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Hydrolysis of grape glycosides by enological yeast β-glucosidases
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1623
Hydrolysis of grape glycosides by enological yeast p-glucosidases I. Rosl, P. Domizio, M. Vinella and M. Salicone Dipartimento di Biologia, Difesa, Biotecnologie Agro-Forestali, Universita della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy
Abstract Three enological yeast strains, belonging to the species Debaryomyces hansenii, Debaryomyces polymorphus, and Saccharomyces cerevisiae, characterized by an exocellular p-glucosidase activity, were examined for their ability to hydrolize a glycosidic extract from grape juice. The enzymatic preparations (culture supernant fluid) of the different yeasts released different amounts of terpenols such as linalol, a-terpineol, geraniol, nerol, citronellol and benzyl and 2phenylethyl alcohol. The extent of release of the flavour compounds was related to yeast species. When an enzymatic preparation (concentrate culture supernatant) of Debaryomyces hansenii was incubated with a wine containing glycosidic precursors, significant production of monoterpenols and benzyl and 2-phenylethyl alcohol was observed.
1.
INTRODUCTION
Terpenes are a class of compounds responsible for the varietal aroma of many grapes, wines, and other fruits. Among the terpenes, the monoterpenols (linalol, nerol, geraniol, a-terpineol and citronellol) are the most active, from an olfactory point of view, due to their low sensory threshold. For example, linalol has a sensory threshold of 100 |Lig/l, while that of nerol and a-terpineol is three to four times higher (1). However, most of the terpenols in grapes are found in glycosidically bound forms which are almost odourless. Studies conducted to identify the glycosidic part of these flavour precursors, have shown that it is mostly formed by 6-0-a-arabinofuranosyl-p-D-glucopyranosides, 6-0-a-L-
1624 rhamnopyranosyl-p-D-glucopyranosides (rutinosides) (2, 3), 6-0-p-apiofuranosylP-D-glucopyranosides (4) and, to a much lesser degree, p-D-glucopyranosides (2, 3). The aglyconic part is primarily made up of monoterpenols and benzyl and 2phenylethyl alcohol. To release this potential reserve of aroma, studies have been conducted using enzymes with glycosidase activity . Earlier studies showed that hydrolysis of glycosidically bound terpenes occurs in two sequential steps (5). In the first, an a-L-arabinofuranosldase, a-L-rhamnopyranosidase and p-D-apiosidase must break the bond between the glucose and the terminal sugar (rhamnopyranose, arabinofuranose and apiofuranose). In the second step, a p-glucosidase must release the volatile aglycon from the glucose. Enzymatic preparations from plants (grape and sweet almond) and from microorganisms (moulds and yeasts) were studied to evaluate their capacity to hydrolyze the aromatic precursors found in grape and other fruits. Plant-produced p-glucosidases were characterized by a restricted specificity with respect to aglycon, were not very active between pH 3 and 4 and were inhibited by a glucose concentration over 1% (6, 7) .The p-glucosidase of fungal origin reacted strongly to a glucose concentration above 1-1.5 % (8). The P-glucosidases produced by the yeasts {Candida molischiana, C. wickeramii, Saccharomyces cerevisiae) were less sensitive to glucose and had a wider specificity for aglycon (9, 10). In a recent study (11) conducted to show the p-glucosidase activity in yeasts of enological origin, we found a strain of Debaryomyces hansenii which could produce an exocellular p-glucosidase whose activity was not inhibited by high ethanol and glucose concentrations and was not greatly influenced by acidic pH and low temperatures. Currently, we are studying the environmental conditions needed to increase the production of exocellular p-glucosidase by this strain of yeast as well as a strain of Debaryomyces polymorphus and Saccaromyces cerevisiae, which in a previous study, exhibited exocellular p-glucosldase activity. The aim of this work is to verify the capacity of exocellular p-glucosidase, produced by three different yeasts, to hydrolyze the glycosidically bound aroma compounds , in light of their use in juice processing and winemaking.
1625 2. MATERIALS AND METHODS 2.1 Yeasts The strains used were Debaryomyces hansenii 4025, Deb. polymorphus 3631 and Saccharomyces cerevisiae 1014. All strains were obtained from the Industrial Yeast Collection of Dipartimento di Biologia Vegetale, deH'Universita di Perugia (DBVPG). The strains were maintained at 4° C on slopes of yeast malt agar (YM). 2.2 Medium and culture conditions The basal culture medium was Yeast Nitrogen Base (YNB, Difco) : 0.67%, buffered with phosphate tartrate (100 mM, pH 5.0); the carbon source was arbutin (0.5%) for Saccharomyces cerevisiae 1014 and Debaryomyces polymorpiius 3631 and glucose (0.5%) for Debaryomyces iiansenii 4025. Aerobic culture was performed in Erienmeyer flasks filled to one tenth of their volume and shaken at 150 rev min""" on a giratory shaker for 24 h at 25 °C. 2.3 Enzymes After centrifugation (4000 rev min""", 10 min, 4 °C), the culture supernatant fluid of Debaryomyces Iiansenii 4025, Deb. polymorphus 363^ and Saccharomyces cerevisiae 1014 and the culture supernatant of Debaryomyces hansenii 4025 concentrated 50-fold by ultrafiltration in an Amicon cell (PM 10 filter) were used for hydrolysis assay. AR 2000 (Gist-Brocade, DAL GIN, Milano, Italy), a commercial preparation of pectolytic enzyme, which is a soluble powder, was also used. 2.4 Enzyme assay p-glucopyranosidase, a-rhamnopyranosidase and a-arabinofuranosidase activities were assayed by measuring the amount of p-nitrophenol (pNP) liberated from the substrates p-nitrophenyl-p-D-glucopyranoside, p-nitrophenyl-a-Lrhamnopyranoside or p-nitrophenyl-a-L-arabinofuranoside. According to the previously described method (11), 0.2 ml of each enzymatic preparation was mixed with 0.2 ml of a 5 mM solution of the appropriate glycoside in 100 mM citratephosphate buffer (pH 5.0). The reaction mixture was incubated at 30 °C for 1 h and subsequently 1.2 ml of carbonate buffer (0.2 mM, pH 10.2) were added to stop the reaction. The pNP liberated was measured by spectophotometry at 400 nm in a LBK Ultraspec 4050 spectophotometer. All assay were performed in duplicate and
1626 averaged. One unit of enzymatic activity (U) was defined as jimol of p-nitrophenol released min"'', under the above conditions. 2.5 Protein assay The protein concentration of samples was determined with the Bio-Rad protein reagent with bovine serum albumine as standard (Bio-Rad Laboratories, Richmond, Ca, USA) 2.6 Experiments with grape glycosides a) Isolation of the glycosides 200 ml aliquots of a mixture of Traminer grape juice and water (1:1) orTraminer wine were passed through a reverse-phase CI8 adsorbent column (5g) (Mega Bond Elut, VARIAN, Harbor City, CA, USA) that was activated by flushing with 40 ml of methanol and 40 ml of water. The column was washed with methylene chloride (100 ml) in order to remove free terpenols. Glycosides were then recovered by elution with methanol (100 ml) and stored at -18 °C. Before using, this fraction was concentrated to dryness under vacuum at 30 °C . b)Enzymatic hydrolysis of the glycosides The glycosidic sample obtained from 200 ml of grape juice was dissolved in 50 ml of culture supernatant fluid (pH 4) of Debaryomyces hansenii 4025, Deb. polymorphus 363^ and Saccharomyces cerevisiae 1014. The glycosidic sample obtained from 200 ml of Traminer wine was suspended in 50 ml of lOOmM citratephosphate buffer (pH 5.0) and hydrolyzed with the concentrated supernatant of Debaryomyces hansenii 4025 and with the commercial pectolytic enzymatic preparation (AR 2000). The two enzymatic preparations, having the same [3glucosidase activity (0.7 U), were also used to hydrolyze the terpene glycosides found in 50 ml of Traminer wine (pH 3.1) in which free terpenols were measured. All samples were incubated at 25 °C for 48 h. Controls were run as above but without enzymatic preparation. All assays were performed in triplicate. 2.7 Analysis Liberated monoterpenols were: a) isolated by dynamic headspace analyzer, b) separated by gas chromatography and c) detected and evaluated by ion trap detector.
1627 a) Dynamic headspace sampling of monoterpenols (12) The volatile monoterpenols in the headspace were isolated by dynamic headspace analyzer LSC 2000TM (TEKMAR Inc., Cincinnati, OH, USA). The sample vessel, after conditioning at 25°C for 10 minutes, was purged with helium at 40 ml min-"" for 30 min to isolate headspace monoterpenols. The compounds isolated by purging were then trapped in a TenaxTM , 12x1/8" column. The trapped volatile monoterpenols, after a 10 min dry purge, were desorbed at 220 °C for 20 min, using helium gas at 1 ml min"'', and cryogenically focused at the first 10 cm capillary column which was cooled down to -100 °C by liquid nitrogen. The volatile compounds, condensed in the first 10 cm of the capillary column, were rapidly vaporized at 220 °C, and transfered to the capillary column for the analysis.
b) Gas chromatographic analysis A gas chromatograph (VARIAN 3300, Palo Alto, CA, USA) with an Ion trap detector (Finningan Mat ITD 700) was used to analyze of volatile monoterpenols. A fused-silica capillary column, Supelcowax 10, 60 m x 0.32mm, 0.25 mm film thickness, was used. The helium gas flowrate was 1ml min-""- The initial column temperature of 60 °C was maintained for 10 min after which the temperature was programmed to increase 3°C min-i to the final temperature of 195 °C which was maintained for 10 min. The peak areas of monoterpenols were calculated by peak integration module in the ion trap detector. Figure 1 shows the separation of some volatile monoterpenols and two cyclic alcohols, released by the culture supernatant fluid of Debaryomyces hansenii 4025 from glycoside extract of Traminer grape juice, after 48 h at 25 °C. Figure 2 shows the volatile compounds present in glycoside extract of Traminer grape juice after 48 h at 25 °C, without yeast enzymatic preparation. The identifications in the legend were determined by comparing the results with mass spectra of reference substances. c) Ion trap detection (ITD) Electron impact mass spectra of monoterpenols enzymatically hydrolyzed from the glycosides were recordered by coupling the Supelcowax 10 fused-silica capillary column to ITD. The transfer line was mainatained at 220 °C. The source temperature was 230 °C. Mass spectra were scanned between 50 and 80 eV in the 50-250 m/z range at 2-s intervals. The electron multiplier voltage was 1350 V.
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1630 E3 Saccharomyces cerevisiae 400
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COMPOUNDS
Figure 3.
Monoterpenols and aromatic alcohols released from a grape glycoside extract added to the culture supernatant fluid of three yeasts 1=Linalool, 2=a-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol. (LSD value, **, *** significant at 1 and 0.1%levels)
1631 E3i Sacch. cerevisiae B Deb. polymorphus H Deb. hansenii
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Figure 4
Total amount of monoterpenols (1) and aromatic alcohols (2) released by: a) culture supernatant fluid of three yeasts from grape glycoside extract b) Deb. hanseneii and AR2000 enzymatic preparations from wine (pH 3.1); c) Deb. hanseneii and AR2000 enzymatic preparations from wine glycoside extract in citrate-phosphate buffer (pH 5.0) (LSD value,*, **, *** significant at 5, 1 and 0.1%levels)
1632
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Monoterpenols and aromatic alcohols released by Deb. hanseneii and AR2000 enzymatic preparations from: a) wine (pH 3.1); b) wine glycoside extract in citratephosphate buffer (pH 5.0) 1 =Linalool, 2=a-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol. (LSD value, **, *** significant at 1 and 0.1 % levels)
1633 2.8 Chemicals p-nitrophenyl-p-D-glucopyranoside, p-nitrophenyl-a-L-rhamopyranoside, pnitrophenyl-a-L-arabinofuranoside were obtained from Sigma (St. Louis, MO, USA). All other chemicals used were of reagent grade. 3. RESULTS AND
DISCUSSION
The production and properties of yeast-produced p-glucosidases is of interest because of the potential to increase the availability of specific enzymatic preparations which could be used in winemaking to increase the varietal aroma of the wine. There is also interest in the possibilty of arranging selected cultures of yeasts characterised by the presence of active p-glucosidase during and/or after alcoholic fermentation. The hydrolysis values of the terpene glycosides of Traminer grape juice, added to the culture supernatant containing the exocellular p-glucosidase produced by Saccharomyces cerevisiae 1024, Debaryomyces polymorphus 3631 and Debaryomyces hansenii 4025, are reported in Figure 3. More linalol and a terpineol were released by the enzyme present in the supernatant of Deb. polymorphus 3631, while more 2-phenylethyl alcohol was released by the enzyme of Deb. hansenii. The enzymatic preparations of Deb. polymorphus 3631 and Sacch. cerevisiae 1014 released the most monoterpenols (Figure 4, a). The smaller quantity of monoterpenols released by the exocellular enzyme produced by Deb. hanseniii 4025 could be due to the lack of a-L-arabinofuranosidase and a - L rhamnopyranosidase enzymatic activity as reported in Table 1. These results confirm previous findings, i.e. that most of the monoterpenols are found as diglycosides (2, 3). Therefore, the enzymatic preparations of Deb. polymorphus 363^ and Sacch. cerevisiae 1014, which have a-L-arabinofurosidase and a-L-rhamnosidase activity, can release more monoterpenols into the medium. Furthermore, the enzymes produced by these yeasts showed a rather wide specificity. They could attack the glycosides with tertiary alcohols as aglycon, as well as, those with primary alcohols. This behaviour has rarely been found in the enzymatic preparations of either plant or microbial origin studied to date. Having shown that p-glucosidase, produced by Deb. hansenii 4025, had some interesting properties that could be used in winemaking (11), we carried out trials to analyze the hydrolytic behaviour of the culture supernatant fluid, concentrated 50 times, and compared the results to those from the commercial
1634 pectolytic enzyme preparation (AR2000). Recent studies have shown that this enzyme, produced by Aspergillus niger, efficiently releases the monoterpenols from the terpenic glycosides present in white and red aromatic grape must (13). Figure 5 reports the amounts of monoterpenols and benzyl and 2-phenylethyl alcohol released by the two enzymatic preparations added to Traminer wine (pH 3.1) and to a citrate-phosphate buffer (pH 5.0) containing glycosides extracted from the Traminer wine. The enzymatic preparation of Deb. hansenii 4025 released more linalol, a-terpineol, benzyl and 2-phenylethyl alcohol, while the commercial enzyme released more citronellol, nerol and geraniol. This behaviour was noted in both wine and buffer. In the wine, however, the total amount of alcohols (monoterpenols, benzyl and 2-phenylethyl alcohol) released by the two enzymatic preparations was less than in the buffer (Figure 4, b and c); this demonstrates that pH is an important factor for enzymatic activity. Nevertheless, the enzymatic preparation of Deb. hansenih 4025 appears to be less affected by a low pH in the wine because it released more alcohols (Figure 4, b). Table 1 Glycosidase activities in culture supernatant fluid of Debaryomyces hansenii 4025, Debaryomyces polymorphus 363^ and Saccharomyces cerevisiae 1024
Enzyme
Deb. hansenii Deb. polymorphus Sacch. cerevisiae
Substrate pNPp-D-Glucopyranoside 0.50^-51 b 0.60a-33b 1.10a.-5lb
pNPa-L-Arabino-
pNPa-L-Rhamno-
furanoside
pyranoside
0.005^-0.27^ 0.010^-0.05^
0.03^-0.15b 0.03^-0.17b
^Activity expressed as units (U) b Activity expressed as specific activity (U mg-"' protein) 4.
CONCLUSIONS
The data presented here confirm the interest in utilizing yeasts with P-glucosidase activity for enriching the aroma of grape juice and wines by the
1635 hydrolysis of glycosidic precursors. Further study is in progress to purify the pglucosidases of these yeasts and to study the biochemical properties and the possibilities to utilize these enzymes or the yeasts themselves under juice processing and winemaking conditions.
5. REFERENCES 1 2 3 4 5 6 7 8 9 I 0 II 12 13
P. Ribereau-Gayon, J.N. Boidron and A. Terrier, J. Agr. Food Chem. 23 (1975) 1042. P.J. Williams, C.R. Strauss, B. Wilson and R.A. Massy-Westropp, Phytochemestry 21 (1982) 2013. P.J. Williams, C.R. Strauss, Phytochemestry 22 (1983) 2039. S.G. Voirin, R.L Baumes, S.M. Bitteur, Z.Y. Gunata and C.L Bayonove, J. Agr. Food Chem. 38 (1990) 1373. Z.Y. Gunata, S.M. Bitteur, J.M. Brillouet, C.L Bayonove and R.E. Cordonnier, Carbohydr. Res. 184 (1988) 139. C.L Bayonove, Y.Z. Gunata and R.E. Cordonnier, Bulletin de I' O.I.V. 643 (1984)741. A.P. Aryan, B.Wilson, C.R.Strauss, and P.J. Williams, Am. J. Enol. Vltic. 38 (1987) 182. R.E. Cordonnier, Y.Z. Gunata, R.L Baumes and C.L Bayonove, Conn. Vigne Vin23 (1989)7. Y.Z. Gunata, C.L Bayonove, A. Arnaud, and P. Galzy, J. Sci.Food Agric. 50 (1990)499. P.H. Darriet, J.-N. Boidron and D. Dubourdieu, Conn. Vigne Vin 22 (1988) 189. L Rosi, M. Vinella and P. Domizio, J. Appl. Bacteriol. (1994) in press. M. Bertuccioli, in preparation C. Bayonove, Y.Z. Gunata, J.C. Sapis, R.L Baumes, I. Dugelay and C. Grassin, VigneVini 9 (1993) 33.
Research supported by National Research Council of Italy, Special project RAISA, Sub-project No. 4, Paper N. 1491
1623
Hydrolysis of grape glycosides by enological yeast p-glucosidases I. Rosl, P. Domizio, M. Vinella and M. Salicone Dipartimento di Biologia, Difesa, Biotecnologie Agro-Forestali, Universita della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy
Abstract Three enological yeast strains, belonging to the species Debaryomyces hansenii, Debaryomyces polymorphus, and Saccharomyces cerevisiae, characterized by an exocellular p-glucosidase activity, were examined for their ability to hydrolize a glycosidic extract from grape juice. The enzymatic preparations (culture supernant fluid) of the different yeasts released different amounts of terpenols such as linalol, a-terpineol, geraniol, nerol, citronellol and benzyl and 2phenylethyl alcohol. The extent of release of the flavour compounds was related to yeast species. When an enzymatic preparation (concentrate culture supernatant) of Debaryomyces hansenii was incubated with a wine containing glycosidic precursors, significant production of monoterpenols and benzyl and 2-phenylethyl alcohol was observed.
1.
INTRODUCTION
Terpenes are a class of compounds responsible for the varietal aroma of many grapes, wines, and other fruits. Among the terpenes, the monoterpenols (linalol, nerol, geraniol, a-terpineol and citronellol) are the most active, from an olfactory point of view, due to their low sensory threshold. For example, linalol has a sensory threshold of 100 |Lig/l, while that of nerol and a-terpineol is three to four times higher (1). However, most of the terpenols in grapes are found in glycosidically bound forms which are almost odourless. Studies conducted to identify the glycosidic part of these flavour precursors, have shown that it is mostly formed by 6-0-a-arabinofuranosyl-p-D-glucopyranosides, 6-0-a-L-
1624 rhamnopyranosyl-p-D-glucopyranosides (rutinosides) (2, 3), 6-0-p-apiofuranosylP-D-glucopyranosides (4) and, to a much lesser degree, p-D-glucopyranosides (2, 3). The aglyconic part is primarily made up of monoterpenols and benzyl and 2phenylethyl alcohol. To release this potential reserve of aroma, studies have been conducted using enzymes with glycosidase activity . Earlier studies showed that hydrolysis of glycosidically bound terpenes occurs in two sequential steps (5). In the first, an a-L-arabinofuranosldase, a-L-rhamnopyranosidase and p-D-apiosidase must break the bond between the glucose and the terminal sugar (rhamnopyranose, arabinofuranose and apiofuranose). In the second step, a p-glucosidase must release the volatile aglycon from the glucose. Enzymatic preparations from plants (grape and sweet almond) and from microorganisms (moulds and yeasts) were studied to evaluate their capacity to hydrolyze the aromatic precursors found in grape and other fruits. Plant-produced p-glucosidases were characterized by a restricted specificity with respect to aglycon, were not very active between pH 3 and 4 and were inhibited by a glucose concentration over 1% (6, 7) .The p-glucosidase of fungal origin reacted strongly to a glucose concentration above 1-1.5 % (8). The P-glucosidases produced by the yeasts {Candida molischiana, C. wickeramii, Saccharomyces cerevisiae) were less sensitive to glucose and had a wider specificity for aglycon (9, 10). In a recent study (11) conducted to show the p-glucosidase activity in yeasts of enological origin, we found a strain of Debaryomyces hansenii which could produce an exocellular p-glucosidase whose activity was not inhibited by high ethanol and glucose concentrations and was not greatly influenced by acidic pH and low temperatures. Currently, we are studying the environmental conditions needed to increase the production of exocellular p-glucosidase by this strain of yeast as well as a strain of Debaryomyces polymorphus and Saccaromyces cerevisiae, which in a previous study, exhibited exocellular p-glucosldase activity. The aim of this work is to verify the capacity of exocellular p-glucosidase, produced by three different yeasts, to hydrolyze the glycosidically bound aroma compounds , in light of their use in juice processing and winemaking.
1625 2. MATERIALS AND METHODS 2.1 Yeasts The strains used were Debaryomyces hansenii 4025, Deb. polymorphus 3631 and Saccharomyces cerevisiae 1014. All strains were obtained from the Industrial Yeast Collection of Dipartimento di Biologia Vegetale, deH'Universita di Perugia (DBVPG). The strains were maintained at 4° C on slopes of yeast malt agar (YM). 2.2 Medium and culture conditions The basal culture medium was Yeast Nitrogen Base (YNB, Difco) : 0.67%, buffered with phosphate tartrate (100 mM, pH 5.0); the carbon source was arbutin (0.5%) for Saccharomyces cerevisiae 1014 and Debaryomyces polymorpiius 3631 and glucose (0.5%) for Debaryomyces iiansenii 4025. Aerobic culture was performed in Erienmeyer flasks filled to one tenth of their volume and shaken at 150 rev min""" on a giratory shaker for 24 h at 25 °C. 2.3 Enzymes After centrifugation (4000 rev min""", 10 min, 4 °C), the culture supernatant fluid of Debaryomyces Iiansenii 4025, Deb. polymorphus 363^ and Saccharomyces cerevisiae 1014 and the culture supernatant of Debaryomyces hansenii 4025 concentrated 50-fold by ultrafiltration in an Amicon cell (PM 10 filter) were used for hydrolysis assay. AR 2000 (Gist-Brocade, DAL GIN, Milano, Italy), a commercial preparation of pectolytic enzyme, which is a soluble powder, was also used. 2.4 Enzyme assay p-glucopyranosidase, a-rhamnopyranosidase and a-arabinofuranosidase activities were assayed by measuring the amount of p-nitrophenol (pNP) liberated from the substrates p-nitrophenyl-p-D-glucopyranoside, p-nitrophenyl-a-Lrhamnopyranoside or p-nitrophenyl-a-L-arabinofuranoside. According to the previously described method (11), 0.2 ml of each enzymatic preparation was mixed with 0.2 ml of a 5 mM solution of the appropriate glycoside in 100 mM citratephosphate buffer (pH 5.0). The reaction mixture was incubated at 30 °C for 1 h and subsequently 1.2 ml of carbonate buffer (0.2 mM, pH 10.2) were added to stop the reaction. The pNP liberated was measured by spectophotometry at 400 nm in a LBK Ultraspec 4050 spectophotometer. All assay were performed in duplicate and
1626 averaged. One unit of enzymatic activity (U) was defined as jimol of p-nitrophenol released min"'', under the above conditions. 2.5 Protein assay The protein concentration of samples was determined with the Bio-Rad protein reagent with bovine serum albumine as standard (Bio-Rad Laboratories, Richmond, Ca, USA) 2.6 Experiments with grape glycosides a) Isolation of the glycosides 200 ml aliquots of a mixture of Traminer grape juice and water (1:1) orTraminer wine were passed through a reverse-phase CI8 adsorbent column (5g) (Mega Bond Elut, VARIAN, Harbor City, CA, USA) that was activated by flushing with 40 ml of methanol and 40 ml of water. The column was washed with methylene chloride (100 ml) in order to remove free terpenols. Glycosides were then recovered by elution with methanol (100 ml) and stored at -18 °C. Before using, this fraction was concentrated to dryness under vacuum at 30 °C . b)Enzymatic hydrolysis of the glycosides The glycosidic sample obtained from 200 ml of grape juice was dissolved in 50 ml of culture supernatant fluid (pH 4) of Debaryomyces hansenii 4025, Deb. polymorphus 363^ and Saccharomyces cerevisiae 1014. The glycosidic sample obtained from 200 ml of Traminer wine was suspended in 50 ml of lOOmM citratephosphate buffer (pH 5.0) and hydrolyzed with the concentrated supernatant of Debaryomyces hansenii 4025 and with the commercial pectolytic enzymatic preparation (AR 2000). The two enzymatic preparations, having the same [3glucosidase activity (0.7 U), were also used to hydrolyze the terpene glycosides found in 50 ml of Traminer wine (pH 3.1) in which free terpenols were measured. All samples were incubated at 25 °C for 48 h. Controls were run as above but without enzymatic preparation. All assays were performed in triplicate. 2.7 Analysis Liberated monoterpenols were: a) isolated by dynamic headspace analyzer, b) separated by gas chromatography and c) detected and evaluated by ion trap detector.
1627 a) Dynamic headspace sampling of monoterpenols (12) The volatile monoterpenols in the headspace were isolated by dynamic headspace analyzer LSC 2000TM (TEKMAR Inc., Cincinnati, OH, USA). The sample vessel, after conditioning at 25°C for 10 minutes, was purged with helium at 40 ml min-"" for 30 min to isolate headspace monoterpenols. The compounds isolated by purging were then trapped in a TenaxTM , 12x1/8" column. The trapped volatile monoterpenols, after a 10 min dry purge, were desorbed at 220 °C for 20 min, using helium gas at 1 ml min"'', and cryogenically focused at the first 10 cm capillary column which was cooled down to -100 °C by liquid nitrogen. The volatile compounds, condensed in the first 10 cm of the capillary column, were rapidly vaporized at 220 °C, and transfered to the capillary column for the analysis.
b) Gas chromatographic analysis A gas chromatograph (VARIAN 3300, Palo Alto, CA, USA) with an Ion trap detector (Finningan Mat ITD 700) was used to analyze of volatile monoterpenols. A fused-silica capillary column, Supelcowax 10, 60 m x 0.32mm, 0.25 mm film thickness, was used. The helium gas flowrate was 1ml min-""- The initial column temperature of 60 °C was maintained for 10 min after which the temperature was programmed to increase 3°C min-i to the final temperature of 195 °C which was maintained for 10 min. The peak areas of monoterpenols were calculated by peak integration module in the ion trap detector. Figure 1 shows the separation of some volatile monoterpenols and two cyclic alcohols, released by the culture supernatant fluid of Debaryomyces hansenii 4025 from glycoside extract of Traminer grape juice, after 48 h at 25 °C. Figure 2 shows the volatile compounds present in glycoside extract of Traminer grape juice after 48 h at 25 °C, without yeast enzymatic preparation. The identifications in the legend were determined by comparing the results with mass spectra of reference substances. c) Ion trap detection (ITD) Electron impact mass spectra of monoterpenols enzymatically hydrolyzed from the glycosides were recordered by coupling the Supelcowax 10 fused-silica capillary column to ITD. The transfer line was mainatained at 220 °C. The source temperature was 230 °C. Mass spectra were scanned between 50 and 80 eV in the 50-250 m/z range at 2-s intervals. The electron multiplier voltage was 1350 V.
1628
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1630 E3 Saccharomyces cerevisiae 400
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Debaryomyces polymorphus Debaryomyces hansenii
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COMPOUNDS
Figure 3.
Monoterpenols and aromatic alcohols released from a grape glycoside extract added to the culture supernatant fluid of three yeasts 1=Linalool, 2=a-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol. (LSD value, **, *** significant at 1 and 0.1%levels)
1631 E3i Sacch. cerevisiae B Deb. polymorphus H Deb. hansenii
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Figure 4
Total amount of monoterpenols (1) and aromatic alcohols (2) released by: a) culture supernatant fluid of three yeasts from grape glycoside extract b) Deb. hanseneii and AR2000 enzymatic preparations from wine (pH 3.1); c) Deb. hanseneii and AR2000 enzymatic preparations from wine glycoside extract in citrate-phosphate buffer (pH 5.0) (LSD value,*, **, *** significant at 5, 1 and 0.1%levels)
1632
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3 4 5 COMPOUNDS
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3 4 5 COMPOUNDS Figure 5
Monoterpenols and aromatic alcohols released by Deb. hanseneii and AR2000 enzymatic preparations from: a) wine (pH 3.1); b) wine glycoside extract in citratephosphate buffer (pH 5.0) 1 =Linalool, 2=a-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol. (LSD value, **, *** significant at 1 and 0.1 % levels)
1633 2.8 Chemicals p-nitrophenyl-p-D-glucopyranoside, p-nitrophenyl-a-L-rhamopyranoside, pnitrophenyl-a-L-arabinofuranoside were obtained from Sigma (St. Louis, MO, USA). All other chemicals used were of reagent grade. 3. RESULTS AND
DISCUSSION
The production and properties of yeast-produced p-glucosidases is of interest because of the potential to increase the availability of specific enzymatic preparations which could be used in winemaking to increase the varietal aroma of the wine. There is also interest in the possibilty of arranging selected cultures of yeasts characterised by the presence of active p-glucosidase during and/or after alcoholic fermentation. The hydrolysis values of the terpene glycosides of Traminer grape juice, added to the culture supernatant containing the exocellular p-glucosidase produced by Saccharomyces cerevisiae 1024, Debaryomyces polymorphus 3631 and Debaryomyces hansenii 4025, are reported in Figure 3. More linalol and a terpineol were released by the enzyme present in the supernatant of Deb. polymorphus 3631, while more 2-phenylethyl alcohol was released by the enzyme of Deb. hansenii. The enzymatic preparations of Deb. polymorphus 3631 and Sacch. cerevisiae 1014 released the most monoterpenols (Figure 4, a). The smaller quantity of monoterpenols released by the exocellular enzyme produced by Deb. hanseniii 4025 could be due to the lack of a-L-arabinofuranosidase and a - L rhamnopyranosidase enzymatic activity as reported in Table 1. These results confirm previous findings, i.e. that most of the monoterpenols are found as diglycosides (2, 3). Therefore, the enzymatic preparations of Deb. polymorphus 363^ and Sacch. cerevisiae 1014, which have a-L-arabinofurosidase and a-L-rhamnosidase activity, can release more monoterpenols into the medium. Furthermore, the enzymes produced by these yeasts showed a rather wide specificity. They could attack the glycosides with tertiary alcohols as aglycon, as well as, those with primary alcohols. This behaviour has rarely been found in the enzymatic preparations of either plant or microbial origin studied to date. Having shown that p-glucosidase, produced by Deb. hansenii 4025, had some interesting properties that could be used in winemaking (11), we carried out trials to analyze the hydrolytic behaviour of the culture supernatant fluid, concentrated 50 times, and compared the results to those from the commercial
1634 pectolytic enzyme preparation (AR2000). Recent studies have shown that this enzyme, produced by Aspergillus niger, efficiently releases the monoterpenols from the terpenic glycosides present in white and red aromatic grape must (13). Figure 5 reports the amounts of monoterpenols and benzyl and 2-phenylethyl alcohol released by the two enzymatic preparations added to Traminer wine (pH 3.1) and to a citrate-phosphate buffer (pH 5.0) containing glycosides extracted from the Traminer wine. The enzymatic preparation of Deb. hansenii 4025 released more linalol, a-terpineol, benzyl and 2-phenylethyl alcohol, while the commercial enzyme released more citronellol, nerol and geraniol. This behaviour was noted in both wine and buffer. In the wine, however, the total amount of alcohols (monoterpenols, benzyl and 2-phenylethyl alcohol) released by the two enzymatic preparations was less than in the buffer (Figure 4, b and c); this demonstrates that pH is an important factor for enzymatic activity. Nevertheless, the enzymatic preparation of Deb. hansenih 4025 appears to be less affected by a low pH in the wine because it released more alcohols (Figure 4, b). Table 1 Glycosidase activities in culture supernatant fluid of Debaryomyces hansenii 4025, Debaryomyces polymorphus 363^ and Saccharomyces cerevisiae 1024
Enzyme
Deb. hansenii Deb. polymorphus Sacch. cerevisiae
Substrate pNPp-D-Glucopyranoside 0.50^-51 b 0.60a-33b 1.10a.-5lb
pNPa-L-Arabino-
pNPa-L-Rhamno-
furanoside
pyranoside
0.005^-0.27^ 0.010^-0.05^
0.03^-0.15b 0.03^-0.17b
^Activity expressed as units (U) b Activity expressed as specific activity (U mg-"' protein) 4.
CONCLUSIONS
The data presented here confirm the interest in utilizing yeasts with P-glucosidase activity for enriching the aroma of grape juice and wines by the
1635 hydrolysis of glycosidic precursors. Further study is in progress to purify the pglucosidases of these yeasts and to study the biochemical properties and the possibilities to utilize these enzymes or the yeasts themselves under juice processing and winemaking conditions.
5. REFERENCES 1 2 3 4 5 6 7 8 9 I 0 II 12 13
P. Ribereau-Gayon, J.N. Boidron and A. Terrier, J. Agr. Food Chem. 23 (1975) 1042. P.J. Williams, C.R. Strauss, B. Wilson and R.A. Massy-Westropp, Phytochemestry 21 (1982) 2013. P.J. Williams, C.R. Strauss, Phytochemestry 22 (1983) 2039. S.G. Voirin, R.L Baumes, S.M. Bitteur, Z.Y. Gunata and C.L Bayonove, J. Agr. Food Chem. 38 (1990) 1373. Z.Y. Gunata, S.M. Bitteur, J.M. Brillouet, C.L Bayonove and R.E. Cordonnier, Carbohydr. Res. 184 (1988) 139. C.L Bayonove, Y.Z. Gunata and R.E. Cordonnier, Bulletin de I' O.I.V. 643 (1984)741. A.P. Aryan, B.Wilson, C.R.Strauss, and P.J. Williams, Am. J. Enol. Vltic. 38 (1987) 182. R.E. Cordonnier, Y.Z. Gunata, R.L Baumes and C.L Bayonove, Conn. Vigne Vin23 (1989)7. Y.Z. Gunata, C.L Bayonove, A. Arnaud, and P. Galzy, J. Sci.Food Agric. 50 (1990)499. P.H. Darriet, J.-N. Boidron and D. Dubourdieu, Conn. Vigne Vin 22 (1988) 189. L Rosi, M. Vinella and P. Domizio, J. Appl. Bacteriol. (1994) in press. M. Bertuccioli, in preparation C. Bayonove, Y.Z. Gunata, J.C. Sapis, R.L Baumes, I. Dugelay and C. Grassin, VigneVini 9 (1993) 33.
Research supported by National Research Council of Italy, Special project RAISA, Sub-project No. 4, Paper N. 1491