Qualitative and quantitative screening of the ß-glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must.

The aim of the present work was to screen a pool of 75 yeasts belonging to the species Saccharomyces cerevisiae and Saccharomyces uvarum in order to select the strains endowed with ß-glucosidase activity. The first screening was a qualitative assay based on chromogenic substrates (arbutin and esculin). The second screening was the quantitative evaluation of the ß-glucosidase activity via a p-nitrophenyl-ß-d-glucopyranoside assay. The measurement was performed on three different cell preparations, including the extracellular compartment, the cell lysates and the whole cells. This study pointed out the high frequency of ß-glucosidase activity in S. uvarum strains. In particular, we retrieved three promising S. uvarum strains, CRY14, VA42 and GRAS14, featuring a high enzymatic activity, exploitable for winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: In yeasts, ß-glucosidase activity has been extensively described, especially in non-Saccharomyces species, while there is only little evidence of this activity in strains belonging to the Saccharomyces species. In winemaking, ß-glucosidase plays essential roles in the hydrolysis of glyco-conjugated precursors and the release of active aromatic compounds. This study provides new insights into the ß-glucosidase activity in strains belonging to Saccharomyces cerevisiae and Saccharomyces uvarum species, which are the most important strains in wine industry. Our results point out a marked enzymatic activity for the tested S. uvarum strains. These strains could be exploited for their potential ability to enhance the aroma profiles of wine. In addition, they could be potential sources for the commercial production of enzymes to be applied in winemaking.

Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding.

AIMS: The present work proposes a two-step molecular strategy to select inter- and intra-species Saccharomyces hybrids obtained by spore-to-spore mating, one of the most used methods for generating improved hybrids from homothallic wine yeasts. METHODS AND RESULTS: As low spore viability and haplo-selfing are the main causes of failed mating, at first, we used colony screening PCR (csPCR) of discriminative gene markers to select hybrids directly on dissection plate and discard homozygous diploid colonies arisen from one auto-diploidized progenitor. Then, pre-selected candidates were submitted to recursive streaking and conventional PCR in order to discriminate between the hybrids with stable genomic background and the false-positive admixtures of progenitor cells both undergone haplo-selfing. csPCRs of internal transcribed spacer (ITS) 1 or 2, and the subsequent digestion with diagnostic endonucleases HaeIII and RsaI, respectively, were efficient to select six new Saccharomyces cerevisiae × Saccharomyces uvarum hybrids from 64 crosses. Intragenic minisatellite regions in PIR3, HSP150, and DAN4 genes showed high inter-strain size variation detectable by cost-effective agarose gel electrophoresis and were successful to validate six new intra-species S. cerevisiae hybrids from 34 crosses. CONCLUSIONS: Both protocols reduce significantly the number of massive DNA extractions, prevent misinterpretations caused by one or both progenitors undergone haplo-selfing, and can be easily implemented in yeast labs without any specific instrumentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a method for the marker-assisted selection of several inter- and intra-species yeast hybrids in a cost-effective, rapid and reproducible manner.