APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1980, p. 908-912 0099-2240/80/04-0908/05$02.

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Vol. 39, No. 4

Microbial Production of Pectin from Citrus Peel

TAKUO SAKAI* AND MINORU OKUSHIMAt Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Mozu- UmeMachi, Sakai, Osaka 591, Japan

A new method for the production of pectin from citrus peel was developed. For this purpose, a microorganism which produces a protopectin-solubilizing enzyme was isolated and identified as a variety of Trichosporon penicillatum. The most suitable conditions for the pectin production were determined as follows. Citrus (Citrus unshiu) peel was suspended in water (1:2, wt/vol), the organism was added, and fermentation proceeded over 15 to 20 h at 30C. During the fermentation, the pectin in the peel was extracted almost completely without macerating the peel. By this method, 20 to 25 g of pectin was obtained per kg of peel. The pectin obtained was special in that it contained neutral sugar at high levels, which was determined to have a molecular weight suitable for practical applications.

Pectin is a polysaccharide having properties such as gelation and emulsion stabilization which make it useful in the manufacture of food, cosmetics, and medicine. It is a normal constituent of food and may therefore be safely ingested. Citrus peel, a by-product of the citrus processing industry, is a suitable source of pectin. Pectin is usually extracted by placing the peel in vats of water, bringing the mixture to a boil as a slurry, and adding concentrated hydrochloric or sulfuric acid to adjust the pH to about 2.0. Filtration of the extract is a tedious process because the extract, containing pectin and disintegrated peel, is corrosive and viscous. The peel of mandarin oranges (including Citrus unshiu, which totals more than 80% of the citrus fruit produced in Japan) is not suitable as a raw material in this process because the peel is fragile and becomes pasty on heating, preventing the separation of pectin from the residues (5). Thus, in Japan, pectin is not manufactured, although nearly 0.5 x 106 tons (ca. 45.36 x 104 tonnes) of citrus peel, containing about 5% pectin on a fresh-weight basis, is produced each year. We attempted pectin production by using C. unshiu peel as raw material and in so doing developed a new microbial method which can extract pectin from citrus peel without macerating the peel.
MATERIALS AND METHODS Chemicals. All chemicals used were obtained from Wako Pure Chemical Industries Ltd., Osaka, and were of certified reagent grade. Citrus peel was taken from citrus fruit (C. unshiu) harvested within 1 month. Microorganism. A strain of Trichosporon penicillatum, SNO-3, was isolated from infected vegetable
t Present address: Yakult Research Institute, Nishinomiya,

tissue, obtained in Sakai City, Osaka, as a protopectinsolubilizing strain. The organism was maintained on an agar slant containing 2% glucose, 0.2% pectin, and 0.1% yeast extract, pH 5.0. Taxonomical studies. Strain SNO-3 was taxonomically characterized by a systematic method and identified by the descriptions of Lodder and Kregervan Rij (4). Staining of pectin in peel tissue. The pectin in the peel tissue was qualitatively determined by staining with ruthenium red (BDH) Chemicals, Poole, Dorset, England) by the method described by Hanke and Northcote (3). Cultivation of organisms. For the seed culture, the microorganism was aerobically grown in a medium containing 2% glucose, 0.4% peptone, and 0.2% yeast extract, pH 5.0, at 30C for 24 h. Fermentation conditions. Fermentation for the extraction of pectin was carried out with broth containing 30 g of citrus peel (C. unshiu) shredded to about a 1-cm width and 100 ml of sterilized water. The broth was inoculated with 5% of the seed culture and incubated at 30C on shaker. Determination of pectin extracted by fermentation. After the fermentation, the culture filtrate was poured into 3 volumes of ethanol. The precipitated pectin was collected by centrifugation, washed with ethanol, and dried in vacuo. As a control, 0.02% Osuban (a disinfectant, benzalkonium chloride solution, from Daigo Eiyo Ltd., Osaka, Japan) was added to noninoculated fermentation broth to prevent contamination. In this experiment, although the peel was not sterilized, contamination rarely occurred when the organism was added.

Hy6go, Japan.

RESULTS AND DISCUSSION Taxonomic studies on the yeast strain SNO-3. Diagnostic tests of the strain SNO-3 were performed by the methods of Lodder and Kreger-van Rij. The morphological and physiological characteristics of strain SNO-3 agreed with those described by Carmo-Sousa (1) for T. penicillatum, except for the ability to grow on
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glycerol and the lack of growth stimulation by thiamine and pyridoxine. We identified this strain as a variety of T. penicillatum and named it SNO-3. Fermentation conditions. Strain SNO-3 grew well in the extract of citrus peel as the sole nutrient source. The organism apparently assimilated the water-soluble carbon and nitrogen compounds in the peel, since nothing was present in the medium other than citrus peel. The amount of pectin extracted was dependent on the concentration of peel in the medium. As the ratio of peel to water decreased, the total amount of pectin extracted increased, but the pectin concentration in the culture filtrate decreased (Table 1). In pectin production, the pectin concentration in the broth is important, because the isolation procedure proceeds more easily with pectin solutions of higher concentrations. The peel/water ratio of 1:2 to 1:3 was found to be favorable. Pectin was extracted effectively between 25 and 30C (Table 2), although the microorganism grew well between 25 and 37'C. A temperature of 30C was chosen as the best temperature for the fermentation. The effect of the fermentation time on pectin extraction was examined. Pectin began to appear after 5 h and increased with fermentation time, and after 20 to 25 h the amount of pectin extracted reached
TABLE 1. Effect of the ratio ofpeel to water on pectin extraction by T. penicillatum SNO-3' Pectin extracted (g/100 g of peel) Concn in Peel:water (wt/vol) Water solu- Microbial Total broth (%) oa
bleb

1.9 0.2 2.9 0.3 3.1 0.4 3.5 0.3 a Fermentation was carried out at 30C under standard conditions, except that the peel and water were varied as indicated. b Extracted without inoculation. ' Extracted by fermentation.
1:2.3 1:3.3 1:4.0 1:5.0

soluble' 1.7 2.6 2.7 3.2

0.8 0.9 0.8 0.7 for 12 h ratios of

its maximum (Fig. 1). A small amount of pectin was extracted by water without inoculation; however, a much greater amount of pectin was extracted after inoculation with the microorganism. The increased amount of pectin extracted by the microorganism is termed microbial-soluble pectin (Fig. 1). The pectins extracted at various fermentation times were isolated, and their viscosities were determined. During the course of fermentation, viscosity of the pectins did not vary. This result suggests that the extracted pectin is stable in the fermentation broth under the conditions tested. In Fig. 2, photographs of peels before and after fermentation are shown. The peels did not disintegrate during fermentation, so that the separation of pectin from the residues was very easy. Pectin in plant tissues was located by staining with ruthenium red. The photographs shown in Fig. 3 are photomicrographs of thin-sliced specimens of peels treated with ruthenium red. The peel contained plenty of pectin before fermentation (Fig. 3A and B). Figures 3C and D, taken after fermentation, indicate that the pectin in peel was extracted completely during fermentation. Raw materials of pectin. Vegetable matters, which are wastes from industry, were tested for their suitability as raw materials for pectin extraction. Peels or segment covers of various citrus fruits were found to be good raw materials, whereas vegetables such as carrots, wax gourds, and radishes were poor ones (Table 3). Physical and chemical properties of isolated pectin. The properties of microbially extracted pectin and pectin obtained by the conventional method were compared after they
3 N
CD

0D

cj< 0_'
1-1

Zm

(DO

TABLE 2. Effect of temperature on pectin extraction by T. penicillatum SNO-3a Pectin extracted (g/100 g of peel) Temp (0CIMirbilsou Total Water solubleh Microbial solu ble~
2.8 2.3 0.5 2.9 2.6 0.3 1.7 1.4 0.3 a Fermentation was carried out for 12 h under standard conditions, except that the temperature was varied as indicated. b Extracted without inoculation. c Extracted by fermentation. 25 30 37

I2
40

x a,
c

1-

0-

12

16 20 26

Fermentation period (hour) FIG. 1. Time course of pectin extraction during fermentation. The fermentation was carried out under standard conditions. Symbols: 0, water-soluble pectin; A, microbial-soluble pectin; A, total pectin extracted; 0, relative viscosity of pectin (0.3%o solution, at 37C).

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SAKAI AND OKUSHIMA

APPL. ENVIRON. MICROBIOL.

FIG. 2. Photographs of C. natsudaidai peel before and after the fermentation. (A) The peel before the fermentation; (B) The peel after the fermentation at 30'C for 12 h.

~~~~~~~~~~~e2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~l
A
K-i

4,.

FIG. 3. Photomicrographs of thin-sliced specimens of C. natsudaidai peel treated with ruthenium red. A and B are the specimens prepared from the peel before the fermentation, and C and D are the specimens prepared from the peel after the fermentation at 30'C for 12 h. Pectin substance is present at the darker areas indicated by the arrows. A and C are the specimens prepared from flavedo layer, and B and D are from the albedo layer of the peel.
were

purified by deionization by Amberlite IR120, treatment with active carbon, and chromatography on a Sepharose 6B column, and lyoph-

ilization to obtain the purified pectins. The microbially extracted pectin is special in that it contains neutral sugars at higher levels than that

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obtained by the conventional method (Table 4). The molecular weight of our pectin, 100,000, determined by the method described by Smit and Bryant (6), is almost the same as that of commercial pectin from lemon and higher than that of the pectin obtained from C. unshiu by conventional methods. The molecular weight of pectin has a marked influence on its jellying
TABLE 3. Yields ofpectin extracted by T. penicillatum SNO-3 from various origins'
Pectin extracted (g/100 g)

Origin
Navel orange (Citrus

Water sol-

Microbial

uble'

solubleC

Total

sinesis)
0.4 2.5 2.9 Segment cover 0 2.7 2.7 Grapefruit Peel 0.2 2.5 2.7 Segment cover 0 2.1 2.1 Mandarin (Citrus unshiu) Peel 0.3 2.6 2.9 Segment cover 0 1.8 1.8 Mandarin (Citrus natsudaidai) Peel 0.1 2.1 2.2 Segment cover 0 1.9 1.9 Lemon Peel 0.9 1.7 2.6 Segment cover 0 1.6 1.6 Apple (K6ogyoku) 0.1 0.1 0.2 Radish 0 0.2 0.2 Carrot 0 0.1 0.1 Wax gourd 0.4 0.3 0.7 a Fermentation was carried out at 30C for 12 h under standard conditions, except that the vegetable matter indicated was used instead of C. unshiu peel. bExtracted without inoculation. C Extracted by fermentation.
Peel

ability (2), and for this reason pectin having a large molecular weight is desirable. From the results of the present study, a new method, illustrated in Fig. 4, was established. In this system, peels are washed, if necessary, and suspended in sterilized water in a fermentor, to which a seed culture of strain SNO-3 is introduced (corresponding to 3 to 5% of the fermentation broth) from a seed tank. After 15 to 20 h of fermentation at 25 to 30C, the residual peels are filtered off, and the resultant filtrate is passed through another filter to remove the microbial cells. The filtrate is concentrated, and the pectin is precipitated in ethyl alcohol, collected, and dried. By this procedure, 20 to 25 kg of pectin (corresponding to 100 to 110% of the yield obtained by the conventional method) is obtained per ton of the mandarin peels which are a by-product of the citrus processing industry.
Evaporator
Seed tank
(Citrus Peel)

U Washer
02~

Dried

(Waste Peel
Dried

cm

(Yeast) CellI

Alcohol distiller
process

FIG. 4. Schematic diagram of manufacturing ofpectin from citrus peel.

TABLE 4. Some properties ofpectin

t

(%)tof1Ash

Relative Methoxyl Esterified Galactu. groupb carboxyl ronic solution group (%) acid b(%)
%

Neutral pH of Ugarb 0.5%

Mol wt

Element analysis'
C

solu- (xlT-:f)

1.53 9.24 63.1 85.0 5.7 3.96 2.6 102c 40.86 5.76 0.80 Commercial product (>200) (from lemon) From C. unshiu peel 23.2 68.2 3.24 105 40.27 5.77 0.61 8.58 73.8 1.46 Extracted by ND fermentation (>200) 80.3 66.1 10.5 4.34 50 9.13 38.27 5.40 0.41 3.5 1.23 Extracted by acid (>200) Samples which were not treated with a cation-exchange resin were used for the analysis. b Values expressed on an ash- and moisture-free basis. c Calculated by the equation of Smit and Bryant (6). Figures in parentheses were determined by Sepharose 6B gel filtration.

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LITERATURE CITED

APPL. ENVIRON. MICROBIOL.

4. Lodder, J., and N. J. W. Kreger-van Rij. 1967. Characteristics and methods used in the classification, p. 635. In J. Lodder (ed.), The yeasts, a taxonomic study. North-Holland Publishing Co., Amsterdam. 5. Miyazaki, H., and K. Terada. 1974. Processing of citrus peel and extraction of peel components. Shokuhin Kogyo (in Japanese). 17:81-87. 6. Smit, C. J. B., and E. F. Bryant. 1967. Properties of pectin fractions separated on diethylaminoethyl-cellulose columns. J. Food Sci. 32:197-199.

1. Carmo-Sousa, L. do. 1970. Trichosporon Behrend, p. 1309-1352. In J. Lodder (ed.), The yeasts, a taxonomic

study. North-Holland Publishing Co., Amsterdam. 2. Fogarty, W. M., and 0. P. Ward. 1974. Pectinase and pectic polysaccharides. Prog. Ind. Microbiol. 13:59-119. 3. Hanke, D. E., and D. H. Northcote. 1975. Molecular visualization of pectin and DNA by ruthenium red. Biopolymers 14:1-17.