PARTⅤ SOLVOLYSISSEPARATION AND CHARACTERIZATION OF PECTIN
FROM JUICE PROCESSING RESIDUE EXTRACTEDBY SUB-CRITICAL WATER
Munehiro Hoshino1, Masahiro Tanaka*2, Akihiro Terada2,Mitsuru Sasaki2, Motonobu Goto2
1

ASCII Co. Ltd., 2425 Tabara, Kawasaki-mati Tagawa-Gun, Fukuoka, 827-0004, Japan
2

Graduate School of Science and Technology, Kumamoto University,
2-39-1 Kurokami, Kumamoto 860-8555, Japan.

E-mail: 074d9104@st.kumamoto-u.ac.jp
Abstract: Citrus Junos is an evergreen tree whose fruit processed into juice and often
preferred to vinegar as an ingredient in sauces and salad dressing for its special flavor. Most
food processing residues are disposed without being recycled. These residues are mainlycomposed of the part of peel, juice sack and seed. Especially, peel includes valuablehydrocarbons such as terpenoid and carbohydrate such as pectin and cellulose. Sub-criticalwater has solubility and reactive selectivity to polar substance carbohydrate such as pectin andcellulose based on pressure and temperature. Pectin is composed of an acidic polysaccharide
and several types of neutral sugars. The main chain consists of a-1, 4-linked D-galacuronic
acid, which is partly methyl esterified. Conventionally using mild acid or chelating agent,
pectin can be extracted and separated from the waste of beet pulp and citrus fruits such aslemon and apple pomace, which is an effective utilization of industrial waste. In this work, weaimed to develop an effective separation method for pectin using subcritical water from theresidue of Citrus junos and to examine the characteristics of pectin. Experimental apparatus
we used was semi-continuous flow reactor. Experimental condition follows: at thetemperature range of 110-160oC, at the pressure range of 4-30MPa. As a result, the yield ofpectin was more than 75% at the temperature range from 120-140℃ and pressure range from4-30MPa. And they lack the amount of impurities. Extracted at higher temperature 160℃,
molecular weight of pectin was lower molecules. On the other hand, pectin extracted at 120℃
and 140℃ were almost the same molecular weight of commercial pectin. This result
indicates that different molecular weight pectin was able to recover by changing the extraction
temperature.

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1. Introduction
Citrus fruits are grown in subtropical and tropical areas. The origin of citrus fruit isconsidered to be China. Since early 1900’s, development of transportation have increased
citrus product and processing in countries with suitable climate for citrus culture [1]. It has
been important to deal with increase juice processing residue. These residues are mainlycomposed of the part of peel, juice sack and seed. Especially, peel includes valuable
compounds such as terpenoid, pectin, and flavonoid. The traditional citrus by-product of cattle
feed pellets and essential oil are still most important utilization of juice processing residue.
Pectin is composed of an acidic polysaccharide and several types of neutral sugars [2].
The main chain consists of a-1, 4-linked D-galacuronic acid, which is partly methyl esterified.
It is impossible to extract free pectin with water from cell tissues because it exists in an
insoluble form known as protopectin. Having this characteristic, conventionally using mildacid [3] or chelating agent, pectin can be extracted and separated from the waste of beet pulp
and citrus fruits such as lemon and apple pomace, which is an effective utilization ofindustrial waste. Recently, pectic origosaccharide has become a more important material in
the health food market, because it has many bioactivities, such as hypocholesterolemic
activity and elevated blood pressure inhibitory effect.
Citrus Junos, called yuzu in Japan, is an evergreen tree harvested mainly in the southernpart of Japan, whose fruit processed into juice and often preferred to vinegar as an ingredientin sauces and salad dressing for its special flavor. Most food processing residues are disposed
without being recycled.
Recently, an environmentally-friendly extraction and fractionation technology for naturalmaterials has been developed which uses sub-critical water. Sub-critical water has someunique properties. The hydrogen bond between water molecules weakens by raising thetemperature, and the permittivity can be changed greatly. Ion product of water (Kw) isincreased dramatically with the temperature rising to around 270℃. At these properties,
sub-critical water has solubility and reactive selectivity to polar substance carbohydrate suchas pectin and cellulose based on pressure and temperature. This technique has been applied torecover protein and amino acid[4], and phenolic compounds [5] from natural products. And
also, the hydrothermal treatment has been demonstrated by several studies to effectively
convert cellulosic [6] and lignocellulosic biomass [7] into useful products.
In this work, extraction and separation of pectin was carried out from Citrus junos juice
processing residues using green solvent such as sub-critical water. Furthermore, the optimum
extraction condition was investigated under the various temperature and pressure conditions,and effect of temperature and pressure were investigated on the molecular weight of pectin.
2. Experimental
For the yuzu flavedo starting material, the residue from supercritical CO2 extraction at- 224 -PARTⅤ SOLVOLYSIS
60℃ and 20MPa was used. The residue was freeze dried, ground, and sieved. Flavedo with aparticle size of 170–450mm was obtained as the raw material. This pretreatment of the raw
material may eliminate the mass transfer limitation during the reactive extraction process,which help to understand dissolution behavior of materials in the subcritical water.
Reagents used for quantitative analysis of pectin were as follows: D-galacturonic acid,sodium chloride, sulfuric acid, and acetic acid (Wako Pure Chem. Ind. Ltd.,) and3,5-dimethylphenol (Tokyo Kasei Kogyo Co., Ltd.).
The pectin was quantitatively analyzed in terms of its galacturonic acid content
according to the dimethylphenol method [8]. The extraction sample (125mL) was mixed with
5% NaCl (125mL) and then 2mL of H2SO4 was added to the mixture in an ice bath. The
mixture was mixed well and placed in a water bath at 70 ℃

for 10minuites.

3,5-dimethylphenol–acetic acid reagent (0.1%, 100mL) was added and the reaction was stirred
at room temperature for 10minuites. Absorbance of the mixture at 400 and 450nm wasmeasured. The absorbance of the galacturonic acid and its methyl ester were calculated fromthe difference in the absorbance at 400nm subtracted from the absorbance at 450nm.
The molecular weight distribution were determined by gel filtration chromatographyusing OHpak SB-805HQ column (8.0×300mm, Showa Denko K.K) maintained 30℃ Theeluent was 0.1M NaCl solution at a flow rate of 1.0mL /min, and monitored by a differential
refractometer (Jasco Co., Ltd., Model RI-930)
A schematic diagram of the semi-continuous flow extractor is shown in Fig.1. The
maximum working conditions of the apparatus are 450℃ and 45MPa.
T2Filter

Reactor

HPLC pump

Degassed water

GC oven

P1 Back pressure regulator

T1
Cooling water

Cooling bath

7ml / fraction

Fig.1 Schematic diagram of semi continuous sub-critical water flow reactor
A 0.5 g sample of dried flavedo was charged in the extractor (SUS 316; 8.7mm
i.d.×118mm length, 7.0mL), and the extractor was capped with gasket filters (average poresize 20mm) to prevent sample particles from flowing out. Distilled water was degassed andcontinuously delivered through a heating coil placed in an oven into the extractor by ahigh-pressure pump (Jusco Co., Ltd., Model PU-980) at water flow rates of 0.5, 1.0, and2.0mL/min. The pressure in the extractor was controlled by a back-pressure regulator(AKICO Co., Ltd., Model HBP-450) at 4, 10, 20, and 30MPa. The extraction temperature wasrepresented by the temperature measured at the inlet and outlet of the extractor. Prior to- 225 -The 5th ISFR (October 11-14, 2009, Chengdu, China)
extraction, degassed water (60mL) was delivered to the extractor at the temperature of 80oC toremove the air and water soluble substance. At first, one fraction was collected. And then, thereaction temperature was immediately raised from 80℃ to various temperatures such as 110,120, 130, 140, 150, and 160℃, respectively. Its reactive solutions were fractionated each 7ml
volume. Ten fractions were collected at each temperature and pressure conditions.
3. Results and discussion
Fig. 2 shows the effect of temperature on the extraction behaviour at the pressure of4MPa and the water flow rate of 0.5mL/min. At the highest temperature of 160oC, pectin
extraction ended fastest. As the extraction temperature was lower, pectin was extracted moregently. This result indicated that the temperature accelerate separation of pectin from cell wallunder the hydrothermal condition. Pectin functions as adhesive material to bond adjacent cellwalls on contact with hydrogen bond [2]. Dielectric constant of water, ε, drastically decreases
with increase in temperature.In other words, it was thought that dielectric constant of water isconcerned to dissolve pectin in water.
Fig. 3 shows effect of pressure on the pectin yield at various temperatures. At lower
temperature of 120℃, the pectin yield was higher as the pressure was lower. Though, theextraction pressure did not influence the yield of pectin at the higher temperature region.
Moreover, the yield of pectin was decreased over the temperature of 140℃. This indicatesthat pectin molecules begin to be decomposed at the temperature higher than 140oC. As aresult, highest yield was obtained under the temperature of 140℃ at each pressure. It is

2.528080.00]
Yield of pectine [%Pressure:4MPa, Water flow rate:0.5ml/min
4.5110℃
4120℃
130℃
3.5140℃
150℃
3160℃

7576.88
73.75

70

70.63
67.50

65

64.38
61.25

60

58.13

1.5

55
5
101resu

0.5
01

2

3

45678

9Fraction number

Fig.2 Effect of temperature onthe extraction behavior

1020]
Pa[M055.0015esPr

Galacturonic acid [mg/ml]

thought that the important factor was not pressure but temperature when extraction andseparation of polar substance with hydrogen bond is carried out from natural product using
sub-critical water.

2530120125130135140145oTemtpera[ure150C]

Fig.3 Effect of pressure on the pectinyield at various temperatures

Fig. 4 shows effect of water flow rate on the yield of pectin under the temperature of 140- 226 -PARTⅤ SOLVOLYSIS℃ and pressure of 4 MPa. The water flow rate did not influence the cumulative yield ofeleven fractions. Those yields were approximately 76%. Though, the pectin concentration perone fraction that was beginning of pectin elution was higher as the water flow rate wassmaller.
Temperature:140oC, Pressure:4MPa
18080

1607060

14050

120

40
30

100

100

0.5ml/min
1.0ml/min
2.0ml/min
Temperature

2002

4

68

Temperature [ oC]

Cumulative yield [%]

9080
1060

Fraction number

Fig.4 Effect of water flow rate on the yield of pectin
Fig. 5 shows the gel filtration chromatogram of pectin extracted under the variousconditions. The average molecular weight of pectin extracted under the mild acidic condition
was approximately 5.7×104. On the other hand, molecular weight of pectin extracted under
the hydrothermal conditions were distributed mainly into three size which wereapproximately 63.5×104, 3.5×104, and 1.5×104, respectively. Under the hydrothermalcondition, the pectin molecule was smaller as the extraction temperature was higher. This
result indicates that different molecular weight pectin was able to recover by changing theextraction temperature. Some researchers have reported the decomposition of acidic
polysaccharide. Miyazawa et al. have reported recovery of mono and origomers from polygalacturonic acid under the hydrothermal condition [9] .Matsushima et al. successfully
separate alginic acid constituent sugar using quick-heating quick-cooling method under thesub-critical and supercritical water [10]. As prospects, recovery of pectin origosaccharide isexpected changing the reaction conditions.
Mw
106

105

104103

1.5RI response

10000

3.5

1.2

HCl
120oC
140oC
160oC

5.7

8000
6000

63.5

4000
2000
0-2000567

8910111213

1415

Retention time [min]Fig.5 Gel filtration of the pectin extract under the various conditions- 227 -The 5th ISFR (October 11-14, 2009, Chengdu, China)
4. Conclusions
A sub-critical water extraction enabled the separation of pectin effectively
without using chemicals such as acid. And, the yield of pectin was more than 75%.
Different molecular weight pectin was able to recover by changing the extraction
temperature.
References[1] R. J. Braddock, Handbook of Citrus By-products and Processing Technology, Willy Inter
Science, New York, 1999.
[2] F. A. Henglein, Handbuch der Pflanzenphysiologie, SpringerVerlag, Berlin, 1958, 6,407-478.
[3] C. D. May, Carbohydr. Polym., 1990, 12, 79-99.
[4] I. Sereewatthanawut, S. Prapintip, K. Watchiraruji, M. Goto, M. Sasaki, A. Shotipruk,Bioresource Technology, 2008, 99, 555-561.
[5] A. T. Quitain, S. Katoh, T. Moriyoshi, Ind. Eng. Chem. Res., 2004, 43, 1056.
[6] M. Sasaki, T. Adschiri, K. Arai, Bioresource Technology, 2003, 86, 301-304.
[7] Wahyudiono, T. Kanetake, M. Sasaki, M. Goto, Chem. Eng. Technol., 2007, 30, 1113.
[8] R.W. Scott, Anal. Chem., 1936. 51, 936-941.
[9] T. Miyzawa, T. Funazukuri, Ind. Eng. Chem. Res., 2004, 43, 2310-2314.
[10] K. Matsushima, H. Kawanami, Y. Ikushima, M. Nishizawa, A. Kawamukai, K. Hara, Ind.
Eng. Chem. Res., 2005, 44, 9626-9630.

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