Detection of �-Glucosidase and Esterase Activities in Wild Yeast in a Distillery Environment

Giovanna Lomolino1, Federico Zocca1, Paolo Spettoli1 and Anna Lante1,2

ABSTRACT

J. Inst. Brew. 112(2), 97–100, 2006

The study of �-glucosidase and esterase in wild yeast, the enzy-matic activities of which contribute to the distinctive flavours of grape-derived alcoholic beverages, was the aim of this work. The study focused on wild yeast isolated from grape pomace and on identifying strains with interesting characteristics by examin-ing their electrophoretic profiles. Zymograms revealed a high level of polymorphism. Some of these wild yeasts may be of interest for improving the quality of the distillate. This study also highlights the necessity of associating enzymatic properties to various environmental conditions, since these play an impor-tant role in the expression of wild yeast performance.

Key words: �-glucosidase, distillery, esterase, wild yeast, zymo-grams.

INTRODUCTION Flavour is an important attribute of grape-derived alco-

holic beverages and many variables contribute to it. Among these, the enzymatic activities of �-glucosidase and esterase of yeast play an important role as they are involved in either the expression of varietal and fermenta-tive flavours or in their breakdown4,10,15,16. Previous stud-ies1 have revealed the potential of indigenous wine yeasts to produce enzymes that improve the sensory properties of wine. This has encouraged studies on yeast which pos-sess these enzymes, especially for wine cellars3,12–14,18 apart from distilleries, where grape pomace, the largest by-product obtained from must or wine at the end of alco-holic fermentation, is extensively used in producing spirit beverages, called Bagaceiras in Portugal, Marc in France and Grappa in Italy2. In distilleries, wild yeasts are more important than in wine cellars, where the use of selected yeasts is customary. Our work focused on the distillery wild yeast found in grape pomace. The electrophoretic profiles of �-glucosidase and esterase activities extracted from the cytoplasm and periplasm of the cells from nine yeast strains were examined using chromogenic sub-strates.

MATERIALS AND METHODS

Yeast strains

Eight wild yeast strains were isolated from Prosecco grape pomace (2000–2002 vintage). One yeast, Blastosel-Fr95 (Perdomini, Verona, Italy) was a commercial Sac-charomyces cerevisiae employed as an oenological strain to enhance varietal aroma. All yeasts were grown at 30°C and their cells fractionated as described previously17.

Electrophoretic analyses

All electrophoretic procedures were carried out at room temperature in a Mini-Protean 2 apparatus (Bio-Rad Laboratories, Milan, Italy). The cytoplasm and periplasm fractions were electrophoresed in native conditions by polyacrylamide gel electrophoresis (N-PAGE) using gels with a total polyacrylamide concentration of 8.5%. Before loading, the extracts were added to glycerol at a final con-centration of 10% (w/v). Eighty and 50 µg of protein, for detection of esterase and �-glucosidase activities respec-tively were loaded in the gel. N-PAGE was performed at room temperature, at a constant current of 30 mA, until the tracking dye Bromophenol Blue ran off the gel. Gels were stained for proteins by Coomassie R-250. For es-terase activity staining, gels were washed with Phosphate Buffered Saline (PBS), pH 7.5, for 10 min and the various lanes were separated by a cutter. Esterase activity was detected essentially as reported by Gilott and Andre7, with 30 mL of PBS containing 2 mg of 2-naphthyl acetate (Fluka, Buchs, Switzerland), previously emulsified in 1 mL of 10% Triton X-100 (Merck, Darmstadt, Germany). The 0.06% (w/v in PBS) of fast BB salt (Fluka) was used as the dye coupler at 37°C. After 45 min, gels were washed in distilled water and images were acquired on a Biorad Gel Doc 1000 apparatus. To detect �-glucosidase

1 Dipartimento di Biotecnologie Agrarie, Università di Padova, Agri-polis, Viale Università 16, 35020 Legnaro, Padova, Italy.

2 Corresponding author. E-mail: anna.lante@unipd.it

Publication no. G-2006-0619-436 © 2006 The Institute of Brewing & Distilling

TABLE I. Protein content of periplasm and cytoplasm of wild yeast strains.

µg Protein /g cell

Yeast strain Periplasm Cytoplasm

Candida stellata 77/D 1949 ± 156 2079 ± 24 Zygosaccharomyces bailii 86 /S 2758 ± 76 554 ± 5 Saccharomyces ludwigii 99 /R 485 ± 7 4044 ± 83 Candida stellata 136 /Z 3562 ± 205 1296 ± 77 Saccharomyces cerevisiae I-00-006 724 ± 71 5674 ± 148 Saccharomyces cerevisiae I-00-069 812 ± 165 1024 ± 122 Saccharomyces cerevisiae I-00-012 1160 ± 87 4474 ± 86 Saccharomyces cerevisiae I-00-041 900 ± 146 4298 ± 189 Saccharomyces cerevisiae Fr-95 368 ± 9 7578 ± 131

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activity, the technique of Kwon et al.9 was applied, in which esculin is used as substrate. After N-PAGE, gels were soaked in 0.1 M sodium acetate buffer, pH 5.1, for 10 min at room temperature to exchange the buffer sys-tem. Gels were then incubated overnight at 37°C in the same buffer containing 0.1% (w/v) esculin (Sigma) and 0.03% (w/v) ferric chloride. Gels were washed in distilled water and images acquired as reported above.

RESULTS AND DISCUSSION Protein quantification of the periplasm and cytoplasm

and their ratios showed great differences among the vari-ous strains (Table I). This finding suggests that further

investigations on protein content and cell wall thickness would be useful in order to link results to this parameter of yeast characterisation. After Coomassie staining of the nine yeast strains analysed (row A), differing electro-phoretic profiles were observed (Fig. 1). The zymographic technique revealed esterase activity in the cytoplasm frac-tions of almost all strains examined. It failed to detect enzyme activity in the periplasm fraction of the two Sac-charomyces cerevisiae strains I-00-6 and Fr95 (Fig. 1, row B, lanes 5, 9). Taking into account our previous results using various chromogenic substrates11,13, employing only one substrate for esterase activity detection may be a limi-tation, because the presence of major and minor bands8 requires various substrates. In this respect, our approach

Fig. 1. N-PAGE of cytoplasm and periplasm fractions of nine yeast strains. Lane 1: Candida stellata 77/D. Lane 2: Zygo-saccharomyces bailii 86 /S. Lane 3: Saccharomyces ludwigii 99 /R. Lane 4: Candida stellata 136 /Z. Lane 5: Saccharo-myces cerevisiae I-00-6. Lane 6: Saccharomyces cariocanus I-00-069. Lane 7: Saccharomyces cariocanus I-00-012. Lane 8:Saccharomyces cerevisiae I-00-041. Lane 9: Saccharomyces cerevisiae Fr-95. Gels were stained for protein with Coo-massie brilliant blue (Row A), 2-naphthyl acetate for esterase activity (Row B) and esculin for �-glucosidase activity(Row C).

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was preliminary and focused on macroscopic differences between yeast strains selected from the distillery. Candida strains showed the highest esterase activity (Fig. 1, lanes 1, 4), confirming that this yeast is a good producer of es-terase/lipase enzymes employed in the food industry19. The almost identical esterase electrophoretic pattern ob-served in the two cell fractions may be due to the presence of a precursor of the periplasm protein in the cytoplasm fraction, rather than to cross-contamination during purifi-cation. Some identical bands were also observed in the total protein profile (Fig. 1, row A), not only of the two Candida strains but also of the other yeasts, and did not seem to depend on the different protein concentrations of periplasm and cytoplasm (Table I). Although confirmation of the presence of the same precursor in the two cell frac-tions requires supportive data, the experiment was re-peated several times, with the same result (data not shown). Esterase activity, enhanced in Candida strains with respect to other yeasts, is involved in hydrolysis of esters and may be considered positive or negative in its double action. When it hydrolyses varietal esters such as geranil acetate, releasing the alcoholic moiety (geraniol) which has a lower threshold than the ester form6, esterase activity improves the varietal quality of Grappa. This en-zyme may also have a negative effect when it hydrolyses fermentation esters, lowering the aromatic quality of dis-tilled beverages. In the distillery environment, the survival of non-Saccharomyces yeast until the end of pomace fer-mentation may play a more important role than in a wine cellar environment, where the high alcohol concentration preferentially favours Saccharomyces strains. Among S. cerevisiae strains (Fig. 1, lanes 5, 8, 9) two yeasts re-vealed the same electrophoretic patterns in both fractions and between themselves (Fig. 1, lanes 5, 9); S. cerevisiae I00041 differed from the others, even though it main-tained the same profile in its fractions (Fig. 1, lane 8).

Other researchers18 have screened various S. cerevisiae strains for their �-glucosidase activity, but only one yeast displayed this in its intracellular fraction. The zymo-graphic technique revealed �-glucosidase activity in the periplasm fractions of all strains examined, but not in the cytoplasm fraction of Candida stellata 77D, S. ludwigii 99R or S. cerevisiae I-00-041 (Fig. 1, row C, lanes 1, 3, 8). The two fractions showed different profiles not only be-tween strains but also within the same yeast. The three S. cerevisiae strains showed one band with the same elec-trophoretic mobility (Fig. 1, lanes 5, 8, 9). This finding may be important in ascertaining the potential of wild yeast for producing �-glucosidase activity of interest, be-cause in the Blastosel-Fr95 strain (Fig. 1, lane 9), a com-mercial S. cerevisiae used to enhance varietal aroma, the electrophoretic profile is closely related to phenotype characteristics. Several authors have reported5 that the �-glucosidase activity of yeast is inhibited by high glucose concentrations and the low pH of must, but in pomace these parameters are near the optimal value of enzymatic activity and may affect the varietal aroma of Grappa. The differing environmental conditions between cellar and dis-tillery must be taken into account: thus, when choosing a “distillery” yeast, its aromatic contribution may be more important than its fermentative performance. In this re-spect, some wild yeasts isolated from grape pomace may

be of interest in characterising the distillate and in im-proving its quality for proper geographic criteria of terri-torial identification.

CONCLUSIONS These results allow some conclusive remarks. First, the

zymograms of esterase and �-glucosidase revealed a high level of polymorphism, and some of these wild yeasts may be of interest in regard to improving beverage qual-ity. In addition, the electrophoretic patterns of the yeast strains examined were like fingerprints, allowing identifi-cation of strains. Second, with the limitations of a prelimi-nary study focusing on macroscopic differences between wild yeast strains, this work highlights the necessity of associating enzymatic properties to various environmental conditions, since they play an important role in the ex-pression of wild yeast performance.

ACKNOWLEDGEMENTS

The authors thank Professors Viviana Corich and Alessio Giacomini for the isolation of the wild yeast strains. This study was supported by grants from the Ministero dell’Università e della Ricerca Scientifica (MIUR 2004). This work is part of research activities of the Dottorato in Viticoltura, Enologia e Marketing delle Imprese Vitivinicole, supported by the Provincia of Treviso.

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(Manuscript accepted for publication April 2006)

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