Typing of non-Saccharomyces yeasts with enzymatic activities of interest in wine-making

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Abstract

Enzymatic activity of potential interest in wine-making was studied for 182 non-Saccharomyces yeasts isolated from musts before and at the onset of fermentation at wine cellars operating under the La Mancha Appellation of Origin in Spain. Tests were carried out on plates containing differential substrates appropriate for each case (casein, gelatin, polygalacturonic acid, and arbutin) to determine whether each of the isolates exhibited proteolytic, polygalacturonase, and β-glucosidase activities. Nearly 80% of the wild yeasts possessed one or more enzymes of biotechnological interest. Once the enzymatic activities of the isolates had been established, 69 of the isolates that exhibited pronounced enzymatic activity and 11 randomly selected isolates that were devoid of any activity were typed using PCR/RFLP, which gave 13 different molecular profiles. The isolates for each of the profiles were then identified by classical methods. The enzyme β-glucosidase was linked to the species Metschnikowia pulcherrima, and polygalacturonase activity was common in most of the species identified. Proteolytic activity was observed in Pichia membranifaciens and in Metschnikowia pulcherrima. Typing revealed the possibility of intraspecific differences in Pichia membranifaciens, because six different molecular profiles with one or more shared restriction bands were recorded for that species.

Introduction

Most studies dealing with microbiological aspects of wine-making have focused chiefly on yeasts of the genus Saccharomyces, which is employed in alcoholic fermentation. However, during the early stages of wine-making there is substantial growth of non-Saccharomyces yeast species that may impart special characteristics to the wines (Ciani and Picciotti, 1995).

Enzymes from the grapes, from the microorganisms present, and exogenous enzymes in the form of commercial preparations that may be added all play a role in wine-making. Adding commercial enzyme preparations to address problems relating to clarification and filtration or to increase aroma is a common wine-making practice. The enzymes are usually produced by bacteria or by filamentous fungi, but in many cases less defined enzyme blends are used (Esteve-Zarzoso et al., 1998).

For that reason, it is important to ascertain the potential of wild wine yeasts for producing extracellular enzymes of interest in wine-making, in order to be able to alter certain components of the musts and thus enhance the sensory attributes of the wines. Pectinases, proteases, and glycosidases are some of the enzymes secreted by yeasts that are of interest in wine-making because of their technological effects and their contribution to aroma formation.

Pectinolytic enzymes cleave long pectin chains into shorter, more soluble chain segments that facilitate pressing of the grapes, contribute to clarification of the musts, may increase extraction of substances that contribute to colour and aroma during the time the musts remain in contact with the skins, and may enhance filtration of the wines.

Proteins, which are present in grapes in varying amounts, are another component which, like polysaccharides, increases the turbidity of musts and wines. Enzymatic hydrolysis of the proteins into smaller, more soluble nitrogen-containing molecules (peptides and amino acids) is another technical solution to problems bearing on clarification and stabilization of the musts and wines. It also helps prevent incomplete fermentations due to a deficiency of assimilable nitrogen in the must.

Terpenes and such other volatile substances as straight-chain and cyclic alcohols, to name just a few, help improve wine aroma. These volatile, odorous compounds are present in free form in the grapes or otherwise they mainly bind to sugar molecules to form odourless non-volatile glycosidic complexes (Gunata et al., 1988). β-Glucosidase cleaves off aroma precursors bound to the sugar molecules contributing to wine aroma.

The aim of this study was to investigate the enzymatic activities of 182 wild non-Saccharomyces wine yeast strains of most interest to wine-making and to identify some strains with interesting characteristics using classical (Barnett et al., 1990) and molecular (PCR/RFLP) methods.

Section snippets

Yeast strains

Samples of musts were collected from five wine cellars of the La Mancha Appellation of Origin. A total of 17 must samples were taken before fermentation or at the onset of fermentation. Each of the must samples was spread onto CLEN agar (Martin and Siebert, 1992) with added 0.025% sodium propionate (Sigma Chemical Co., St. Louis, USA) and around 10 colonies were randomly selected, obtaining a total of 182 isolates.

None of the selected colonies belonged to the genus Saccharomyces by

Results and discussion

The study of the enzymatic activities of the 182 isolates of non-Saccharomyces wine yeasts revealed that nearly 80% of the yeasts presented at least one enzyme of biotechnological interest. Polygalacturonase was the enzyme most commonly found and was secreted by 45% of the yeasts. Proteases were recorded on the two test substrates employed, with similar percentages 23% on casein and 18% on gelatin, whereas β-glucosidase was only observed in 14% of the yeasts. Some isolates possessed more than

Conclusions

All the genetic profiles of the isolates exhibited one or more of the enzymatic activities studied, with the exception of Debaryomyces hansenii. The enzyme β-glucosidase was closely related to Metschnikowia pulcherrima, while polygalacturonase was common to most of the species identified. The polymorphism revealed by PCR/RFLP was closely related to the phenotypic characteristics and enabled proper identification of those species which could not be identified on the basis of classical methods

Acknowledgements

The authors wish to express their gratitude to FEDER for funding this research, which was performed in the framework of project 1FD97-0165. Thanks are also due to Junta de Comunidades de Castilla la Mancha for a grant concession to M. Fernández.

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