This study evaluated the population dynamics of non-Saccharomyces biota during spontaneous fermentation of organic musts. Non-Saccharomyces yeasts were found to be present at high levels during all fermentations. A total of 543 yeast colonies were isolated, 190 from Lysine-Medium (LM) agar, 254 from Wallerstein Laboratory Nutrient (WLN) agar and 99 from YPD agar. To estimate yeast population dynamics during spontaneous fermentation a genotypic approach was applied. PCR-RFLP of the ITS1-5.8S rDNA-ITS2 region and sequence determination of the D1/D2 region of the 26S rRNA gene enabled identification of the yeast isolates at the species level. Hanseniaspora uvarum, Metschnikowia fructicola and Candida zemplinina predominated, while Issatchenkia terricola, Issatchenkia orientalis and Pichia sp. were identified with a lower frequency. Hanseniaspora uvarum, M. fructicola and C. zemplinina represented 43%, 31% and 11% of the total non-Saccharomyces population isolated, respectively. Some yeast isolates were shown to be closely related to Hanseniaspora spp. and Candida spp. on the basis of the D1/D2 sequences. Based on those results, the coexistence of different Hanseniaspora and Pichia species in grape musts was supposed, and their complete identification was achieved using additional molecular markers. Moreover, strain typing and differentiation was carried out by RAPD-PCR. High strain polymorphisms were observed in the different species. For some strains, appreciable properties were demonstrated both in vitro by the API-ZYM test and in must. In must microvinification some strains showed good fermentation performances, low production of acetic acid and a partial capability to degrade malic acid.
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