1994) Aluminuminduced rapid root inhibition and changes in cell-wallcomponents of squash seedlings. Plant Physiology 106,971–976.
Yang, Z.B,, Eticha, D., Rao, I.M., Horst, W.J. (2010)
Alteration of cellwall porosity is involved in osmotic
stress-induced enhancement of aluminium resistance incommon bean (Phaseolus vulgaris L.). Journal of
Experimental Botany 61, 3245–3258.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P07

Annual characterization and variations of airborne insect
Panyapon Pumkaeo1, Junko Takahashi1,2 and Hitoshi Iwahashi1
Division of Science of Biological Resources, The United Graduate School of Agricultural Science,
Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
2. National Institute of Advanced Science and Technology (AIST), 1-1, Higashi, Tsukuba,
Ibaraki, 305-8566, Japan

1.

INTRODUCTION
Bioaerosols are atmosphere particles, mists or dust of µm
range, associated with metabolically active or inactive viable
particles. They contain living organism’s included
microorganisms such as viruses, bacteria, and fungi also plantmaterial as well as pollen.
The presence of microorganism in aerosols has been known
for centuries, However, most studies have only focus onbacteria to have a potential impact on public health andecosystems. Therefore, in this study, insect abundance andcommunity composition were determined by cytochromeoxidase I (COI region) using Next generation sequencing andcompared on particle size distribution.
MATERIALS AND METHODSThe Aerosol samples collected on the filter membrane using
Anderson’s air sampler at the rooftop of Gifu Field Science
Center building, Gifu University.
The sample was divided into three subgroup andtwo subgroups total 6 samples according to the size of its
bioaerosols, large subgroup contains bioaerosols whose
diameter is bigger than 3.3μm, and small subgroup contains
those smaller than 3.3μm. (during 2017–2018; 24 samples overfour seasons)
The DNA was extracted by using soil DNA extraction kits.
and PCR amplication of cytochrome oxidase I (COI region)
using mlCOIintF and HCO2198 primers and followed by highthroughput sequencing.

Fig. 1: Taxonomic composition of each sample of outdoor
airborne insect communities. (size <3.3 μm)

RESULTS
The seasonal distribution in insect abundance of large
particle size (> 3.3 μm) were not observed. About 530 OTUs
were Identified to the species level as they showed more than
98% sequences similarity with GENBANK sequences.
Hemiptera sp. remained the dominant insect species in all
samples.(Fig 1 and 2) Potentially of fungal plant pathogen
strain were found using mlCOIintF and HCO2198 primers suchas Calonectria colhounii strain H125.
The insect community composition on smaller size particle
was rather stable seasonally and did not differ from that on large
size particle.

Fig. 2: Taxonomic composition of each sample of outdoor
airborne insect communities. (size >3.3 μm)
DISCUSSION
The Heteroptera (Insecta: Hemiptera), or true bugs, are oneof the major insect groups with respect to the very diverse
habitat preferences. Due to the high habitat diversity and therelatively high environment specificity, Hemiptera can be auseful bio-indicator of various environmental parameters, suchas habitat structure and vegetation coverage.
Our findings suggest that aerosol sampling might be helpful
for early warning screening of species and also plant pathogen
detection.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P07
ACKNOWLEDGMENTS

REFERENCES

This study is acknowledged for Gifu Field Science Centerbuilding, Gifu University as the sampling place.

Park. J, Ichijo. T, Nasu. M, Yamaguchi. N. (2016)
Investigation of bacterial effects of Asian dust events throughcomparison with seasonal variability in outdoor airborne
bacterial community. Scientific Reports | 6:35706
Ishikawa. T, Masayuki U. Saito , Keiko Kishimoto-Yamada,Toshihide Kato , Osamu. (2015) Inventory of theHeteroptera (Insecta: Hemiptera) in Komaba Campus of the
University of Tokyo, a highly urbanized area in Japan.
Biodiversity Data Journal 3: e4981
Srivastava, A., Singh, M., & Jain, V. K. (2012). Identificationand characterization of size-segregated bioaerosols at
Jawaharlal Nehru University, New Delhi . Nat Hazards ,485-499.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P08

The relationship between major chemical components and genomic
structures in tea accessions
Hiroto Yamshita1,2, Tomoki Uchida1, Hideyuki Katai3, Lina Kawaguchi4, Atsushi J. Nagano4, Akio Morita1 and Takashi
Ikka1
1. Faculity of Agriculture, Shizuoka University, Ohya, Shizuoka, Japan
2. United Graduate School of Agricultural Science, Gifu University, Yanagito, Gifu, Japan
3. Shizuoka Prefectural Research Institute of Agriculture and Forestry, Tea Research Center, Kurasawa, Kikugawa,Shizuoka, Japan
4. Faculty of Agriculture, Ryukoku University, Yokotani, Seta Oe-cho, Otsu, Shiga, JapanINTRODUCTION
Tea is one of the most popular beverages worldwide. Tea
plant (Camellia sinensis (L.) O. Kuntze) contains many
functional ingredients such as theanine and catechins.
Consequently, the production area and yield of tea are
increasing worldwide, but especially in Asia. Tea is a woody
plant in the Theaceae family of angiosperms. The origin of tea
plants is considered to be southwestern China and surrounding
regions (Tanaka, 2012). In general, there are two varieties of tea
plants; var. sinensis (Chinese type), and var. assamica (Assamtype). These two varieties have different phenotypiccharacteristics and different origins (Yang et al., 2016). For
example, C. sinensis var. sinensis has small leaves and is able
to withstand colder climates, while C. sinensis var. assamica
has large leaves and is sensitive to cold temperatures. C.
sinensis var. sinensis is mainly cultivated in China and Japanfor green tea production and C. sinensis var. assamica is mainly
cultivated in India and Sri Lanka for black tea production.
Assam hybrids, which are crosses between var. sinensis andvar. assamica, have larger leaves and are more cold-tolerant
than var. assamica, and so these hybrids are mainly utilized forbreeding and for black tea production in Japan and China.
However, the relationship between genetic differentiation andphenotypic characteristics such as major chemical components
traits in these tea varieties is still unclear. And, it is important
for tea breeding to control the content of chemical components,
which contribute tea quality, in tea young shoots. Here, we
performed the phenotyping of major chemical components offirst flush and genotyping by double-digest restriction siteassociated DNA sequencing (ddRAD-seq) in tea accessions,and genome wide association study to identify these locus
associated with these traits.

MATERIALS AND METHODS
Plant materials and the measurement of major chemicalcomponents for tea—Tea accessions were obtained from the
Tea Research Center, Shizuoka Prefectural Research Instituteof Agriculture and Forestry, Kikugawa city, Shizuoka, and theBotanical Research Gardens of the ICHIMURA Foundation for
New Technology, Atami city, Shizuoka. The 167 accessions
comprised three subspecies: 96 Japanese var. sinensis, 42 exotic
var. sinensis (originating from China and Taiwan); and 29
Assam hybrids (introduced from India and Nepal to Japan). To
measure the content of major chemical components, young
leaves at the same developmental stage in the first flush season
were harvested from 150 accessions grown under the same
cultivation environment at the Tea Research Center. 11 Free
amino acids, 8 catechins, caffeine and chlorophyll as major
chemical components for tea were measured by HPLC system(unpublished) and absorption spectrometry (Porra et al., 1989).

Genotyping by ddRAD-seq — Genomic DNA was extracted
from young leaves of each tea accession using a DNeasy Plant
Mini Kit (Qiagen) following the manufacturer’s instructions,and then ddRAD-seq was conducted as described elsewhere(Sakaguchi et al., 2015). Genomic DNA was digested with Bgl
II and EcoRI-HF. Sequencing of 50-bp single-end reads and theindex sequences of the library was conducted using one lane of
Hiseq2500 (Illumina, San Diego, CA, USA). Reads werepreprocessed using Trimmomatic-0.33. After preprocessing,
the remaining reads were mapped to the tea reference draft
genome (Wei et al., 2018) using Bowtie2, and then the SNPs
were called using Stacks (ver. 1.37). These raw SNPs data were
filtered against the following thresholds: SNP call rate within alocus ≥ 0.7 and minor allele frequency (MAF) ≥ 0.05. The
filtered SNP data were imputed using R package missForest andused for subsequent population analyses. The RAD-Seq data
have been deposited in the DDBJ Sequence Read Archive(Accession number: DRA008166).
Population analysis — To clarify the genetic structure, weused the Bayesian clustering algorithm, hierarchical cluster
analysis (HCA), and principal component analysis (PCA). The
Bayesian clustering analysis was performed using
STRUCTURE ver. 2.3.4 (Pritchard et al., 2000). In thisanalysis, we evaluated one to eight genetic subgroups (K) withten runs per K value. For each run, the initial burn-in period was
set to 50,000 with 20,000 Markov chain Monte Carlo iterations.
The values of ΔK (Evanno et al., 2005) were calculated to infer
the optimum number of subgroups. The components of eachsubgroups, i.e., ancestral components determined by thisSTRUCTURE analysis, were compared with the origin andpedigree information for the accessions.
Genome Wide Association Study — Genome wideassociation analysis (GWAS) was performed by using mixed
linear model (MLM) implemented in the “GWAS” function of
the R package rrBLUP ver. 4.6. In total, 10,943 SNPs among
150 accessions were used for the GWAS after selected SNPs
without missing rates ≥ 0.3 and MAF < 0.05. The principal
components and a kinship matrix were included in the GWAS
calculation based on MLM. A principal component analysis(PCA) was conducted using the prcomp function of R to
estimate the population structure. The first eight principal
components from the plot explaining the total variation among
SNPs were selected. The kinship matrix was computed by using
the “A.mat” function of the R package rrBLUP ver. 4.6. To
illustrate the localization of associated SNPs by GWAS, wecreated Manhattan plots of all anchored to linkage groups foreach traits.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P08

RESULTS AND DISCUSSION
SNPs identification by ddRAD-seq and genetic structures ofworld tea accessions — To acquire high-resolution genotypic
information for the 167 tea accessions, we conducted SNPs
genotyping by the ddRAD-seq method. After preprocessing thedata, 1,269,648 SNPs were initially identified by the Stacks
pipeline. Further filtering (SNP call rate within a locus ≥ 0.7,
MAF ≥ 0.05) returned 13,715 in all 167 accessions To verify
the locations of the detected SNPs on linkage groups, scaffolds
with these SNPs were mapped to the reference tea genetic map(Wei et al., 2018). In total, 11,257 of the detected SNPs in allaccessions were anchored across 15 linkage groups. To clarify
the genetic structure and the degree of relatedness among tea
accessions with different genetic backgrounds, we first used theBayesian clustering approach with high-density SNP markers.
To infer the optimal number of subgroups (K), we calculated
ΔK values. We obtained the highest ΔK values in the order of
K = 2 and K = 3. At K = 2, the 167 worldwide tea accessions
were divided into var. sinensis and Assam hybrids, reflecting
the two main subspecies of tea plants. The ΔK method of
Evanno et al. (2005) often results in K = 2, because there is avery low likelihood of K = 1 in all analyses. Accordingly, there
was a very low likelihood of K = 1 in our analyses, and thechanges of likelihood values based on K also became moderate
at K = 3. Therefore, we regarded K = 3 as the optimalclassification in the genetic structure analysis of worldwide tea
accessions. This classification of K = 3 more accuratelyreflected the genetic background of the tea accessions and was
supported by the results of PCA and HCA. Comparisons of theaccessions with the three ancestral components at K = 3
revealed the main composition of each subgroups: (1) Japanese
var. sinensis; (2) Japanese and exotic var. sinensis; and (3)Assam hybrids and exotic var. sinensis. In a previous study,
genotyping of tea accessions by SSR markers revealed marked
genetic differentiation between Japanese var. sinensis andexotic accessions including var. assamica (Taniguchi et al.,2014). A possible reason for this result was that there was a bias
in the SSR markers that were identified based on

Fig. 1 The boxplots of major chemical components
among three genetic subgroups. Different letters
indicates significantly difference (Steel-Dwass test, P <0.05).

polymorphisms between the Japanese and Chinese accessions.
Our analyses clarified the genetic structure of each accession
without biases from their genetic backgrounds by using highdensity SNPs markers.
Genetic variation of major chemical components in tea
shoots and GWAS — The variation of each major chemicalcomponents in the tea first flush were widely observed; thecontent of theanine, epigallocatechin gallate (EGCG), caffeine
and total chlorophyll ranged from 4.7 to 41.0 mg g−1 D.W., 38.9
to 96.4 mg g−1 D.W., 20.0 to 49.4 mg g−1 D.W. and 1.5 to 6.0
mg g−1 D.W (Fig.1). When compared the content of major
chemical components among three genetic subgroups, thecontent of EGCG and caffeine in Group3 (subgroup of Assamhybrids) was higher than Group1 and Group2 (subgroup of var.
sinensis) (Fig.1). Finally, GWAS based on each major chemicalcomponents identified some candidate locus. These results
suggested that these locus may contain the gene(s) controllingthe content of chemical components in tea shoots.

ACKNOWLEDGMENTS
We thank Satoko Kondo, technical assistant, from Ryukoku
University for conducting the ddRAD-seq analyses in thisstudy.

REFERENCES
Evanno G, Regnaut S and Goudet J. (2005) Detecting thenumber of clusters of individuals using the software
STRUCTURE: A simulation study. Mol Ecol.14(8):2611–
2620.
Porra RJ, Thompson WA and Kriedemann PE (1989)
Determination of accurate extinction coefficients andsimultaneous equations for assaying chlorophylls a and b
extracted with four different solvents; verification of theconcentration of chlorophyll standards by atomic absorption
spectroscopy. Biochim Biophys Acta 975: 384–394
Pritchard JK, Stephens M and Donnelly P. (2000) Inferenceof Population Structure Using Multilocus Genotype Data.
Genetics. 155(2):945–959.
Sakaguchi S, Sugino T, Tsumura Y, Ito M, Crisp MD,
Bowman DMJS, Nagano AJ, Honjo MN, Yasuji M, Kudoh H,
Matsuki Y, Suyama Y and Isagi Y (2015). High-throughput
linkage mapping of Australian white cypress pine (Callitris
glaucophylla) and map transferability to related species. Tree
Genet Genomes.11(6).121
Tanaka J. (2012) Japanese tea breeding history and the future
perspective. In: Chen L, Apostolides Z, Chen ZM. Grobal tea
breeding Springer, berlin. 227–239.
Taniguchi F, Kimura K, Saba T, Ogino A, Yamaguchi S andTanaka J. (2014) Worldwide core collections of tea (Camelliasinensis) based on SSR markers. Tree Genet Genomes.
10(6):1555–1565.
Yang H, Wei C, Liu H, Wu J, Li Z. (2016) Genetic
Divergence between Camellia sinensis and Its Wild Relatives
Revealed via Genome-Wide SNPs from RAD Sequencing. 1–
21.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P09

Screening and molecular breeding of amylose utilizing yeast forbioethanol production
Annisyia Zarina Putri and Tomoyuki Nakagawa
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu City 501-1193, JapanINTRODUCTION
Amylases have a significant commercial importanceand they employed about 25–33% of the world enzyme market(Nguyen et al., 2003). Because of the importance of amylases,
isolation of new microorganisms, which are suitable foramylase production, could provide potential new sources of the
enzyme (Aullybux and Puchooa, 2013). Recently, theof the yeast strain was done by enrichment static or shaking
culture at 30ºC.
Amylolytic activity of the strains was observed on Amy/YNB
plate. Yeast cultures were incubated at 30ºC for 24-36hrs. Afterincubation of the yeast strains, Iodine vapor was used to detectdegradation of amylose. The degradation of amylase wasindicated by the clear zone of starch hydrolysis surrounding thecolony.

hydrolysis of starch by α-amylase has been increasingly used asa key step to produce carbohydrate substrate for bio-ethanol

Determination of Amylase Activity:

production. Starch is the best substrate for production of yeast

Amylase activity was carried out by measuring thecells in a large scale due to its low price and easily available

amount of sugar released using DNSA method. Amylase

raw material in most regions of the world. Because most ofactivity was determined by incubating a mixture of 1 ml of eachyeasts from environments are generally recognized as safe

enzyme source and 1% soluble starch dissolved in 0.5 M

compared to bacteria, interest in amylolytic yeasts has increased

phosphate buffer, at pH 5.5, at 30°C for 10 min. The reaction

in recent years as their potential value for conversion of starchy

was stopped by adding 1 ml of 3, 5-dinitro salicylic acid, andbiomass to single-cell protein and ethanol has been recognizedthen followed by boiling for 10 min. The final volume was(Gupta R et al., 2003).

made up to 12 ml with distilled water and the reducing sugar

In recent years, the interest in production of enzymes

released was measured at 540 nm. One unit of amylase activity

has increased due to several potential applications, such as thewas defined as the amount of enzyme that releasing 1μmole

production of bioenergy and biofuels, and application in textile

glucose equivalent per minute under the standard assay

and paper industries (Soccol et al., 2003). Many studies haveconditions. Reducing sugar (Glucose or maltose) concentration

been published seeking new microorganisms to produce

was determined from a standard curve under same condition

enzyme with higher specific activity and efficiency. Several

using glucose

strategies are available for improving the production andefficiency of cellulases, including optimization of thefermentation process, genetic difications and mutagenesis.
The purpose of this study is to screen, and breeding
amylase producing yeast from different sources for bioethanol

Determination of Ethanol Activity:
The fermentation samples were analyzed by highperformanceliquidchromatography.

Tomonitorthefermentation, about 2 mL of sample was drawn at 144-216

production and also describe some of the biochemical

hours, centrifuged at 10,000 rpm for 10 min. The liquid wasproperties of the α-amylase purified from the culture broth.

immediately filtered through 0.2 μm into HPLC vials.

Total of around 190 strain yeast isolates were obtained from soil,
fruits, vegetables, etc. on the starch agar medium.

RESULTS
Screening of amylase producing yeast strains

MATERIALS AND METHODSIn this work, we screened amylolitic yeast strainsfrom yeast strains stocked in our laboratory. As a result, 7 yeast

Screening of Amylase Producing yeast strains:
YNB medium containing 2% amylose (Amy/YNB medium)was used for cultivation of amylolytic yeast strains. Screening

strains, GY 16, GY 73, GY 74, GY 76, GY 169, GY 183 andGY 184, could grow on Amy/YNB medium. Moreover, they
showed clear zones on the Amy/YNB plate (Fig. 1). Especially,

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P09
the two yeast strains (GY 16 and GY 73) exhibited the largest

the ethanol production during fermentation steps for 144-216

clear zones (Fig. 1).

hours (Fig. 3).

EtOH
3
2
1
0
-1

16

73

74

76

169

183

184

Fig. 3 Ethanol level from HPLCDISCUSSION
Fig. 1 Amylolytic activity of yeast transformants in plate
assays.
Moreover, we identified these strains using 16SrRNA sequences. Strain GY 16 was identified as Cryptococcus
flavus, strain GY 73 was Ustilago trichophora, and other strains
were classified in Papiliotrema laurentii.

In this work, we identified 7 strains of the amylolitic
yeasts, GY 16, GY 73, GY 74, GY 76, GY 169, GY 183 andGY 184, and these strains belongs to C. flavus, U. trichophora,and P. laurentii. Moreover, the strains had enough amylase
activities. In the next work, we will purify amylases from theseyeast strains and will identify amy genes of the strains.

Determination of Amylase Activity:

REFERENCES

In this research, amylase activities of the strains
grown on Amy/YNB medium at 30ºC for 24 hours were

Aullybux, A., & Puchooa, D. (2013). α-Amylase Productioon

mesured. All strains had enough levels of amylase activity (Fig.

Low-Cost Substrates by Naxibacter sp. Isolated from2). Among these strains, GY 73 had the highest activity (Fig. 2).

MauritianSoils.

Microbiology

Research

JournalInternational, 3(4), 478-491.
Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B.
Microbial α-amylases: a biotechnological prospective.
Process Biochem. 2003; 38: 1599–1616. doi: 10.1016/
S0032-9592(03)00053-0.
Nguyen, C.V.; Smulikowska, S.; Mieczkowska, A., 2003.
Effect of linseed and rapeseed or linseed andrapeseed oilon performance, slaughter yield and fatty acid depositionin edible parts of the carcass in broiler chickens. J. Anim.
Fig. 2 Amylase activities of the amylolitic yeast strains.
Determination of Ethanol Activity:
Finally, we observed ability of ethanol production of
the amylolitic yeast strains. As a result, all strains did not show

Feed Sci., 12:27-288
Soccol C R, Luciana P S, Vandenberge (2003). Overview ofapplied solid state fermentation in brazil. Biochem. engg.
13: 205-218

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P10

The influence of particle size distribution on the mechanical properties ofwood plastic composite
Arif Delviawan, Yoichi Kojima, Hikaru Kobori, Shigehiko Suzuki and Kenji Aoki
Faculty of Agriculture, Shizuoka University, JapanINTRODUCTION
Wood plastic composite (WPC) is one of bio-composite
product that made from wood-based materials and polymers(Gardner et al. 2015). It is made by a variety of production
techniques (Najafi and Eslam 2011) under the specific heat andpressure conditions (Rahman et al. 2013). WPC has advantages
such as lower maintenance requirement, better in thermal
properties and durability compared other wood-based materials(Garcia et al. 2009). One of the largest commercial applications
for WPC is exterior use, such as outdoor decking applications(Gardner et al. 2015), automotive interior substrates, furniture,
packaging, housing (English and Falk. 1995), and cladding
applications (Friedrich and Luible 2016).
The characteristics of wood flour (WF) as a raw material are
important in the manufacturing of WPC. The quality andfunctionality of WPC should be improved to meet theincreasing public demand. The wet ball milling process
fibrillates the surface fibers of WF. The degree of fibrillation
increased with increasing wet milling time (Isa et al. 2014).
Although various studies have been conducted, there are still
unsolved factors such as the particle size distribution as afunction of wet milling time. It is also necessary to consider the
relationship between this factor and the properties of WPC. The
objective of this study is to evaluate the influences of particle
size distribution due to different wet milling times and themechanical properties of WPC.

MATERIALS AND METHODS
Commercial WF of Japanese red pine (Pinus densiflora),
Polypropylene (PP, J107G, Prime Polymer Co., Ltd., Japan)
and maleic anhydride-grafted PP (MAPP, Kayabrid 006PP-N,
Kayaku Akzo Co., Ltd., Japan) were used as a filler, matrix andcompatibilizer, respectively. WF (13.5 g) was milled with 200
ml of water by a ball mill (Pulverisette 5, Fritsch, Germany). A
rotating speed of 200 rpm was applied with the different wet
milling times (0, 10, 20, 30, 40, 60, and 120 min). After wet
milling, two kinds of drying conditions were applied; sevendays of freeze-drying at a temperature of −45 °C by a freeze
dryer (FDU-1200, Eyela, Japan) and 24 h of heat drying by anoven dryer (SOFW-450S, Ettas, Japan) at a temperature of
80 °C. Then, dried WF was refibrillated for 1 min by a blender(IFM-800DG, Iwatani, Japan) and screened by mesh to beclassified into 90–180 µm of mesh size. A vibrational
acceleration of ± 20 G was applied for 30 min for each condition
by a compact vibrating shaker (VSS-50D, Tsutsui, Japan). The
micro-structure of WF was investigated through a scanningelectronmicroscope(JSM-6510LV,
JEOL,Japan)
examination. The particle size distribution of WF after milling,
drying, and screening was analyzed by a laser diffraction
particle analyzer (LA-950S2, Horiba, Japan).
The ratio of the weight of WF, PP, and MAPP in thecompound was 25:74:1, respectively. PP and MAPP weremixed by a kneader under 190 °C with 30 rpm for 3 min. Then,WF was added to mix them for 10 min with an extruder machine(Laboplast Mill 30 R 150, Toyo Seiki Seisakusho Ltd., Japan).
After kneading, the WPC compound was crushed by a crusher
machine (Pulverizer SA-23, Stolz Co. Ltd., Japan). Melt

kneading was carried out for 5 min at a temperature of 190 °C
and a screw rotation speed of 50 rpm by a twin-screw small
kneader (Micro 5 cc Twin Screw Compounder, DSM Xplore,
Netherlands). After that, the injection molding machine (Micro
5.5 cc Injection Moulding Machine, DSM Xplore, Netherlands)
was used to make WPC test specimens. The dimensions of abending test specimen were 50 mm in length, 2 mm inthickness, and 6 mm in width. The specimens were conditioned
for 5 days or more in a constant temperature and humidity room
(20° C, RH 65%) before testing. The bending and tensile tests
were conducted to evaluate the performance of WPC according
to the JIS A-5741 standard. The three-point bending flexural
test was conducted using a universal testing machine (BT-805,
Yasui Kikai, Japan). The speed of the test was 5 mm/min, andthe span was 32 mm. The number of replications for eachcondition was 5.

RESULTS AND DISCUSSION
The particle distribution of dried-WF after the screening
process is shown in Fig. 1. The WF obtained with heat drying
condition can be seen in Fig. 1(a) and the dried WF under thefreeze-drying condition can be seen in Fig. 1(b). Deviation ofparticle size distribution of the produced WF depends on the
milling conditions. The particle size distribution for the freezedrying condition was wider than that for the heat drying
condition.
At 0 to 30 min of wet milling time, there was no peak for thelarger particle size near 1000 µm. However, from 40 up to 120
min, the peak intensity decreased with increasing the wet
milling time. Under those conditions, the peak intensity for thefreeze-drying condition was higher than that for the heat drying
condition. For the particle size near 100 µm, the intensity
decreased as the wet milling time increased up to 30 min.
However, the intensity for a particle size near 100 µm from 40–
120 min increased with increasing the wet milling time. The
highest intensity of milled WF for the heat drying condition wasobserved at 120 min, which might be due to aggregation. In thecase of the smaller particle size near 20 µm, heat and freezedrying conditions showed the same trend. As the wet milling
time increased, the intensity near 20 µm increased. Under thiscondition, the intensity for freeze-drying was higher than thatfor the heat drying condition.
The correlation coefficient (R) between the intensity at any
particle size (Ip) and mechanical properties (M) are calculated
as the following Equation,
𝑅𝑅𝑀𝑀,𝐼𝐼𝐼𝐼 =���)� )(𝐼𝐼𝐼𝐼𝑡𝑡 −𝐼𝐼𝐼𝐼∑(𝑀𝑀𝑡𝑡 −𝑀𝑀���)2� )2 ∑(𝐼𝐼𝐼𝐼𝑡𝑡 −𝐼𝐼𝐼𝐼�∑(𝑀𝑀𝑡𝑡 −𝑀𝑀

� , 𝐼𝐼𝐼𝐼𝑡𝑡 , and 𝐼𝐼𝐼𝐼
� are any mechanical property atwhere 𝑀𝑀𝑡𝑡 , 𝑀𝑀
wet milling time t, mean mechanical property, intensity at p µm
on the particle size distribution of wet milling time t and its
mean intensity, respectively. R is calculated for the intensity at
any particle size versus flexural modulus. Because the particle
size distribution pattern on freeze-dried and heat-dried WF
were different, R for freeze-drying and heat drying wereseparately calculated. Note that R is the parameter to evaluate

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P10
only the ‘linear’ relationship between intensity and mechanicalproperties.
The correlation vector of flexural modulus for each WF size
was observed. The results showed that the flexural modulus ata particle size smaller than 100 µm had negative correlation.
Inversely, particle size from 100 – 900 µm had positive
correlation for these flexural modulus. For heat dried WF,
correlation vector for flexural modulus at smaller particle lessthan 30 µm had a negative correlation. However, particle sizearound 40 – 100 µm and around 200 µm showed positive andnegative correlation, respectively. The highest correlation
between flexural modulus was observed at a particle size 77.3
µm as a representative of the smaller size of WF. The intensity
at 678.5 µm, which also indicated a higher correlation between
flexural modulus, decreased with increasing wet milling time.
Figure 2 shows the relationship between the flexural modulus
and the intensity of small and large particles. As shown in Fig.
2(a), an excessive percentage of small particles resulted in thedecrease of flexural modulus. In the case of large particle, as
shown in Fig. 2(b) the flexural modulus decreased during
decreasing the intensity The lowest flexural modulus wasobserved under the condition of heat drying with a wet milling
time of 120 min. the wet milling condition is the same for bothfreeze and heat drying.
Therefore, the actual amounts of smaller particles for thesame milling time must be identical. It is suggested that theaggregation of smaller particles during heat drying reduces theapparent intensity for the small particles. The fact that theflexural modulus for a wet milling time of 120 min with heat
drying showed a similar value as that for freeze-drying,indicating that the aggregation of smaller particles did notstrongly influence the flexural modulus of WPC.

ACKNOWLEDGMENTS

Rahman, K. S., Islam, M. N., Rahman, M. M., Hannan, M. O.,
Dungani, R., and Khalil, H. P. S. A. (2013) Flat-pressedwood plastic composites from sawdust and recycled
polyethylene terephthalate (PET): physical and mechanicalproperties. SpringerPlus 2(629):1-7.

Fig. 1: The particle size distribution after the screened
process for 90 to180 µm obtained by (a) heat drying
condition and (b) freeze-drying condition.

The authors are grateful to the Toclas corporation for all of
collaborating, supporting, and providing the materials for thisresearch.

REFERENCES
English, B. W., and Falk, R. H. (1995) Factors that affect theapplication of woodfiber-plastic composites. In: Proc of
Woodfiber-plastic composites: Virgin and recycled woodfiber and polymers for composites, May 1–3, 1995, Madison,
WI, pp 189–194.
Friedrich, D., and Luible, A. (2016) Investigations on ageing ofwood-plastic composites for outdoor applications: A metaanalysis using empiric data derived from diverse weathering
trials. Constr Build Mater 124:1142-1152.
García, M., Hidalgo, J., Garmendia, I., and Jaca, J. G. (2009)
Wood–plastics composites with better ﬁre retardancy anddurability performance. Composites Part A 40:1772-1776.
Gardner, D. J., Han, Y., and Wang, L. (2015) Wood–Plastic
Composite Technology. Springer Int Pub 1(3):139-150.
Isa, A., Toyoda, T., Suzuki, S., Kojima, Y., Ito, H., Makise, R.,
and Okamoto, M. (2014) The effects of wet-milled woodflour on the mechanical properties of WF/polypropylene
composites. J Wood Chem Technol 34:20-30.
JIS A5741–2016 (2016) Wood-plastic recycled composite (inJapanese). Japanese Standards Association, Tokyo, pp 1172–
1191.
Najafi, A., and Eslam, H. K. (2011) Lignocellulosic
filler/recycled HDPE composites: Effect of filler type on
physical and flexural properties. Bioresources 6(3):24112424.

0 min

0 min

0 min
0 min

Fig. 2: The relationship between flexural modulus andintensity at (a) 77.3 µm and (b) 678.5µm.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P11

A novel peptide derived from rice α-globulin decreased cholesterol
micellar solubility in vitro and inhibited cholesterol absorption in rats
Maihemuti Mijiti, Wang Jilite, Huang Bingyu and Satoshi Nagaoka
Gifu University, Gifu JapanINTRODUCTION
It is well known that the appearance of coronary heart diseasedepends on the serum cholesterol level. To diminish the riskof such lifestyle related diseases, early prevention throughchronic treatment with low-cost medicines have attracted
increased attention. Numerous studies have focused on thebeneficial effects of soy protein on hypercholesterolemia andcardiovascular disease. On the other hand, remarkably fewer
reports have focused on the atheroprotective effects of riceprotein.
Rice α-globulin reduces serum cholesterol
concentrations in rats fed a hypercholesterolemic diet bypromoting excretion of fecal neutral sterols (1). This
experiment was designed to identify the peptides which havebile acid-binding activity and inhibit the cholesterol absorption
from rice α-globulin.

MATERIALS AND METHODS
Peptide array— In this study, we used a peptide array toevaluate the bile acid-binding activity of peptide derived fromrice α-globulin. A peptide array was constructed using
previously reported methods (2, 3). In the peptide array, bile
acid-binding ability was evaluated by the binding ability oftaurocholic acid and fixed peptides on cellulose membrane,then binding peptides with taurocholic acid were detected byimmunoassay.
Bile acid binding— The abilities of selected peptides, SPH(soybean protein peptic hydrolysate), CTH (casein tryptichydrolysate), and cholestyramine (Sigma, St. Louis, USA) tobind with taurocholate were measured by a method described
previously. (13) Mixtures containing 1.85 kBq oftauro[carbonyl-14C] cholic acid (sodium salt) (Amersham
International, Buckinghamshire, UK), 0.1 mol/l sodium
taurocholate in 2ml of 0.1 mol/l Tris-HCl buffer (pH 7.4), andbinding substance (selected peptides, SPH, CTH orcholestyramine, 5 mg/ml) were incubated at 37℃ for 2 h, andthe radioactivity in the supernatant (15.000×g for 15 min) was
measured by liquid scintillation counting.
Micellar solubility— The micellar solubility of cholesterol
with the selected peptides, SPH, CTH, and cholestyramine invitro was measured by previously described method. (13)[14C]-labeled micellar solutions (1.0 ml) were prepared at thefollowing concentrations, and were mixed by sonication: 0.74
kBq [4-14C]-cholesterol (2.1 Gbq/mmol, NEN, Boston, MA),
0.1 mmol/l cholesterol (Sigma), 6.6 mmol/l sodium
taurocholate (Sigma), 1 mmol/l oleic acid (Sigma), 0.6 mmol/l
phosphatidylcholine (Sigma), 0.5 mmol/l monoolein (Sigma),and 15 mmol/l sodium phosphate (pH 7.4). After incubation at
37 ℃ for 24 h, selected peptides, SPH, CTH, orcholestyramine (5 mg/ml) was added to the micellar solution,
dispersed by sonication, incubated at 37 ℃ for 1 h, andcentrifuged at 100.000×g for 60 min at 37 ℃. The supernatant
was collected for determination of [14C]-cholesterol by aliquid scintillation counter.

Intestinal cholesterol absorption in rats. Selected candidate
peptide (MRFRDR) were synthesized with purity greater than
95% and remaining trifluoroacetic acid was removed. 7-weekold Wistar male rats (Japan SLC, Hamamatsu, Japan) weredeprived of food for 48 h, without restriction of their access towater. Next, the rats received the test solutions by intragastricintubation with a polyethylene catheter. One hour later. Blood
was collected by cardiac puncture for separation of the serum,
and the liver and intestines were quickly excised. The test
solutions were MRFRDR, or control. The [3H]-cholesterol
incorporated into the serum, liver, and intestine was extractedwith hexane.

RESULTS
Analysis of the 6-mer peptides derived from s rice α-globulin
with bile acids binding affinity by using the peptide array― To
find the peptide derived from rice α-globulin with bile acidbinding capacity, we constructed 6-mer peptides, the arrayconsisting of 181 sequences plus positive control sequence(VAWWMY) plus negative control sequence (GGGGGG).
Using the peptide array, 15 spots (quintuplicate x3 arrays) were
evaluated for each unique sequence. So, there are some peptides
with higher bile acid binding activity than positive control
peptide (VAWWMY) (Fig.1).

VAWWM

Fig.1 Fluorescent intensity of taurocholic acidbinding to overlapping peptides 6 residues in length,
covering globulin. The sequence is in descendinghydrophobicity order from left to right. The
sequence VAWWMY described in the figure is the
positive control, GGGGGG is the negative control.
Bile acid binding capacity― MRFRDR had a significantly
greater ability to bind bile acid than CTH in vitro. Among thefive candidates, the binding capacity of MRFRDR was greaterthan the other peptides.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P11
Micellar solubility— The micellar solubility of cholesterol was
significantly lower in the presence of MRFRDR, of SPH, andof cholestyramine than in the presence of CTH.
Cholesterol absorption in vivo— Incorporation of [3H]cholesterol into the serum, liver, and intestines was
significantly lower in the MRFRDR than in the control group(Fig.2).

even in in vivo tests using animals. For that reason we
examined the effects of MRFRDR on cholesterol absorption inrats (in vivo) using 3H-cholesterol. The result indicated,
MRFRDR significantly suppresses cholesterol absorption in
the blood, upper small intestine and all over the small intestinecompared with the control group containing no peptide in theadministration solution, and significantly suppresses the totalcholesterol absorption as well (Fig.2) .
In conclusion, starting from the peptide array screening, wewere successful in finding novel bile acid binding peptides(MRFRDR) that interact with bile acid in soluble-form andstrongly lower to micellar solubility of cholesterol. And the
peptide (MRFRDR) significantly inhibited cholesterol
absorption in rats. Our results are consistent with Iwami et al.
(8) where they report successful inhibition of bile acid reabsorption in the ileum with bile acid binding peptides.We
would also like to note that this is the first report of peptide
array screening technology applied to the optimization of bile
acid binding peptides. We believe these findings could
contribute to the future investigation and design of therapeutic
peptides and functional food constituents for the early
prevention of life style related diseases.

REFERENCES

Fig.2 Effect of MRFRDR on the distribution of 3Hcholesterol in serum, liver and intestine.

Statistical significance compared to control by Student’s t-test(**: P<0.01)

DISCUSSION
Rice protein consumption with modulation of plasmacholesterol level has been demonstrated in some studies (5).
And also rice α-globulin reduces serum cholesterol
concentrations in rats fed a hypercholesterolemic diet bypromoting excretion of fecal neutral sterols (1). The bile acidbinding 6-mer peptide VAWWMY (Val-Ala-Trp-Trp-MetTyr) was previously reported as the soystatin obtained fromthe soybean glycinin (11S globulin) A1aB1b subunit ofsoybean protein (6, 7). In this study, we screened the peptide
library covering all possible those peptide. As a result, we
succeeded in finding novel peptides with high bile acidbinding capacity. Unfortunately, all candidates’ peptides
didn’t showed good results in the bile acid binding capacityand micellar solubility in vitro. But some of them showed
good results, like MRFRDR showed higher bile acid binding
ability and disruption of bile acid micelles so peptide canlower micellar solubility of cholesterol. The lowering of
micellar solubility of cholesterol in small intestine is known as
one of the effective strategies to inhibit cholesterol absorption.
Cholesterol forms micelles with fatty acids, 2-monoglycerides
and bile acids. The mixed micelle cholesterol passes throughthe unstirred water layer (UWL) to reach the small intestinal
epithelium. Mixed micelles are integral to the process of
passing cholesterol UWL. It was thought that MRFRDR could
inhibit the passage of UWL and reduce cholesterol absorption
in the small intestine by reducing cholesterol micellesolubility. Therefore, MRFRDR is considered to have thepotential to exhibit cholesterol absorption inhibitory effect1.Tong, LT., Fujimoto, Y., Shimizu, N., Tsukino, M., Akasaka,T., Kato, Y., Iwamoto, W., Shiratake S., Imaizumi, K., Sato,M. (2012) Rice α-globulin decreases serum cholesterol
concentrations in rats fed a hypercholesterolemic diet andameliorates atherosclerotic lesions in apolipoprotein Edeficient mice. Food Chem., 132: 194–200.
2. Kato, R., Noguchi, H., Honda, H., and Kobayashi, T. Hidden
markov model-based approach as the first screening ofbinding peptides that interact with MHC class II molecules.
(2003) Enzyme Microb. Technol., 33: 472-481
3. Kato, R., Kaga, C., Kunimatsu, M., Kobayashi, T., andHonda, H. Peptide array-based interaction assay of solidbound peptides and anchorage-dependant cells and its
effectiveness in cell-adhesive peptide design. (2006) J.
Biosci. Bioeng., 101: 485-495.1.
4. Nagaoka, S., Miwa, K., Eto, M., Kuzuya, Y., Hori, G., andYamamoto, K., Soy Protein Peptic Hydrolysate with Bound
Phospholipids Decreases Micellar Solubility andCholesterol Absorption in Rats and Caco-2 Cells. J. Nutr.,
129, 1725–1730 (1999).
5. Morita, T., Oh-hashi, A., Kasaoka, S., Ikai, M., Kiriyama,S. (1996) Rice protein isolates produced by the twodifferent methods lower serum cholesterol concentration inrats compared with casein. J. Sci. Food Agric., 71:415–424.
6. Choi, S. K., Adachi, M., and Utsumi, S., Identification of
the Bile Acid-binding Region in the Soy Glycinin A1aB1b
Subunit. (2002) Biosci. Biotechnol. Biochem., 66: 23952401
7. Nagaoka, S., Nakamura, A., Shibata, H., Kanamaru, Y.,
Soystatin (VAWWMY), a Novel Bile Acid-Binding
Peptide, Decreased Micellar Solubility and Inhibited
Cholesterol Absorption in Rats (2010) Biosci. Biotech.
Biochem., 74: 1738-1741.
8. Iwami, K., Kitagawa, M., and Ibuki, F., Effect of dietary
proteins and/or their digestive products on intestinal
taurocholate absorption. (1990) J. Nurt. Sci. Vitaminol. 36,
S141-S14

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P12

The effect of frozen storage on textural quality of cooked pasta
Rachmad Adi Riyanto and Takahisa Nishizu
The United Graduated School of Agricultural Science, Gifu University, JapanINTRODUCTION
Pasta is popular worldwide and considered as a staple foodof many nations. It is a simple product, traditionally, theprimary ingredient of pasta is wheat semolina, where durum
wheat semolina and water are mixed and kneaded to obtain adough that is then shaped and, most commonly, dried (Carini,
Curti, Minucciani, Antoniazzi, & Vittadini, 2014). Dry pasta isconsumed mainly around the world because of its securestorage and cooking, long shelf-life and versatility whencombined with other ingredients to yield a variety of final
products (Hager, Zannini, & Arendt, 2012; Lucisano, Cappa,
Fongaro, & Mariotti, 2012; Marti & Pagani, 2013). However,
nowadays, consumers pay more and more attention to ready-toeat convenience and healthy food (Diez et al., 2009). Therefore,frozen cooked pasta offer a feasible alternative.
Olivera and Salvadori (2009) investigated the influence of
freezing on the quality of cooked organic pasta and found that
fast-frozen pasta quality was closer to the fresh pasta than theslow frozen one. However, quite a few literature are available
concerning the detailed evaluation of products by different
precooking methods. The effects of freezing and storage life offrozen foods have been studied for several years (Fellows,1997). Recently starch-based food products have been analyzed
during freezing and frozen conditions (Giannou, Kessoglou, &Tzia, 2003; Gormley, Walshe, Hussey, & Butler, 2002; Ribotta,
Leon, &Anˇon, 2001), but there are few pieces of information
about the relationship between the quality of cooked and frozen
pasta as in ‘‘ready-to-eat’’ meals (Kobs, 2000).
One of the critical parameters to assess cooked pasta quality,
concerning consumer acceptability, is textural properties(Carini, Vittadini, Curti, & Antoniazzi, 2009). The texture is aparameter that could imitate the sensory as of the critical quality
attributes that determine consumer acceptance of the product(Luo et al. 2015; Ajila et al. 2010; Olivera and Salvadori, 2009).
The texture of foods is mostly determined by the moisture andfat contents and the types and amounts of structuralcarbohydrates (cellulose, starch, and pectic materials) andproteins that are present. Changes in texture are caused by lossof moisture, fats, formation or breakdown of emulsions andgels, hydrolysis of polymeric carbohydrates, and coagulation orhydrolysis of proteins (Fellows, 1997). The retention of texture
similar to that of the fresh product is an essential criterion for
acceptance of a frozen convenience pasta by the consumer(Hatcher, 2004). The quality of the frozen cooked pasta wasevaluated via texture profile analysis (TPA).
The effect of frozen storage on the textural quality of cooked
pasta has not been fully investigated, especially for long-term
storage. Therefore, the aims of this study were to determine thetexture properties of cooked pasta during frozen storage for 6
weeks and observe microstructure changes by SEM.

MATERIALS AND METHODS
The commercial dried pasta “Ma Ma” with diameter size
1.6mm from Nisshin Foods Inc was used as the sample. The
dried pasta was cut into 11cm length and boiled in the water
bath with polypropylene reaction tube size 15ml at 100oC for 7
minutes. After boiling, pasta was drained and wiped using

tissue paper to reduce the water on the surface for 1 minute, andthen kept on the freezer at -20oC before being analyzed.
Moisture analysis Frozen cooked pasta was kept outside thefreezer for 10 minutes before moisture analysis. Samples weredried in the oven at 121oC for 72 hours. The moisture content
was determined using the oven-drying method.
Texture analysis Frozen cooked pasta was kept outside thefreezer for 10 minutes before texture analysis. Texture
properties were measured by a TA-XT2i Texture Analyzer(Stable Micro Systems, London, England). The texture analyzer
was equipped with a light knife blade (A-LKB) probe and themeasurements were carried out at room temperature using a 5
kg compression cell. A sample of pasta was placed onto themeasurement plate of the texture analyzer. The measurement
assay parameters were: pretest speed: 1.0 mm/s; test speed: 1.0mm/s; posttest speed: 1.0 mm/s. The strain was 100%, so thesample was cut completely.
Scanning electron microscopy (SEM) The section profile offrozen cooked noodles with different frozen storage wereanalyzed using SEM (S-4300, Japan Electron Optics
Laboratory CO., Ltd., Tokyo, Japan). Firstly, the frozen cooked
pasta were soaked in glutaraldehyde with a solution of 2.5%(w/w) for 90 minutes for chemical fixation. Then rinsed with
distilled water. After that, the samples were immersed in
Osmium 50% for 60 minutes then rinse with distilled water andcontinue eluted in graded ethanol series (30%, 50%, 60%, 70%,
80%, 90%, 95%, and 100%) for 15 min at each gradation. After
that, remove the ethanol and immerse the sample on the t-butyl
alcohol for 15 minutes. The pasta was freeze-dried afterward
and the dehydrated samples were coated with osmium gas for
15 seconds. The images of pasta were observed at 60 times and
250 times respectively at an accelerating voltage of 5.0 kV.
Statistical analysis All the determinations and analysis were
performed in triplicate at least. One-way ANOVA (analysis ofvariance) and Duncan’s test were conducted by using thesoftware SPSS 16.0 for windows (SPSS Inc., Chicago, IL,
USA). Significance was defined at P < 0.05.

RESULTS

Fig. 1: Moisture content (% db) of cooked pasta after
freezing

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P12

Fig. 2: Textural properties of cooked pasta after freezing

showed that the moisture content decrease during frozen
storage. This is because the water was transformed into ice
crystals, and during the frozen storage, the ice crystal could be
sublimated into the gas as known as freezer burn phenomena.
These phenomena affect the textural properties of frozen
cooked pasta.
The texture characteristics of frozen cooked pasta play anessential role in quality, which determines the acceptance ofconsumers on the product (Olivera & Salvadori, 2009). In thisstudy, textural profile analysis (TPA) was used to evaluate thequality of cooked pasta. The textural parameters of cooked
pasta during frozen storage were measured and summarized inFig. 2. The results showed that during the frozen storage thetexture of cooked pasta gradually decreased, the lowest force
was at the 6 weeks of freezing storage.
To have tangible evidence of microstructure changes offrozen cooked pasta during the frozen storage visually, thescanning electron microscopy (SEM) was employed. The crosssection of frozen cooked pasta with different storage time were
exhibited in Fig. 3. The cross-section of frozen cooked pasta
stored after 1 week presented a smooth and continuous status.
However, clear differences can be noticed after 6 weeks, when
the surface became rougher along with large pores, which may
be related to the ice crystals formation.

ACKNOWLEDGMENTS
1 weekI would like to thank the MEXT scholarship for sponsoring
my study and thank Food Processing and Chemistry Laboratory
at the University of Gifu for the use of equipment and inparticular Prof. Takahisa Nishizu for his guidance on thisproject.

REFERENCES

2 weeks

6 weeks
Fig. 3: Scanning electron microscope images of cooked
pasta after freezing

DISCUSSION
Water, playing as an active substance, is regarded as a criticalrole in the quality of food, especially for frozen food. As frozen
cooked pasta possess a high water content of over 60%, andmost of the water can transform into ice crystals (Carini et al.,2010). In this study, the moisture content was analyzed using
the oven-drying method, as presented in Fig. 1. The results

Carini, E., Vittadini, E., Curti, E., & Antoniazzi, F. (2009).
Effects of different shaping modes on physico-chemical
properties and water status of fresh pasta. Journal of FoodEngineering, 93, 400–406.
Carini, E., Curti, E., Minucciani, M., Antoniazzi, F., &Vittadini, E. (2014). Pasta. In R. D. P. F. Guine, & P. M. dos
Reis Correia (Eds.). Engineering aspects of cereal andcerealbased products (pp. 211–238). Boca Raton: CRC Press.
Fellows, P. (1997).Food processing technology—principles
and practice.London: Woodhead Publishing Ltd.
Hatcher, D. W. (2004). Influence of frozen noodle processing
on cooked noodle texture. Journal of Texture Studies, 35(4),429–444.
Hager, A. S., Zannini, E., & Arendt, E.,K. (2012). Gluten-freepasta: Advances in research and commercialization. Cereal
Foods World, 57, 225–229.
Luo, L. J., Guo, X. N., & Zhu, K. X. (2015). Effect of steamingon the quality characteristics of frozen cooked noodles. LWT– Food Science and Technology, 62(2), 1134–1140.
Olivera, D. F., & Salvadori, V. O. (2009). Effect of freezing rate
in textural and rheological characteristics of frozen cookedorganic pasta. Journal of Food Engineering, 90(2), 271–276.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P13

Effect of forest age on slope stability evaluated by a distributed landslide
model
Vany Fadhilah1, Al Nuriza Rahmadania1 and Fumitoshi Imaizumi2
1. Graduate School of Integrated Science and Technology, Shizuoka University, Japan
2. Faculty of Agriculture, Shizuoka University, JapanINTRODUCTION
Since ancient times, people have suffered numerous
landslide disaster. Landslide can be simply described as the
movement of a mass of rock, earth or debris down a slope.
Landslides, specifically in Japan, generally occur along gently
to moderately sloping ground which is also important as theseareas include residential and agricultural use. Additionally,landslide also frequently occurred after clearcutting forest on
steep area (Imaizumi, 2008). Because of these conditions,landslide disaster is harmful for human. Therefore, the study oflandslide phenomenon is necessary to protect the slopes fromfuture land sliding and failures.
In preventing landslide, the stability of slope is an important
issue to be concerned. There are some natural factors that
responsible for slope stability, those are geomorphic factors,
soil properties, hydrology, vegetation and seismicity (Sidle etal. 1985). However, vegetation has been widely recognized thatit plays an important role in increasing slope stability. throughroot (Stokes et al. 2008; Kreng et al. 2015).
Previous studies have tried to evaluate contribution ofvegetation on the slope stability by interpreting spatial
distribution of underground roots and physical test of individualroot. However, such studies cannot evaluate contribution ofvegetation on the slope stability in hillslope-scale, because ofdifficulty in interpretation of root network in wide areas.
The overall aim of this study is to evaluate effect of forest
age on slope stability. The specific objectives of this study are(i) to interpret temporal changes in landslide ratio and (ii) toevaluate temporal changes in root cohesion after harvesting,through GIS analysis and back calculation of a physicalparameter using a distributed landslide model.

STUDY SITE
The study area is about 17.6km2 located in the Ikawa
University Forest catchment, southern side of the Southern
Japanese Alps, central Japan (Fig.1). Geologically,
characteristic of the study area is sedimentary rocks withalternate strata of sandstone and shale of the late Cretaceous
period (Maita, 2001). The range of topography is from 900 ma.s.1 in the south to the 2406 m a.s.1 peak of Mt. Aonagi in thenorthwest, leads to gradient of 35-40o comprising about 50%of the entire catchment. The study area is hilly covered bybrown forest soil and podzol. Also, it has high average annual
precipitation calculated approximately 2800 mm from 1993 to
2002. The most abundant rainfall events mainly occur from theearly summer in June through the typhoon season in August to
early October. In this study area, forest practice such as forest
harvesting specifically clear-cutting is frequently occurred in
artificial forest which cover 17% of area, while the rest area isnatural forest. This artificial forest is comprised by Cryptomeria
japonica (Japanese cedar, sugi), Chamaecyparis obtusa(Japanese cyprus, hinoki) and Larix kaempferi (karamatsu). A
significant proportion of the trees in the secondary growthforest was harvested in the 1950s and 1960s. After clearcutting, branches and leaves of harvested trees were left on the

ground to protect the slopes from surface erosion. These
harvesting operations conducted in the Ikawa University Forest
are common in Japanese forestry (Tsukamoto 1987; Imaizumi
et al. 2017).

METHODOLOGY
1. Aerial Photograph Interpretation
Assessing the location and area of landslide in the Ikawa
University Area was obtained by using monochrome aerial
photographs for two different years (1980 and 1984). New
occurrences of landslide were identified by comparing thosesuccessive aerial photographs. In this study, landslides wereidentified as all mass movement from hillslopes to channels,
while other mass movements move down channels were
excluded. Landslide area ratio was calculated through dividing
landslide area that occurred in every forest age by forest area inevery forest age.
2. Back Calculation of Root Cohesion
The model framework of slope stability used for this study is
a distributed landslide model. This model proposed by Okimura
et al. (1985) which are consisted of groundwater levelcalculation and infinite slope analysis. Groundwater level iscalculated using Darcy’s law (Shuin et al. 2014). Darcy’s law
possibly calculates groundwater flow through aquifer to thecross-sectional area perpendicular. While infinite slope analysis
is calculated as used by Shuin et al. (2014) which defines that asafety factor of 1.0 as unstable slope. In this study, we used
cohesion, which is a parameter representing root and soil
strength, as varied parameter when calculating the safety factor.
Therefore, slope stability generated by various root cohesion
would be obtained, and by back-calculation derived value forroot cohesion of real landslide occurrences in every forest age
class (O’Loughlin CL, 1974).

Fig. 1:
Location and forest type of the Ikawa University Forest
RESULT AND DISCUSSION

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P13
1. Temporal changes in landslide ratio after harvesting
New landslide occurrences were identified in the study area
specifically in artificial forest on series of aerial photographs
taken from 1980-1984 with total 29 new landslides. A total of
29 landslides occurred in the various forest age. Relationshipbetween landslide ratio and forest age as shown in the Fig. 2
shows that the immediate effect of landslide occurrence inforest age less than 10 years after harvesting was the largest.
However, the effect significantly decreased in the forest age
more than 10 years. It was probably caused by root decay of
harvested trees that existed in the area. Roots begin to decay a
few years after cutting, with the most strength disappearing
within 10 years. Consequently, root decay in 1–10 years oldforests accelerates the occurrence of landslides (Tsukamoto,1987). It is in accordance with prior study in Sanko catchment
Japan (Imaizumi et al. 2008) that forest stands age 1 to 10 years
after harvesting impact greatly on landslide occurrence. In
addition, Fig. 2 also shows that no landslide occurrence foundedin the forest age more than 15 years old. This shows that thestability of the slope in this area almost entirely recovers within
15 years.

Fig 3: Root cohesion of obtained by back calculation
reaching certain time. This study could adequately predict rootcohesion calculated by landslide model without fieldmeasurements which are time consuming and regional coverage
is difficult to obtain. This result of this study could contributeto help predict and mitigate landslide disaster specificallyaround the artificial forest industry on steep terrain.
Furthermore, it could also be used as consideration inestablishing forest management.
ACKNOWLEDGMENTS
This study was supported by the Ministry of Education,
Culture, Sports, Science and Technology, Japan.

Fig. 2: Landslide area ratio obtained by aerial photographinterpretation
2.Temporal changes in root cohesion after harvesting
Relationship between root cohesion and estimated landslidearea shows that estimated landslide area decreased withincreased cohesion. It is in accordance with prior study that
increasing effective soil cohesion will decrease potential oflandslide area (Shuin, 2014).
To clarify how gradual increased root cohesion on forest age
which impact on real landslide occurrence, back calculated ofroot cohesion was conducted as shown as Fig. 3. It shows thatroot cohesion gradually increased with increasing age of forest.
However, the result of this study also shows that root cohesion
for artificial forest age 5 years and 10 years after harvesting was
not very distinct. It is in accordance with prior study (Imaizumi
et al, 2008) that root re-growth and total root strength were notso evolved yet in the early 10 years. While, root cohesion
significantly increased when forest age more than 10 years andslowly increased when forest age more than 16 years after
harvesting. It is probably the effect of the dynamic root strength
values in Imaizumi et al. (2008) that regrowth of newly planted
was described by a sigmoid relationship. Therefore, the root
strength will so weak in the early, significant in the middle timeand being stagnant when

REFERENCES
Imaizumi, F., Sidle, R.C. and Kamei, R., 2008. Effects of forest
harvesting on the occurrence of landslides and debris flows in
steep terrain of central Japan. Earth Surface Processes andLandforms: The Journal of the British GeomorphologicalResearch Group, 33(6), pp.827-840.
Imaizumi, F., Suzuki, O. and Togari‐Ohta, A., 2017. Seasonalchanges in the sediment flux on steep hillslopes in a humid
diurnal frost environment. Earth Surface Processes andLandforms, 42(3), pp.389-398.
Kreng Hav Eaba, Suched Likitlersuangc,n, Akihiro Takahashi.
2015. Laboratory and modelling investigation of rootreinforced system for slope stabilization. Soils andFoundations 2015;55(5):1270–1281
Maita H, Shizuoka River Work Office. 2001. Ohi River.
Source-to-sink sedimentary cascades in Pacific Rim Geosystems. 36-43
O’Loughlin CL (1974) The effects of timber removal on thestability of forest soils. J Hydrol NZ 13:121–134
Shuin, Y., Otsuka, I., Matsue, K., Aruga, K., Tasaka, T. And
Hotta, N., 2014. Estimation of Shallow Landslides Caused by
Heavy Rainfall Using Two Conceptual Models. International
Journal of Erosion Control Engineering, 7(3), pp.92-100.
Sidle, R.C., Pearce, A.J. and O'Loughlin, C.L., 1985. Hillslope
stability and land use. American Geophysical Union.
Stokes, A., Norris, J.E., Van Beek, L.P.H., Bogaard, T.,
Cammeraat, E., Mickovski, S.B., Jenner, A., Di Iorio, A. andFourcaud, T., 2008. How vegetation reinforces soil on slopes.
In Slope stability and erosion control: ecotechnologicalsolutions (pp. 65-118). Springer, Dordrecht.
Tsukamoto Y. 1987. Evaluation of the effect of tree roots on
slope stability. Bulletin of the Experiment Forests, Tokyo (InJapanese with English abstract).

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P14

Evaluation and characterization of carotenoids and β-carotene in ricelandraces from Northeast India
Devendra Kumar Maravi1, Muthuvel Jothi1, Gundimeda Jwala Narasimha Rao1,2, Lingaraj Sahoo1
1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039,
India
2. Crop Improvement Division, ICAR-National Rice Research Institute, Cuttack, Odisha, 753006, India

INTRODUCTION
Carotenoids are one of the most diverse classed of naturalcompounds. Plant carotenoids are composed of C40
isoprenoids skeleton with or without epoxy, hydroxyl and ketogroups. Carotene, such as ɑ- and β- carotene and lycopene arehydrocarbons, whereas xanthophyll are oxygenated derivativesof carotenes and includes the compounds β-cryptoxanthin,lutein and zeaxanthin (Giuliano et al. 2008; Stanley and Yuan,2017). They have fundamental roles in human nutrition as
antioxidant and precursor of vitamin A and their consumption
is associated with protection from a range of diseases. Vitamin
A deficiency is one of the leading causes of micronutrient
malnutrition in developing countries. Carotenoids carrying list
on unsubstituted beta-ionone ring, such as alpha and betacarotene, are nutritional vitamin A precursor. This is prompted
as series of efforts to evaluate and breed high provitamin-A
levels into staple crops.
Biosynthesis of carotenoids in plants take place within theplastids, chloroplast of photosynthetic tissues and chromoplastof fruits and flowers. Chlorophyll, tocopherols, plastoquinone,
gibberellins and carotenoids all share a common biosynthetic
precursor geranyl geranyl-diphosphate (GGPP) which isderived from plastid isoprenoid metabolism. It has been
established that four enzymes in plant mainly involves in βcarotene biosynthesis (Jourdan et al. 204). Phytoene synthase,
phytoene desaturase, ζ-carotene desaturase and lycopene
cyclase catalyzes the β-carotene synthesis from GGPP (Fig. 1).
The first step in carotenoid biosynthesis is the condensation of
two molecules of GGPP to produce phytoene by the enzyme
phytoene synthase (PSY). PSY is firmly associated with thechromoplast membrane in its active form (Yao et al. 2018). The
cyclization of lycopene by two different lycopene cyclases
specific for 〈- and ∑-ionone end-groups of LCY marks abranching point in the pathway where one branch leads to βcarotene and its oxygenated derivative lutein, while the otherforms -carotene and the derived xanthophylls, such aszeaxanthin, antheraxanthin, violaxanthin and neoxanthin.
Carotenoids are very minor constituents in cereal grains.
Besides their nutritional and health benefits, they are
responsible for the attractive yellow to pale yellow colors of
durum wheat (Roncallo et al. 2012). Rice grains are known to
devoid of carotenoids in their endosperms. Rice, an important
staple crop, is produced around the world and consumed by halfof the world’s population. Asian countries are much more
dependent on rice for food energy and protein intake than othercountries. Evaluation and characterization of important
carotenoids from rice landraces is necessary. North-East (NE)
India, the probable origin of rice has diverse genetic resources.
Many rice landraces of NE India were not yet characterized forsourcing of pro-vitamin.

We explored the possibility of finding β-carotene in grains ofrice landraces of Northeast India in order to use in conventional
plant breeding or biofortification programs, aiming to increase
the total carotenoids and β-carotene contents. We determined
the total carotenoid and β-carotene in rice landrace samples.
Our results found β-carotene in few rice landraces of Northeast
India.

MATERIALS AND METHODS
Chemicals: HPLC grade methanol, tetrahydrofuran,acetonitrile, water were purchased from Merck (Germany). The
β-carotene standard were purchased from Sigma (USA).
Carotenoid extraction and HPLC analysis: Indigenous ricecultivar were collected from Northeast Region of India(Assam). Rice were de-howled manually and milled usingmortar pestle in liquid nitrogen. The carotenoids were extracted
from 500 mg of seed powder with acetone (2 ml the first timeand 1 ml the second time). Half the proportion of petroleum
ether was added to combined extract. The phase extraction was
performed by adding the water, upper clear phase eluted andevaporated under vacuum.
Spectrophotometer absorbance was measured at 470 nm. The
total carotenoid (µg/g) was calculated based on the
spectrophotometer reading, taking the extinction coefficient(2592) in petroleum ether.
The petroleum ether is evaporated in vacuum and extract
were re-dissolved in chloroform and 20µl were used for HPLC
analysis (Perkin Almer). For HPLC, C18 reverse-phase column
were used with following solvent system: solvent A=
acetonitrile (ACN): Tetrahydrofuran (THF): water (H2O),
10:4:6; solvent B= ACN: THF: H2O, 10:8.8:1.2 with a linear
gradient of 100% A for first 3 min and slowly to 100% B for
10min, with the total run time of 25min.
Nuclear Magnetic Resonance - the rice carotenoids samples
were analyzed on Bruker AVANCE III 600 MHz NMR
instrument using 1D -1H and 13C spectroscopy in CDCL3.
Fatty acid compositional analysis from rice endosperm werealso analyzed based on the NMR spectra.

RESULTS
Quantification of total carotenoids and β-carotene: Rice
endosperm from individual landraces were analyzed
quantitatively by spectrophotometry and qualitatively for βcarotene by high performance liquid chromatography (HPLC).
Estimation of carotenoids from rice seeds showed total
carotenoids level ranging from 2.24- 6.79 µg/g (Table 1). The
rice variety B and SS showed lower carotenoid content inendosperm while GS showed high content of carotenoids(Table). The HPLC revealed the β-carotene content indifference rice varieties collected from northeast India. Among
all rice varieties MS and BA showed lower quantity, 1.325,
2.713 µg/g respectively (Table 2) and in SS has high content of
β-carotene (4.221 µg/g).

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P14
Table 1 Total carotenoid content in rice endosperm.
Rice Variety
GS
B
BS
MZ
SS
NB

λ450
0.088
0.031
0.064
0.075
0.064
0.088

Concentration (µg/g)
6.790
2.392
4.938
5.695
2.237
6.685

Table 2 β-carotene content in rice endosperm
Peak
Area
Varieties
RT
Conc. (µg/g)
GS18.705
9.696
3.992
B
18.6286.589
2.7127
BS
18.60123.8009.8007
MZ
18.6883.219
1.3255SS
18.44310.2524.2206
NB-

spectra of the six rice variety revealed characteristics featuresof carotenoid spectra. The spectra of all samples revealed anumber of resonances in the region o 6.0-7.0 ppm, which
represent different olefinic proton along the conjugated chains(Fig. 3). 1H NMR spectra confirm the presence of β-carotene in
all rice varieties however signal intensities are significantly
low.

Fig. 3 Representative 1H NMR spectra of β-carotene standard.

DISCUSSION

Fig.1 HPLC chromatogram showing the β-carotene peaks in the
carotenoid extracts from rice varieties in of Northeast India.

Carotenoids are one of the most diverse natural compounds.
Carotenoids are very minor constituents in cereal grains.
Carotenoids are not reported in rice endosperm yet. Several
genetic engineering approaches has been reported in rice andother crop to enhance the β-carotene content in rice grains. In
present study, we observed carotenoids and β-carotene in ricelandrace endosperms. The BS and GS rice variety showed
higher β-carotene content as well carotenoids. Breeding of theserice landraces with high yielding rice variety can augment
biofortification programs. Further metabolomics approaches
will be done to reveal the metabolomics pathway involves in βcarotene biosynthesis.

ACKNOWLEDGMENTS
We acknowledge the Department of Biotechnology (DBT),
New Delhi, India for Program Support Project funds forcarrying out the research work. GJN Rao thank DBTNERBPMC for funding Visiting Research Professor at IIT
Guwahati.

REFERENCES

Fig. 2 Comparison of total carotenoids and β-carotene content
in rice verities
Table.3 Fatty acid profiling in rice varieties
Linoleneic Linoleic Oleic
Stearic
acid
acid
acid
acid
GS
2.0
24.57
24.91
48.52
B
2.11
26.18
47.92
23.28
BS
4.78
24.1
25.88
46.02
MZ
2.12
31.97
39.026
26.18
SS
1.75
29.8
44.61
23.84
NB
3.4
28.5
44.35
24.72
1H NMR spectroscopy- Fatty acid composition were
measured with peak integration. GS and BS showed higher
oleic acid 48.52, 46.02% respectively (Table 3). The 1H NMR

Giuliano, G., Tavazza, R., Diretto, G., Beyer, P., Taylor, M.A. (2008)
Metabolomic engineering of carotenoid biosynthesis in plants.
Trends in Biotechnology 26: 3.
Stanley, L. and Yuan, Y.W. (2017) transcriptional regulation of
carotenoid biosynthesis in plants: so many regulators, so little
consensus. Frontiers in Plant Science 10:1017.
Jourdan, M., Gangne, S., Laurent, C.D., Maghraoui, M., Huet, S., Suel,A., Hamama, L., Briard, M., Peltier, D., Geoffriou, E. (2014)
Carotenoid content and root color of cultivated carrot: a candidategene association study using an original broad unstructured
population. PLOSone 0116674.
Yao, D., Wang, L., Li, Q., Ouyang, X., Li, Y., Wang, C., Ding, L., Hou,
L., Luo, M., Xiao, Y. (2018) Specific upregulation of a cotton
phytoene synthase gene produces golden cottonseeds with enhanced
provitamin A. Scientific Reports 8:1348.
Roncallo, P.F., Cervigni, G.L., Jensen, C., Miranda, R., Carrera, A.D.,
Helguera, M., Echenique, V. (2012) QTL analysis of main andepistatic effects for flour color traits in durum wheat. Euphytica
185:77–92

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P15

Magnetic cellulose nanofibers/chitosan based edible nano-coating withcurcumin loading: Influence of filler materials on perishable food products
Tabli Ghosh1, Yoshikuni Teramoto2, Kohei Nakano3 and Vimal Katiyar1
1. Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam-781031, Assam, India
2. Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan
3. The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, JapanINTRODUCTION
From very early days, edible coating based postharvest
management system has attained significant attention as acommercially viable mode of produce deliverables. The
application of edible coating is generally applied as a thin layerof materials on food products for improved shelf life, andquality. For edible coating, cellulose, starch, chitosan, and gum
arabic based polysaccharides are generally used for enhanced
food property. However, chitosan obtained from deacetylationof chitin, is very effective in maintaining the quality of foodmaterials during the product life for its antimicrobial property.
Moreover, the biocomposites and blend preparation fromavailable biopolymers can provide additional benefits.
Among available bionanomaterials, cellulose nanofiber(CNF) is considered as a remarkable candidate in themultifaceted application for their multifunctional properties
such as surface property, aspect ratio, mechanical property, etc.
which further make them a better candidate for stringent foodpackaging sectors. The specific material is considered as one of
the emerging bionanomaterials to be used in the developmentof edible nano-coating in addition to various biopolymers(Ghosh et al., 2019).
Moreover, the noteworthy properties of CNF and otheravailable biopolymers such as non-toxicity, biodegradability,
biocompatibility, are utilized to obtain the tailor-madeproperties to improve shelf life of perishable food products.
Further, CNF can be modified through the incorporation of ironparticles to offer iron fortified products as edible coating
materials. In this regards, the work investigates thedevelopment of non-toxic magnetic CNF (mgCNF) andchitosan based edible nano-coating materials with loading of
curcumin for tailored administration of nutraceuticals and for
improved properties of cut pineapple fruits, where thefabrication of mgCNF can be obtained following several
methods.

MATERIALS AND METHODS
The fabrication of mgCNF is obtained through single step
coprecipitation method, which is used as a nanofiller material
for the fabrication of edible coating materials. The developmentof edible nano-coating on cut pineapple was obtained throughfollowing dip coating method. The developed materials arefurther taken for various property analysis such as mechanical,
transparency, color properties, and others.

RESULTS
The modified nanofiller mgCNF are used as effective fillers
for iron fortification of food products. Further, the fabricationof filler materials can be confirmed via FETEM micrograph asshown in Fig. 1. The neat CNF (Trade name: BiNFi-s CNF)
have a cloudy appearance when dispersed in water, whereas the
fabricated mgCNF provide spherical shaped particles attachedon CNF molecules.

Fig. 1: FETEM micrograph of (a) BiNFi-s CNF and(b) mgCNF
In the present study, the nanofiller materials are utilized todevelop chitosan based biocomposites for improved properties.
The mechanical properties (tensile strength, and Young’s
Modulus), thermal properties in terms of temperature at which
50% of sample weight get degraded (T50), transparency (UV
and visible region), and color properties are shown in Table 1.
The color properties are expressed in terms of L values(brightness to darkness), a* values (+a* is red and –a* is green),
and b* values (+b* is yellow and –b* is blue).
Characteristics
Mechanical Property
Thermal Properties
Transparency
Color Properties

Components
Tensile strength = ~ 57 MPa
Young’s Modulus =~2348 MPa
T50= ~ 340 °C~ 18% (UV Region)~ 45% (Visible Region)
L = ~28.69
a*= ~3.34
b*= ~2.48

Table. 1: Properties of 1.5% mgCNF/Chitosan based
biocomposites materials
Table. 1 represents the properties of 1.5% mgCNF/chitosan
based biocomposites materials, where, the tensile strength has
been found to improve by ~89% in comparison to neat chitosan.
Further, Young’s modulus is also improved by ~80% whencompared with neat chitosan. The observed transparency ofneat chitosan is ~85% which is reduced to ~ 18% in the UV
region. In the same way, transparency has been reduced to~45% from ~89% (neat chitosan) in the visible region (Ghoshet al., 2019).
Further, Fig. 2 represents the surface morphology of 1.5%
mgCNF/Chitosan based biocomposites materials withcurcumin loading, where the uniform dispersion of mgCNF
nanofiller in chitosan matrix has been observed.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P15
coating for food products provide improved tensile strength,
young’s modulus, thermal stability, optical properties andothers. As well known the perishable food products such as
fruits and vegetables have very less product life, so the usedmaterials are beneficial to prolong the shelf life of cut
pineapple.

ACKNOWLEDGMENTS

Fig. 2: FESEM micrograph of 1.5% mgCNF dispersed
chitosan with curcumin loading for edible coating.
Interestingly, the developed edible nano-coating materials
are applied on cut pineapple, where various properties areanalyzed to check the quality of stored food products in termsof respiration rate, total soluble solids, microbial count, etc.

DISCUSSION
The investigation shows that the developed mgCNF
dispersed chitosan based edible nano-coating materials are very
effective to be acted as commercially viable mode of producedeliverables. The used postharvest management system withthe targeted materials are very beneficial for offering improved
packaging properties. The formulated materials as edible nano-

The authors are thankful to Centre of Excellence for
Sustainable Polymers (CoE-SusPol) for the experimental
facilities. Further, the authors are thankful to central instrument
facility and IIT Guwahati for the research facility. The authors
are very grateful to Gifu University, Japan. Miss Ghosh is verymuch thankful to the department of science and technology,
Government of India, for awarding INSPIRE Fellowship for
persuing doctoral degree.

REFERENCES
Ghosh, T., Teramoto, Y., and Katiyar, V. (2019). Influence ofNontoxic Magnetic Cellulose Nanofibers on Chitosan Based
Edible Nanocoating: A Candidate for Improved Mechanical,
Thermal, Optical, and Texture Properties. Journal of
agricultural and food chemistry 67:4289-4299.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P16

Poly (L-lactic acid)/modified gum arabic biocomposites for targeted foodpackaging applications
Purabi Bhagabati, Tabli Ghosh and Vimal Katiyar
Department of Chemical Engineering, Indian Institute of Technology Guwahati, North Guwahati, Assam, India-781039

INTRODUCTION
Poly (L-lactic acid) (PLLA) is an aliphatic polyester which isderived from renewable resources such as potato, Corn sugar,
and sugarcane. It has played a central role in replacing fossilbased polymers for certain applications. It is a biodegradable
thermoplastic which can be used as a packaging materials
including the area of foods and is also a hygienic productbecause of its good biodegradability, compatibility andenvironmental friendly (Lim et al. 2008, Auras et al. 2004).
Gum Arabic (GA) or Acacia gum is the exudates from the
Acacia Senegal and Acacia Seyal trees. It is widely used forindustrial purposes such as stabilizer, emulsifier, thickener andan encapsulating in the food industry, and to a lesser extent inceramics, lithography, cosmetics, textiles and other application(Verbeken et al. 2003). The prime motivation of the work is todevelop tough and flexible films of PLA/mGA biocomposites
for specified food packaging applications. Chemical
modification of Gum Arabic via in-situ polymerization of lactic
acid would form oligomeric lactic acid grafted Gum Arabic(OLLA-g-GA) (Tripathi et al. 2016). Effect of the modified GAof varying grafting percentage would affect the hydrophilicityof the GA and so about the dispersion within the PLLA matrix
in the biocomposites. The work reports the effect of thechemical modification of GA on crystal morphology, thermal,
mechanical and optical properties of its PLLA biocomposites
responsible for its potential candidacy for food packaging
applications.

MATERIALS AND METHODS
L-lactic Acid and poly(lactic acid) (PLLA) (granular form;
2003D) were procured from Jiaan Biotech and Nature Works,
respectively. Gum Arabic(GA) from Acacia tree, HPLC
Chloroform (SPECTROCHEM PVT. LTD) were purchasedfrom Sigma Aldrich. StartSYNTH Microwave Synthesis
Labstation, Magneto Rheometer, Analytical Balance,
Sonicator, Magnetic hot plate stirrer, UTM, Color
Spectrophotometer, DSC, POM, TGA.
Fabrication of LA-g-GA in STARTsynth Microwave
Synthesis Labstation.
Preparation of LA-g-GA
Table. 1: Composition of Lactic Acid and Gum Arabic
Modified GA (mGA)
L-lactic
Gum Arabic
samplesAcid
1% LA-g-GA
25ml
0.25 g3% LA-g-GA
25ml
0.75 g
5% LA-g-GA
25ml
1.25 gChemical modification GA to synthesize OLLA-g-GA wascarried out in StartSYNTH Microwave Synthesis Labstation.
Lactic acid and Gum Arabic were taken in three neck roundbottom flask as per the composition presented in Table 1.
The temperature of the heating tape connected between glassrod outlet and condenser was maintained at 100ºC in order toavoid condensation of byproducts. The amount of oligomer
formed and the water collected was measured.

Preparation of PLLA/LA-g-GA Nanocomposite films
PLLA/LA-g-GA nanocomposite films with 2, 5 and 7 wt%
of LA-g-GA filler (1%, 3%, 5%), and 1 neat PLLA film wereprepared by solution casting method. The solution of LA-g-GA
was prepared by dissolving LA-g-GA with PLLA granules inchloroform with vigorous stirring magnetically at room
temperature for 5-6 hours. The composition of PLLA granules,
LA-g-GA and chloroform for different films is given in Table
2. The solution was poured into Teflon petri dishes to obtain thefilm after evaporation of chloroform. The formed films were
peeled off from the Petri dishes after 12 hours and further driedin a vacuum oven for 12 hours to remove the remaining
chloroform from the films.
Table. 2: Composition of PLLA, oligomer, chloroform
Concentration
PLA
Oligomer Chloroform
granulesDesignation
1% LA-gGA
3% LA-gGA
5% LA-gGA
NPLA

2%
5%
7%
2%
5%
7%
2%
5%
7%
-

1.96 g
1.90g
1.86g
1.96g
1.90g
1.86g
1.96g
1.90g
1.86g
2g

0.04 g0.10 g0.14g
0.04g
0.10 g0.14g
0.04g
0.10 g
0.14g
-

60 ml
60ml
60ml
60ml
60ml
60ml
60ml
60ml
60ml
60ml

RESULTS AND DISCUSSION
Trans-crystallization phenomenon
The crystallinity of a semi-crystalline polymer like PLLA hasa tremendous impact upon other physio-mechanical properties.
Addition of fillers of varying size and shape as additive of
PLLA instinctively affects its crystallinity. While nanoparticles
are more effective in altering the crystalline morphology of basepolymer matrix, addition of microparticles in unmodified form
has no significant impact. However, a strategic covalentmodification of microparticles may act as a great way to cause
alteration of these properties.
Trans-crystallization phenomenon was observed at theinterphases of these mGA particles, which can be interpreted
from the second image of each sample. Further crystal growthfrom these nucleating sites led to formation of a single large
spherulite of diameter >800 µm and at 30 minutes, which
superficially looks like a single spherulite that was nucleated bya single mGA particle. However, there is a distinct variation in
the rate of crystallization in the samples and they are as follows:
GA1>GA3>GA5. This indicates that a higher grafting
concentration of OLLA enhances rate and degree ofcrystallization in the composite. It is noteworthy to mention thatno such nucleation phenomenon was observed in case of PLLA
composites with unmodified GA. The loosely bound particles
are incapable to transfer load to the polymer matrix and thispromotes stress initiation under mechanical testing ofrespective samples. For detailed understanding, mechanicalProceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P16
performance is evaluated by a destructive mechanical test under
tensile mode.
Mechanical properties
The tensile strength of the fabricated films sample represents
the ability for its load bearing capacity and ultimate tensile
strength and % elongation at break values of the samples arepresented in Fig 2. While, NPLLA samples show maximum
UTS of 29 MPa, its composites with GA1, GA3 and GA5 show
relatively closer UTS of 26.7, 28.1 and 26.8 MPa, respectively.
In each case of the PLA/mGA composites, the lowest
concentration of each set of mGA (i.e. 2wt%) showed a better
tensile property in comparison to its higher concentration
counterparts.

Fig 2: Ultimate tensile strength (MPa) and percentage
elongation break of NPLA and its biocomposites of mGA
samples
PLLA is highly brittle in nature and showed percentage
elongation at break of NPLA close to 11%. However,incorporation of the mGA into PLLA matrix caused asignificant increment in the values of percent elongation atbreak. A maximum of 60% elongation at break was achieved in
PLA/5%(GA5) sample, which infers a significant increment invalue from NPLA samples. The PLA/2%(GA5) sample render
great improvement in its percentage elongation by 263% from
NPLA, which also showed UTS value apparently close to
NPLA, i.e. 26.8 MPa. An approximately 7.5% reduction in thetensile strength value and the appreciable 263% increment inductility may logically be supported as the optimized
composition for the composites.
Thermal analysis
The DSC analysis of NPLA and its composites may throw
light into the in-depth understanding on the thermal properties
upon addition of mGA in PLA matrix. The second heating
thermograms of all sample consisted of a single inflection peakcorresponding to Tg, two melting endotherm peak (Tm1 andTm2) and a cold crystallization exothermic peak (Tcc) that lied
between Tg and Tm. While a decrease in Tg of composite with
2wt% mGA caused little to insignificant reduction in Tg,
appreciable reduction in Tg was observed in case of composites
with 5wt% and 7wt% mGA concentration.

Fig. 3: DSC second heating thermograms of NPLA andits biocomposites of mGA samples
While the low molecular weight OLLA is capable toplasticize the brittle PLA matrix, the covalently attached
OLLA-g-GA particles offer good reinforcement to the matrix,
which represents anti-plasticization effect. Existence of goodinterphase interaction between the polymer matrix and themGA is the responsible factor for the reduced Tcc of thecomposites over NPLA. The seemingly higher percentage
crystallinity and Tcc value in most of the composites over
NPLA support the nucleation phenomenon as observed in the
POM analysis.
The heat sealing property of the PLA/mGA biocomposites
with higher mGA content is relatively higher than the NPLA,
which is much beneficial for food packaging applications.
Optical properties
The optical characteristics of NPLA and biocomposites werecharacterized with the UV-vis spectrophotometer and thedatacolor to understand the effect of mGA onto the PLLA films.
Excellent transparency and color of films endorse its
application in the packaging of food items.

ACKNOWLEDGMENTS
The authors sincerely acknowledge the Department ofChemicals and Petrochemicals, Ministry of Chemicals andFertilizers, Government of India funded Centre of Excellence
for Sustainable Polymers (CoE-SusPol) at IIT Guwahati forresearch facilities to perform this research work.

REFERENCES
Auras, R., Harte, B., &Selke, S. (2004). An overview ofpolylactides as packaging materials. Macromolecular
bioscience, 4(9), 835-864.
Lim, L. T., Auras, R., &Rubino, M. (2008). Processing
technologies for poly (lactic acid). Progress in polymer
science, 33(8), 820-852.
N. Tripathi & V. Katiyar, Journal of Applied Polymer Science,
133, 21 (2016).
Verbeken, D., Dierckx, S., &Dewettinck, K. (2003). Exudate
gums: occurrence, production, and applications. Applied
Microbiology and Biotechnology, 63(1), 10-21

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P17

Controlling the molecular architecture to tailor the thermal and mechanicalproperties and water vapor transmission rate of the biodegradable block
copolymers: Versatile materials for food packaging
Neha Mulchandani1, Kazunari Masutani2, Shinichi Sakurai2, Yoshiharu Kimura2 and Vimal Katiyar1
1. Department of Chemical Engineering, Indian Institute of Technology Guwahati, Kamrup, Assam, India
2. Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, JapanINTRODUCTION
Biodegradable aliphatic polyesters such as poly(lactic acid)(PLA) and poly(ε-caprolactone) (PCL) have received
significant attention in the past decades owing to theirremarkable characteristics and end-use applications (Pisani etal., 2018). PLA is widely recognized as a biobased material
with a significant potential to replace the petroleum derived
plastics. However, the inferior thermal resistance and the brittle
nature of PLA have restricted its use in widespread commodityand engineering applications(Hirata, Masutani, & Kimura,2013). On the other hand, PCL is known to be a flexible
polymer possessing high impact strength, while having a
relatively lower tensile strength (Rojo, Martín, Calvo, &Cocero, 2009). In order to improve the thermal resistance of
PLA, stereocompelxation (crystalline form between theenantiomeric PLA chains) has been widely adopted, which
improves the melting temperature of stereocomplex crystallites
to ~230 ℃. Stereocomplexation may be achieved by blending
the enantiomeric chains i.e. poly(L-lactic acid) (PLLA) andpoly(D-lactic acid) (PDLA) or by stereoblock formation (Gupta& Katiyar, 2017).
To develop the materials with tailored properties, PLA andPCL are used in combination. Blending of PLA and PCL often
leads to the macro-phase separation resulting in the inferior
overall mechanical properties (Lv et al., 2016). In this regard,
block copolymerization of PCL and PLA has been regarded as
an effective approach to develop the materials with intended
properties.
With the aim of designing biodegradable polymers forversatile food packaging applications, the current research
focuses on synthesizing triblock and pentablock copolymers
consisting of soft middle segments and hard end segments,
incorporating the concept of stereocomplexation to improve theoverall material properties. The triblock copolymers (BCB) are
synthesized by two-step ring opening polymerization (ROP)
and the pentablock copolymers (ABCBA) are synthesized bythree-step ROP by using PCL as a macro-initiator. The thermal
and mechanical properties along with the water vapour
permeability have been detailed underlying the impact ofmolecular architecture on the properties of the materialindicating their potential for versatile food packaging
applications.

MATERIALS AND METHODS
L- and D-Lactides (99% purities) were acquired fromMusashino Chemical Laboratories Ltd., Tokyo, Japan.
ε-caprolactone was purchased from Nacalai Tesque, Kyoto,
Japan, and distilled prior to use. 1,12-Dodecanediol was
obtained from Wako Pure Chemical Industries Ltd., Tokyo,
Japan. Tin octoate (Sn(Oct)2, 95%) was purchased from Sigma
Aldrich and used after distillation under high vacuum.
Dichloromethane and methanol were procured from Nacalai

Tesque, and 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was
obtained from Central Glass Co. Ltd., Yamaguchi, Japan.
Synthesis of BCB/ACA and ABCBA/BACAB type
copolymers: In the first step, PCL-doil was synthesized which
was then used as a macroinitiator to synthesize PLLA-b-PCLb-PLLA and PDLA-b-PCL-b-PDLA by adding L-and
D-lactides respectively. The block copolymers weresynthesized such that the block length of PLLA/PDLA terminal
segments was 20 kDa and that of PCL was 10 kDa. The triblock
copolymers were further used as macroinitiators to form
ABCBA/BACAB type pentablock copolymers where the block
length of all the segments was ~10 kDa. The molecular weightof the block copolymers was confirmed by 1H-NMR
measurements. The enantiomeric block copolymers were thenblended (50:50) in order to form enantiomeric blends
designated as BCB/ACA blends and ABCBA/BACAB blends.
Additionally, PLLA was synthesized (Mn ~ 50 kDa) tocompare the properties of the block copolymers with thehomopolymer.
1H-NMR
spectra were determined using AV600
spectrometer (Bruker, Germany) where the samples weredissolved in deuterated chloroform (CDCl3).
Differential Scanning Calorimetry measurements wereconducted using DSC 214 Polyma (NETZSCH).
The mechanical properties of the samples were determinedusing a universal testing machine (Kalpak Instruments andControls) equipped with a load cell of 500 N and the cross head
speed being 5 mm/min.
The water vapor permeability was measured using
PERMATRAN-W by using the ASTM standard E398-03
where the relative humidity (RH) was 90%.

RESULTS
Differential Scanning Calorimetry: The thermal transitions inthe synthesized block copolymers are ascertained bydifferential scanning calorimetry (DSC) measurements. The
heating profile indicates the presence of individual melting
peaks of PCL and PLLA/PDLA designating the presence ofindividual crystalline domains. Further, the stereocomplex
crystallites are present in the enantiomeric triblock blend as
well as the pentablock copolymer and its blend which leads tothe improved Tm of the resulting copolymers. The cold
crystallization temperature (Tcc) of the pentablock copolymer isfound to be ~76 ℃ as compared to the triblock copolymer (Tcc
~ 84 ℃). This indicates the relatively faster crystallization incase of pentablock copolymers.
Mechanical Properties: The tensile strength and elongation atbreak of the synthesized homo/block copolymers are shown in
Fig. 1. The ultimate tensile strength is found to improve in caseof the stereoblock copolymers. However, the elongation (atbreak) of stereoblock copolymer is reduced pertaining to its
brittle nature. The elongation at break of the triblock copolymer
is significantly enhanced to (~300%) with approximately

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P17
similar tensile strength. The improvement in elongation is
attributed to the presence of soft (PCL) segments.

exploited for the flexible biodegradable food packaging
applications. Further, the fresh produce requires relatively
higher water vapor transmission rate in order to respire whereas
the packaged food requires low water vapor permeability, andthe developed biodegradable block copolymers may serve as
candidates for these applications possessing versatile
characteristics.

ACKNOWLEDGMENTS

Fig. 1: Ultimate tensile strength and elongation at break of
the synthesized homo/block copolymers
Water Vapor Transmission Rate: The water vapor transmission
rate of the homopolymer and block copolymers is shown in
Table 1. The WVTR of the synthesized PLLA is 3.77 which isincreased by incorporating PCL segments as seen in the case oftriblock and pentablock copolymers. Further, the WVTR isreduced on blending the enantiomeric block copolymers which
may attributed to the increasing crystallinity of the materials
due to the stereocompelxation creating a tortuous pathway for
the water vapor to pass through.
Table 1: Water vapor permeability of the synthesizedhomo/block copolymers
Identity
WVTR (gm.mm.m-2.day-1)
PLLA
3.77 ± 0
BCB
7.9 ± 2.6
BCB blend
4.7 ± 1.0ABCBA
7.8 ± 0.3
ABCBA blend
4.9 ± 2.9

DISCUSSION
The thermal and mechanical properties along with the water
vapor transmission rate are found to be significantly influenced
by the molecular architecture of block copolymers. The
elongation (at break) is found to be enhanced upon the additionof PCL segments into the chains of PLLA/PDLA which may be

The authors acknowledge the Centre of Excellence for
Sustainable Polymers (CoE-SusPol) funded by the Departmentof Chemicals and Petrochemicals (DCPC); Central Instruments
Facility (CIF) at Indian Institute of Technology Guwahati (IIT
Guwahati); Kyoto Institute of Technology (KIT), Japan forproviding the research and analytical facilities; and Departmentof Science and Technology (DST) for providing the INSPIRE
fellowship (IF 160861) for the doctoral research.

REFERENCES
Gupta, A., & Katiyar, V. (2017) Cellulose Functionalized High
Molecular
WeightStereocomplex
Polylactic
Acid
Biocomposite Films with Improved Gas Barrier,Thermomechanical Properties. ACS Sustainable Chemistry &Engineering 5(8):6835-6844.
Hirata, M., Masutani, K., & Kimura, Y. (2013) Synthesis of
ABCBA Penta Stereoblock Polylactide Copolymers by TwoStep Ring-Opening Polymerization of l- and d-Lactides withPoly(3-methyl-1,5-pentylene succinate) as Macroinitiator (C):Development of Flexible Stereocomplexed Polylactide
Materials. Biomacromolecules 14(7):2154-2161.
Lv, Q., Wu, D., Xie, H., Peng, S., Chen, Y., & Xu, C. (2016)
Crystallization of poly(ε-caprolactone) in its immiscible blend
with polylactide: insight into the role of annealing histories.
RSC Advances 6(44):37721-37730.
Pisani, S., Dorati, R., Conti, B., Modena, T., Bruni, G., & Genta,
I. (2018) Design of copolymer PLA-PCL electrospun matrix forbiomedical applications. Reactive and Functional Polymers
124:77-89.
Rojo, S. R., Martín, Á., Calvo, E. S., & Cocero, M. J. (2009)
Solubility of Polycaprolactone in Supercritical Carbon Dioxidewith Ethanol as Cosolvent. Journal of Chemical & Engineering
Data 54(3):962-965.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P18

Fabrication of nano cellulose from marine green algae residue and theireffects as filler in developing biodegradable polymer composite
Kona Mondal1, Shinichi Sakurai2, Purabi Bhagabati1, Vaibhav. V. Goud1 and Vimal Katiyar1
1. Department of Chemical Engineering, Indian Institute of Technology Guwahati, India
2. Department of Biobased Materials Science, Kyoto Institute of Technology, Kyoto, JapanINTRODUCTION
Green algae are widely available around the world and its
exploitation for the production of biodiesel, bioethanol, andagar has become an important industry in recent years aiming
to conserve the fossil fuels and minimizing global warming.
Also, it became the alternative source for third generation
biodiesel production. Furthermore, petroleum-based plastics
are reason for occurring global warming, plastic waste, andmany other hazards. Contrariwise, biodegradable polymers
with identical properties have been found as a great researchoutcome for substitution of petroleum-based plastics. Thus, thegreen or sustainable polymer would be the best possible
replacement for conventional plastics to protect our globe fromthe environmental hazard. However, the industrial processingof green algae generates a large quantity of solid wastes, whichconstitutes a source of serious environmental problems.
Further, these algae wastes contain polysaccharide such as
cellulose which is a natural biodegradable polymer with someunique characteristics of renewability, biodegradability, hightensile strength and stiffness, cost effectiveness and light weight(Sucaldito and Camacho 2017). Although, other lignocellulosic
biomass sources are available in environment for extractions ofcellulose, in spite of those algae are of special interest, ideallysuitable for a variety of applications, including polymer
reinforcement (Guo et al. 2017). Additionally, algae derivatives
consist of several valuable components such as apolysaccharide, antioxidants, pigments, fatty acids, proteinsand vitamins, and minerals. On the otherside, the demand offood packaging is increasing more with the growing population.
Nano Cellulose (NC) has emerged as a biobased material thathas captured considerable research and industrial interest. They
feature a high aspect ratio, low density, high mechanicalstrength, and low coefficient of thermal expansion. In addition,
they display abundant hydroxyl groups that facilitate NC
functionalization and allow strong inter fibrillar hydrogenbonding.
In the present work, utilization of green algae waste as a raw
material to produce value added nano celluloses (NC) has been
investigated, and the effect of the as-isolated NC as a filler
material in biodegradable polymers (poly(Lactic acid) (PLA)and poly(ɛ-caprolactone)(PCL)) have been studied. Further,washed algae biomass residue (WABR) is also used as filler inboth the polymer matrix to check the effect of WABR.

0.5, 1 and 2wt% respectively. WABR composite and NC
nanocomposite specimens have been developed by solution
casting. Thermal properties and crystalline behaviour of thespecimens have been examined using a differential scanningcalorimeter (DSC) and a polarized optical microscope (POM).
The DSC isothermal and non-isothermal study has been done
for both composite specimens. The isothermal crystallization of
PLA and PCL composite specimens are conducted at 110˚C and45˚C respectively. The POM observations have been conductedwith the isothermal condition at 125˚C for PLA and at 45˚C for
PCL composite specimens. To observe the spherulite growth
behavior in both of the polymer composite specimens POM
study has been done.

RESULTS AND DISCUSSIONSIt has been observed that NC acts as a good nucleating agent
in both types of polymer composite specimens as compared to
WABR. It has also been found that WABR can act as anucleating agent. Non-isothermal DSC study has shown the
reduction in cold crystallization temperature in PLA/NC
nanocomposite specimens (in the second heating process) from
135.8˚C to 119.3˚C (Fig.1a) whereas NC helped to increase thecrystallization temperature in the PCL/NC nanocomposite
specimens from 29.4˚C to 35˚C (in the cooling process frommelt) (Fig.2a&b). Both results indicate the improvement ofcrystallization in the nanocomposite specimens. The DSC
isothermal crystallization study confirms the improved
crystallization in PLA/NC nanocomposite specimens (Fig.1c)
whereas in case of PCL/NC crystallinity decreased after
addition of NC (Fig.2c). It also been observed that withincreasing the filler loading percentage crystal formation occurs
rapidly (Fig.1b). The POM study has also shown accelerated
crystallization for both of the polymers. Overall, we
successfully demonstrated a high-end utilization of abioresource from the marine green algae residue.

MATERIALS AND METHODSPLA granules (grade: 4032D, with weight average molecularweight (Mw) ~200 kDa and content of L-lactic acid/Dlacticacid: 98.6/1.4) have been purchased from Nature Works®
LLC., USA. Further, PCL has been synthesized in thelaboratory with Mw ~80 kDa. Algae biomass residue (ABR)
has been subjected to washing for removal of dirt andextraneous matter followed by drying and grounding which issupplied by TERI, India. Fabrication of NC has been done
through acid hydrolysis (sulphuric acid) method. Both thefillers WABR and NC loading amount in polymer matrix are 0,

Fig.1a:Non-isothermal DSC thermogram of PLA/NC
nanocomposites

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P18

ACKNOWLEDGEMENT
The authors are very grateful to Excellence of Sustainable
Polymers (CoE-SusPol), CIF at IIT Guwahati and Kyoto
Institute of Technology, Japan for providing research facilities.
We also thankful to TERI (The energy and Resource Institute)DBT (Department of Biotechnology, Gov. of India) Centre of
Excellence on Biofuels and Biocommodities for funding the
project.
Fig.1b: Isothermal DSC
nanocomposites at 110˚C

thermogramof

PLA/NCREFFERENCES

Fig. 1c: Crystallinity curve of PLA/NC anocomposite

Fig.2a: Non-isothermal DSC thermogram of PCL/NC
nanocomposites

Fig.2b: Isothermal DSC thermogram of PCL/NC
nanocomposites at 45˚C

Fig. 2c: Crystallinity curve of PCL/NC anocomposite

Sucaldito, M. R., & Camacho, D. H. (2017). Characteristics
of unique HBr-hydrolyzed cellulose nanocrystals from
freshwater green algae (Cladophora rupestris) and its
reinforcement in starch-based film. Carbohydrate polymers
169: 315-323.
Guo, J., Uddin, K. M. A., Mihhels, K., Fang, W., Laaksonen,P., Zhu, J. Y., & Rojas, O. J. (2017). Contribution ofresidual proteins to the thermomechanical performance of
cellulosic nanofibrils isolated from green macroalgae. ACS
Sustainable Chemistry & Engineering 5(8): 6978-6985.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P19

A sensitivity analysis of Piano Key Weir discharge capacity based on 3D
numerical modeling
Le Anh Tuan1, 2 and Ken Hiramatsu3
1. The United Graduate School of Agricultural Science, Gifu University
2. Gifu University Rearing Program for Basin Water Environmental Leaders
3. Faculty of Applied Biological Sciences, Gifu University
geometry based on these parameters. Therefore, the process is
significantly dependent on the mesh resolution. For the
INTRODUCTION
numerical approximation of the advection terms, a first orderscheme was employed for an initial period of time, follow by
Piano key weirs (PKW) are new cost-effective free-flow
an explicit second order scheme with gradient preserving using
spillways, designed to substantially increase the dischargea Restart simulation.
capacity of dams. However, their structure involves a lot ofgeometrical parameters and the hydraulic behavior is complex.
Physical modeling usually offers different characteristics to
RESULTSderive adequate hydraulic design and gain insight into thehydrodynamics (Novak, et al. 2010). However, design, building
In this study, a sensitivity analysis of the main configurations
and operation of physical models may take long periods of time.
in FLOW-3D based on the prototype discharge results wasWith the improvement of computational power, 3D numericalcarried out. They were:modeling turned out to be an interesting opportunity as it could- Momentum advection model (1st order vs 2nd order withenable to model various geometries within a reasonable delay.
monotonicity preserving)
However, the accuracy of computational fluid dynamics (CFD)- VOF method (automatic vs split Lagrangian method);
is an important matter of concern. The objective of the current- Maximum turbulence mixing length (MTML);
study is to evaluate the accuracy of CFD to predict the headThe numerical results are presented in Table 1. In order todischarge behavior of PKW, including a discussion regarding
evaluate if the flow rate obtained in the numerical simulations
model setup and the influence of different turbulence closure
corresponded to those of the experimental tests, the relative
methods on solution results.
differences between prototype and numerical discharges were
evaluated (δ) (Fig. 1).

EXPERIMENTAL DATAIn this study, 28 simulations were performed using acommercially available CFD solver (FLOW-3D) forEscouloubre PKW in France (Epricum, et al. 2013). The PKWof Escouloubre, operated by “Electricite de France (EDF)” issituated in the Aude River in the Pyrenees in the south ofFrance. It is used as a spillway on the rest lake of thehydropower plant of Escouloubre and is currently used as a bypass for the penstock of the hydropower plant of Nentilla. The
PKW is composed of a central inlet key, two outlet keys and,on both weir sides, two rectangular labyrinth inlet keys without
overhang or bottom slope. Additional details regarding the
PKW geometries are contained within Machiels (2012).

NUMERICAL MODEL
The majority of simulations utilized the Reynolds Average
Navier-Stokes (RANS) equations by means of the finite volume
method, coupled with RNG k-ε turbulence modelling, being k
the turbulent kinetic energy and ε the turbulent dissipation rate,
both modelled by their respective transport equations.
However, within the RNG model, there are two options for theuser to control the maximum turbulence mixing length(MTML), one is to allow for dynamical control by the solver,
the other is for the user to prescribe a set value. For the latter acommon rule of thumb is to choose 7% of the channel depth.
Besides, the upstream head used in the head-discharge
relationships corresponds to a subcritical flow condition withrelatively low turbulence, the simulations were also conductedwith no turbulence model to decrease the calculation times.
FLOW-3D® uses an advance algorithm for free surface
tracking, TruVOF, developed by Hirt and Nichols (1981). Afterdefining both geometry and a Cartesian, staggered grid, thesoftware uses FAVORTM method to generate area fractions foreach cell face in the grid by determining which corners of theface are inside of a defined geometry and reconstructing the

DISCUSSION
In general, the model has demonstrated a good correlation
with the prototype results. However, there are discrepancies in
the numerical results with the low head water over weir (i.e. H= 0.11; 0.19m), where the region near the weir will becharacterized by sharp changes in momentum (especially at theoverflow nappe) and thin geometry (the overhang crests are
only centimeter thick). This will require a fine mesh to resolve.
Some other main conclusions were obtained:- The transition from a laminar regime model to a turbulence
had influence on the discharge results. It is the least accurate
simulation based on discharge capacity.
- The second order momentum advection with monotonicity
preserving did not show more accurate results but with morecomputational effort. This is contrast to studies of (Miguel, etal. 2014) and (Crookston, et al. 2017).
- The sensitivity analysis for the RNG k-ε turbulence modelfor dynamically computed and constant (7% of P and of H)
MTML found a better agreement using dynamically computed
MTML when estimating Q for PK weirs. In contrast, Savage etal. (2016) employed constant MTML values to estimate Q for
labyrinth weirs.
- The results of this PK weir CFD validation study indicatedthat the 3 dimensional CFD simulation, employing either LES
or RNG k-ε turbulence models are suitable for the discharge
estimation of PK weirs with mean relative errors of 2 to 5% (incondition of refined enough cell size).
- The obtained discharges were highly dependent on cell size.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P19

Machiels, O. 2012. "Experimental study of thehydraulic behaviour of Piano Key Weirs." PhD
Thesis, University of Liege (Belgium).
Miguel, R.Silva, Lucia, T.Couto, and Antonio,
N.Pinheiro. 2014. "Complementary spillway of
Salamonde dam. Physical and 3D numericalmodelling." 3rd IAHR Europe Congress. Porto Portugal.
Novak, P, Guinot V, Jeffrey A, and Reeve D.E. 2010.
Hydraulic modelling - An Introduction. London and
New York: Spon Press.
Savage, B, Crookston, B.M, and Paxon, G. 2016.
"Physical and numerical modeling of large
headwater ratios for a 15 degree labyrinth spillway."

REFERENCES
Crookston, B.M, Anderson, R.M, and Tullis, B.P. 2017.
"Free-flow discharge estimation method for Piano
Key weir geometries."
Epricum, S, Silvestri, A, Dewals, B, Arcchambeau, P,
Pirotton, M, Colombie, M, and Faramond, L. 2013.
"Escouloubre Piano Key Weir: Prototype versus
scale models." Labyrinth and Piano Key Weir II PKW 2013. London: Taylor&Francis Group.
Hirt, C.W., and B.D. Nichols. 1981. "Volume of fluid(VOF) method for the dynamics of the freeboundaries." Journal of Hydraulic Engineering 138(9): 796-802.

Table 1. PK weir numerical simulations results
Head over PKW (m)Qprototype (m3/s) (Q1)
QRNG k-e dynamic - automatic - 1st order(Q2)
QRNG k-e dynamic-split Lagrangian -2ndorder with monotonicity preserving (Q3)
QRNG k-e (0.07P) - split Lagrangian - 2ndorder with monotonicity preserving (Q4)QRNG k-e (0.07H) - split Lagrangian - 2ndorder with monotonicity preserving (Q5)
QLES - split Lagrangian - 2nd order withmonotonicity preserving (Q6)
QLaminar - automatic VOF – 1st order
(Q7)

1.2

0.11
δ(%)

2.8

0.19
δ(%)

5

0.26
δ(%)

10

1.49

24.16

3.27

16.79

4.98

-0.4

10.07

0.7

1.53

27.5

3.28

17.14

5.03

0.6

10.39

3.9

1.51

25.83

3.28

17.14

5.01

0.2

10.47

4.7

1.53

28

3.28

17.14

5.01

0.2

10.69

6.9

1.52

26.67

3.28

17.14

5.04

0.8

10.19

1.9

1.5

25

3.32

18.57

4.99

-0.2

10.46

4.6

Fig. 1. Relative error between prototype and numerical flow rates

0.48

δ(%)

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P20

Nitrogen deposition of bulk precipitation, throughfall and stemflow in a
Lucidophyllous forest near Gifu Park in Central Japan
Ruoming Cao1,2, Siyu Chen2, Shinpei Yoshitake2 and Toshiyuki Ohtsuka2
1. The United Graduate School of Agricultural Science, Gifu University
2. River Basin Research Center, Gifu University

INTRODUCTION
Atmospheric nitrogen (N) deposition inputs into forest
ecosystems by wet deposition (rainfall and snowfall), dry
deposition (gases) and cloud deposition (cloud or fog). It wasreported that cloud deposition usually occurred in highelevation forest ecosystems and was often neglected in mostforest ecosystems with low elevation. N deposition (wet anddry deposition) can originate from various natural sources(lightning, forest fires and lava flows) and anthropogenic
sources (energy combustion, agricultural activities andbiomass burning). It was gradually increased at the global
scale mainly due to considerable fossil fuel combustion (NOxemission) and agricultural activities (NH3 emission). Chronic
N deposition significantly alters N cycle of forest ecosystems
with beneficial or detrimental ecological effects, such as,promotion of plant growth, or soil acidification and Nleaching via seepage water or runoff.
The role of precipitation in transferring nutrients to forest
ecosystems has long been recognized. From the perspective of
the hydrological cycle of forest ecosystems, precipitation isrouted to the forest floor by throughfall (TF) and stemflow(SF). TF is defined as the precipitation that directly passes
through a canopy or intercepted by plant surfaces andsubsequently drips from a canopy. SF is defined as the part ofprecipitation that flows along leaves and branches and then ischanneled to the bole of plants. TF and SF represent theultimate N deposition to the forest floor.
Many studies about bulk precipitation (BP), TF and SF on
natural forest ecosystems that is far from N emission sourceswere reported. However, studies of TF and SF on urban forest
ecosystems exposure to intensive human activities are rare.
The ecology of a natural forest can’t be simply applied tourban forests. Net TF (TF + SF – BP) can indirectly mirror
wash-off of dry deposition gathered on the canopy surface andcanopy exchange (uptake or leaching) of N deposition. It wasreported that wash-off of dry deposition at the pollution
exposed site contributed a lot compared with sheltered siteusing net TF approach (Rodrigo et al., 2003). Effects ofhuman activities on N fluxes in biogeochemical cycle of urban
forest ecosystems were still unclear.
The present study was designed to estimate theconcentrations and fluxes of dissolved N in BP, TF and SF
over three years in the Mt.Kinka forest site, central Japan,
which is an evergreen broad-leaved forest site of exposure topollution of human activities. The study site is near Gifu Parkand surrounding with mountain trails. We hypothesized thatdry deposition would have substantial effects on N fluxes inthe study site. We attempted to address the following
questions to test the hypothesis: (1) What are the dynamics
and characteristics of the concentrations and fluxes ofdissolved N in BP, TF and SF? (2) What are the temporalvariations of dissolved N fluxes in net TF?

MATERIALS AND METHODS

Study site—The study site is a subtropical evergreen broadleaved forest located at Mt.Kinka near Gifu Park, Gifu city incentral Japan. A 0.7 ha study plot (70 m × 100 m) wasestablished on the lower slopes of Mt. Kinka (ca. 60 m a.s.l.,
35°26′ N, 136°47′ E) in 1989. The annual mean temperature is
15.8 °C and the annual mean precipitation is 1827.5 mm
during the period from 1981 to 2010. Dominant species in thisstudy site is Castanopsis cuspidata (91.9 % of basal area in2017).
Experimental setup—we set up samplers to collect BP (3replicates), TF (9 replicates), and SF (15 replicates) from July
2016 to June 2019. BP was collected in bottles (20 L)
equipped with funnels (collection area: 0.0346 m2) that were
set up in areas near the study plot without a tree canopy. The
TF collectors were the same as those for BP, except for thevolume of the bottles (12 L) and the fact that they were evenly
distributed within the study plot. An SF collector consisted ofa polyethylene film surrounding a tree trunk (like a collar), a
tube connecting the film to a rain gage (HOBO RG3), and areservoir tank (24 L). SF collectors were set up on evergreen
dominant tree species according to different diameter at breast
height (DBH) classes of Castanopsis cuspidata (DBH > 20
cm, DBH > 30 cm, DBH > 40 cm, and DBH > 50 cm) anddeciduous tree species (3 replicates each). The volumes ofwater samples were measured by cylinder or rain gauge andwater subsamples were collected by 100 ml bottles at twice
per month.
Chemical analysis—After being filtering by 0.45μm
membrane filter, we measured the concentrations of total
dissolved nitrogen (TDN), NH4+–N and NO3– + NO2––N(DIN) by a QuAAtro 2-HR nutrient analyzer using acolorimetric method and continuous flow analysis. In eachanalysis run, standard solutions with 0.5 mg L−1 concentration(NO2––N and NO3––N) were used to check the recovery ofNO3− during the oxidation process, with the oxidation
efficiency kept over 90%. The concentration of DON was
calculated by difference between TDN and the sum of NH4+–
N and NO3– + NO2––N (DIN).
Calculation—N fluxes were calculated based on the
average concentration (mg L−1) and water depth (mm) in eachsampling time. Annual N fluxes were calculated by the sum of
N fluxes in each sampling time during whole year. Net TF wasused to evaluate the interactions of canopy to N deposition.
Net TF was performed in terms of flux data in each samplingtime: Net TF = TF + SF – BP.

RESULTS AND DISCUSSION
For net TF, DIN fluxes in each sampling time followed aclear seasonal pattern in each sampling year, with the highest
positive net fluxes in summer. And DON fluxes revealed
negative value in some sampling time (Fig. 1).
In this study site, the positive DIN fluxes in net TF
indicated that dry deposition played a vital role in DIN fluxes

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P20
for tree canopy interactions. Generally, DIN is easy to be
consumed by the tree canopy reported by many studies (e.g.
Aguillaume et al., 2017). Direct foliage or bark uptake,epiphyte uptake, and microbial action can cause theconsumption of DIN. Enrichment of DIN by the canopy also
was reported possibly due to wash-off of dry deposition
gathered by the surface of canopy (e.g. Izquieta-Rojano et al.,2016). The positive DIN fluxes in net TF in this study
coincided with the previous study (Rodrigo et al., 2003),
which reported the more importance of wash-off of dry
deposition in pollution exposed site. The possible source ofdry deposition in the study site may come from transportationand energy consumption around the study site. Clear seasonalpattern of DIN fluxes in net TF was in accordance withemission from considerable electricity production during
summer in Gifu city.
Negative value of DON fluxes in net TF indirectly impliedthat there was consumption of DON in canopy exchange
process. Leaching of DON from a canopy is a common
phenomenon (Lovett and Lindberg, 1993). In contrast, it wasalso reported that plants can consume some organic N directlyfrom solution without microbial mineralization (e.g. IzquietaRojano et al., 2016).

Aguillaume, L., Izquieta-Rojano, S., García-Gómez, H.,
Elustondo, D., Santamaría, J.M., Alonso, R., Avila, A.,
2017. Dry deposition and canopy uptake inMediterranean holm-oak forests estimated with a
canopy budget model: A focus on N estimations.
Atmospheric Environment 152, 191–200.
Izquieta-Rojano, S., García-Gomez, H., Aguillaume, L.,
Santamaría, J.M., Tang, Y.S., Santamaría, C., Valiño,
F., Lasheras, E., Alonso, R., Àvila, A., Cape, J.N.,
Elustondo, D., 2016. Throughfall and bulk depositionof dissolved organic nitrogen to holm oak forests in the
Iberian Peninsula: Flux estimation and identification ofpotential sources. Environmental Pollution 210, 104–
112.
Lovett, G.M., Lindberg, S.E., 1993. Atmospheric depositionand canopy interactions of nitrogen in forests. Canadian
Journal of Research.
Rodrigo, A., Àvila, A., Rodà, F., 2003. The chemistry of
precipitation, throughfall and stemflow in two holm oak(Quercus ilex L.) forests under a contrasted pollution
environment in NE Spain. Science of the Total
Environment 305, 195–205.

REFERENCES

2.5
DON flux

DIN flux

Net throughfall (kg N ha–1 episode–1 )

2
1.5
1
0.5
0
-0.5

J A S O N D JF M A M J J A S O N DJFMA M J J A S O N D J F M A M J
2016

2017

2018

2019

-1
-1.5
-2

Fig. 1: Dissolved organic nitrogen (DON) and dissolved inorganic nitrogen (DIN) fluxes (kg N ha–1 episode–1) in net
throughfall from July 2016 to June 2019. Net throughfall = throughfall + stemflow – bulk precipitation.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P21

Various swidden activities play complementary roles for local foodsecurity in montane northern Laos
Cahyo Wisnu Rubiyanto1 and Isao Hirota2
1. The United Graduate School of Agricultural Science, Gifu University, Japan
2. Faculty of Applied Biological Sciences, Gifu University, JapanINTRODUCTION
In Laos, agricultural and forestry plays important role ineconomic sector. The farming system in Laos decided into two
major practices; lowland farming (rain-fed or irrigated farming)and upland farming (Shifting cultivation or swidden system).
Shifting cultivation is the most common agriculture practice in
Laos because the landscape of Laos about 80% is mountainous.
One of the landmark policies in Laos during 1990s to clarify
the the shifting cultivation in village forest, the village
boundary, classifying the villages forest and land to mitigate
illegal logging activities is The Land and Forest Allocation(LFA) (Fujita, 2008). LFA was used to control shifting
cultivation because shifting cultivation is one of the factor of
deforestation. Shifting cultivation must be eliminated by theyear 2000 because the objectives of this policy are to alleviate
poverty and to introduce more sustainable management ofagricultural resources. In fact, in 2019, the indigenous people innorthern Laos are continuing shifting cultivation to get
sufficient in rice.
Sifting cultivation practice have been decline in some area of
Laos. However, there are negative impacts of replacing shifting
cultivation to other stable ecological system; changes in thepatterns of forest–field landscapes (Castella, 2013), and also to
the social and human capital (Vliet, 2012). Whereas, shifting
cultivation provides a lot of products and livelihood activities.
Rice crop in swidden field is typically mixed with vegetables
and some fruits that it could be covered their food security inhousehold level. In addition, to understand the food security innorthern Laos, this study has main objective to conduct local
food security of indigenous people in four villages of Sone
District, Houaphan Province.

MATERIALS AND METHODS
The livelihood change is described based on statistic data andby interviewing head of household in study site. The collectionof both quantitative and qualitative data were collecting throughindividual interviews. To gain an understanding of the village
context and to support the interview findings, the study area was
documented through direct observation and photographs of thevillage. In addition to participatory observation relating toresearch objectives, one hundred twenty-five households werechosen and interviewed with semi-structured questionnaire.
The quantitative data were processed using simple statistics (inexcel) to support the qualitative data.

RESULTS
Average rice production per household are shown in table. 1,
and the value of various swidden activities in the study area isshown in table.2 and fig.1.

Table 1. Average rice production per household and its
sufficiency in the four villages of Sone District, Houaphan
Province.

Items
Bong
Houay Su Houay Sanguan Houay Lao
Paddy
Household engaged (%)
40.62
15.79
45.71
0.00
Production (kg/HH/y)574.3898.31
1028.57
0.00
Estimated price of produced
172.3129.49
308.570.00
rice (USD/HH/y)*UplandHousehold engaged (%)
Production (kg/HH/y)Value (USD/HH/y)*Sufficiency Rice
Self-sufficiency ratio in rice
Insufficient value in rice
(USD/HH/y)*87.50
1813.75
544.1392.11
2500.26750.0865.71
1521.00
456.3095.00
3222.00
966.60

68.75

92.10

57.14

85.00

223.8961.59

327.82144.99

Swidden activities
NTFPs
1293.171403.511397.31606.09Commercial crops
118.74353.9376.35
897.84
Livestock500.38465.77655.131275.30
Source: Field Survey 2019
Note:
Total sample: Bong (35), Houay Su (38), Houay Sanguan (35), Houay Lao (20)*price (0.3USD/kg)

Table 1. shows that upland rice in swidden field is the mostcommon agricultural practice for the local people in northern
Laos. However, some household, like in Houay Sanguan village
tried to replace the swidden to the paddy field and others
activities. Overall, more than 50% the households at the village
had enough sufficiency in rice and the others not. The range
insufficient value of rice between 60 US dollars to 327.82 US
dollars per household per year. It indicates that the householdwith less activities on swidden has a risk to vulnerability to thefood security for each household in the village.
The value of various swidden activities in the study area aredifferent in each village. These indicates the strategy for eachhousehold at the village level to encounter the sufficiency rice.
Almost in the all villages, non-timber forest products (NTFPs)
have significant value to cover their food security. However,the value from livestock and commercial crop in Houay Lao
village more significant than value from NTFPs (Fig.2). It
examines the market and road access in Houay Lao village are
well available. Otherwise, the household in remote village withpoor access are still depending on NTFPs products for theirmain activities to get cash income and make ends meet.

DISCUSSION
This study clarified that shifting cultivation can provide a lotof products for their food security. Upland rice activity is still
important for the household to get rice sufficiency in eachhousehold. Generally, paddy is the backbone of food security
to the household (Castella and Erout, 2002). However,increasing of landowner of paddy field in the study area are notsignificantly related to the rice sufficiency in each household.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P21
They who replacing the upland field (shifting cultivation) tolowland field (paddy) could not fulfill their rice needs.
Household that have met their rice needs begin to diversify theirlivelihood with cash crops and cattle and buffalo raising like in
Houay Lao and Houay Su village. However, household thathave not met their rice needs begin to collecting NTFPs as thebest way to get cash income and cover their rice needs inadvance like in Bong and Houay Sanguan village. In the end,
this study need further research in livelihood strategies and its
diversity to understand the strategy for the poor people tosurvive and met their needs.

ACKNOWLEDGMENTS
The authors would like to express sincere gratitude to allvillagers and officers of Sone district, Houaphan province. This
research could not be done without their cooperation

REFERENCES
Castella, J-C., Erout, A. 2002. Montane paddy rice: thecornerstone of agriculture production system in Bac Kan

Province, Viet Nam. The Agriculture Publishing House(book): Doi Moi in the Mountain, Vietnam.
Castella, J-C., Lestrelin, G., Hett, C., Bourgoin, J., Fitriana,
Y.R., Heinimann, A. & Pfund, J-L. 2013. Effects oflandscape segregation on livelihood vulnerability: movingfrom extensive shifting cultivation to rotational agriculture
and natural forests in northern Laos. Human Ecology, 41:
63–76.
Fujita, Y. and Phanvilay, K. (2008). Land and Forest Allocationin Lao People’s Democratic Republic: Comparison of Case
Studies from Community-Based Natural Resource
Management Research. Society and Natural Resources,21(2), pp. 120-133.
Vliet, N.V., Mertz, O., Heinimann, A., Langanke, T., Pascual,U., Schmook, B., Adams, C., Schmid-Vogt, D., Messerli, P.,
Leisz, S., Castella, J-C., Jorgensen, L., Birch-Thomsen, T.,
Hett, C., Bech-Brunn, T., Ickowitz, A., Vi, K.C., Yasuyuki,
K., Fox, J., Padoch, C., Dressler, and W., Ziegler, A.D. 2012.
Trends, drivers, and impacts of changes in swidden
cultivation in tropical forest-agriculture frontiers: A global
assessment. Global Environmental Change, 22: 418-429.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P22

Dynamics of dissolved organic carbon (DOC) and soil carbon
sequestration in a deciduous forest
Islam Md Rashidul1, 3, Yasuo Iimura2, Takeo Onishi1, Shinpei Yoshitake3, Ashik Tahmidul1, 3 and Toshiyuki Ohtsuka3
1. Faculty of Applied Biological Science, Gifu University, Japan.
2. School of Environmental Science, The University of Shiga Prefecture, Japan
3. River Basin Research Centre, Gifu University, Japan.
1. Lysimeters: We set small free tension lysimeters of each 137
cm2 in 7 July, 2018 at 0 cm (litter layer) and 60 cm depth with
INTRODUCTION
five points each in three different place viz. Ridge, middle anddown slope respectively. Lysimeters have been used to measure
Growing concern about climate change evoked interest in the
DOC dynamics in litter layer (0cm) and deep soil layer (60cm)
role of dissolved organic matter (DOM) in the global C balanceof three soil profiles to quantity of DOC input to construct C(Hedges, 2002). DOC is often considered the most labile
accumulation at certain depth of soil.
portion of organic matter in soil and these fluxes are small
2. Small watershed techniques: To construct runoff dynamics
compared to the C fluxes due to primary productivity and(daily discharge) in both growing and snow melting period aheterotrophic respiration in terrestrial ecosystems. DOC is one
par shall flume type flow meter was setup in the 4-hectareof the most actively cycling soil organic pools and its
watershed on 29 August, 2018 and first water Samples weresignificance is translocation and stabilization of organic mattercollected in 25 September, 2018. An auto sampler was up forin forest soils (Fujii et al. 2009) whereas the DOC export fluxes
daily water sample collection in order to measure daily DOC.
with runoff might represent a substantial contribution to the net
3. Laboratory analysis: EC, pH, DOC, C and N density, Bulk
C budget of ecosystems (Kindler et al. 2011). We studied in thedensity: samples are filtered by 0.45um filter (Whatman,
Takayama Forest, is a part of the Asia Flux network, the carbon
GF/C), the concentrations of DOC were measured with a total
fluxes and budgets within it have been measured regularly sinceorganic carbon analyzer (TOC-V, Shimadzu, Japan). Total
October 1993. Biometric-based carbon flux measurements havecarbon and nitrogen contents of soil (dry weight base) werebeen conducted intensively in the Takayama Forest, so thatmeasured with a CN-analyzer (SUMIGRAPH NC-22F).
where and how the forest stores C is well known (Ohtsuka et al.
4. Statistical analyses: All data are presented as arithmetic
2007). Yet there are no previous studies about the contributionmeans. One-way ANOVA was used to detect significant
of DOC fluxes to the carbon accumulation in the soil of thisdifferences (P < 0.05). Analyses were performed with Statistics
study site, and the contribution of DOC fluxes to the large soil
10.
organic carbon pool remains unknown. Although, the 1–2 m
RESULTS AND DISCUSSION
depth snow generally has melted at the end of April, the studyof melt water draining dynamics during the snowmelt period
Soil chemistry: The soil at the study site has a very thick andwas insufficient. Plot based Biometric NEP measurement has
dark colored A horizon with high C content, particularly in thesome uncertainty due mainly to the model estimation error of
2A horizon, (FAO 2014) is used as a criterion for andisol (a soil
annual soil respiration and root respiration. Errors in measuring
order that is rich in soil carbon and allophane and/or organoor estimating any one of these fluxes can lead to relatively large
aluminum) along with Japanese volcanic ash soils.
errors in the net exchange. Therefore, we need to measure othera. Difference between three soil profiles: Slope position atfluxes such as C in precipitation, and losses of C such as elution
watershed scale varies greatly. Thickness of black soil layer isof DOC by soil water flow. However, we used small watershed
lower in ridge portion but gradually increases to middle andtechniques which has been extremely useful for quantitative
down slopes (>60cm) and large surface layer (A) around 32 cm
study of element fluxes and cycling with appropriate hydrologic
in down slope was found. Soil organic carbon at 5-10 cm depth
control and precise estimates of chemical inputs and outputs can
is higher in ridge slope and gradually decreases to down slopes
be obtained. Watershed based aquatic C dynamics is not only
(151.9, 142.4 and 113.3 C g kg-1). Higher C: N ratio
measuring C sequestration in soil organic matter but also for(High N content) and high moisture contents was found in down
aquatic C export from forests, (Bormann and Likens, 1967).
slope than ridge and middle slopes (Table, 1). 60 cm depth
The investigations of linking the DOC fluxes in temperatereaches both surface and subsurface soil of Ridge and middle
forest ecosystems to the soil organic carbon in Japan wereslopes but down slope within surface soil due to large A and 2A
limited (Shibata et al. 2001). Laboratory experiment found thatsoil horizon. Litter leachates DOC production, sorption andadded to a subsoil was retained 60–90% of DOC by sorption
water volume is significantly higher in ridge and middle slopes(Kaiser and Zech, 1997). However, in field level accumulation
than down slope profile.
of carbon in soil by DOC fluxes and their leaching loss from
1. Water dynamics: Inter annual and seasonal variability isdifferent forested ecosystem is rare and their contribution to net
higher among three soil profiles. In 2018, monthly water foundC balance is uncertain.
in litter leachate 0.87±0.16, 1.06±0.16 and 0.64±0.08 litter in
The present study focuses with the objectives are:ridge, middle and down respectively. But in contrast the in 2019
1. To estimate soil C accumulation and aquatic C export using
growing season (3 month) water volume was found 1.97±0.29,
water-shed technique.
2.65±0.63 and 2.08±0.44 litter respectively. No significant
difference was found among soil profiles in case of deep soil
MATERIALS AND METHODS
layer (60cm). Monthly water depth was around 3-10 cm among
them.
Study site: Takayama forest is located in the central region of
2. DOC dynamics: DOC concentration in Growing season
the main island of Japan, far from the heart of city, a permanent(2018) 2 fold and (2019) 3 fold larger than winter season but inplot of 1 ha (100 m * 100 m) was set on a west-facing slope isboth year Ridge and middle was significantly different fromin forest belonging to the river basin research center, Gifu
down slopes. Mean concentration in growing season 2018 wasuniversity, Japan.
found 16.86±0.0.83, 15.56±1.49 and 10.11±0.74 mg L-1 andProceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P222019 was found (25.01±3.81, 23.25±1.38 and 14.81±1.41 mg
L-1) and winter season (Dec, 2018-april 2019) was found10.18±1.47,10.64±0.64 and 7.07±0.42 mgL-1. No significancedifference was found in deep soil layer both in seasonality andinter-annual variation. Average monthly concentration was
found 5.04±0.54, 4.93±0.42 and 3.19±0.42 mg L-1.
3. DOC input to soil: DOC fluxes of ridge and middle in litterleachates were also significantly different from down slopes inboth year (2018 and 2019). 2018, monthly DOC input to soil
was 8.52±2.22, 10.93±2.35 and 3.91±0.608 and 2019 was
found 37.68, 49.48 and 21.16 kgha-1. But no significancedifference at 60 cm depth, where the monthly flux (2018) was
found 0.618±0.246, 0.4528±0.22 and 0.317±0.076 and 2019
was 0.2215, 0.2652 and 0.7147 kgha-1 respectively.
4. DOC adsorption: DOC storage at 60 cm depth of ridge andmiddle was significantly different from down slopes in bothyear (2018 and 2019). But the no significant different was foundfor sorption rate (92%) for three sites. DOC accumulation was
lower in 2018 may- 2019 April around 60 kgha-1 and 2019
(may- July) was found 107 kgha-1.
5. Stream water dynamics: Water year (August 2018 - 2019
July), we found irregular discharge in studied watershed.
Discharge was only found during high rainfall occurred during
July to September. During dry periods and Snow melt period
we found no discharge in par-shall flow meter.
6. Stream DOC dynamics: Stream DOC concentration was 10
fold smaller than the DOC input to litter leachate. Monthly
DOC concentration was found 2.08 mg L-1 and DOC flux was
found 5.33 kg ha-1 7 month-1.
Table. 1 Chemical properties of three soil profiles (0-90 cm)

correlated. Thus it indicates SOC and DOC production at threesite largely depends on the slope position (Fig.1) but thesorption of DOC at 60 cm depth is uniform around >90% at
three site indicates variability of Sorption rate (%) in Takayama
andisol soil is negligible. At watershed scale, upper andmiddle portion contains much C due to high input of
DOC than lower position (down) but C stability is morethan upper sites due to lower decomposition. DOC

retention in mineral sub-soils within the ranges of 0-90 cm
depth in 17 sites around 40-370 kg ha-1 year-1 (Currie et al.
1996). Moreover, Kawahigashi (2011) reported that andisols,
adsorbing more than 90% of DOC, and that DOC rarely leaches
from andisols to stream. In growing season (2018 and 2019) wefound 65 kgha-1 5month-1 and 107 kgha-1 3month-1 (May-July)and in winter season 20 kgha-1 5month-1. Amount of DOC
fluxes is vital for much C accumulation (Fig.1). Therefore, atwatershed scale, DOC plays a critical role in C sequestration insurface and subsurface soils due to its high sorption capacity
and DOC concentration is very lower in lateral flow comparedto litter leachate probably high adsorption in surface soil thus
aquatic export is negligible (5.33 kgha-1 7month-1) compared toheterotrophic respiration.
B

Fig.1, Relationships between (A) DOC concentration and
Surface C content (B) EC (mS/m) and DOC flux of three soil
profiles.

REFERENCES

Table. 2 Monthly mean litter leachate DOC conc. (mg L-1),
water volume (L) and Flux; Storage (kg ha-1) at three profiles

b. Soil profiles at watershed scale: Soil heterogeneity andthickness of A horizon variability, we used three soil profile inwatershed scale. The distinct soil layers and their SOC contents
signifies the importance of slope position in hilly areas. The C:
N ratios at three sites were strongly positively correlated. Slopewise variation not only for decomposition but also fortemperature, pH, EC, plant litter, root exudates, hydrologicalinputs and other chemical properties of soils. With pH and EC
both SOC and DOC production at surface soil was strongly

Bormann, FH., Likens, GE., (1967) Nutrient cycling, Science,
177: 424-9.
Currie, W. S., Aber, J. D., McDowell, W. H., Boone, R. D., andMagill, A. H. (1996) Vertical transport of dissolved organic
C and N under long-term N amendments in pine andhardwood forests. Biogeochemistry, 35(3), 471-505.
Fujii, K., Uemura, M., Hayakawa, C., Funakawa, S., Kosaki,T., and Ohta, S. (2009) Fluxes of dissolved organic carbon in
two tropical forest ecosystems of East Kalimantan,
Indonesia. Geoderma, 152(1-2), 127-136.
Hedges, J. I., (2002) Why dissolved organic matter?,
Biogeochemistry of Marine Dissolved Organic Matter.
Academic Press, San Diego, pp. 1–33.
Kaiser, K., and Zech, W., (1997) Competitive sorption ofdissolved organic matter fractions to soils and related mineralphases. Soil Science Society of America Journal, 61(1), 6469.
Kindler, R., Siemens, J. A. N., Kaiser, K., Walmsley, D. C.,
Bernhofer, C., Buchmann, N., and Heim, A. (2011)
Dissolved carbon leaching from soil is a crucial componentof the net ecosystem carbon balance. Global Change
Biology, 17(2), 1167-1185.
Ohtsuka, T., Mo, W., Satomura, T., Inatomi, M., and Koizumi,
H. (2007) Biometric based carbon flux measurements and net
ecosystem production (NEP) in a temperate deciduous broadleaved forest beneath a flux tower. Ecosystems, 10(2), 324334.
Shibata, H., Mitsuhashi, H., Miyake, Y., & Nakano, S. (2001)
Dissolved and particulate carbon dynamics in a cooltemperate forested basin in northern Japan. Hydrological
Proceses, 15(10), 1817-1828.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P23

A paired catchment study of nitrogen dynamics in a cool-temperate mixed
deciduous broad-leaved and coniferous evergreen forest, central Japan
Tahmidul Ashik1, 2, Takeo Onishi2, Rashidul Islam1, 2, Cao Ruoming1, 3 and Toshiyuki Ohtsuka1
1. River Basin Research Centre, Gifu University
2. Faculty of Applied Biological Science, Gifu University
3. The United Graduate School of Agricultural Science, Gifu University
throughfall and NO3-N (mg/l) leaching from forest soil. It is thefirst time to study nitrogen dynamics in Kuraiyama forest. The
INTRODUCTION
present study shows the quantity of leaching of N from soils to
streams in the pair catchment and pathways and reasons of
Nitrogen (N) which is considered an essential nutrient forhigher N leaching from planted coniferous forests thanplants, can be deposited into forest ecosystem from thesecondary deciduous forests.
atmosphere via the hydrological pathway or dry deposition
process. Plant can uptake nitrogen for growth by root
absorption from soil (NH4+ and NO3-) and from atmosphere
MATERIALS AND METHODS(NO2- and NH3+) (Rennenberg and Gessler, 1999). Paired
catchment studies involve the use of two catchments with
Study site— The study site is the Kuraiyama experimental forest
similar characteristics in terms of slope, aspect, soils, geology,
at Gifu University (137°11´-137°14´E and 35°58´-36°01´N),area, climate and vegetation located adjacent or in close
which is located in Gero City, Gifu Prefecture, Japan.
proximity to each other. Following a calibration period, where
Experimental plots are divided in two catchments (deciduous
both catchments are monitored.
and coniferous evergreen). Two permanent plot (20m x 20m)
In previous studies, the annual runoff of two types of forests athave set in both catchments.
the same basin was measured by long-term observation while
the forest was being converted from a deciduous broadleaf typeRiver water sample collection—Auto sampler had set up along
to an evergreen coniferous type (Swank, and Douglass, 1974).
the watershed in each catchment for collecting river water
In Kuraiyama forest biogeochemistry studies area is divided
sample.
into two catchment, viz. A (Deciduous forest type) and B(Coniferous forest type). Catchment area for deciduous
Field survey—For estimating the Nitrogen inputs into the forest(0.71km2) and coniferous forest (0.61km2) is about same,
through water flux, we have set up sampler in 9th April 2019 tooaverage slope (21.1 ), maximum and minimum elevation,collect water.
geological type, soil type (Nohi rhyolite), precipitation and1. Lysimeter for collecting litter leachate (horizontal area
maximum and minimum temperature are also same for both0.0144m2)
type of forest. The difference of two catchments is only
2. Throughfall collector, consists of a funnel (horizontal area
vegetation difference, one catchment is artificially planted
0.0346m2)
coniferous type and another is broad leaved deciduous type.
3. Bulk precipitation collector, consists of a funnel
The deciduous forest floor is covered with a high density of(horizontal area 0.0346m2)
bush, sasa bamboo grass and a little layer and coniferous forest
floor is uncovered.
Observing soil mineralization—It will be observed during the
In Kuraiyama forest hydrological characteristics studies ofgrowing season (May – November) and snow season
runoff water under watershed had indicated that annual(December – April)discharge in deciduous forest (5.39mm/day) is higher than the
coniferous forest (4.76mm/day) because of no leaves in
Observing soil and air temperature—Stow Away Tidbit Temp.
broadleaf deciduous forests during winter season, the more
Logger have been set up for soil and air temperature.
snow can reach the ground (Rahmat et al. 2019). But peakdischarge is higher in coniferous forest (1.69m3/s/km2) than theChemical analysis—pH and EC were measured by using a pH
deciduous forest (1.61m3/s/km2) during rainy season and afterand EC meter (Horiba, D-54) after collecting the runoff andrainfall ended the discharge in coniferous forest decreased very
water sample. The concentrations of NH4+, NO3-, NO2- wererapidly. According to Komatsu et al. (2011) it can be concluded
measured using a ion chromatograph analyzer and total dissolvethat the annual runoff in broadleaf deciduous is higher than thenitrogen (TDN) were measured using nutrient analyzer
coniferous evergreen in the region with high winter(QaAAtro 2-HR) by colorimetric methods.
precipitation. It also estimate that organic matter in coniferous
forest was concentrated only in surface soil due to theaccumulation of hardly decomposable fallen leaves and in
RESULTSdeciduous forest organic matter was distributed to the wholesoil profile due to the dead and decomposed understory. And
Soil observation— Soil sample were collected from the four
soil permeability is higher in deciduous forest.
sample site from broadleaved deciduous and evergreen
In Kuraiyama the EC in watershed water is higher in deciduous
coniferous forest catchment. Soil pH, bulk density, NO3-N
forest because of ionic character (Higher amount of Na+, K+,concentration and NH4+ concentration had been measured. The
Ca2+ and Mg2+ ion present in water). And nitrate nitrogen
pH of evergreen forest soil and deciduous forest soil is 5.62 ±concentration is higher in coniferous forest. In broad leaved
0.18a and 5.45 ± 0.091a respectfully which are almost same.
deciduous forest mineralization rate is higher than the
There are significant difference in bulk density between
coniferous forest because we found that the NH4+ concentration
evergreen forest soil (0.679 ± 0.043a g/cm3) and deciduous
is higher in deciduous forest. The rate of NO3- leaching depends
forest soil (0.410 ± 0.011b g/cm3). The NO3-N and NH4+
on soil drainage, rainfall, nitrate present in the soil, and plant
concentration are almost same in both catchment. The NO3-N
uptake. There is no previous studies on nitrogen deposition by
concentration in evergreen forest is 0.075 ± 0.024a mg/l andProceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P23
deciduous forest is 0.096 ± 0.008a mg/l and for NH4+
concentration in evergreen forest is 0.099 ± 0.015a mg/l and for
deciduous forest is 0.146 ± 0.041a mg/l. There is no significant
difference among them.
River water hydrological characteristics— Auto sampler
had been set previously in the both catchment for collecting
river water sample every day. PH and EC is higher inbroadleaved deciduous forest than the evergreen coniferous
forest. All the nutrient conc. (Na+, K+, Ca2+, Mg2+, Cl-) arehigher in deciduous forest than the evergreen forest river water.
Only NO3-N (Fig. 1) and SO42- concentration is higher in theevergreen forest river water.
Dynamics of nitrogen in forest—In throughfall theconcentration of NO3-N and NH4+ is higher in evergreen forest
than the deciduous forest (Table 1). For average NO3-N fluxes,
in evergreen forest (0.137±0.047 ka/ha/month) which is higher
than the deciduous forest (0.089±0.055 kg/ha/month) flux. And
for average NH4+ fluxes, in evergreen forest (0.935±0.533
kg/ha/month) which is lower than the deciduous forest(1.171±0.653 kg/ha/month) flux from May to July 2019 (Fig.
2). In litter leachate the concentration of NO3-N and NH4+ ishigher in deciduous forest than the evergreen forest (Table 1).
For average NO3-N and NH4+ fluxes, in evergreen forest (NO3N = 0.129±0.054 kg/ha/month and NH4+ = 0.731±0.376
kg/ha/month) which is higher than the deciduous forest (NO3-N= 0.105±0.041 kg/ha/month and NH4+ = 0.610±0.238
kg/ha/month) fluxes from May to July 2019 (Fig. 2).

Fig. 1: NO3-N concentration in evergreen and deciduous
Forest river water.

Fig. 2: Monthly flux (kg/ha) in evergreen and deciduous
Forest (A) Throughfall Flux and (B) Litter Leachate FluxDISCUSSION
Soil characteristics are same among the broadleaved deciduousand evergreen coniferous forest catchment. The pH, NO3-N and
NN4+ concentration is almost same in the both catchment. Only
the bulk density is higher in evergreen forest than coniferous
forest, and higher bulk density indicates the low soil porosity
and soil compactness. In river water in deciduous forest
catchment the presence of Na+, K+, Ca2+, Mg2+ ion are higher
than the evergreen forest and for that the EC is higher in
deciduous forest. NO3-N concentration is higher in evergreen
forest. The annual NO3-N and NH4+ deposition is higher in
evergreen forest than the deciduous forest with a high seepageof NO3-N and NH4+ compared to deciduous forest (Matson etal. 2002). In my experiment I only get 3 month data and I foundthat the throughfall deposition of NO3-N and NH4+ are almost
same in both catchment. And for litter leachate the NO3-N and
NH4+ flux is higher in evergreen coniferous forest. It is higher
because the evergreen forest floor is uncovered and thedeciduous forest floor is covered with sasa bamboo and bushand these understory needs extra nutrient for survive. Now myresearch is still running.

REFERENCES

Table 1: Monthly NO3-N and NH4+ conc. of Evergreen
and Deciduous forest from May to July 2019.

Komatsu, H., Kume, T., and Otsuki, K. (2011) Increasing
annual runoff-broadleaf or coniferous forests?. Hydrological
Process 25, 302-318.
Matson, P., Lohse, K.A., and Hall, S.J. (2002) The globalizationof nitrogen deposition: consequences for terrestrial
ecosystems. Ambio 31, 113-119.
Rahmat, A., Ariyanto, D.P., Noda, K., Onishi, T., Ito, K., andSenge, M. (2019) Hydrological characteristics under
deciduous broadleaf and evergreen coniferous forests incentral Japan. International Journal of GEOMATE 16, 217224.
Rennenberg, H., and Gessler, A. (1999) Consequences of N
deposition to forest ecosystems-Recent results and future
research needs. Water Air Soil Pollution 116, 47-64.
Swank, W.T., and Douglass, J.E. (1974) Streamflow greatly
reduced by converting deciduous hardwood stands to pine.
Science 185, 857-859.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P24

Evaluation of wave power generation potential in coastal area using wave
model
Ryota Matsuura1, Tomonao Kobayashi 2 and Jun Yoshino 2
1. Department of Energy Engineering, Graduate School of Natural Science and Technology, Gifu University
2. Environmental and Renewable Energy Systems Division, Graduate School of Engineering, Gifu University

INTRODUCTION
In accordance with the basic energy plan, the amount of
renewable energy introduced is increasing at an accelerating rate
(METI, 2018). Japan is considered an island country and has ahigh abundance of marine energy, which is one of the renewableenergies (NEDO, 2014). The ocean basic plan is also promoting
the use of ocean energy (Cabinet Office, 2018). The introductionof wave power generation is delayed compared to otherrenewable energy. The reason is that energy density is low and
it is difficult to extract energy, which is a big hurdle tocommercialization.
Therefore, in this study, the wave energy potential in the coastal
area was estimated, and the appropriate land of wave power
generation and profitability of wave information was calculated.
Attempts to use ocean energy have existed for a long time.
Maeda and Kinoshita et al. (1979) estimate the wave energy
around Japan, and Katayama et al. (2014) rearranged the wave
energy along the coast of Japan by increasing the number oftarget sites and the statistical period. These wave energies are
ideal physical quantities that do not take into account thecharacteristics of the generator. Therefore, various wave
characteristics required from the viewpoint of mechanicalengineering and naval engineering were evaluated and arranged.

POTENTIAL OF WAVE POWER GENERATIONALONG JAPANESE SHORELINE(1) Computational method
First, tried to select a suitable site for wave power generation
throughout Japan. In this selection of suitable locations, the wave
power at each point was calculated, and points with high wave
power were extracted. The wave power was estimated using thesignificant wave height and the significant wave period every
two hours of the nationwide ocean wave information network(NOWPHAS) (MLIT, 2018). The target period was five years
from 2012 to 2016. The wave power used for the evaluation wasan Omni-directional wave power that represents the amount of
power that can be generated at that point. This is the amount of
power generation on the premise that wave power can bereceived for waves coming from all directions without taking thewave direction into consideration. This wave power WE (kW /m) is expressed by the following formula.
𝜌𝜌ｇ2
𝐻𝐻 2 𝑇𝑇
64𝜋𝜋 1/3 1/3= 0.492𝐻𝐻1/3 2 𝑇𝑇1/3

WE =

(1)

Where ρ: seawater density (kg/m3), g: gravitational acceleration(m/s2), H1/3: significant wave height (m), T1/3: significant wave
period (s).
(2) Results and discussions
The Fig.1 shows the average value and standard deviation of
Omni-directional wave power estimated at the NOWPHAS
observation point. As shown in this figure, the coastal area of the
Sea of Japan, such as Sakata, generally has large average wave
energy, but its standard deviation is also large. On the other hand,the standard deviation of wave energy is relatively small on the
Pacific side, and the wave state is stable. Comparing the wave
energy at each point, the points with the highest wave power are
Sakata 11.9 kW / m, Kashima 9.85 kW / m, and Wajima 9.74
kW / m, respectively. However, Sakata and Wajima have large
seasonal variations of waves as indicated by the standard
deviation. From the viewpoint of wave power generation, it isdesirable that the amount of power generation is large and there
is no seasonal variation. From this point, Sakata and Wajima are
not suitable for wave power generation due to large seasonalvariations. On the other hand, Kashima has large wave energy
and small standard deviation. Therefore, large capacity andstable power generation can be expected. From this result, it was
found that the suitable site for wave power generation is the areaaround Kashima.

ESTIMATION OF ENERGY DISTRIBUTIONSAROUND HIGH-POTENTIAL AREA
(1) Computational methoda)Target area
In the previous section, the area around Kashima was selected
as a suitable site for wave power generation from the coastal area

Fig.1 Average value of Omni-directional wave power at each point and time variation standard deviation (2012~2016year)
The bar graph shows the average wave power (left axis), and the line graph shows the standard deviation (right axis).

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P24(2) Results and discussions

Table.1 Calculation condition
Lattice spacing
longitude
latitude
Number of lattices

Large area

medium area

small area

1/2°
115～180°0～65°
130×130

1/8°
138～143.5°
33～38°
44×40

1/20°
140～142°35.5～37°
40×30

of Japan due to the magnitude of wave energy and seasonal
stability. Here estimated how the wave energy is distributed inthis area. Table.1 shows the conditions in each calculation area
when conducting wave estimation. In the estimation, thecalculation was performed by nesting three areas from thewestern Pacific (large area) to the Kashima area (small area).
The target period was one year in 2016. Sea surface wind data
were GSM-JP and MSM objective analysis data from the Japan
Meteorological Agency (MLIT, 2016).
b) Wave prediction model
In this study, the third generation wave model, SWAN
(version40.91ABC) was used. Output parameters were added tothe small area based on this SWAN (The SWAN Team, 2013).
The reason is to find the wave parameters from the viewpoint of
mechanical engineering and ship engineering.
Here, as new wave parameters, the Omni-directional wave
power, WE(Maximum directionally resolved wave power),
WEθWEmax (maximum wave power direction), θWEmax (Directionof maximum directionally resolved wave power), SW (Spectral
width), and d(Directionality coefficient) are calculated inaccordance with IEC standard TS62600-101 (Wave Energy
Resource Survey and Evaluation)．

a)Omni-directional wave power
Fig.2 shows the average Omni-directional wave power
distribution in 2016 for a small area. In Kashima and Hitatinaka,
the Omni-directional wave power is 10～15 kW/m along thecoast. From equation (1), the wave energy is proportional to theperiod. In this region, the wave energy is large because the swellgenerated in the Pacific is easy to reach. In particular, the point
indicated by the black circle (the sea area around Choshi) has anenergy of 13～20 kW/m. Therefore, the sea around Choshi is apromising area for wave power generation.
b) Maximum directionally resolved wave power
Maximum directionally resolved wave power imposes
direction restrictions on the Omni-directional wave power.
Therefore, it can be evaluated as a more realistic power
generation amount. The Fig.3 shows the distribution of theannual average Maximum directionally resolved wave power
and the Direction of maximum directionally resolved wave
power in a small area. The black circle is 140.9 ° east longitude
and 35.7 ° north latitude, the Omni-directional wave power is
15.8 kW / m, and the Maximum directionally resolved wave
power is 11.2 kW / m. By limiting the wave direction, energy
was reduced by 29%.

CONCLUSIONSIn this study, the appropriate location was selected using wave
parameters required from the viewpoint of mechanicalengineering and ship engineering. First, the wave energy of thewhole country was calculated using NOWPHAS, and highenergy areas were extracted. As a result, the area aroundKashima with high annual average wave energy and little
seasonal variation was suitable for wave power generation. Next,
wave estimation was made around Kashima and a wave potential
map was created. From this potential map, the sea around Choshi
was promising for wave power generation. When the maximum
wave energy direction was taken into account, the energy was
reduced by 29% in this sea area due to direction restrictions.

REFERENCES

Fig.2 One-year-averaged Omni-directional power
distribution (small area, 2016)

Fig.3 One-year-averaged Maximum directionally resolved
wave power distribution (small area, 2016)

Ministry of Economy Trade and Industry (METI): Fifth Energy
Basic Plan, 2018
New Energy Industrial Technology Development Organization(NEDO): NEDO Renewable Energy Technology White Paper
Second Edition, Chapter 6, 2014
Cabinet Office: Basic Ocean Plan, 2018
Hisada Maeda, Ken Kinoshita: Wave Power Generation,
Production Research Vol. 31, No. 11, 1979
Hiroyuki Katayama, Haruo Yoneyama, and Kenichiro
Shimosako: Evaluation of wave power along the coast ofJapan for selection of suitable locations for wave power
generation, Journal of Japan Society of Civil Engineers B3(Ocean Development), vol.70, No.2, I_73-I_78, 2014.
Port Bureau, Ministry of Land, Infrastructure, Transport andTourism (MLIT): National Oceanographic Information
Network 「 https://nowphas.mlit.go.jp/prg/pastdata/ 」
2018.10.10 Search
Ministry of Land, Infrastructure, Transport and Tourism
Meteorological Operations Support Center: Objective
Analysis Data, 2016
The SWAN Team : SWAN USER MANUAL SWAN Cycle Ⅲ
version40.91ABC,2013

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P25

A new Pythium species causing lettuce crown rot
Akihiro Hayano1, 2, Kensuke Yamada2, Ayaka Hieno2, Haruhisa Suga3 and Koji Kageyama2
1. Graduate School of Natural Science and Technology, Gifu University, Gifu 501-1193, Japan
2. River Basin Research Center, Gifu University, Gifu 501-1193, Japan
3. Life Science Research Center, Gifu University, Gifu 501-1193, JapanINTRODUCTION

members of clade J, resulting in a single lineage. (Fig. 2)

In 2016, the lettuce showing in the above-ground and root rot
was observed in a lettuce field in Kagawa prefecture (Fig. 1a).
The crown was rotted. (Fig. 1b) This disease occurred from
October to December, and multiple species of Pythium andPhytophthora were detected. Among the isolated Pythium
species one isolate did not match any existing Pythium species
in BLAST search of the rDNA-ITS region. This study reveals
that the isolate will be a new species causing lettuce disease.

abFig. 1. Wilting and crown rot in of lettuce Kagawa prefecture.
a. wilting.
b. rotted crown.

MATERIALS AND METHODS
The crown of diseased lettuce was placed on Pythium genus
selective medium to isolate this pathogen. The pathogenicity test
was carried out at 20 ° C by placing a mycelial agar disc on asection of crown of lettuce. Molrcular phylogenetic analyses
were conducted based on the sequencing the rDNA-ITS region,cox I, cox II, and β-tubulin. In morphological observation, apiece of turf grass infected with the isolate was put in water toinduce the formation of reproductive structures.

Fig. 2. Neighbor-joining phylogenetic tree based on thesequences of cox I, cox II and β-tubulin and rDNA-ITS
region of Pythium clad J.
Morphological characterization and growth temperature
The morphological characteristics were confirmed; sporangia
were spherical, average 27.3µm (18.7-35.6µm) (Fig. 3a),oogonium were smooth, spherical, 32.5µm (25.6-37.2µm) (Fig.
3b), and aplerotic oospores were unfilled, 27.6µm (21.030.2µm) (Fig. 3c). The antherdia were diclinous and theantheridial cells were clavate and crook-necked. (Fig. 3b) One
to three the antheridia were attached per oogonium. Mycelial
growth was observed between 5℃ and 25℃. (Fig. 4)

RESULTS
Isolation
An isolate with fast-growing and non-septate hyphae likePythium species was obtained from the root.
Pathogenicity test
The inoculated crown of lettuce developed a brown watersoaked lesion. Moreover, this isolate was re-isolated from thelesion.
Molecular phylogenetic analyses
When the sequence of the rDNA-ITS region was analyzed, it
belonged to the molecular phylogenetic clade J of Pythium
species classified by Lévesque and de Cock (2004). Additionally,
the sequences of cox I, cox II and β-tubulin were examined, andmultigene phylogenetic tree was constructed with the otherabcBars=20µm

Fig. 3. Morphological characteristics.
a. Spherical sporangia.
b. Crook-necked antheridial cells and spherical oogonium.
c. Aplerotic oospore.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P25

Fig. 4. Growth temperature reaction.

Fig. 5. Growth pattern on PCA.

DISCUSSION
The isolate from the rotted lettuce medulla showedpathogenicity to the crown of lettuce, and re-isolated from thelesioned part, suggesting that it would be a pathogen in lettuce.
P. irregulare and P. uncinatum have been reported as pathogens
that cause similar disease of lettuce. In molecular phylogenetic
analyses, it belonged to molecular clade J of the geuns Pythiumand showed a phylogenic monophyly. In addition, comparing to
P. perplexum, which is a morphologically related species, theisolate was differed by diclinous antheridia, larger size of the
reproductive structures and lower growth temperature. These
results indicate that the isolate will be a new Pythium species
causing lettuce disease.

REFERENCES
Lévesque C.A and Cock W.A.M.D (2004) Molecular phylogeny
and taxonomy of the genus Pythium. Mycol. Res. 108 : 1363
– 1383.
Feng W., Hieno A., Kusunoki M., Suga H. and Kageyama K.
(2019)
LAMP Detection of Four Plant-Pathogenic
Oomycetes and Its Application in Lettuce Fields. Plant Dis.
103 : 298 – 307.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P26

Future climate projections for storm surge associated with typhoon Jebi
(2018) by a pseudo global warming downscaling
Masaya Toyoda, Jun Yoshino and Tomonao Kobayashi
Environmental and Renewable Energy Systems Division, Graduate School of Engineering, Gifu University

INTRODUCTION
Typhoon Jebi (2018), which attacked Japan in 2018, was acategory 5 super typhoon (Fig. 1). The maximum wind speed
was 55 m/s, breaking the previous wind speed record of 45 m/s.
It caused a storm surge of 329 cm in Osaka and 233 cm in Kobe
(Japan Meteorological Agency (JMA), 2018). Both storm surges
exceeded the existing highest tide, which was recorded during
Typhoon Nancy (1961). The total damage caused by Jebi wasestimated to exceed 1 trillion yen (Mori et al., 2018), and there
were numerous destructive events such as storm surge induced
flood damage to the runway of Kansai International Airport(Mori et al., 2018) among recent typhoon disasters in Japan, Jebi
produced remarkable storm surge destruction. In the future, as
global warming progresses, coastal disasters caused byintensifying typhoons are likely to be also intensified.
In this research, future climate experiments for Typhoon Jebi
(2018) are conducted, based on the pseudo- global warming
downscaling technique using a storm surge model coupled witha high-resolution typhoon model. The purposes of this research
are to evaluate the impacts of global warming on typhoon
intensity (central pressure, maximum wind speed) and structure(radius of maximum wind speed), and to quantitatively assess
the impact of global warming on maximum potential storm
surges in Osaka Bay. In addition, the worst-case track scenario
under the future climate is also investigated, taking into
consideration typhoon intensity and the radius of maximum
wind speed.

COMPUTATIONAL METHOD

a time period of 1 day (24 hours) from 15:00 UTC on 3
September 2018 to 15:00 UTC on 4 September 2018.
(3) Present climate and future climate experiments
In this study, first of all, present climate experiments arecarried out using NCEP FNL data as initial, boundary andassimilation conditions. We conduct future-climate experiments
by applying the method of pseudo-global warming experiments(PGWE; Sato et al., 2007) to estimate future changes of intensity
and structure of typhoons in comparison with present climate
observations. The input conditions for the pseudo-global
warming experiment are created by adding monthly average
global warming difference (GWDs) to NCEP FNL. The GWDs
are the difference of monthly mean fields between the futureclimate present climates. Temperature, Sea surface temperature,geopotential height, east-west wind speed, north-south windspeed and relative humidity are used as the3D monthly mean
fields derived from the general circulation model GCMs. In thisstudy, the monthly mean fields in the future climate are averaged
during 2080-2099 by a total of 8 GCMs of the CMIP5 RCP8.5
scenario. In addition, the monthly mean fields in the present
climate are averaged during 2000-2019 by 15 GCMs of theCMIP5 historical scenario (2000-2005) and 8 GCMs of theCMIP5 RCP8.5 scenario (2006-2019).

RESULTS AND DISCCUSION(1) Present-climate experiment (CNTRL)First, we will discuss the results of the present-climate
experiment for Typhoon Jebi (Fig. 2) before discussing the(1) High-resolution typhoon model (HTM)
The high-resolution typhoon model (hereafter HTM) is used
to simulate the intensity and size changes of overall life of
typhoons. HTM is based on the mesoscale meteorological modelMM5. The automatic movable nesting technique and the several
kinds of physical parameterizations (e.g. the ocean mixed layer,
dissipative heating, and sea-spray processes) are introduced into
MM5 to express realistic typhoon intensity and structure
accurately (Yoshino et al., 2013). The triply nested
computational domains used in this study have a horizontal grid
spacing of 27-km (D1), 9-km (D2), and 3-km (D3), respectively.
The movable nesting technique is applied to both D2 and D3.
(2) Storm surge model (SSM)
The storm surge model (SSM) used in this study is composedof the nonlinear long-wave equation system, which predicts 2dimensional distributions of sea-level anomalies and ocean
current vectors. The typhoon atmospheric fields estimated by theETM are inputted as the boundary conditions every 15 min.
Computational domains for the SSM are set with a horizontal
resolution of 1 km located around Osaka Bay. The topography
data is sourced from the global earth surface relief
dataset ETOPO1 (produced by NOAA) with a horizontal grid of
1 min, and the global land use dataset, with a horizontal grid of
30 sec, is provided by the USGS. The SSM computation covers

5, Aug. 0:00UTC
Extratropical cyclone

4, Sep. 3:00UTC
Landfall (950 hPa)

31, Aug. 0:00UTC
Peak time (915 hPa)

28, Aug. 0:00UTC
The typhoon occurrence

Fig. 1 Track of Typhoon Jebi (2018)

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P26
m

Fig. 2 Time series of the central pressure of Typhoon
Jebi simulated by the HTM (present: red line, future:orange line), derived by the JMA best track (blue dottedline), the star is the peak time and the circle is thelandfall time.
m

Fig. 4 Distribution of the worst storm surge caused by
Typhoon Jebi simulated by PGWE.
investigated the reason of this typhoon course became the worst
course for Osaka bay (Fig.4). The worst storm surge course islocated about 80 km from Osaka Bay, and this distance will beconsistent with the value of the RMW of Jebi (80.9 km) under
the future climate. Therefore, in this case, Osaka and Kobe areconsidered to be just within the area of maximum wind speed, asa result, sea-level anomaly becomes the largest in all tracks oftyphoon.

CONCLUSION

Fig. 3 Distribution of the storm surge caused
by Typhoon Jebi simulated by present climate
results of the pseudo-global warming experiment (PGWE).
According to the JMA best track (Fig. 3; blue dotted line), theobserved central pressures at peak time (5,760 min) and at
landfall time to Japan (10,260 min) are 915 hPa and 950 hPa,
respectively; the HTM results are 916 hPa and 948 hPa,
respectively (Fig. 2; red line). The simulated typhoon intensities
during the whole life cycle shows a good agreement with theobserved values. The RMW at the time of storm surge wasestimated to be 77.9 km, and the HTM simulated value was 70.0
km. The HTM results reproduced the observed structures in Jebiwith a high accuracy.
The result for maximum sea level anomaly in Osaka Bay fromthe SSM is 282 cm (Fig. 2), which is close to the observed valueof 277 cm at Osaka. Our numerical simulation technique canreproduce the mature intensity and structure of Super Typhoon
Jebi and the storm surge at landfall time with high accuracy,
which leads us to expect a certain precision for the PGWE.
(2) Maximum potential storm surge in the future climate
We will discuss the result of storm surge in the worst case
(Figs. 3). The maximum sea level anomaly reached the highest
value (328 cm) at Osaka when the typhoon followed a course0.3°
further to the west than the actual typhoon course. In this time,
the future change is +46.0 cm. Moreover, as the sea levelanomaly increased in the Osaka bay, the area with a maximum
sea level anomaly of 150 cm or more extended to the northernpart of Shikoku Island. This suggests that the damage caused by
future typhoons would be more widespread than under thepresent climate conditions. In addition, we

In this study, a pseudo-global warming experiment using acoupled high-resolution typhoon and storm surge model was
performed for the case of Typhoon Jebi (2018). Results showthat global warming causes typhoon intensity to increase at bothpeak and landfall times; typhoon associated storm surges also
increase at Osaka Bay. Although the increase of the typhoon
intensity at the time of landfall is small compared with the peaktime, it needs to be careful because it is higher than the present
climate. In addition, the worst case storm surge would be about
50 cm larger, and the worst course for a typhoon to take would
be 0.3° west of the actual course. By applying the method of thisresearch to other bays (e.g. Tokyo bay, Ise bay), it is expectedthat it will be possible to assess the global warming impact onstorm surges in each coastal area considering typhoon intensity
and structure.

ACKNOWLEDGEMENTS
This study was supported by JSPS Research Fellow(No.17J04771) and JSPS KAKENHI Grant (18H01542).
REFERENCES
Mori, N., T. Yasuda, T. Arikawa, T. Kataoka, S. Nakajo, K.
Suzuki, Y. Yamanaka (2018): Typhoon Jebi Post-Event
Survey of Coastal Damage in the Kansai Region, Japan.
Coastal Engineering Journal, submitted
Yoshino, J., S. Arakawa, M. Toyoda, and T. Kobayashi (2015):
Inter-comparison of global warming scenarios for typhoon
intensity change using a High-Resolution Typhoon Model,
JJSCE B2 (Coastal Engineering), Vol.71, No.2, 1519-1524(in Japanese).
Sato, T., F. Kimura, and A. Kitoh (2007): Projection of global
warming onto regional precipitation over Mongolia using a
regional climate model, Journal of Hydrology, 333, 144-154.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P27

Prediction of object-movement by image processing with
spectrum analysis
Takanori Ishii1 and Rina Takada 2
1.
Department of Energy, Engineering, Graduate School of Natural Science and Technology, Gifu University
2.
Department of Civil Engineering, Faculty of Engineering, Gifu University

INTRODUCTION
In recent years, the amount of photovoltaic power generation
has increased worldwide in order to realize a recycling-oriented
society. On the other hand, photovoltaic power generation is
subject to adverse effects on the power system because theamount of power generation varies greatly depending on theweather conditions1). In particular, solar radiation is attenuatedby the generation, development and annihilation of clouds, themovement of the clouds, and dust in the atmosphere such as
aerosols, resulting in large temporal and spatial fluctuations2).
Improving the accuracy of cloud movement prediction has thepotential to bring about further development of stable power
supply. Currently, there are two main types of cloud movement
prediction: long-term and short-term. Long-term fluctuations
throughout the year and throughout the day have periodicity andare relatively easy to predict. However, short-term fluctuations in
the tens of minutes to several hours ahead have not yet reached atechnique for predicting with high accuracy3). In the future, if ashort-term prediction of cloud movement can be realized, it will
be possible to adjust the power generation more delicately andtake a step closer to the solution to the instability of renewableenergy. In this study, we focused on short-term cloud movement
prediction, and tried to develop the technology of cloud
movement prediction using the time evolution method. In thispaper, as a preparation stage, we created pseudo data of cloud
images and made predictions. Furthermore, we compared andconsidered the cloud movement prediction with the original
pseudo images.

CONTENTS(1)

Pseudo-data of cloud images

We investigate the characteristics about the forecast analysis ofcloud movements by making the pseudo-data of the cloud images
and analyzing its. The number of pixels about the pseudo-data
image is 256×256. Make 24 pseudo-data images (a and b) in total,and 24 forecast images (c) by time development of original
pseudo-data. After that, the motion of the pseudo image and thepredicted image was synthesized (d).
(2) Forecast method
In this research, the forecast of cloud movements is performed
based on the pseudo-data which I described in 2.1. Clouds movehorizontally, develop and decay gradually. This spatiotemporalfluctuation is analyzed and forecasted on the spectral space. The
image data uses 3D parameters in the x, y, and z directions
defined in the .dat file. The intensity of the image data and theadvection in the horizontal direction are represented byconverting the image data to the spectral space. We try to forecast

the image data by time development of them and converting
spectral space to the image.
(3) Transformation and inverse transformation to spectral
space
In this research, Fourier transform is performed on the image
plane in order to analyze the intensity of the image data and theadvection in the horizontal direction. The amplitudes and thephases in the spectral space which has the wave number plane(kx-ky plane) are calculated. If one-dimensional Fouriertransform is extended to 3 dimension, Fourier transform forimages can be similarly defined.
F(u, v, ω)1 ∞ ∞=� � 𝑓𝑓(𝑥𝑥, 𝑦𝑦, 𝑡𝑡)𝑒𝑒 −𝑗𝑗(𝑢𝑢𝑢𝑢+𝑣𝑣𝑣𝑣+𝜔𝜔𝜔𝜔) 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑
2𝜋𝜋 −∞ −∞(1)

where, F(u, v, ω) is an arbitrary tertiary signal, imaginary unit
is j = √−1 and ω is angular frequency. u and v are wave
number. Spectral space data can be converted to image data by
performing inverse Fourier transform to the wave number planeof spectral space data.
(4) Extrapolation method
We try to forecast the images by extrapolating amplitudes andphases into the time direction on the spectral space after Fouriertransform. Least squares method (LSM) is used in the time series
direction as extrapolation method.

RESULTS
Location change of the cloud, intensity change of the cloud andsize change of the cloud about the pseudo-data were analyzed.
These clouds are considered as the state change of the actual
clouds. Each forecast result was shown as (1) to (3).
(1) Pseudo-data forecast about location change of the cloudFig. 1 shows the forecast result of the pseudo-data aboutlocation change of the cloud. Location change of the pseudocloud can be accurately forecasted according to Fig. 1. It isconsidered that the linear approximation by LSM was able toaccurately forecast location change of the pseudo-cloud because
the pseudo-data changes regularly and linearly.
(2) Pseudo-data forecast about intensity change of the cloudFig. 2 shows the forecast result of the pseudo-data about
intensity change of the cloud. Intensity change of the pseudocloud can be accurately forecasted according to Fig. 2. It is also
considered that the linear approximation by LSM was able toaccurately forecast intensity change of the pseudo-cloud because
the pseudo-data changes regularly and linearly.
(3) Pseudo-data forecast about size change of the cloudFig. 3 shows the forecast result of the pseudo-data about sizechange of the cloud. Size change of the pseudo-cloud cannot beProceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P27
from teachers, members of the Renewable Energy Laboratory,
accurately forecasted according to Fig. 3. Size change of theand past seniors. We would like to express my gratitude to thepseudo-cloud means that a nonzero value enters into a new region
people concerned and work hard for future research results.
that was previously zero on the spectral space. The amplitude at
the point where it was originally zero continues to be zero even
REFERENCES
in the subsequent forecast. For this reason, size change of thepseudo-cloud cannot be accurately forecasted and noise is added．

DISCUSSION
We knew that this method can cope with location change of thecloud and intensity change of the cloud in the pseudo-data
analysis. However, it cannot cope with size change of the cloud.
Introducing a method to reduce noise or a new extrapolation
method is cited as future issues and prospects. It is expected thatimage analysis will lead to the development of comprehensive
system which can quantitatively understand various informationsuch as a cloud amount, a cloud type and a cloud movement. If it
becomes possible to combine these forecast methods withinformation from meteorological models or information fromsolar power generation measurement data, the forecast for theamount of solar power generation will be more accurate. First ofall, after confirming that the behavior of the pseudo-data of thecloud images works correctly, we would like to use it for themeteorological satellite image obtained from Himawari-8. We
would like to verify the accuracy of the prediction technology bycomparing the predicted images with the actual cloud motion.

2)

3)

Naoto Ishibashi, Tatsuya Iizaka, Tohru Katsuno : Photovoltaic
Power Generation Forecasting Technology for Supporting Energy
Management Systems, Fuji Electric Technical Report (2013).
Hideaki Ohtake, Takumi Takashima, Takashi Oozeki : Forecast of
Solar Irradiance obtained from a Local Forecast Model and its
Validation of Forecast Errors, Journal of Japan of Energy andResources, Vol.36, No.4(2015).
Japan Meteorological Agency：Widespread use of numerical
forecasts, https://www.jma.go.jp/jma/kishou/know/whitep/1-3-1
0.html(Viewed August 2019)．

1.0
0.8

Relative strength

1)

0.6
0.4
0.2
0.0

0

pseuo data
predicted data

ACKNOWLEDGMENTS
1.0
Relative strength
Fig.2(a) 1st image

Fig. 3(a) 1st image

200
Horizontal coordinate

Fig.1(d) Object shifting

The writing of this article was completed with great cooperation

Fig.1(a) 1st image

100

0.8
0.6
0.4
0.2
0.0

0

100
pseuo data
predicted data

200
Horizontal coordinate

Fig.2(d) Object increasing its intensity

Fig. 1(b) 24th image Fig. 2(b) 24th image

Fig. 3(b) 24th image

Relative strength

1.0
0.8
0.6
0.4
0.2
0.0

Fig. 1(c) 48th image

Fig. 2(c) 48th image:

Fig. 3(c) 48th image

Relative strength

0100
pseuo data
predicted data

200
Horizontal coordinate

Fig. 3(d) Object becoming bigger

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P28

A new idea of energy-saving activated sludge wastewater treatment
process by promoting co-growth of algae and bacteria

MATERIALS AND METHODS
Activated sludge and the reactors
The mixed liquid in the aeration tank was collected from
Hokubu Wastewater Treatment Plant in Gifu, Japan. The
activated sludge in the liquid was allowed to settle to separate
from the supernatant. The supernatant was used as a liquidmedium, whereas the settled sludge was used for preparing aculturing medium with different suspended solid (SS)concentrations.
300 mL Erlenmeyer flasks, each with a working volume of
250 mL, were used as reactors. The first set of reactors wereused to evaluate the effect of SS concentrations on co-growingof algae and bacteria. The concentrations of SS were 0 mg/L
(R1), 500 mg/L (R2), 1000 mg/L (R3), 2000 mg/L (R4) and5000 mg/L (R5), respectively. The second set of reactors were
designed with the same SS concentration (2000 mg/L) but withdifferent glucose concentrations: 0 mg/L (G0), 150 mg/L (G1)
300 mg/L (G2) and 600 mg/L (G3). These reactors were used
for evaluation of the effect of biodegradable organic source on
the co-growth of algae and bacteria.

Chlorophyll-a Concentration (μg/L)

Faisal Arsyad1 and Fusheng Li2
1. Graduate School of Natural Science and Technology, Gifu University, Japan
2. River Basin Research Center, Gifu University, Japan
Operation and analysis
All the reactors were placed on a shaker at 70 rpm installed
INTRODUCTION
in an incubator (Eyela FLI-2000). The incubation conditions fortemperature, illumination, and dark/light cycle were 25°C,
The primary pollutant of domestic wastewater is organic
13000 lux, and 12/12 hours, respectively. The liquid from eachmatter, including proteins, amino acids, peptides,
reactor was sampled twice a week for measurement of pH, DO,carbohydrates, fats, and fatty acids. Other pollutants in
EC, chlorophyll-a, DOC, nitrate, nitrite, and ammonium.
domestic wastewater include nutrients such as phosphorus andpH and DO were measured by using a digital pH meter andnitrogen. In domestic wastewater, the ratio of carbon, nitrogen,
DO meter, respectively. Chlorophyll-a was measured byand phosphorous is generally suitable for the growth offollowing the trichromatic method. Dissolved Organic Carbon
activated sludge bacteria (Davies, 2005). Activated sludge(DOC) was measured by a total organic carbon analyzer
process is dependent on oxygen availability because aerobic(Shimadzu, Japan). Nitrate, nitrite, and ammonium were
heterotrophic bacteria require oxygen to degrade themeasured by liquid chromatography-mass spectrometry (LCbiodegradable organic matter. According to Bennett (2011),
MS) (Shimadzu, Japan).
about 54%-90% of total supplied energy for WWTP is used in
the aeration process. For energy saving, it is necessary to find
new methods that could minimize the oxygen supply for the
RESULTS AND DISCUSSION
activated sludge process.
Promoting co-growth of algae and bacteria is a possible
Effect of SS concentration on co-growth of algae and bacteria
method for energy-saving in the activated sludge process.
Chlorophyll-a is generally used as a measurement index for
Algae, through their photosynthetic action, release oxygen thatquantification of algae concentration. As shown in Fig. 1 there
can probably be used by aerobic heterotrophic bacteria.
were no significant increase of chlorophyll-a in all reactors
Bacteria also release CO2 that can probably be useful for algae
from day one to day seven. This indicates a lag phase for algaein the photosynthesis process. Hence, the energy requirement
to grow. Algae in R2, R3, R4, and R5 reactors showed
for air supply can be lowered. The idea of algae photosynthesis
significant increases for chlorophyll-a. The chlorophyll-aapplication can be used as a pretreatment before the activatedconcentrations in the R1, R2, R3, R4, and R5 reactors at the end
sludge process or combined in the process. However, the coof the incubation period was 0.014, 0.088, 0.089, 0.280, andgrowth of algae and bacteria still have some significant
0.404 μg/L, respectively. The concentration of chlorophyll-a
problems. The main problem is the competition for nutrientchanged along with SS concentrations and more chlorophyll-a
uptake.
was generated for the reactors with higher SS concentrations.
In this study, we investigated the growth and oxygen
production potential of algae in the mixed liquid of the aeration
0,5tank. This research includes two specific items: determinationof the growth of algae and dissolved oxygen (DO) production
0,4
potential during co-growth of algae and bacteria, and the
R1clarification of the relationship between algae, heterotrophic
0,3
R2
bacteria, and nitrifying bacteria during the process of co-growthR3of algae and bacteria.
0,2
R4
R5

0,1
0

0

5

10

15

20

25

30

Time (Day)

Fig. 1 Time profile of chlorophyll-a
The average of DO concentration during incubation in R1,
R2, R3, R4, and R5 reactors was 6.29, 5.62, 4.98, 3.97, and 1.65
mg/L, respectively (Fig. 2). In the R5 reactor with the highest
SS concentration (5000 mg/L), DO was the lowest and viceversa in the reactor R1 without SS (0 mg/L). The negative
relationship between SS concentration and DO is probably due
to the fact that more SS may contain a large number of aerobic
heterotopic bacteria. The measured DO was the sum of DO
released by algae and DO consumed by aerobic heterotrophic
bacteria. Therefore, it is highly conceivable that more aerobic
heterotrophic bacteria existing in the higher SS concentration
reactors consumed more released oxygen from algae, thus
leading to lower concentrations of DO detected in the reactors.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P28
The significant growth of algae in the R1 reactor elevated thepH value as shown in Fig. 3, which may be due to the overutilization of CO2 by algae photosynthetic process that induced
the conversion of HCO3- to CO2. According to the reaction: H++ HCO3- ⇌ CO2 + H2O, H+ consumed on HCO3- to CO2
conversion. The decrease of H+ in the liquid led to an increasein pH (Zhang et al., 2014).

Algal diversity was observed roughly by microscopic
observation. Chlorella sp. was found to be the dominant species
in all reactors. The diversity was obviously higher in thereactors added with higher concentrations of SS. It is thus
conceivable that sludge in the mixed liquid of the aeration tank
may contain more diversified algal species than those in the
supernatant.
0.6

Chlorophyll-a Concentration (μg/L)

8

6
5
4
3
2
1
R1

R2

R3

R4

Effect of biodegradable organic matter concentration on cogrowth of algae and bacteria
Different concentrations of glucose were added to observe
the effect of biodegradable organic matter concentration on cogrowth of algae and bacteria. A lag phase in G1, G2, and G3
reactors occurred until day 13 of incubation (Fig. 4). The lagphase was longer compared to G0, the reactor without glucose
addition where algae started to grow after ten days of
incubation. The longer lag phase in G1, G2, and G3 reactors
occurred probably due to that the higher concentration of
biodegradable organic matter promoted aerobic heterotrophic
bacteria to grow and dominate in the reactors. The existence of
aerobic heterotrophic bacteria in G1, G2, and G3 were reflectedwith the observed lower DO concentrations in these reactors ascompared to G0 (Fig 5). The competition for nutrients between
heterotrophic bacteria and algae might be a major reason behindthe observed lag time difference.

0.2
0.1

9
R1
R2

7

R3
R4

5

R5

0

5

10

15
20
Time (day)25

Fig. 3 pH during incubation.

30

0

5

10

15

Time (Day)

20

25

30

Fig. 4 Time profile of chlorophyll-a in the reactors addedwith glucose.
The results of the present study indicated algal growth in themixed liquid connected from the aeration tank of a biologicalwastewater treatment plant was confirmed. The co-growth ofalgae allowed maintenance of DO at certain levels even without
aeration. The co-growth of algae seemed to be inhibited bypromoted growth of heterotrophic bacteria when abiodegradable substrate is supplied.
6
5
4

G0

3

G1

2

G2
G3

1
0

11

G0
G1
G2
G3

0.3

R5

Fig. 2 Average DO concentration during incubation.

pH

0.4

0

0

3

0.5

DO (mg/L)

DO concentration (mg/L)

7

0

5

10

15
20
Time (Day)25

30

Fig. 5 DO time profile in the reactors added with glucose.

REFERENCES
Bennett, A. (2011). Wastewater treatment: Bubbling up for
major energy saving. Filtration and Separation 48:42–43.
Davies, P. S. (2005). The biological basis of wastewater
treatment. West of Scotland: Strathkelvin instruments Ltd.
Zhang, Q., Wang, T., Hong, Y. (2014). Investigation of initial
pH effects on growth of an oleaginous microalgae Chlorella
sp. HQ for lipid production and nutrient uptake. Water
Science and Technology 70:712-719.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P29

A new species of Pythium with spiny oogonia isolated from forest soil inJapan
Daichi Iijima1, Ayaka Hieno1, Haruhisa Suga2 and Koji Kageyama1
1. River Basin Research Center, Gifu University
2. Life Science Research Center, Gifu University
P. helicandrum CBS 393.54
P. prolatum CBS 845.68
P. anandrum CBS 285.31
No.24
P. senticosum CBS 122490
80/84
P. dimorphum CBS 406.72
100/99 P. undulatum CBS 157.69
P. ultimum var. ultimum CBS 398.51
99/99
56/60

INTRODUCTION
The genus Pythium belongs to the Pythiaceae of the oomycetes
which is different from true fungus. The genus is diployd andaseptate in vegetative hyphae and form zoospores in a vesicle
that extends from the sporangium. Kirk et al. (2008) reported
that there are about 150 species of Pythium reported in theworld, but the number of species has been increasing year by
year due to the recent reports of new species. Observation of
the characteristics of asexual and sexual reproductive organs inorder to clarify the characteristics of new species to be notfound in known species in the genus Pythium and molecularphylogenetic analyses based on the sequence of cox 1 gene,
rDNA ITS region and other regions are essential. A taxonomic
key provided by van der Plaats Niterink (1981) is used tocompare morphological features between species. The spine on
the surface of oogonium are one of the important morphologicalfeatures. In molecular phylogenyetic analysis, clades based onr DNA ITS region by Lévesque and de Cock (2004) are always
used. Among the Pythium genus divided into 10 (A-K) clades,
there are 6 species belonging to clade H, of which 5 species
except for P. undulatum have spines in the oogonium.
Therefore, clade H is a clade representing the genus Pythium
which has spines in oogonium. In this study, we report a new
species of a strain that has spines on oogonium isolated from aforest soil.

MATERIALS AND METHODS
The genus Pythium was isolated from a forest soil in
Tosashimizu City, Kochi Prefecture by the bait isolation by
Kenton et al. (2018). We analyzed the rDNA ITS region andcox 1 gene of the isolated strain, and created a molecularphylogenetic tree. We also observed the morphologicalcharacteristics of the sporangium, oogonium, antheridium andoospore.

RESULTS
Phylogenetic analyses
The total size of the rDNA ITS region of the isolate was
776bp, and BLAST search revealed that it belonged to themolecular strain clade H of the genus Pythium classified by
Lévesque and de Cock (2004). The strain was shown to beclosely related to P. anandrum by the molecular phylogenetic
tree of the rDNA ITS region, but showed monophyly different
from P. anandrum (Fig. 1). The phylogenetic tree combining
the rDNA ITS region and the cox 1 gene was also monophyly(Fig. 2).
Morphological characteristics
Mature sporangia are ellipsoid, lemon-shaped, and egg-shapedwith papilla at the apex (Fig. 3a-b). Occasionally, externallyproliferating spores were seen (Fig. 3c). The size of thesporangia was 23.2-85.4 × 25.2-30.1 μm, with an average of
40.1 × 22.5 μm. There were no chlamydospore. Oogonium

0.05

Fig. 1 Maximum Likely phylogenic tree based on the sequenceof rDNA ITS region. Numbers over branches represent MP(left) and ML (right) bootstrap values (1000 replicates).
P. dimorphum CBS 406.72
P. undulatum CBS 157.69
P. senticosum CBS 122490
P. helicandrum CBS 393.54
P. prolatum CBS 845.68
P. anandrum CBS 285.31
No.24
P. ultimum var. ultimum CBS 398.51

100/100
83/83
87/95

0.02

Fig. 2 Maximum Likely phylogenic tree based on combined
sequence of the rDNA ITS region and cox 1gene. Numbers

over branches represents MP (left) and ML (right)
bootstrap values (1 000 replicates).

is spherical and has conical spines on the surface (Fig. 3e-h).
The diameter of the oogonium was 19.3-30.2 (average 25.3)
μm, and the length of the spines was 3.2-7.3 (average 4.6) μm.
The antheridium is monoclinous, and one antheridium tooogonium (Fig. 3e). The antheridium just before bonding is
inflated (Fig. 3e). Antheridum stlak is curved.Oospore isaplerotic (Fig. 3f-g). Rarely, two oospores are formed in theoogonium (Fig. 3g). The diameter of the oospore was 17.5-24.5(average 21.0) μm. The mature oogonium is empty (Fig.
3h).

DISCUSSION
The isolated strain was molecularly phylogenetic monophyly
with the rDNA ITS region and cox 1 gene. In addition, P.
anandrum, which is closely related in molecular phylogeny,
did not form an antheridum, and P. senticosum, which ismorphologically similar, did not form a proliferatingsporangium. These results indicate that the isolate will be a
new species

REFERENCES

Kenton, S., Tyler J, D., Ellen, C., Chris, B. (2018) Detectionof Phytophthora cinnamomi in Forest Soils by PCR on DNA
Extracted from Leaf Disc Baits. Plant
Health Progress 19(3):193-200.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P29

a

b

c

d

e

f

g

h

Fig. 3. Morphological characters of
the isolate. a, mature lemon-shapedsporangium with papilla; b, ellipsoidsporangium ;c, externallyproliferating sporangium; d,sporangium and vesicle withzoospores ; e, broad contact
monoclinous antheridium on
ornamented oogonium; f, aplerotic
oospore in ornamental oogonium; g,
two oospores in an oogonium ; h,abative oospore.
Bars=20μm

Kirk PM, Cannon PF, Minter DW, Stalpers JA （2008）
Ainsworth & Bisbyʼs Dictionary of the Fungi, tenth edition.
CAB International, Wallingford, pp.771.
Lévesque CA, de Cock AWAM （2004） Molecularphylogeny and taxonomy of the genus Pythium. Mycol Res
108: 1363－1383.

van der Plaats-Niterink AJ. 1981. Monograph of the genus
Pythium. Study Mycol 21:1–242.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P30

Bacterial community structure in fixed-bed activated carbon adsorbers
treating Nagara River Water
Hiroki Maruyama1, Wenjiao Li2, Yasushi Isiguro3, Toshiro Yamada4 and Fusheng Li3
1. Graduate School of Natural Science and Technology, Gifu University, Japan
2. Graduate School of Engineering, Gifu University, Japan
3. River Basin Research Center, Gifu University, Japan
4. Faculty of Engineering, Gifu University, Japan
Table 1 Characteristics of all five GACs used in this study

A long-term down-flow mode experiment was conductedusing GAC columns packed with five types of GAC, namely
F400, F600, GL830, CW130 and GM830, respectively. The
empty bed contact time (EBCT) was controlled identical for all
five adsorbers as 20 minutes and the total run time was 13,000
hours. The raw water used in this study was sampled fromNagara River that contained low concentration of organic
matter and various minerals necessary for the formation of
BAC. Sampling from adsorber was conducted from threedifference bed levels: top, middle and bottom, respectively.
After sampling, water was filtered through 0.2μm membranefilter along with the raw water, and the filtrate was analyzed for
dissolved organic carbon (DOC), UV absorbance at 260nm(UV260), fluorescence spectrum (EEM).
At the end of water feeding, three layers of BAC were takenout gently from each adsorber based on the designed three water
sampling levels and, from each layer, DNA was extracted fromthe detached biofilm based on mechanical stirring. In addition
to bacterial density and structure, gene Intl 1 involved in thetransmission of antibiotic resistance bacteria and genes (ARGs)
in the biofilm was also quantified, together with four types oftargeted ARGs (tet G, tet X, tet M, sul 1). The community
structure of bacteria was analyzed through high throughput
sequencing (Huang et al., 2018).

GM830 GL830
Coal-based

F600

CW130
Wood-based

Pore volume
3(cm /g)~0.8(submicro)

0.248

0.246

0.253

0.284

0.387

0.8~0.2(micro)

0.194

0.162

0.215

0.031

0.091

2~
(meso)

0.162

0.161

0.100

0.037

0.002

Surface area2
(m /g)

1168

1140

1163

1619

1112

Pore volume
3(cm /g)0.6037

0.569

0.568

0.351

0.351

RESULTSThe largest accumulative adsorption amount for dissolved
organic matter was found with F400 and the smallest with F600
(Fig. 1). F400 has more micro-pores and meso-pores than F600
and the pore volume in these two size regions were reduced due
to adsorption (Fig. 2).
12

DOC

10

UV260

8
6

30
20

4

10

2
0

40
UV260 (1/m/g-AC)

MATERIALS AND METHODS

F400

Pore size

DOC (mg/g-AC)

In order to provide safe drinking water, the use of fixed bed
granular activated carbon (GAC) as an advanced water
purification unit is increasing in drinking water treatment plants(DWTP). GAC is effective in adsorbing a wide range ofhazardous organic compounds, taste and odors, and precursorsof chlorination and ozonation by-products. The application of
GAC without pre-chlorination for surface water treatment
allows microorganisms to develop on the GAC surface, thencechanging GAC adsorbers into biological ones (BAC). In
addition to the function of adsorption, we can also expectorganic matter degradation as well as ammonia nitrification.
However, little is known about the microorganism species inthe biofilm of BAC, so with their roles and contributions to therisk relating to some newly emerging microbial agents of great
health concern, such as bacterial and viral pathogens andantibiotic resistance bacteria and genes. The main objective of
this study was to identify the structure and composition ofbacterial community in the biofilm of BAC adsorbers treating
surface water for a long term. The changes of bacterial density
and species along the fixed bed depth and with GAC types werealso investigated.

GAC
Origin

F400

F600

GL830 CW130 GM830

0Fig. 1 Accumulative adsorption amounts of dissolved
organic matter on five GACs. (The accumulative
adsorption amount is calculated based on flow rate
and the influent and effluent concentrations over the
total runtime of 13,000 hours)
0.3
Pore volume (cm3/g)

INTRODUCTION

F400

F600

0.2
0.1
0
beforeafter～0.8nm (submicro)
2nm～ (meso)

beforeafter

0.8～2nm (micro)

Fig. 2 Changes in pore volume of three different pore size
ranges of F400 and F600 before and after use.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P30
In the treated water from the outlet of the adsorbers, Gammaproteobacteria, unidentified_Actinobacteria, and Alphaproteobacteria were detected (Fig. 3a). Following the direction ofwater flow in the fixed bed, the relative abundance of Gammaproteobacteria was found getting higher (Fig. 3a). In thebiofilm detached from BAC, Alphaproteobacteria, Gammaproteobacteria, Deltaproteo-bacteria and Nitrospira weredetected for all three targeted BAC adsorbers (Fig. 3b). Deltaproteobacteria and Nitrospia, their relative abundance was
obviously higher for the adsorber of F400 (Fig. 3b). This classincludes the bacterial species of purple sulfur bacteria widelydistributed in natural environments such as soil was wellreported. The class of Unidentified-Actinobacteria generally
refers to actinomycete, a group of bacterial species including
those necessary for industrial production through their role indecomposing various organic compounds such as antibiotics
and amino acids. In addition to various genera,
Alphaproteobacteria also include many purple non-sulfur
photosynthetic bacteria.

100%

(a)

DISCUSSION
In the biofilm of all targeted BAC adsorbers and also thetreated water, the presence of Alphaprobacteria, reported to bemainly consisted of purple non-sulfur photosynthetic bacteriathat may be capable of decomposing organic matter, wasconfirmed, which probably contributed to the performance of
BAC. Deltaproteobacteria were detected in the biofilm but notin the water after BAC treatment. Higher abundance ofNitrospira (nitrifying bacteria) was found in the BAC of F400,
which showed the best performance in the uptake of dissolved
organic matter.

REFERENCE
Huang K., Xia H., Wu Y., Chen J., Cui G., Li F., Chen Y., Wu
N., Effects of earth-worms on the fate of tetracycline andfluoroquinolone resistance genes of sewage sludge duringvermicomposting. Bioresource Technology, 259:32–39,
2018.
(b)

Relative abundance (%)

80%

60%

40%

20%

0%

Gammaproteobacteria
Alphaproteobacteria
Thermoleophilia
Bacteroidia
Acidimicrobiia
unidentified_Acidobacteria
Clostridia
Armatimonadia
Negativicutes
Chloroflexia
unidentified_Chloroflexi
Ignavibacteria
Rubrobacteria
unidentified_Chlamydiae

unidentified_Actinobacteria
Deltaproteobacteria
Nitrospira
Bacilliunidentified_Oxyphotobacteria
Anaerolineae
Nitrososphaeria
unidentified_Bacteria
unidentified_Gemmatimonadetes
Deinococci
Verrucomicrobiae
Dehalococcoidia
unidentified_Calditrichaeota
Acidobacteriia

Fig. 3 Relative abundance of bacterial community structure in the treated water (a) and biofilm (b) of BAC at the class level(W: water, B: biofilm; F4: F400, F6: F600; T: top layer, M: middle layer, B: bottom layer)

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P31

Efficient multi-site operation of optical communication between satellite
and ground
Naoki Akiyama1 and Akane Kusabuka2
1. Gifu University
2. CHUBU Electric Power Co.,Inc.

INTRODUCTION
In space communication, in addition to the conventional
communication using electromagnetic waves, the establishmentof communication technology using laser light has beenpromoted in recent years. In this space optical communication,
the frequency of the carrier wave is high. It is excellent in termsof miniaturization and weight reduction of equipment, and highspeed and large capacity communication. However, when thistechnology is applied to satellite-to-ground communications,the effects of clouds blocking the laser beam cannot be avoided.
Asai et al.(2017) estimated the transmittance of laser light byreproducing clouds in the atmosphere using a weather forecast
model. Their estimation results reproduce the actual
measurements with high accuracy.
Therefore, in this study, we estimated the atmospherictransmittance for the whole area of Japan using their method.
Furthermore, by making the ground station multi-site, we study
to improve the probability of stable communication andevaluate the efficiency improvement effect.

MATERIALS AND METHODS

Prefecture, the correlation coefficient is small and the annual
average atmospheric permeability is high. Establishing a site atsuch a point is thought to lead to higher efficiency.

Fig. 1: Annual average atmospheric transmittance
distribution throughout the Japanese archipelago

In this research, first, the meteorological field is reproducedby meso-meteorological model WRF. Using the result obtained
from the calculation, the atmospheric transmittance is output by
the atmospheric transmittance estimation model. The target
period is one year from January to December 2016. The
horizontal resolution of the estimation model is 0.05° × 0.05°
and the target wavelength of the laser beam is 1550 nm.

RESULTS AND DISCUSSION
Figure 1 shows the distribution of annual averageatmospheric transmittance in Japan in 2016. This figure showsthat there is a clear difference in the distribution of atmospherictransmittance between the Sea of Japan and the Pacific Ocean.
This is thought to be due to the difference between the Sea ofJapan climate and the Pacific Ocean climate. The regions withhigh annual average atmospheric transmittance are the Okazaki
Plain in Aichi Prefecture, Yamanashi Prefecture, a part ofNagano Prefecture, the Osaka Plain, and the remote islands in
Okinawa Prefecture, which are about 60% to 70%. Comparing
the results of 2016 and 2011, the first candidate for the standard
for multi-site conversion was Hokuto City, Yamanashi
Prefecture.
Laser light is attenuated by clouds. Therefore, the efficiency
improvement of the system by multi-site (installing multiple
ground sites) is examined.
Figure 2 shows the results of calculating the correlation
coefficient around Hokuto City using the estimatedatmospheric permeability. The site switching cycle is 1 hour. In
this figure, a region with a high correlation coefficient spreads
in the vicinity of Hokuto City, and the correlation coefficientdecreases with increasing distance. In other words, the greater
the distance between each ground site, the higher the efficiency
improvement effect can be expected. In Obihiro City, Miyazaki
Prefecture, Miyazaki City, and Miyakojima City, Okinawa

Fig. 2: Correlation coefficient of transmittance based on
Hokuto City (Red-dotted point)
In order to evaluate the probability that communication canbe performed stably, it is necessary to set the atmospherictransmittance that allows stable communication. However, theestimated atmospheric transmittance does not take into accountall communicable states such as momentary cloud breaks.
Therefore, using the observation result of the solar radiation
intensity by the PV module sensor, we investigated therelationship between the probability of actual communication
being possible and the atmospheric transmittance, and derived
the "atmospheric transmittance that can be considered as stablecommunication". .
Fig. 3 is a CI (clear index) graph showing the degree of solar
radiation intensity. For ideal clear weather, the CI is 1.0. Figure
4 shows a scatter plot that plots the annual average airpermeability using this result on the horizontal axis and theprobability that the CI exceeded the threshold value of 0.6 perProceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P31
hour on the vertical axis. Regarding solar radiation fluctuations,data from April 2016 to March 2017 was used, and the target
time was 9:00 to 15:00.
In order to perform satellite-to-ground opticalcommunication smoothly, it is required that the thresholdexcess rate is at least 50%. From Fig. 4, when the probability
that the CI exceeds the threshold value of 0.6 is 0.5, that is, 50%,
the maximum value of hourly average atmospherictransmittance is 0.7. In addition, when the hourly averageatmospheric transmittance is 70% or more, the probability that
CI per hour exceeds 0.6 is plotted in a range where all points
except two are higher than 50%. In other words, the condition
required for atmospheric transmittance for stablecommunication is that the probability that the CI exceeds 0.6 is
50% or more, and it means that the hourly average atmospherictransmittance is 70% or more on the model. Therefore, in thisstudy, we evaluate the actual efficiency improvement effect ofmulti-site by setting the condition that the stablecommunication is possible as “atmospheric permeability 70%
or more”.

Miyakojima City, it improved to about 0.86. This is 223.3 daysa year at one point in Hokuto City when converted to thenumber of days in a year (366 days). When combined with
Obihiro City, 285.5 days a year, when combined with Miyazaki
City, 289.1 days a year, and when combined with Miyakojima
City, 314.8 days a year.

Fig. 5: Excess rate of 70% atmospheric transmittance atany point

Fig. 3: Solar radiation intensity time series graphFig. 6: Distribution of excess rate of 70% atmospherictransmittance when multi-site is made based on Hokuto City
(Red-dotted point)

REFERENCES
Fig. 4: Atmospheric transmittance and excess of CI 0.6
Figure5 shows how many hours the atmospherictransmittance exceeds 70% in one year (24 hours x 366 days =8784 hours) at any point. In Hokuto City, Yamanashi
Prefecture, the excess rate is as high as the atmosphericpermeability shown in Figure1, which is considered to beappropriate for the multi-site standard.
Figure 6 shows the distribution of excess rate of 70% air
permeability when multi-sites are established based on Hokuto
City. In the case of only one point in Hokuto City, the ratio thatcan be said to be able to communicate stably in a goodcommunication environment is about 0.61. On the other hand,
it was found that multi-sites were improved at two points, andwhen combined with Obihiro City, it improved to about 0.78,
combined with Miyazaki City, about 0.79, and combined with・WRF Model Users Page:http://www.mmm.ucar.edu/wrf/
・ Stephens, G. L.: Radiation Profiles in Extended Water
Clouds. II: Parameterization Schemes. J Atmos Sci, 35, 21232132, 1978.
・ Asai, J., T. Kobayashi, J. Yoshino, H. Yoshida, Y.
Takayama: Estimation of atmospheric transmittance for opticalcommunications between satellites and ground，Proc. of 61st
Space Sciences and Technology Conf. ， 2A06 ， 2017 (inJapanese)・Akiyama, N., T. Kobayashi, J. Yoshino, H. Yoshida, Y.
Takayama: Estimation of atmospheric transmittance for
optical communications between satellites and multi-sites onground, Proc. of 62nd Space Sciences and Technology
Conf.，1G06，2018 (in Japanese))Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P32

Vermicomposting of fruit and vegetable waste with the addition of excess
activated sludge: A feasible approach leading to final products of higher
utilization value
Wenjiao Li 1 and Fusheng Li 2
1. Graduate School of Engineering, Gifu University, Gifu, Japan
2. River Basin Research Center, Gifu University, Gifu, Japan
cabbage, lettuce, carrot, potato, and EAS were added
respectively. For the remaining five substrate compartments,
INTRODUCTION
100 g (wet basis) of the mixed substrates of each type of FVWwith the EAS (FVW:EAS = 3:2, wet basis) were addedFruit and vegetable waste (FVW) are generated in large
respectively. All reactors were kept in dark and were run underquantity during production, processing and consumption.
25 °C.
Several conventional methods are used for treatment and/or
Analysis
recycling of FVW, such as incineration, landfill, anaerobic
The earthworms were taken out by hand and weighted every
digestion, feeding and composting. Compared to thesethree days after washing with pure water and wiping with tissue.
methods, vermicomposting has the advantages of effective
Dehydrogenase activity (DHA), a parameter reflecting thestabilization of the organic wastes through the joint function of
overall activity of existing bacteria, was determined by using
earthworms and microorganisms in decomposition of easily
the triphenyl tetrazolium chloride (TTC) method. Total carbon
decaying organic constituents, and of high utilization value forand total nitrogen were measured using a nitrogen and carbon
its final product as fertilizer. On the other hand, excess activatedanalyzer (SHIMADZU, Japan). Total phosphorus was
sludge (EAS), as the main byproduct of sewage treatment
measured by using the molybdenum blue-absorption method
process, is also an organic waste. However, since EAS is mainlywith a spectrophotometer at the wavelength of 880 nm.
consisted of microorganisms and can be also treated byvermicomposting, it is reasonable to consider that the wide
RESULTS AND DISCUSSION
variety of microorganisms contained in EAS may enhance themicrobial activities and contribute positively to thedecomposition of FVW since the complex microbial
Effect of EAS on earthworms
communities are reported to play a key role during the
The body weight of earthworms increased in the initial periodvermicomposting (Chen et al., 2018). In addition, the richand then declined gradually in the end of vermicomposting forcontent of proteins contained in EAS may promote the growthmost treatments. Such a decline was corroborated by otherof earthworms and specific bacterial species, thus improving
studies and can be explained as the ageing of substrate
the stabilization efficiency of FVW and also may improve thematerials. In general, earthworms in the treatments for FVW
utilization value of the final product.
with the addition of EAS exhibited a distinct trends of weight
Accordingly, the overall objective of this study was togain, as compared to the earthworms in the treatments for FVW
investigate the effect of EAS on vermicomposting of FVW,
alone (data not shown). The growth rate of earthworms differed
which included the following specific aims: 1) to clarify thewith the types of FVW during the vermicomposting. There was
effect of EAS on earthworms; 2) to evaluate the effect of EAS
a negative growth rate occurred in the treatment for banana
on decomposition efficiency of FVW; 3) to clarify the effect ofpeels, cabbage, potato and its mixture with EAS but a positive
EAS on the utilization value of final product.
growth rate in the treatment for others. The maximum growthrate was found in the treatment for lettuce with the addition ofEAS (27.7 mg/worm/day), which is about twice the value of the
MATERIALS AND METHODS
treatment for lettuce alone (14.3 mg/worm/day). In addition,
mixing EAS also promoted the cocoon production in treatment
Materials
for banana peels, cabbage, carrot and potato as the number of
The epigeic earthworm species Eisenia foetida was chosen forcocoons in the reactors treating banana peels, cabbage, carrot
this experiment due to its wide tolerance against environmental
and potato mixed with EAS (2, 2, 3, and 3, respectively) was
variables. Five types of FVW, namely banana peels, cabbage,larger than that treating respective FVW alone (0, 0, 0, and 2,
lettuce, carrot and potato, were cut into pieces with a width
respectively).
about 1 cm (and a thickness of 2 mm for carrot and potato)
Effect of EAS on pH and electrical conductivity
before experiment. In order to avoid the threat to earthworms
The pH value in the substrate compartments increased fromfrom leachate generated during vermicomposting, a supporting
the initial range of 5.51 - 6.07 to the final range of 6.13 - 8.65
bed was designed. The bed material was a mixture of soil andat the end of vermicomposting. For the bed materials, where thethe final product from previous vermicomposting of FVW. For
initial pH was 5.76, a variation of pH in the range of 5.41 - 7.80
EAS, dewatered sludge from a slaughterhouse wastewater
was recorded. The variation of pH is probably in reflection oftreatment plant in Gifu, Japan was used.
the changes in the processes of organic acid production andExperimental set up
mineralization during vermicomposting.
Eleven plastic containers were used as vermicomposting
The changes in the electrical conductivity (EC) before andreactors. Each reactor was formed with two compartments,
after vermicomposting are showed in Table 1. EC decreased
namely a substrate compartment and a bed compartment. The
substantially in the substrate compartments of all reactors after
two compartments were separated by a plastic plate opened
vermicomposting. The maximum decreasing rate was measured
with eight holes for earthworms to move freely between the twoin the treatment for lettuce and photo as 76.1%, followed by thecompartments. For the bed compartments, 100 g (wet basis) bed
treatment for carrot as 75.5%. The minimum decreasing ratematerial was added and then 10 earthworms with an individual
was found in the treatment for EAS as 36.7 %. The decrease ofweight of 350-500 mg were invited. For the substrate
EC during vermicomposting could be explained by reason thatcompartments of six reactors, 100 g (wet basis) of banana peels,

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P32

Effect of EAS on decomposition efficiency
The total carbon decreased in all reactors except thetreatment for banana peels, lettuce and carrot. The total carbon
was lost as CO2 during vermicomposting owing to theconsumption of the available carbon as a source of energy forearthworms and microorganisms. A higher decreasing rate in
the substrate compartment was observed in the treatment for
FVW with the addition of EAS (data not shown). This may becontributed to the better growth of earthworms in the treatment
with the EAS.
Looking into the decomposition efficiency from a view of
microbial activity, a lower DHA was observed in the treatment
for FVW except for carrot, but a higher DHA in the treatment
for FVW with the addition of EAS (data not shown). An
extremely higher DHA was found in the treatment for carrot
and a lowered DHA was found in treatment for its mixture with
EAS. Different types of FVW probably related to different
decomposition efficiencies of phenolic compounds duringvermicomposting, thus causing the different values of DHA.
All results indicated that the EAS enhanced the decomposition
efficiency of FVW by improving the microbial activities of
FVW or by providing higher nutrients for earthworms.
Effect of EAS on utilization value of final productAs displayed in Fig. 1, a significantly higher total nitrogen
was observed in both the substrate and bed compartments of thetreatment for FVW with the addition of EAS after
vermicomposting, except the bed compartment of the treatment
for carrot. It may imply that earthworms were provided with afavored condition for nitrification and/or nitrogenmineralization. The loss of organic carbon, reduction in dry
mass of the substrates, and the addition of nitrogenous
substances etc. could be the major causes for the increase of
nitrogen during the vermicomposting. The total phosphorus
showed similar results with the total nitrogen, as could be seen

Total nitrogen (g/kg)

80

FVW

EAS

FVW + EAS

60
50
40
30
20
10

60

Total nitrogen (g/kg)(a) Substrate compartment

70

0Banana peels

Cabbage

Lettuce

CarrotFVW(b) Bed compartment

PotatoEASEAS

FVW + EAS

50
40
30
20
10
0

Banana peels

Cabbage

Lettuce

CarrotPotatoEAS

Fig. 1 Total nitrogen in the (a) substrate compartment andthe (b) bed compartment after vermicomposting. Data arepresented as mean and standard deviation (n = 3).
16

Total phosphorus (g/kg)

Banana peels
Banana peels + EAS
CabbageCabbage + EAS
Lettuce
Lettuce + EAS
CarrotCarrot + EAS
PotatoPotato + EAS
EASElectrical conductivity (mS/m)
Substrate compartment
Bed compartment
Changes
Changes
Initial Final
Initial Final
(%)
(%)
455
146.8
-67.7
72.6
159.4
119.6
589
161.2
-72.6
72.6
162.4
123.7
347
86.1
-75.2
72.6
162.2
123.4
-56.2
72.6
162.3
123.6
308
134.9
-76.1
72.6
77.2
6.3
258
61.6
244
67.9
-72.2
72.6
124.0
70.8
420
102.9
-75.5
72.6
114.5
57.7
311
89.4
-71.3
72.6
127.2
75.2
529
126.6
-76.1
72.6
159.6
119.8
387
131.8
-65.9
72.6
173.0
138.3
197
124.7
-36.7
72.6
157.8
117.4

from Fig. 2. The higher phosphorus concentrations can beassumed as the result of the reason that the added EAS
encouraged the abundance of enzymes from earthworms andthe activity of microorganisms, thus enhanced themineralization and mobilization of phosphorus.
In summary, all data revealed that adding EAS into thevermicomposting of FVW could be a feasible option to enhancethe decomposition efficiency of FVW and to enrich theutilization value of the final product of vermicomposting.

Total phosphorus (g/kg)

the decomposition of organic substrates can lead to a great lossof dissolved ions (Huang et al., 2017). In contrast, the EC in all
bed compartments increased from the initial level of 72.6 mS/m
to 77.2 - 173.0 mS/m after vermicomposting. It is worthy of
special notice that the increasing percentage of EC in thetreatment for FVW with the addition of EAS was higher thanthat in the treatment for FVW alone except for cabbage. For thetreatments of lettuce and carrot with or without the addition of
EAS, the increasing percentage was a little bit lower comparedto other treatments. The lower increasing percentage may be inassociation with the higher growth rate of earthworms in these
two treatments (27.7 and 21.0 mg/worm/day). A larger amountof leachate was released from the treatments for lettuce andcarrot due to their higher water content and was transferred tothe bed compartment where some ions in the leachate wereconsumed by earthworms for their body growth.
Table 1 Changes of electrical conductivity in all substratecompartments and bed compartments. The positive values
mean increasing percentages, and the negative values mean
decreasing percentages.

14

FVW

(a) Substrate compartment

EAS

FVW + EAS

12
10
8
6
4
2
0
12

Banana peels

Cabbage

Lettuce

CarrotFVW(b) Bed compartment

PotatoEASEAS

FVW + EAS

10
8
6
4
2
0

Banana peels

Cabbage

Lettuce

CarrotPotatoEAS

Fig. 2 Total phosphorus in the (a) substrate compartment
and the (b) bed compartment after vermicomposting. Data
are presented as mean and standard deviation (n = 3).

REFERENCES
Chen, Y., Chang, S.K.C., Chen, J., Zhang, Q., Yu, H. (2018)
Characterization of microbial community succession duringvermicomposting of medicinal herbal residues. Bioresour.
Technol. 249, 542–549.
Huang, K., Xia, H., Cui, G., Li, F. (2017) Effects of earthworms
on nitrification and ammonia oxidizers in vermicomposting
systems for recycling of fruit and vegetable wastes. Sci. Total
Environ. 578, 337–345.

Proceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P33

Inhibition of Arsenic transfer from contaminated soil to vegetation bywood ash
Shiamita Kusuma Dewi1, Huijuan Shao2, Shunichiro Mitsunaga 3 and Yongfen Wei2
1. Graduate School of Natural Science and Technology, Gifu University, Japan
2. River Basin Research Center, Gifu University, Japan
3. Faculty of Engineering, Gifu University, JapanINTRODUCTION
Arsenic (As) is a toxic metalloid which is ubiquitous innature. Because of its wide abundance, humans are always
exposed to it that exists in food, water, air, and soil. Especially,
Arsenic in the soil can be transferred to plants and hence
threaten human health via food chain. In Japan, it is reported
that there were 14 Areas (391 ha) of soils contaminated with As
over 15 mg kg-1 (Ministry of the Environment, 2010).
Wood ash is the residual powder left after the combustion ofwood, such as burning wood in a home fireplace or an industrial
power plant. Since wood ash is composed of organic matter, Ca,
Mg, Fe and other minerals, while Smith et al (2002) reported
that the presence of Ca+ and Na+ led an increase in the retentionof As and also Fe-oxides represented the role major sink for As
sorption in soil; and Gadepalle et al (2007) also indicated thatapplication of organic matter reduced the mobility of arsenic insoil, therefore wood ash is considered to be an effective material
to influence the behavior of As in the soil-vegetation system bychanging soil chemical/physical characteristics.
In this study, to investigate the inhibition effect of wood ash
on As transfer from soil to vegetation, indoor pot experiment
was designed and carried out. At the same time, the distributionof As in different parts of vegetation was also examined.

Fig. 2 Experimental design
Pot experiment and analysis – Komatsuna (Brassica rapa
var. perviridis) was used in the pot experiment since it is a leaf
vegetable that grow very fast and can actually be cultivated year
round. Komatsuna was cultivated inside the room with constanttemperature 25° C in Gifu University from Dec. 14, 2018 toApril 2, 2019, and no additional fertilizer was added during thisperiod. After harvest, the vegetation was washed and separated
into three parts including leaf, stem and root, then further hotdried in oven at 80°, followed by shattering by crusher anddigested with nitric acid in autoclave. As concentrations of thedigested samples from different parts were measured using
inductively coupled plasma mass spectrometry (ICP-MS,
Agilent 7700X).

MATERIALS AND METHODS
Soil and additive samples – The mixture of two commercial
products: Gardening soil and Decomposed granite soil withratio of 3:2 by dry-weight, as shown in Fig. 1 is used as soil
media. Two different shapes of wood ash, namely powder andgranular shape, were used in the study. Wood ash are madefrom Cryptomeria or Japanesse red-cedar wood. The properties
of soil and wood ash are summarized in Table 1.

7 days21 days

35 days

60 daysFig. 3 Komatsuna in different growth stages
Table 1 Properties of soil and wood ash used in this study
Specific
Electrical
Organic
surface
SamplepH
Conductivity
Matterarea(mS/m)(%)(m²/g)A

B

C

Fig.1 Soil sample (B: mixture of gardening soil anddecomposed granite soil) and Gardening soil A and
Decomposed granite soil C
Experimental design (Fig. 2) – Na2HAsO4 was used sincearsenate was the main As species in the aerobic soil. The soil
sample was in advance adjusted to four different As
contamination levels with Na2HAsO4 (0, 50, 150, and 300 mgkg-1), and then powder ash and granular ash were separately
added to the soil at three different ratios (0, 1%, 5% and 10%,w/w). 600 g of soil was placed in each plastic pot, having aheight about 10 cm and a perimeter about 15 cm. No additive
applied soil was used as control for comparison. Each treatment
had two replicates. Totally, 56 pots were prepared.

Soil

6.08

81.2

12.361

4.014

PowderWood Ash

12.78

457

0.743

17.418Granular
10.15
214.3
7.202
0.082
Wood Ash
All values represent the mean for each sample. pH and EC were
measured using pH and EC meter (sample: water = 1:5).
Organic matter was measured by Loss-on-Ignition method.
Specific surface area was measured using 3Flex micrometrics.

RESULTS AND DISCUSSION
Komatsuna growth status – As shown in Fig. 4 (Granularwood ash was taken as an example), the addition of wood ash
seemed to be promoting the growth of Komatsuna cultivated inProceedings of International Symposium on a New Era in Food Science and Technology 2019UGSAS-GU & BWEL Joint Poster Session on Agricultural and Basin Water Environmental Sciences 2019: P33
soil with As concentration of 50 mg/kg. However, in the caseof higher As contamination (150 mg/kg and 300 mg/kg), it
looked like that the addition of the ash conversely suppressed
the growth of the Komatsuna. On the other hand, no obvious
difference was found between the influences of two different
ashes on Komatsuna growth.
2

Root

1.8

Stem

Leaf

1.6

Dry weight (g)

1.4
1.2
1
0.8

et al. (2008) described that Ca2+ and Mg2+ can induce theadsorption of phosphate and arsenate in soil.
Competition between Arsenic and Phosphorus – As shown in
Fig. 5-6, arsenic addition showed a positive effect on As
transfer to Komatsuna. But according to Fig. 7, we found that
arsenic addition had a negative effect on phosphorus transfer toKomatsuna from soil; the lowest phosphorus transfer
corresponded to the highest contamination level of As (300mg/kg). This might be due to arsenate is a structural analogueof phosphate, replacing phosphate arsenic entered plant cells
via phosphate transporters and also interfered phosphate’s
metabolism (Zhao, 2010).
25

0.6
0.2
0

As: 50
mg/kg

As: 0

As: 150
mg/kg

As: 300
mg/kg

Fig. 4 Dry weight of different parts of Komatsuna under
different addition ratio of granular wood ash (G) anddifferent contaminated As level
Arsenic amount accumulated in different parts of vegetation– As shown (Fig. 5, Fig. 6), As amount accumulated in root was
the highest, followed by leaf and stem follow each As
contaminated level. And obviously, the addition of powder
wood ash decreased As accumulation in Komatsuna as
compared to that of control, except the addition of 1% powder
ash at 50 mg/kg
As amount in the vegetation (mg/pot)

0.3
Root

0.25

Stem

20

Root

Stem

Leaf

15
10
5
0

As: 50mg/kg

As-transfer

As: 150 mg/kg

As: 300
mg/kg

Fig. 7 Phosphorus amount accumulated of different parts ofKomatsuna under different addition ratio of granular woodash (G) and different contaminated As level
CONCOLUSION
In this study, the inhibition effect of wood ash on As transferfrom soil to Komatsuna was investigated via an indoor pot
experiment. The obtained results showed that wood ash had thecapacity to reduce the amount of As accumulated in
Komatsuna.

Leaf

0.2
0.15
0.1

REFERENCES

0.05
0

As: 150
mg/kg

As: 50mg/kg

As-transfer

As: 300
mg/kg

Fig. 5 Arsenic Amount of different parts of Komatsuna
under different addition ratio of powder wood ash (P) anddifferent contaminated As level
0.35

As amount in the vegetation (mg/pot)

P amount in the vegetation (mg/pot)

0.4

0.3
0.25

Root

Stem

Leaf

0.2
0.15
0.1
0.05
0

As: 50mg/kg

As: 150
mg/kg

A t

f

As: 300
mg/kg

Fig. 6 Arsenic Amount of different parts of Komatsuna
under different addition ratio of granular wood ash (G) anddifferent contaminated As level
As contamination level (Fig. 5) and the additions of 5%
granular ash at 50 mg/kg, 1% granular ash at 300 mg/kg As
contamination levels, respectively (Fig. 6). The minerals
included in the wood ash such as Fe, Al, K, Ca and Mg (datanot shown) is considered to be a responsible reason for causing
an increase in the As retention in the soil. Similarly, Stachowicz

Gadepalle VP, Ouki SK, Van HerwijnenR, Hutchings T(2007) Immobilization of heavy metals in soil using naturaland waste materials for vegetation establishment on
contaminated sites. Soil Sedim Contam 16:233-251
Ministry of the Environment (2010) Enforcernent Status of
Agricultural Land-Soil Pollution Prevention Law in fiscal
2008,
MinistryoftheEnvironment,Japan,http:llwww.enugo.jplpre$s/fleviewLphp?serial=16311&hou-id=12979
Smith E, Naidu R,Alston AM (2002) Chemistry of inorganic
arsenic in soils. II. Effect of phosphorous, sodium, andcalcium on arsenic sorption. J Environ Qua l31:557–563
Stachowicz M, Hiemstra T, van Riemsdijk WH (2008) Multicompetitive interaction of As (III) and As(V) oxyanionswith Ca2+, Mg2+, PO3−4, and CO2−3 ions on goethite. J
Colloid Interf Sci 320: 400-414
Zhao FJ, McGrath SP, Meharg AA (2010) Arsenic as a Food
Chain Contaminant: Mechanisms of Plant Uptake andMetabolism and Mitigation Strategies. The Annual Reviewof Plant Biology 61:535-59j

The United Graduate School of Agricultural Science, Gifu University
1-1 Yanagido, Gifu 501-1193, Japan
Tel: +81-58-293-2984 (or, 2985)
E-mail: renno@gifu-u.ac.jp
Home Page: http://www.ugsas.gifu-u.ac.jp/eng/

Promotion Office of Gifu University Rearing Programfor Basin Water Environmental Leaders, Gifu University
1-1 Yanagido, Gifu 501-1193, Japan
Tel: +81-58-293-2085
E-mail: bwel@green.gifu-u.ac.jp
Home Page: https://www.green.gifu-u.ac.jp/BWEL/eng/index.html

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