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Isolation of two glucodisaccharides from Japanese wines
JotrRr~,~t Or FERMENTATIONAND BIOENGINEERING Vol. 67, No. 3, 148-152. 1989
Isolation of Two Glucodisaccharides from Japanese Wines KOTOYOSHI NAKANISHI* AND KOKI YOKOTSUKA
Institute of Enology and Viticulture, Yamanashi University, Kofu 400, Japan Received 2 September 1988/Accepted l0 December 1988 The composition of oligosaccharides in four varieties of Japanese white table wines was examined using two separation methods, charcoal column chromatography and paper chromatography. Laminaribiose and gentiobiose were isolated as the principal constituents of the oligosaccharides from all the wines tested. The concentration of each of these sugars in the wines ranged from 15 mg/1 to 25 rag~1. When fermentation was done with a synthetic medium, these amounts of laminaribiose and gentiobiose were also isolated from the fermentation broth using the same separation techniques. The results suggested that both sugars were released from yeast cells into the wines during the fermentation. The amounts of both sugars released from yeast were closely associated with the conditions of the fermentation and independent of the strain of the yeast used for the fermentation.
(9). The sample solution was put on a charcoal (Nakarai Chemicals Ltd.; 60-150mesh) column (2.2×25 cm) and eluted with distilled water to remove the monosaccharides and salts. It was then eluted stepwise with 10%, 20%, and 30% aqueous ethanol at the rate of about 120 ml/h. The sugar content in the effluent was measured by the phenol sulfuric acid method (10). Paper chromatography Paper chromatography was done twice with filter paper (Toyo Roshi no. 51). The solvent system was n-butanol-pyridine-water ( 6 : 4 : 3 ) . The sugars were detected with p-anisidine hydrochloride. The amounts of each of the oligosaccharides extracted from the paper chromatogram were measured by the SomogyiNelson method (11). Acid hydrolysis Each of the oligosaccharides extracted from the paper chromatogram was partially hydrolyzed with acid to analyze its composition. One ml of the solution containing the oligosaccharides and 1 ml of 2 N HC1 were mixed, and left at 90-100°C for 2 h. The mixture was evaporated to dryness by bubbling with nitrogen gas, and then dissolved in 1 ml of distilled water. The sugar was measured by the Somogyi-Nelson method before and after acid hydrolysis. Also the sugar after acid hydrolysis was measured by the glucose oxidase method and gas liquid chromatography (12). The acid hydrolysis products were examined by paper chromatography. Enzyme hydrolysis Anomer analysis of each of the oligosaccharides obtained was done using pure yeast aglucosidase and almond p-glucosidase (Sigma Chemical Company). The reaction mixture consisted of 0.25 ml of the substrate solution, 0.25 ml of McIlvaine buffer (pH 6.0 for a-glucosidase and pH 5.0 for ]~-glucosidase) and 0.5 ml of the enzyme solution. The final concentrations of oligo. A and oligo. B were 0.15°//oo and 0.08%, respectively. The concentration of a-gluosidase was 0.31 units for both oligo. A and oligo. B and that of the fl-glucosidase was 0.91 units for the oligo. A and 4.6 units for the oligo. B. The reaction was run at 35°C for 4 h, and the hydrolyzates from each reaction mixture were examined by paper chromatography. Microorganisms and culture condition Saccharornyces cerevisiae OC-2 and W-3 (wine yeasts), Saccharomyces cerevisiae Kyokai no. 9 (sake yeast), and
Wines contain small amounts of residual mono-, oligo-, and poly-saccharides other than glucose and fructose. Almost all of these sugars are usually unfermentable and are originally present in grape juices. These sugars are believed to have either desirable or undesirable effects on the quality of wines during aging. Many reports on the residual sugars in wines have been published (1, 2). However, information on the residual oligosaccharides is very limited (3-5). Particularly, there has not been any such report on Japanese wines. The compositions of residual oligosaccharides and monosaccharides in wines have been directly analyzed by paper-, thin layer-, gas-, and liquid chromatographies (1, 6, 7). Although these methods are quick and easy, the fact that the concentration of the individual oligosaccharides is usually very small compared to that of the monosaccharides makes it difficult to examine the composition of the oligosaccharides. In this work, charcoal column chromatography methods were used, and the oligosaccharides were detected and isolated by paper chromatography. This report mainly describes the isolation and properties of two oligosaccharides obtained from the four different Japanese white table wines. In addition, some data on the origin of both sugars are also presented. MATERIALS AND M E T H O D S Wines For the analysis of residual oligosaccharides, four kinds of white table wine made at our Institute of Winery in 1983 from Koshu, Chardonnay, Riesling, and Pinot Gris grapes were used. All the grapes were harvested from our Institute's vineyard. General analyses of the four wines was made using the methods in Kokuzeicho Shoteibunsekihochukai (8). The results are shown in Table 1. Charcoal column chromatography Seven hundred ml of the wines was concentrated to approximately onefifth of the original volume. The concentrated sample was treated with saturated neutral lead acetate solution, glacial acetic acid, and disodium hydrogen phosphate according to the methods for the analysis of reducing sugar in wine * Corresponding author. 148
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TABLE 1. Compositions of the four wines used for the analysis of the residual oligosaccharides Alcohol Extract (%) (%)
Wines Koshu Riesling Chardonnay Pinot Gris
12.5 11.7 11.3 10.3
3.66 4.88 4.59 4.71
Reducing su0ga (%)r
Total acid (%)
pH
1.06 2.06 1.82 1.80
0.66 0.64 0.59 0.60
3.20 3.32 3.39 3.56
b a k e r ' s yeast (Oriental Yeast Co., Ltd.) were used. Basal m e d i u m consisted o f a c a r b o n source, 2 g o f asparagine, 1 g o f KH2PO4, and 3 g o f MgSO4 7H20 per 1 1 o f tap water. F o r the seed culture, yeast extract was a d d e d to the m e d i u m at the concentration o f 0 . 1 % . The initial p H o f the m e d i u m was adjusted to 3.5, 5.5, or 7.5 with 10% tartaric acid or 1 N N a O H solution. Five ml o f cell suspension obtained f r o m seed culture was inoculated into a 500 ml flask containing 300 ml o f the medium. The flask was anaerobically incubated at 25°C for 15-20d. The supernatant o f the culture b r o t h was used as a sample. Chemicals Kojibiose and nigerose were d o n a t e d by P r o f . Seiya C h i b a ( H o k k a i d o Univ.). Laminaribiose was d o n a t e d by Associate P r o f . Isao K u s a k a b e (Univ. of Tsukuba). Other standard glucodisaccharides were obtained from commercial sources. Glucose oxidase (Glucose CTest W a k o ) was purchased from W a k o Pure Chemicals, Ltd., T o k y o . Other chemicals used were o f analytical grade and o b t a i n e d commercially. RESULTS AND DISCUSSION
The composition of the residual oligosaccharides An experiment to elucidate the c o m p o s i t i o n o f residual oligosaccharides in wine was done by charcoal column c h r o m a t o g r a p h y and paper c h r o m a t o g r a p h y . The total a m o u n t o f sugar equivalent to glucose in each fraction is presented in Table 2. In the four wines tested, almost all the residual sugars were eluted with water and the total a m o u n t o f sugar eluted with ethanol solution was a b o u t 34 % . There were no significant differences in the elution profiles o f the four wines. Each ethanol fraction was concentrated and used for paper c h r o m a t o g r a p h y (Fig. 1). A few spots considered to be oligosaccharides from their Rf values were observed. Two m a j o r spots, represented by A and B on the c h r o m a t o g r a m , were detected in 10% and 20% ethanol fractions, and were c o m m o n to the four wines. However, the separation o f A f r o m B in b o t h the ethanol fractions was not very clear. The two sugars were tentatively n a m e d oligo. A and oligo. B; they were isolated.
SFIO
30 20 10 30 20 FS 20 10 30 20 10 30 K R C P
FIG. I. Paper chromatogram of oligosaccharides of wines. Each fraction shown in Table 2 was concentrated and spotted on the paper. Abbreviations: K, Koshu; R, Riesling; C, Chardonnay; P, Pinot Gris; A, oligo. A; B, oligo. B; S, Standard (Rha., rhamnose; Xyl., xylose; Ara., arabinose; Glc., glucose; Gal., galactose; GaA., galacturonic acid; F, fructose); 10, 20 and 30, 10%, 20°//o, and 30% ethanol fraction.
Isolation of oligo. A and oligo. B To obtain a high concentration o f oligo. A and oligo. B, b o t h sugars were isolated with a scale-up system using 3.5 l o f the four wines. The concentrated wine sample (300ml) was pretreated as described before and was then put on a charcoal column (2.5 x 60 cm). After the column was washed with 900 ml o f water, the elution with ethanol was done at the concentration of 5%, 10%, 15%, and 20%. The eluent was collected in 100-ml fraction tubes using a fraction collector. The recovery o f the sugars eluted with aqueous ethanol was a b o u t 3 - 4 % , and this result was similar to that shown in Table 2. In addition, there were no significant differences in the elution profiles. Observation with paper c h r o m a t o g r a p h y showed that spots corresponding to oligo. A and oligo. B were mainly detected in the 10% ethanol and 15°//00 ethanol fractions, respectively. These fractions contained few oligosaccharide contaminants. Each fraction containing oligo. A and oligo. B was collected and concentrated to 10 ml under reduced pressure. Five hundred pl o f the concentrated samples were spotted on a paper and developed two times. Both the edges and the middle part o f each resulting paper strip were cut off and sprayed with the reagent to find the position o f the
TABLE 2. Fractionation of residual sugars in four wines by charcoal column chromatography Wines Fractions Sampleb Water 10~ooEthanol 20~oo Ethanol 30°//ooEthanol
Koshu Volume (ml) 150 325 265 243 250
Sugar (mgp 9275 (100y 7586 (82) 130 (1.4) 93 (1.0) 34 (0.37)
Riesling Volume Sugar (ml) (rag) 160 17120 (100) 375 15050 (88) 255 245 (1.4) 250 175 (1.0) 250 51 (0.30)
" As glucose mg. b Concentrated wine sample (before chromatography). c Yield (sample= 100%).
Chardonnay Volume Sugar (ml) (rag) 155 16275 (100) 355 12715 (78) 250 260 (1.5) 250 240 (1.5) 235 47 (0.29)
Pinot Gris Volume Sugar (ml) (mg) 170 16320 (100) 360 14688 (90) 250 375 (2.3) 250 190 (1.2) 245 96 (0.59)
150
NAKANISHIAND YOKOTSUKA
J. FERMENT.BIOENG.,
bands of oligo. A and oligo. B. Each sugar was extracted from the paper with hot water at about 90°C for 1 h. Each extract was evaporated to a syrup, and was dried with absolute ethanol. Definite amounts of distilled water were added to the dried samples, and the reducing sugars in the solution were measured by the Somogyi-Nelson method. The oligo, A and oligo. B thus obtained gave only a single sugar spot each on the paper chromatogram (Fig. 2). In paper chromatograms developed with several kinds of solvents, each sugar still gave a single spot. Identification of oligo. A and oligo. B Some of the properties of otigo. A and oligo. B obtained from the four wines were examined by the following experiments. To analyze the composition of the sugars, oligo. A and oligo. B were hydrolyzed with acid as described before. The hydrolyzates were analyzed with paper chromatography and the results are shown in Fig. 2. It was found that acid hydrolysis gave glucose as the only product, so oligo. A and oligo. B both consist of glucose. In the above experiment, the amount of the reducing sugar in the sample was measured by Somogyi-Nelson method before and after the acid hydrolysis. It was found that the amount of the sugar in the sample increased about 2 times when treated with acid. This result shows that the degree of polymerization was about 2. The amount of sugar after acid hydrolysis was also measured by the glucose oxidase method and gas chromatography. The results obtained by this method agreed very closely with those of the SomogyiNelson method. These results suggest that the oligo. A and the oligo. B were glucodisaccharides. Various standard glucodisaccharides were paper chromatographed together with oligo. A, oligo. B, and glucose. The Rg values of kojibiose, nigerose, maltose, isomaltose, laminaribiose, cellobiose, and gentiobiose and were 0.69, 0.58, 0.74, 0.59, 0.87, 0.71, and 0.59, respectively. The Rg values of sophorose was reported to be 0.74 (13). The Rg values of oligo. A ranged from 0.85 to 0.87 and agreed very closely with that of the laminaribiose (0.87) and nigerose (0.85). The Rg value of the oligo. B (0.580.60) agreed with that of the isomaltose (0.59) and gentiobiose (0.59). So oligo. A was either laminaribiose (3-0fl-glucopyranosyl-glucose) or nigerose (3-0-a-glucopyranosyl-glucose), and that oligo. B was either gen-
GBBBAA
AG
++
++
a13
a13
FIG. 3. Paper chromatogram of hydrolysis products of oligo. A and oligo. B by a- and fl-glucosidase. Abbreviations: G, glucose; A, oligo. A; B, oligo. B.; A - u , oligo. A+ct-glucosidase; A - t , oligo. A+fl-glucosidase; B a, oligo. B+a-glucosidase; B - t , oligo. B +/~-glucosidase.
tiobiose (6-0-fl-glucopyranosyl-glucose) or isomaltose (6-0a-glucopyranosyl-glucose). Then anomer analysis of both sugars was done using yeast a-glucosidase and almond fl-glucosidase, Oligo. A and oligo. B were separately incubated with a-glucosidase and fl-glucosidase, and then enzymatic hydrolysis products were examined by paper chromatography (Fig. 3). It was found that oligo. A and oligo. B were completely hydrolyzed to glucose with fl-glucosidase. In the case of aglucosidase, no hydrolysis product was observed on the chromatogram. The same results were obtained with all the oligo. A and oligo. B tested. These results suggested that oligo. A and oligo. B have fl-glucosidic linkages. Based on all the experimental results obtained, oligo. A and oligo. B were identified as laminaribiose and gentiobiose, respectively. The iaminaribiose and gentiobiose contents in wine The laminaribiose and gentiobiose were extracted from the paper chromatogram of the four wines which was run in parallel with the above experiment and the amounts of both sugars were measured. Also various amounts of standard laminaribiose and gentiobiose were spotted on the same paper, and the recovery of both sugars which were extracted from the paper were examined. The recovery of TABLE 3. Contents of laminaribiose and gentiobiose in various wines
GABABABABABABABABG K R C P K R C
Wines
P
FIG. 2. Paper chromatogram of acid hydrolysis products of oligo, A and oligo. B isolated from various wines. Abbreviations:K, Koshu; R, Riesling; C, Chardonnay; P, Pinot Gris; A, oligo. A; B, oligo. B; G, Glucose.
Koshu Riesling Chardonnay Pinot Gris
Laminaribiose (mg//) 19 16 18 16
Gentiobiose (mg//) 20 22 24 19
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TABLE 4. Formations of laminaribiose and gentiobiose by various yeasts during the alcohol fermentation with synthetic medium Yeasts
Before fermentation Mediuma Sugar (%)b pH
Alcohol (%)
After fermentation Laminaribiose pH (mg//)
Gentiobiose (mg//)
Wine yeast (OC-2) Wine yeast (W-3) Sake yeastc Baker's yeast
14 14 14 14
3.5 3.5 3.5 3.5
7.2 7.0 7.3 6.8
2.8 2.8 2.9 3.1
15 13 18 13
33 31 38 27
Wine yeast (OC-2)
7 23 14 14
3.5 3.5 5.5 7.5
3.0 12.0 7.2 7.0
3.0 2.7 3.2 3.8
4 17 11 (2
9 37 29 15
a Other components are shown in the text. b Glucose and fructose are in the ratio of 1:1. c Kyokai no. 9.
s t a n d a r d sugars ranged from 90-100%, thus the contents o f the laminaribiose and gentiobiose in wines were measured without correction. In addition, since the recovery o f laminaribiose and gentiobiose by charcoal colu m n c h r o m a t o g r a p h y could not be rigorously calculated, the d a t a on the contents o f b o t h sugars in wines was uncorrected. These results are shown in Table 3. W i t h the four wines tested, there was no significant difference in the contents o f b o t h sugars. A l t h o u g h various disaccharides such as sucrose, lactose, isomaltose, maltose, cellobiose and melibiose are present in wine, the content o f each o f these disaccharides is usually less than 5 mg/l (1). On the other hand, there is a r e p o r t that the contents o f lactose, maltose, and sucrose, which were identified in two A m e r i c a n wines using paper c h r o m a t o g r a p h y , were in the range o f 10-50 m g / l (6). Considering to these reports, the fact that laminaribiose was isolated from wine seems to be o f interest because the presence o f this sugar in wine has not been reported. While, the presence o f gentiobiose in wine has been r e p o r t e d based on paper c h r o m a t o g r a p h y (4), measuring the sugar has not been done. The analytical d a t a on the laminaribiose and gentiobiose contents in wines presented in this report is, therefore, considered to be valuable. The total a m o u n t o f laminaribiose and gentiobiose f o u n d is very small when c o m p a r e d to the concentration o f residual glucose and fructose. Therefore, direct effects of b o t h sugars on the sweetness o f wine m a y be considered to be very little. However, in the cases o f prolonged aging o f wine, it is presumed that these oligosaccharides as well as unfermentable monosaccharides have either desirable or undesirable effects on the quality o f the wines. To clarify how these sugars influence the quality o f wines during aging, m o r e work is needed. The amounts of laminaribiose and gentiobiose released from yeast The contents o f laminaribiose and gentiobiose in wines are higher than those o f other oligosaccharides detected by paper c h r o m a t o g r a p h y . It is, therefore, o f great interest to elucidate the origin o f both sugars in wines. There are two possibilities, that the sugar is originally present in the grape juice or that they are released from the yeast during the fermentation. In the must samples after charcoal column c h r o m a t o g r a p h y , spots corresponding to the laminaribiose and gentiobiose could not be clearly detected on the paper c h r o m a t o g r a m . However, in culture filtrate obtained from alcoholic fermentation o f a synthetic m e d i u m , two clear spots corresponding to the laminaribiose and gentiobiose in their Rg
values were observed on the c h r o m a t o g r a m . Each sugar were extracted from the p a p e r and their properties were examined using the same methods as before. The sugars were confirmed to be laminaribiose and gentiobiose. W i t h every yeast tested, no significant differences in the content of each sugar were observed (Table 4). It was also f o u n d that the laminaribiose content was nearly equal to that found in wine, and that the gentiobiose content was slightly higher than that in wine. These results are sufficient to suggest that the laminaribiose and gentiobiose f o u n d in the wines are released from the yeast during fermentation. It has been reported that the m a n n a n and trehalose f o u n d in wine are p r o b a b l y released f r o m yeast cells during fermentation, by Villettaz et al. (14) and Bertrand et aL (7). Since such a investigation is very limited, the result obtained in this paper is very interesting. Also the extent of the accumulation o f laminaribiose and gentiobiose during the fermentation was examined by varying the initial p H and the sugar concentration o f the medium. These results are shown in Table 4. In both sugars, the a m o u n t o f the sugar increased with an increase in the initial sugar concentration of the medium. However, when the fermentation was done with the m e d i u m at initial p H o f 5.5 or 7.5, the a m o u n t o f b o t h sugars was very small c o m p a r e d to their concentrations in the case o f the m e d i u m with an initial p H o f 3.5 (nearly the same as the p H o f grape juice). The decrease in the amounts o f laminaribiose was very great. On the other hand, the effects o f fermentation temperature on the accumulation of the b o t h sugars were not great. This suggests that the amounts of laminaribiose and gentiobiose released from yeast are closely associated with the conditions o f the fermentation and independent of the difference in the strain o f the yeast used for the fermentation. It is well k n o w n that yeast cells contain both fl-(1-3)- and fl-(1-6)-linked glucan as m a j o r cell wall polymers, and that yeasts have several types o f cell wall associated flglucanases which hydrolyze the fl-(1-3)- and fl-(1-6)glucosidic linkages (15-17). In a few species o f yeast, glucose, laminaribiose, and other laminarioligosaccharides are formed from glucan by the action o f flglucanase during cell wall autolysis (18). Considering these fact, the release o f laminaribiose and gentiobiose from yeast cell to wine may be closely associated with the various types o f fl-glucanase action on the cell wall. To clarify the prosess of the f o r m a t i o n o f laminaribiose and gentiobiose, more work is needed.
152
NAKANISHI AND YOKOTSUKA ACKNOWLEDGMENTS
The authors wish to thank Mr. T. Uesugi and Mr. A. Mimura for their technical assistance. REFERENCES 1. Amerine, M. A. and Ough, C. S.: Methods for analysis of must and wines, p. 41-44. John Wiley & Sons, Inc., New York (1980). 2. Otsuka, K.: Jozoseibunichiran, p. 284-287. Nippon Jozo Kyokal, Tokyo (1977). 3. Esau, P. and Amerine, M . A . : Residual sugars in wine. Am. J. Enol. Vitic., 15, 187-189 (1964). 4. Thaler, H.: Die papierchromatographische untersuchung yon weinverbesserungszuckern. Z. Lebnsmitt. Untersuch., 100, 359366 (1955). 5. Kliewer, W.: Identification and seasonal changes in the concentration of several trace sugars in Vitis vinifera. Am. J. Enol. Vitic., 16, 168-178 (1965). 6. Esau, P. and Amerine, M . A . : Quantitative estimation of residual sugars in wine. Am. J. Enol. Vitic., 17, 265-267 (1967). 7. Betrand, M. A., Dubernet, M. O., and Ribereau-Gayon, P.: Le trehalose principal diholoside des vins. C. R. Acad. Sc. Paris, Serie D, t. 280, 1907-1910 (1975). 8. Murakami, H.: Kokuzeicho shoteibunsekihochukai, 3rd. ed., p. 54-62. Nippon Jozo Kyokai, Tokyo (1974). 9. Amerine, M. A. and Ough, C. S.: Methods for analysis of musts and wines, p. 33-35. John Wiley & Sons, Inc., New York (1980).
J. FERMENT. BIOENG., 10 Fukui, S.: Kangento no teiryoho, p. 45-47. Gakkai Syuppan Center, Tokyo (1982). 11. Fnkui, S.: Kangento no teiryoho, p. 10-12. Gakkai Syuppan Center, Tokyo (1982). 12. Kusakabe, I., Kamiyama, Y., and Yasui, T.: Methyl-fl-Dglucoside as a satisfactory internal standard in gas-liquid chromatographic quantitative-analysis of alditolacetates derived from monosaccharides. Nippon Nogeikagaku Kalshi, 51, 167169 (1977). 13. Aso, K. and Yamauchi, F.: Paper chromatography of Dglucopyranosidic polymer. J. Ferment. Technol., 33, 194-197 (1955). 14. Villettaz, J. C., Amado, R., and Neukom, H.: Comparative structural studies of the D-mannans from a rose wine and Saccharomyces uvarum. Carbohydrate Research, 81, 341-344 (1980).
15. Shiota, M., Nakajima, T., Satoh, A., Shida, M., and Matsuda, K.: Comparison of//-glucan structures in a cell wall mutant of Saccharomyces cerevisiae and the wild type. J. Biochem., 98, 1301-1307 (1985). 16. Farkas, V., Biely, P., and Bauer, S.: ExtraceUlular fl-glucanases of the yeast, Saccharomyces cerevisiae. Biochim. Biophys. Acta, 321, 246-255 (1973). 17. Arnold, W.N.: The structure of the yeast cell wall. J. Biol. Chem., 247, 1161-1169 (1972). 18. Fleet, G . H . and Phaff, H . J . : Glucanases in Schizosaccharomyces. J. Biol. Chem., 249, 1717-1728 (1974).
Isolation of Two Glucodisaccharides from Japanese Wines KOTOYOSHI NAKANISHI* AND KOKI YOKOTSUKA
Institute of Enology and Viticulture, Yamanashi University, Kofu 400, Japan Received 2 September 1988/Accepted l0 December 1988 The composition of oligosaccharides in four varieties of Japanese white table wines was examined using two separation methods, charcoal column chromatography and paper chromatography. Laminaribiose and gentiobiose were isolated as the principal constituents of the oligosaccharides from all the wines tested. The concentration of each of these sugars in the wines ranged from 15 mg/1 to 25 rag~1. When fermentation was done with a synthetic medium, these amounts of laminaribiose and gentiobiose were also isolated from the fermentation broth using the same separation techniques. The results suggested that both sugars were released from yeast cells into the wines during the fermentation. The amounts of both sugars released from yeast were closely associated with the conditions of the fermentation and independent of the strain of the yeast used for the fermentation.
(9). The sample solution was put on a charcoal (Nakarai Chemicals Ltd.; 60-150mesh) column (2.2×25 cm) and eluted with distilled water to remove the monosaccharides and salts. It was then eluted stepwise with 10%, 20%, and 30% aqueous ethanol at the rate of about 120 ml/h. The sugar content in the effluent was measured by the phenol sulfuric acid method (10). Paper chromatography Paper chromatography was done twice with filter paper (Toyo Roshi no. 51). The solvent system was n-butanol-pyridine-water ( 6 : 4 : 3 ) . The sugars were detected with p-anisidine hydrochloride. The amounts of each of the oligosaccharides extracted from the paper chromatogram were measured by the SomogyiNelson method (11). Acid hydrolysis Each of the oligosaccharides extracted from the paper chromatogram was partially hydrolyzed with acid to analyze its composition. One ml of the solution containing the oligosaccharides and 1 ml of 2 N HC1 were mixed, and left at 90-100°C for 2 h. The mixture was evaporated to dryness by bubbling with nitrogen gas, and then dissolved in 1 ml of distilled water. The sugar was measured by the Somogyi-Nelson method before and after acid hydrolysis. Also the sugar after acid hydrolysis was measured by the glucose oxidase method and gas liquid chromatography (12). The acid hydrolysis products were examined by paper chromatography. Enzyme hydrolysis Anomer analysis of each of the oligosaccharides obtained was done using pure yeast aglucosidase and almond p-glucosidase (Sigma Chemical Company). The reaction mixture consisted of 0.25 ml of the substrate solution, 0.25 ml of McIlvaine buffer (pH 6.0 for a-glucosidase and pH 5.0 for ]~-glucosidase) and 0.5 ml of the enzyme solution. The final concentrations of oligo. A and oligo. B were 0.15°//oo and 0.08%, respectively. The concentration of a-gluosidase was 0.31 units for both oligo. A and oligo. B and that of the fl-glucosidase was 0.91 units for the oligo. A and 4.6 units for the oligo. B. The reaction was run at 35°C for 4 h, and the hydrolyzates from each reaction mixture were examined by paper chromatography. Microorganisms and culture condition Saccharornyces cerevisiae OC-2 and W-3 (wine yeasts), Saccharomyces cerevisiae Kyokai no. 9 (sake yeast), and
Wines contain small amounts of residual mono-, oligo-, and poly-saccharides other than glucose and fructose. Almost all of these sugars are usually unfermentable and are originally present in grape juices. These sugars are believed to have either desirable or undesirable effects on the quality of wines during aging. Many reports on the residual sugars in wines have been published (1, 2). However, information on the residual oligosaccharides is very limited (3-5). Particularly, there has not been any such report on Japanese wines. The compositions of residual oligosaccharides and monosaccharides in wines have been directly analyzed by paper-, thin layer-, gas-, and liquid chromatographies (1, 6, 7). Although these methods are quick and easy, the fact that the concentration of the individual oligosaccharides is usually very small compared to that of the monosaccharides makes it difficult to examine the composition of the oligosaccharides. In this work, charcoal column chromatography methods were used, and the oligosaccharides were detected and isolated by paper chromatography. This report mainly describes the isolation and properties of two oligosaccharides obtained from the four different Japanese white table wines. In addition, some data on the origin of both sugars are also presented. MATERIALS AND M E T H O D S Wines For the analysis of residual oligosaccharides, four kinds of white table wine made at our Institute of Winery in 1983 from Koshu, Chardonnay, Riesling, and Pinot Gris grapes were used. All the grapes were harvested from our Institute's vineyard. General analyses of the four wines was made using the methods in Kokuzeicho Shoteibunsekihochukai (8). The results are shown in Table 1. Charcoal column chromatography Seven hundred ml of the wines was concentrated to approximately onefifth of the original volume. The concentrated sample was treated with saturated neutral lead acetate solution, glacial acetic acid, and disodium hydrogen phosphate according to the methods for the analysis of reducing sugar in wine * Corresponding author. 148
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TABLE 1. Compositions of the four wines used for the analysis of the residual oligosaccharides Alcohol Extract (%) (%)
Wines Koshu Riesling Chardonnay Pinot Gris
12.5 11.7 11.3 10.3
3.66 4.88 4.59 4.71
Reducing su0ga (%)r
Total acid (%)
pH
1.06 2.06 1.82 1.80
0.66 0.64 0.59 0.60
3.20 3.32 3.39 3.56
b a k e r ' s yeast (Oriental Yeast Co., Ltd.) were used. Basal m e d i u m consisted o f a c a r b o n source, 2 g o f asparagine, 1 g o f KH2PO4, and 3 g o f MgSO4 7H20 per 1 1 o f tap water. F o r the seed culture, yeast extract was a d d e d to the m e d i u m at the concentration o f 0 . 1 % . The initial p H o f the m e d i u m was adjusted to 3.5, 5.5, or 7.5 with 10% tartaric acid or 1 N N a O H solution. Five ml o f cell suspension obtained f r o m seed culture was inoculated into a 500 ml flask containing 300 ml o f the medium. The flask was anaerobically incubated at 25°C for 15-20d. The supernatant o f the culture b r o t h was used as a sample. Chemicals Kojibiose and nigerose were d o n a t e d by P r o f . Seiya C h i b a ( H o k k a i d o Univ.). Laminaribiose was d o n a t e d by Associate P r o f . Isao K u s a k a b e (Univ. of Tsukuba). Other standard glucodisaccharides were obtained from commercial sources. Glucose oxidase (Glucose CTest W a k o ) was purchased from W a k o Pure Chemicals, Ltd., T o k y o . Other chemicals used were o f analytical grade and o b t a i n e d commercially. RESULTS AND DISCUSSION
The composition of the residual oligosaccharides An experiment to elucidate the c o m p o s i t i o n o f residual oligosaccharides in wine was done by charcoal column c h r o m a t o g r a p h y and paper c h r o m a t o g r a p h y . The total a m o u n t o f sugar equivalent to glucose in each fraction is presented in Table 2. In the four wines tested, almost all the residual sugars were eluted with water and the total a m o u n t o f sugar eluted with ethanol solution was a b o u t 34 % . There were no significant differences in the elution profiles o f the four wines. Each ethanol fraction was concentrated and used for paper c h r o m a t o g r a p h y (Fig. 1). A few spots considered to be oligosaccharides from their Rf values were observed. Two m a j o r spots, represented by A and B on the c h r o m a t o g r a m , were detected in 10% and 20% ethanol fractions, and were c o m m o n to the four wines. However, the separation o f A f r o m B in b o t h the ethanol fractions was not very clear. The two sugars were tentatively n a m e d oligo. A and oligo. B; they were isolated.
SFIO
30 20 10 30 20 FS 20 10 30 20 10 30 K R C P
FIG. I. Paper chromatogram of oligosaccharides of wines. Each fraction shown in Table 2 was concentrated and spotted on the paper. Abbreviations: K, Koshu; R, Riesling; C, Chardonnay; P, Pinot Gris; A, oligo. A; B, oligo. B; S, Standard (Rha., rhamnose; Xyl., xylose; Ara., arabinose; Glc., glucose; Gal., galactose; GaA., galacturonic acid; F, fructose); 10, 20 and 30, 10%, 20°//o, and 30% ethanol fraction.
Isolation of oligo. A and oligo. B To obtain a high concentration o f oligo. A and oligo. B, b o t h sugars were isolated with a scale-up system using 3.5 l o f the four wines. The concentrated wine sample (300ml) was pretreated as described before and was then put on a charcoal column (2.5 x 60 cm). After the column was washed with 900 ml o f water, the elution with ethanol was done at the concentration of 5%, 10%, 15%, and 20%. The eluent was collected in 100-ml fraction tubes using a fraction collector. The recovery o f the sugars eluted with aqueous ethanol was a b o u t 3 - 4 % , and this result was similar to that shown in Table 2. In addition, there were no significant differences in the elution profiles. Observation with paper c h r o m a t o g r a p h y showed that spots corresponding to oligo. A and oligo. B were mainly detected in the 10% ethanol and 15°//00 ethanol fractions, respectively. These fractions contained few oligosaccharide contaminants. Each fraction containing oligo. A and oligo. B was collected and concentrated to 10 ml under reduced pressure. Five hundred pl o f the concentrated samples were spotted on a paper and developed two times. Both the edges and the middle part o f each resulting paper strip were cut off and sprayed with the reagent to find the position o f the
TABLE 2. Fractionation of residual sugars in four wines by charcoal column chromatography Wines Fractions Sampleb Water 10~ooEthanol 20~oo Ethanol 30°//ooEthanol
Koshu Volume (ml) 150 325 265 243 250
Sugar (mgp 9275 (100y 7586 (82) 130 (1.4) 93 (1.0) 34 (0.37)
Riesling Volume Sugar (ml) (rag) 160 17120 (100) 375 15050 (88) 255 245 (1.4) 250 175 (1.0) 250 51 (0.30)
" As glucose mg. b Concentrated wine sample (before chromatography). c Yield (sample= 100%).
Chardonnay Volume Sugar (ml) (rag) 155 16275 (100) 355 12715 (78) 250 260 (1.5) 250 240 (1.5) 235 47 (0.29)
Pinot Gris Volume Sugar (ml) (mg) 170 16320 (100) 360 14688 (90) 250 375 (2.3) 250 190 (1.2) 245 96 (0.59)
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NAKANISHIAND YOKOTSUKA
J. FERMENT.BIOENG.,
bands of oligo. A and oligo. B. Each sugar was extracted from the paper with hot water at about 90°C for 1 h. Each extract was evaporated to a syrup, and was dried with absolute ethanol. Definite amounts of distilled water were added to the dried samples, and the reducing sugars in the solution were measured by the Somogyi-Nelson method. The oligo, A and oligo. B thus obtained gave only a single sugar spot each on the paper chromatogram (Fig. 2). In paper chromatograms developed with several kinds of solvents, each sugar still gave a single spot. Identification of oligo. A and oligo. B Some of the properties of otigo. A and oligo. B obtained from the four wines were examined by the following experiments. To analyze the composition of the sugars, oligo. A and oligo. B were hydrolyzed with acid as described before. The hydrolyzates were analyzed with paper chromatography and the results are shown in Fig. 2. It was found that acid hydrolysis gave glucose as the only product, so oligo. A and oligo. B both consist of glucose. In the above experiment, the amount of the reducing sugar in the sample was measured by Somogyi-Nelson method before and after the acid hydrolysis. It was found that the amount of the sugar in the sample increased about 2 times when treated with acid. This result shows that the degree of polymerization was about 2. The amount of sugar after acid hydrolysis was also measured by the glucose oxidase method and gas chromatography. The results obtained by this method agreed very closely with those of the SomogyiNelson method. These results suggest that the oligo. A and the oligo. B were glucodisaccharides. Various standard glucodisaccharides were paper chromatographed together with oligo. A, oligo. B, and glucose. The Rg values of kojibiose, nigerose, maltose, isomaltose, laminaribiose, cellobiose, and gentiobiose and were 0.69, 0.58, 0.74, 0.59, 0.87, 0.71, and 0.59, respectively. The Rg values of sophorose was reported to be 0.74 (13). The Rg values of oligo. A ranged from 0.85 to 0.87 and agreed very closely with that of the laminaribiose (0.87) and nigerose (0.85). The Rg value of the oligo. B (0.580.60) agreed with that of the isomaltose (0.59) and gentiobiose (0.59). So oligo. A was either laminaribiose (3-0fl-glucopyranosyl-glucose) or nigerose (3-0-a-glucopyranosyl-glucose), and that oligo. B was either gen-
GBBBAA
AG
++
++
a13
a13
FIG. 3. Paper chromatogram of hydrolysis products of oligo. A and oligo. B by a- and fl-glucosidase. Abbreviations: G, glucose; A, oligo. A; B, oligo. B.; A - u , oligo. A+ct-glucosidase; A - t , oligo. A+fl-glucosidase; B a, oligo. B+a-glucosidase; B - t , oligo. B +/~-glucosidase.
tiobiose (6-0-fl-glucopyranosyl-glucose) or isomaltose (6-0a-glucopyranosyl-glucose). Then anomer analysis of both sugars was done using yeast a-glucosidase and almond fl-glucosidase, Oligo. A and oligo. B were separately incubated with a-glucosidase and fl-glucosidase, and then enzymatic hydrolysis products were examined by paper chromatography (Fig. 3). It was found that oligo. A and oligo. B were completely hydrolyzed to glucose with fl-glucosidase. In the case of aglucosidase, no hydrolysis product was observed on the chromatogram. The same results were obtained with all the oligo. A and oligo. B tested. These results suggested that oligo. A and oligo. B have fl-glucosidic linkages. Based on all the experimental results obtained, oligo. A and oligo. B were identified as laminaribiose and gentiobiose, respectively. The iaminaribiose and gentiobiose contents in wine The laminaribiose and gentiobiose were extracted from the paper chromatogram of the four wines which was run in parallel with the above experiment and the amounts of both sugars were measured. Also various amounts of standard laminaribiose and gentiobiose were spotted on the same paper, and the recovery of both sugars which were extracted from the paper were examined. The recovery of TABLE 3. Contents of laminaribiose and gentiobiose in various wines
GABABABABABABABABG K R C P K R C
Wines
P
FIG. 2. Paper chromatogram of acid hydrolysis products of oligo, A and oligo. B isolated from various wines. Abbreviations:K, Koshu; R, Riesling; C, Chardonnay; P, Pinot Gris; A, oligo. A; B, oligo. B; G, Glucose.
Koshu Riesling Chardonnay Pinot Gris
Laminaribiose (mg//) 19 16 18 16
Gentiobiose (mg//) 20 22 24 19
VOL. 67, 1989
OLIGOSACCHARIDES FROM WINE
151
TABLE 4. Formations of laminaribiose and gentiobiose by various yeasts during the alcohol fermentation with synthetic medium Yeasts
Before fermentation Mediuma Sugar (%)b pH
Alcohol (%)
After fermentation Laminaribiose pH (mg//)
Gentiobiose (mg//)
Wine yeast (OC-2) Wine yeast (W-3) Sake yeastc Baker's yeast
14 14 14 14
3.5 3.5 3.5 3.5
7.2 7.0 7.3 6.8
2.8 2.8 2.9 3.1
15 13 18 13
33 31 38 27
Wine yeast (OC-2)
7 23 14 14
3.5 3.5 5.5 7.5
3.0 12.0 7.2 7.0
3.0 2.7 3.2 3.8
4 17 11 (2
9 37 29 15
a Other components are shown in the text. b Glucose and fructose are in the ratio of 1:1. c Kyokai no. 9.
s t a n d a r d sugars ranged from 90-100%, thus the contents o f the laminaribiose and gentiobiose in wines were measured without correction. In addition, since the recovery o f laminaribiose and gentiobiose by charcoal colu m n c h r o m a t o g r a p h y could not be rigorously calculated, the d a t a on the contents o f b o t h sugars in wines was uncorrected. These results are shown in Table 3. W i t h the four wines tested, there was no significant difference in the contents o f b o t h sugars. A l t h o u g h various disaccharides such as sucrose, lactose, isomaltose, maltose, cellobiose and melibiose are present in wine, the content o f each o f these disaccharides is usually less than 5 mg/l (1). On the other hand, there is a r e p o r t that the contents o f lactose, maltose, and sucrose, which were identified in two A m e r i c a n wines using paper c h r o m a t o g r a p h y , were in the range o f 10-50 m g / l (6). Considering to these reports, the fact that laminaribiose was isolated from wine seems to be o f interest because the presence o f this sugar in wine has not been reported. While, the presence o f gentiobiose in wine has been r e p o r t e d based on paper c h r o m a t o g r a p h y (4), measuring the sugar has not been done. The analytical d a t a on the laminaribiose and gentiobiose contents in wines presented in this report is, therefore, considered to be valuable. The total a m o u n t o f laminaribiose and gentiobiose f o u n d is very small when c o m p a r e d to the concentration o f residual glucose and fructose. Therefore, direct effects of b o t h sugars on the sweetness o f wine m a y be considered to be very little. However, in the cases o f prolonged aging o f wine, it is presumed that these oligosaccharides as well as unfermentable monosaccharides have either desirable or undesirable effects on the quality o f the wines. To clarify how these sugars influence the quality o f wines during aging, m o r e work is needed. The amounts of laminaribiose and gentiobiose released from yeast The contents o f laminaribiose and gentiobiose in wines are higher than those o f other oligosaccharides detected by paper c h r o m a t o g r a p h y . It is, therefore, o f great interest to elucidate the origin o f both sugars in wines. There are two possibilities, that the sugar is originally present in the grape juice or that they are released from the yeast during the fermentation. In the must samples after charcoal column c h r o m a t o g r a p h y , spots corresponding to the laminaribiose and gentiobiose could not be clearly detected on the paper c h r o m a t o g r a m . However, in culture filtrate obtained from alcoholic fermentation o f a synthetic m e d i u m , two clear spots corresponding to the laminaribiose and gentiobiose in their Rg
values were observed on the c h r o m a t o g r a m . Each sugar were extracted from the p a p e r and their properties were examined using the same methods as before. The sugars were confirmed to be laminaribiose and gentiobiose. W i t h every yeast tested, no significant differences in the content of each sugar were observed (Table 4). It was also f o u n d that the laminaribiose content was nearly equal to that found in wine, and that the gentiobiose content was slightly higher than that in wine. These results are sufficient to suggest that the laminaribiose and gentiobiose f o u n d in the wines are released from the yeast during fermentation. It has been reported that the m a n n a n and trehalose f o u n d in wine are p r o b a b l y released f r o m yeast cells during fermentation, by Villettaz et al. (14) and Bertrand et aL (7). Since such a investigation is very limited, the result obtained in this paper is very interesting. Also the extent of the accumulation o f laminaribiose and gentiobiose during the fermentation was examined by varying the initial p H and the sugar concentration o f the medium. These results are shown in Table 4. In both sugars, the a m o u n t o f the sugar increased with an increase in the initial sugar concentration of the medium. However, when the fermentation was done with the m e d i u m at initial p H o f 5.5 or 7.5, the a m o u n t o f b o t h sugars was very small c o m p a r e d to their concentrations in the case o f the m e d i u m with an initial p H o f 3.5 (nearly the same as the p H o f grape juice). The decrease in the amounts o f laminaribiose was very great. On the other hand, the effects o f fermentation temperature on the accumulation of the b o t h sugars were not great. This suggests that the amounts of laminaribiose and gentiobiose released from yeast are closely associated with the conditions o f the fermentation and independent of the difference in the strain o f the yeast used for the fermentation. It is well k n o w n that yeast cells contain both fl-(1-3)- and fl-(1-6)-linked glucan as m a j o r cell wall polymers, and that yeasts have several types o f cell wall associated flglucanases which hydrolyze the fl-(1-3)- and fl-(1-6)glucosidic linkages (15-17). In a few species o f yeast, glucose, laminaribiose, and other laminarioligosaccharides are formed from glucan by the action o f flglucanase during cell wall autolysis (18). Considering these fact, the release o f laminaribiose and gentiobiose from yeast cell to wine may be closely associated with the various types o f fl-glucanase action on the cell wall. To clarify the prosess of the f o r m a t i o n o f laminaribiose and gentiobiose, more work is needed.
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NAKANISHI AND YOKOTSUKA ACKNOWLEDGMENTS
The authors wish to thank Mr. T. Uesugi and Mr. A. Mimura for their technical assistance. REFERENCES 1. Amerine, M. A. and Ough, C. S.: Methods for analysis of must and wines, p. 41-44. John Wiley & Sons, Inc., New York (1980). 2. Otsuka, K.: Jozoseibunichiran, p. 284-287. Nippon Jozo Kyokal, Tokyo (1977). 3. Esau, P. and Amerine, M . A . : Residual sugars in wine. Am. J. Enol. Vitic., 15, 187-189 (1964). 4. Thaler, H.: Die papierchromatographische untersuchung yon weinverbesserungszuckern. Z. Lebnsmitt. Untersuch., 100, 359366 (1955). 5. Kliewer, W.: Identification and seasonal changes in the concentration of several trace sugars in Vitis vinifera. Am. J. Enol. Vitic., 16, 168-178 (1965). 6. Esau, P. and Amerine, M . A . : Quantitative estimation of residual sugars in wine. Am. J. Enol. Vitic., 17, 265-267 (1967). 7. Betrand, M. A., Dubernet, M. O., and Ribereau-Gayon, P.: Le trehalose principal diholoside des vins. C. R. Acad. Sc. Paris, Serie D, t. 280, 1907-1910 (1975). 8. Murakami, H.: Kokuzeicho shoteibunsekihochukai, 3rd. ed., p. 54-62. Nippon Jozo Kyokai, Tokyo (1974). 9. Amerine, M. A. and Ough, C. S.: Methods for analysis of musts and wines, p. 33-35. John Wiley & Sons, Inc., New York (1980).
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15. Shiota, M., Nakajima, T., Satoh, A., Shida, M., and Matsuda, K.: Comparison of//-glucan structures in a cell wall mutant of Saccharomyces cerevisiae and the wild type. J. Biochem., 98, 1301-1307 (1985). 16. Farkas, V., Biely, P., and Bauer, S.: ExtraceUlular fl-glucanases of the yeast, Saccharomyces cerevisiae. Biochim. Biophys. Acta, 321, 246-255 (1973). 17. Arnold, W.N.: The structure of the yeast cell wall. J. Biol. Chem., 247, 1161-1169 (1972). 18. Fleet, G . H . and Phaff, H . J . : Glucanases in Schizosaccharomyces. J. Biol. Chem., 249, 1717-1728 (1974).