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Vol. 26
26（2021）（2021），75 ～The Effect of Low Temperature on Biochemical Properties andBioactivity of Selected Malaysian Macroalgae Species
Ong Kuan Hung*1, Kishneth Palaniveloo*1 and Phang Siew Moi*1,2*1 Institute of Ocean and Earth Sciences, University of Malaya, Wilayah Persekutuan Kuala
Lumpur 50603, Malaysia.
* Department of Biotechnology, UCSI University, UCSI Heights, 1, Jalan Puncak Menara
Gading, Taman Connaught, 56000 Cheras, Wilayah Persekutuan Kuala Lumpur.
2

Abstract
Seaweeds are commercial valuable products with high biochemical properties and bioactive compounds. The
effects of low temperature (15℃, 20℃, 24℃ and 28℃) on the biochemical properties and bioactivity ofCaulerpa lentillifera, Ulva sp and Gracilaria sp will be evaluated. The total protein, amino acid, lipid, fatty acid,moisture, ash and carbohydrate content of seaweed cultured under different low temperatures will be compared.
The seaweeds’ metabolite profile changes will be determined using spectrometry and chromatographic
techniques. The antibacterial and antioxidative properties of all seaweed extracts will be evaluated.
Keywords: Temperature, Biochemical, Metabolite, Seaweed, Caulerpa, Ulva, Gracilaria

1. Introduction
Seaweeds are multicellular autotrophic marine organisms, which consist of seagrass and macroalgae. For decades,
coastal communities have widely consumed a variety of seaweed species. Recently, they have increased the commercial
value of seaweed in the global market, both as an edible source for commercial applications and as a reservoir for
pharmaceutical research1,2. Approximately 40% of seaweeds were farmed for food, and over 23 million tonnes ofaggregated production isfrom China and Indonesia1. Many western countries have started to farm seaweed to compensate
for the increased market demand2. Seaweeds are also gaining popularity among vegetarians and vegans dueto greater
nutritional properties as an alternative to terrestrial plant plan. Therefore, many organizations are interested in the
exploratory studies of seaweed’s nutritional value to promote and protect public health 3.
Seaweeds have varying biochemical profiles, leading to different tastes and consumption benefits4. Caulerparacemosa,a Chlorophyte is a top-tier delicacy that is commonly eaten raw as salad in South-East Asia, while Saccharinajaponica, aPhaeophyte is used to make broth in both China and Japan4,5,6. Seaweeds are also rich in carbohydrate content,especiallydietary fibre. Ulva lactuca was found to have 54% dietary fibre (dry weight) which is higher than broccoli leaves (32%of dry weight)7,8. On the other hand, their protein content varies by seaweed groups. For instance, low proteincontents havebeen found in brown macroalgae (5-24% dry weight) while higher protein content was found in green and red macroalgae(10-47% dry weight)9. As food, seaweeds are packed with flavour enhancers, glutamic acid which are responsible for theumami flavour when consuming 4,10,11. Most edible seaweed had been found to contain levels of beneficial fatty acids,suchas eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)10,12,13. Both EPA and DHA are omega-3 fatty acidswhich
are linked to proper foetal development, improving cardiovascular function and preventing mild Alzheimer's disease14.
Furthermore, seaweeds gained popularity in commercial product and pharmaceutical research industries. Alginate havebeen extracted from Phaeophyta since 1881 for wound dressing which promotes wound healing from surgery15.
Fucoxanthins have been found to exhibit antiproliferative effects on multiple cancer cells on animal model16. They also
exhibited positive results in reducing body weight during clinical trials16. Moreover, Caulerpin, unique to the genus
Caulerpa displayed antinociceptive, anti-tuberculosis, antiviral and anti-inflammatory activities in pre-clinical trial17,18,19.
Received date: 2021.11.30
E-mail of corresponding author: s2043080@siswa.um.edu.my

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Like other agriculture products, the iochemical content of seaweed is heavily affected by geolocation and seasonal
factors 20. These environmental factors could affect the yield of seaweed, its metabolites and even its morphology 21. A
study that observed seaweeds from temperate regions showed to have accumulated more total lipid content than seaweedsfrom tropical regions and have higher omega 3 fatty acids13. Furthermore, the biochemical composition of tropicalseaweeds are influenced by variation in light, salinity and water temperature 22. Secondary metabolites are a biologicalresponse toward environmental changes and stresses. Extract from Sargassum collected during monsoon season
demonstrated increased anti-cancer property and Caulerpa during the rainy season also showed a higher concentration of
caulerpin than dry season 23,24. Few studies have shown seaweed could accumulate heavy metal content in the environment
and even suggested them as a bioindicator for pollution 25,26. These variations caused by the environment was always achallenge faced by seaweed farming and this created a demand for the research on the quantitative effect of environmenton the seaweed growth and metabolites level 1,27. Thus, it is importanceto understand the impact of growing factor suchas temperature, salinity, light and water nutrient on seaweed growth and metabolite level. This research will attempt toinvestigate the nutritional and metabolites profile as well as metabolites bioactivity of selected Malaysian seaweed
species under a series of controlled low-temperature conditions (15℃, 20℃, 24℃ and 28℃).

2. Methoda)Seaweed stock establishment:
Fresh Caulerpa lentillifera, Ulva sp. and Gracilaria sp will be obtained from various sources to be cultured underlaboratory conditions. Collected seaweed will be washed clean with artificial seawater before quarantine andacclimatization in an aerated water tank at 28℃, 40 μmol photons m−2 s−1 with 12 h photoperiod. Salinity will bemaintained at 30 ppt. Seaweed growth curve will be determined based on changes in biomass and maximum
photosynthetic efficiency (Fv/Fm) will determined by using chlorophyll fluorometer (>0.5).
b)Experiment design:
To study the effect of temperature, each respective seaweed will be cultured in four different temperatures; 15℃,
20℃, 24℃ and 28℃ in a decreasing pattern. The temperature reduction will be controlled with a marine tank chiller.
One kilogram of seaweed will be cultured in the highest temperature for 30 days before being reduced gradually for aweek at a rate of 1℃ a day to prevent sudden stress in temperature changes. This process will repeat for every temperature
reduction. After 30 days at each temperature, 1.2 kg wet weight of cleaned seaweed will be harvested for biochemical
analysis (1 kg crude extracted in industrial grade ethanol, 200 g oven dried at 60℃ to prevent loss of valuable minerals).
Dried seaweed will be milled by using powder blender and stored airtight under -20℃ for analysis.

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The Effect of Low Temperature on Biochemical Properties
and Bioactivity of Selected Malaysian Macroalgae
Macroalgae Species
Species

Seaweed stock establishment

Experiment
Moisture & Ash

Dried for storing
Crude Extraction

Biochemical
Analysis







Lipid
Fatty acid
ProteinAmino acid
Carbohydrate

Biochemical profiling

Bioactivity assay

Characterization of
metabolites

Antibacterial assay

Antioxidant assay

Fig. 1 Experiment methodology flow chart.

c)Biochemical Analysis:
Seaweed chemical composition (moisture, ash, lipid, fatty acid, protein, amino acid and carbohydrate) will be
determined according to official method of analysis by AOAC 28.
1)
Moisture and ash content:
Moisture content will be estimated by measuring the weight loss of 100g fresh seaweed after drying in hot air ovenat 105°C until constant weight was obtained 29. The moisture content will be expressed as a percent of moisture in the
fresh seaweed mass (% moisture). The ash content was calculated using weight loss of 5g oven-dried seaweed powder
from moisture experiment after incineration at 550°C for 18h using electric muffle furnace. The ash content will be
expressed as a percentage of ash in the seaweed mass (%ash) 28.

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Ong Kuan Hung, Kisheth Palaniveloo, Phang Siew Moi

2)
Lipid content:
Crude lipid content will be determined with modified Folch extraction method described 31. Two-hundred milligramof dried seaweed powder will be weighed and homogenized with 5mL chloroform:methanol (2:1, v/v). The mixture will
be light agitated using vortex mixer at room temperature for 15 minute. The lipid containing chloroform:methanol extract
will be concentrated. The total lipid content will be determined gravimetrically with the weight between the residue anddried seaweed powder. The lipid content will also be expressed as percentage of lipid content of dried seaweed powder(%lipid).
3)
Fatty acid content:
Hundred milligram of lipid extract eluted over hexane: ethyl acetate (9:1) using a silica gel column will be subjectedto
fatty acid methyl ester (FAME) conversion. The concentrated eluent will be trans-methylated using 2.7ml of hexane and0.3ml of 2M sodium methoxide to obtain FAME. The sample will be subjected to continuous stirring with magneticstirrer
for 3h at room temperature. Transmethylation reaction in the sample will be stopped using 1ml chloroform- methanol(1:1) mixture. The sample will be dried using rotary evaporator until yellowish oil is obtained. Fatty acid composition
will be quantified using gas chromatography mass spectrometry (GC-MS) through a Silica BPX70 capillarycolumn (60 m,with a film thickness of 0.25 µm) according to method described in articles29. Input for GC-MS will be adjusted asfollowed: temperature injector-225°C; carrier gas-helium; flow rate-0.75mL/min; capillary column (Rt-2560,100 m, 0.25
mm ID, 0.2 um df) according to method describe in articles 29. Run method will be temperature gradient between 150 to
240 °C for 60 min. Identification and quantification of FAMEs will be accomplished by comparing theretention times ofthe peaks to internal library, National Institute of Standards and Technology (NIST) and the Flavors and Fragrances of
Natural and Synthetic Compounds library (FFNSC). Concentration of FAME will be calculated based onGC area of apeak compared to standards used and expressed as a percentage of individual FAs in the lipid fraction.
4)
Protein content:
Crude protein content will be estimated based on nitrogen composition in the sample analyzed using a CHNS analyzer
multiplied by a conversion factor of five 34. Hundred mg of dried seaweed powder will be packed in tin capsulewith anoxidizer which will be combusted in the analyzer. The analyzer will quantify the % nitrogen content (% N) in thesample
which will convert to % crude protein content (% crude = %N *5). The non-protein nitrogen content (%NPN) will beobtained from precipitated 250mg seaweed powder with 10% trichloracetic acid. Total protein content will be determinedfrom difference between %crude and %NPN and expressed as percentage of protein of dried seaweed powder(%protein)
35.
5)
Carbohydrate content:
Total carbohydrate content will be estimated by weight difference using the following equation 20, 36,
Carbohydrates = [100% - (% protein + % lipid + % ash + % moisture)].
6)
Amino Acid content:
The amino acid content will be determined with automatic online OPA/FMOC derivatization by RP-HPLC using a
Shimadzu LC system (Shimadzu Corporation, Kyoto, Japan) equipped with an LC-20AD pump, DGU-20AS degasser
and photodiode array SPD-M20A (PAD), and fluorescence RF-10AXL (FLD) detectors online and the procedures will
be according to 20,29. Two-gram dried seaweed powder will be hydrolysed with 2ml, 6M hydrochloric acid in screwedcapped test tube for 24h at 110°C. After hydrolysis, the mixture will be evaporated to dryness under vacuum then it will
be reconstituted in 2 ml of 0.1 M hydrochloric acid. Twenty microlitre sample will be derivatized with o-phthalaldehyde(OPA) and 9-fluorenylmethyl chloroformate (FMOC), prepared according to protocol30. Derivatized sample will befiltered through 0.22µm microfiltration membrane to remove microbe contamination. Filtered sample will be injected
onto the RP-HPLC system equipped with an HPLC column (Zorbax Eclipse XDB-C18 (4.6 × 250 mm) conditioned at 27± 0.1 ◦C. The OPA-3-MPA derivatives will be detected by a programmable fluorimeter with excitation (λex) and emission(λem) wavelengths set at 338 and 262 nm, respectively, while the FMOC derivatives will be identified at λex 260nm andλem 315 nm; wavelength change occurred at 18.5 min. The mobile phase used will be a combination of 10 mM disodiumhydrogen phosphate buffer: 10mM disodium tetraborate and 5 mM sodium azide adjusted to pH 8.2 eluent

The Effect of Low Temperature on Biochemical Properties
and Bioactivity of Selected Malaysian
Malaysian Macroalgae
Macroalgae Species
Species

7979(A), and a mixture of acetonitrile, methanol, and water with a ratio of 45:45:10 (v/v/v) as eluent B, at a flow rate of 1.0
mL min−1. The amino acid standards mix (AAS18, Sigma Aldrich, MO, USA) will be prepared by dissolving in 0.1 Mhydrochloric acid then be diluted appropriately to obtain a working solution with norvalin (Merck, MO, USA) used as theinternal standard 20. Concentration of amino acids will be calculated based on peak area with reference to standard andexpressed as a percentage.
d) Biochemical ProfilingTo establish secondary metabolite profile, a series of chromatographic tools, mass spectrometer (MS) will be used.
1) HPLC Profiling:
Moreover, high performance liquid chromatography (HPLC, Shimadzu) will be used to profile extracts from eachexperiment to observe for changes in production of secondary metabolites across the respective seaweeds cultured invarying temperature conditions. One milligram of dried seaweed extract will be dissolved in 1ml, HPLC grade methanol
to prepare a stock solution of 1mg/ml. The 0.22µm PTFE membrane filtered stock solution will be subjected to HPLC
analysis equipped with a Shimadzu C18 (100 mm × 4.6 µm) column and a Photodiode Array Detector. The solvent used
for mobile phase will be water (A) and acetonitrile (B), both will be acidulated with 0.1% formic acid analysed on a
gradient profile. Any compound of interest will be subjected to isolation.
2) Mass Spectrometry (LC-MS/MS)
The crude extracts will be diluted in deionized water and filtered through a 0.45μm nylon syringe filter. A 50ppm of
the filtrate will be prepared in triplicates and used for LCMS analysis. The LCMS analysis will be performed using liquid
chromatography G6550A system (Agilent Technologies, Inc., USA), which consisted of a degasser, a G4220A binary
pump with a pressure gradient unit, a dual AJS ESI MS QTOF detector and a G4226A HiP AutoSampler. A 4.6 x 100
mm Shimadzu C18 column w i l l b e utilized for separation (30 eV at 45°C and 20μL at 0.8 mL/min). A mobile phase of
24% of acetonitrile and 76% of water containing 0.2% formic acid for a separation time of 30 min will be applied following
the previous methods with minor modifications 37. Identification of compounds by the acquisition of mass spectra will be
conducted in both positive and negative modes, respectively, between m/z 100 and 1500.
e) Characterization of Metabolites
Based on the initial HPLC profile, extracts will be selected for targeted isolation. The dried seaweed extracts will be
subjected to column chromatography, a series of thin layer chromatography; derivatized with phosphomolybdic acidsolution 5 wt. % in ethanol were used as a derivatization reagent for observation of phenolics, hydrocarbon waxes,
alkaloids and steroids, and repeated preparative thin layer chromatography (PTLC) for compound isolation. Purification
will involve the use of HPLC. Pure isolates will be subjected to nuclear magnetic resonance spectroscopy (NMR) for 1D(1H, 13C, DEPT-135) and 2D-NMR (HSQC, 1H -1H COSY, HMBC, NOESY) experiments for characterization.
f) Bioactivity
1)
Antibacterial assay:
All extracts and compounds will be selected for antibacterial potential evaluation by determining the Minimum
Inhibitory Concentration (MIC) of extracts or compound according to Nagappan’s study 33. Tested microbe are
Escherichia coli and Staphylococcus aureus and will be preculture in respective growth medium and used for assay.
Colony forming unit (CFU/mL) of test bacteria will be obtained using serially diluted with ten-fold dilution factor in test
tube. Fractions will be diluted by two-fold in growth medium. About 10µl of diluted fraction will be serially added into
each of the sterile 96 well of microdilution tray loaded with 90µl of bacteria solution. Hundred microlitre of bacteria(negative control) and bacteria solution added with imipenem (positive control) will be used for comparison. Mixture andcontrol will be incubated at 37°C for 24h and checked for the presence of whitish sedimentation that indicates deadbacterial cells. Maximum turbidity in positive control will indicate minimum bacterial growth. Minimum inhibition
concentration (MIC) will be determined when a single skipped well occur. Assay will be carried out in triplicate forfractions tested.

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Ong Kuan Hung, Kisheth Palaniveloo, Phang Siew Moi

2)
Antioxidant assay:
Total antioxidant activity of the samples will be determined according to Prieto’s study 38. Selected fractions (100µl)
will be added with 900µl solution comprised of 28 mM sodium phosphate, 0.6 M sulfuric acid, and 4 mM ammonium
molybdate. The mixture will be incubated at 95 ºC for 90 min in a water bath. The absorbance will be measured at 695nm
using UV-Vis 2450 spectrophotometer (Shimadzu, Japan) and a standard curve (between 50 µg/ml and 250µg/ml) will
be determined using ascorbic acid. The total antioxidant activity is expressed as the number of equivalents of ascorbic
acid in milligram per millilitre of extract (mg ascorbic acid/ ml).
The radical scavenging effect of the sample will be determined by 2,2-Diphenyl-1-picrylhydrasyl (DPPH) assay
described by Manoj’s study 39,40. DPPH radical solution (0.5mM) will be prepared in methanol and series of butylatedhydroxytoluene (BHT) and butylated hydroxyanisole (BHA) standard will be prepared using DPPH solution, 0, 100, 200
and 400 µg/ml. One millilitre of DPPH solution and 3ml of sample will be mixed. Sample and standards will be incubatedfor 30min in the dark, then absorbance at 517nm will be measured. Decreasing the absorbance of DPPH solution indicatesan increase in DPPH radical scavenging activity. The radical scavenging effect will be calculated as %DPPH, using
formula 39,40.
% DPPH radical scavenging = [(control absorbance - sample absorbance)/(control absorbance)] × 100
h)Statistical analysis:
Results will be obtained from experiments performed on separate days. All data will be expressed in term of mean
± standard deviation. The means of the carbohydrate, lipid, protein, ash and moisture content will be examined forstatistical significance (p<0.05) between temperature treatments via one-way analysis of variance (ANOVA). The meansof data from antibacterial and antioxidant experiment will be for statistical significance (p<0.05) between temperature
treatments via one-way analysis of variance (ANOVA).

3. Experiment Progress
3.1 Stock establishment
Caulerpa was received from local latok seller who was supplied by a local farmer in Kuala Lumpur, Malaysia. The green
seaweed came in as 3.1kg but after washing and selection, only 2.5 kg of Caulerpa was able to use. Caulerpa was washedaccording to method mentioned and seaweed with intact stolon, rhizoids and fronds was selected. Soft and squishy
Caulerpa was removed or discarded. The green seaweed was washed three times before put into stock tank. Stock tankcondition is 28℃, 40 μmol photons m−2 s−1 with 12 h photoperiod and 30 ppt salinity. Stock tank is a three combinedtank
with aerated and filtered system. Therefore, approximately 1.25 kg of each Caulerpa was added in a tank and total of twotank was used. Weekly photo image was taken to monitor the growth visually, Fig 2 showed the seaweed growth in one
tank fromtime points (1 Sept, 28 Sept, 6 Oct, 21 Oct, 8 Nov and 26 Nov). From Fig 2, seaweed in tank 3 had obvious visual
growthafter a month in stock tank. The Caulerpa was observed to gradually fill up the tank and new stolon, rhizoids andfrondscan be observed along the acclimatization period. Another tank showed the same pattern so it is not showed. On 26
Nov, seaweed in tank 3 was weighted and 2.215kg was recorded. Using weekly specific growth rate (SGR)
equation, %SGR
= [ (weightt – weight0)/( weight0 x weeks) ]x 100%, weekly specific growth rate of 6.43% was achieved. This showed anaverage of 6% increment in fresh seaweed biomass every week for 2 months. A growth curve will be needed to determine
the life cycle stage of the Caulerpa and Fv/Fm will also be used as indication for seaweed stress to standardize the seaweed
growth condition as stock. Currently, the frequency for replenishing artificial seawater will be increased for theseaweed
to establish growing seaweed stock. After successful establish growing seaweed stock, the temperature experiment will
be initiated.

The Effect of Low Temperature on Biochemical Properties
and Bioactivity of Selected Malaysian
Malaysian Macroalgae
Macroalgae Species
Species81
81Fig. 2 Photo image of stock Caulerpa growth, chronologically (1 Sept, 28 Sept, 6 Oct, 21 Oct, 8 Nov and 26
Nov).

a)

b)

c)

Fig. 3 Photo images of new growth of a) stolon, b) rhizoid and c) fronds.

4. Expected Output
As mentioned, seaweeds growth and biochemical level are easily affected by environmental factors (light intensity,
temperature, water nutrient and salinity) 13,20,21. Therefore, it is expected to find changes in seaweed biochemical level(carbohydrate, protein, amino acid, lipid, fatty acid, ash and moisture) due to seaweed growing in low temperature. A
different biochemical levels can lead to changes in seaweed metabolite profile in different temperatures. Therefore, it isexpected to discover new bioactive compound that is produced by the changes in seaweed metabolite profile. This research
will also isolate and characterize the new compound for its bioactivity and aim to develop the commercial potential of thecompound. Studies that found the increased bioactivity of seaweed extract in the rainy season also suggested thatenvironmental changes can increase seaweed bioactive compound level 23,24. Therefore, this experimentis expected to findan increased in bioactive compound level from seaweed cultured in low temperature. Increased bioactive compound leveland production of new bioactive compound also would suggest an increased in the bioactivity level of seaweed extract. It
is to be expected to increase the antibacterial and antioxidant level of the seaweed extract.

5. Acknowledgement
This is supported by the Science and Technology Research Partnership for Sustainable Development (SATREPS)
Program entitled ‘Development of Advanced Hybrid Ocean Thermal Energy Conversion (OTEC) Technology for Low
Carbon Society and Sustainable Energy System: First Experimental OTEC Plant of Malaysia’ funded by Japan Scienceand Technology Agency (JST) and Japan International Cooperation Agency (JICA), and Ministry of Higher Education
Malaysia (MoHE) and led by the Institute of Ocean Energy Saga University (IOES) of Japan, and UTM Ocean Thermal
Energy Centre (UTM OTEC), Universiti Teknologi Malaysia (UTM). Registered Program Cost Centre:
R.K130000.7809.4L887, Project [Cost Centre: Project No IF045-2019].

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Malaysian Macroalgae
Macroalgae Species
Species

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