Dal vigneto al vino finito, identificazione con metodi molecolari di lieviti e batteri lattici

Usually all attempts to characterize the microbial diversity in wine fermentations have employed standard methods of enrichment and isolation to cultivate various microbial constituents before taxonomic identification. This estimation of microbial diversity, in addition to being time-consuming, is often problematic since many microorganisms may not grow on standard laboratory media.
Moreover culture-independent molecular methods allow more rapid profiling of complex populations, or quantification of targeted species, thereby enhancing the information available to the winemaker.

The aim of the present study is to describe the yeast and lactic acid bacteria communities found in the vineyard, the winery and the wine, using culture-dependent and culture independent molecular methods. Samples came from botrytized Picolit grapes during 2013 vintage in Corno di Rosazzo, Friuli Venezia Giulia Region, Italy. On this work, the DNA extracted directly from the must and wine (culture-independent technique) was analyzed by denaturing gradient gel electrophoresis (DGGE), as well as the DNA extracted from isolated colonies that came from the vineyard and winery samples (culture-dependent technique). Both types of DNAs were specifically amplified by PCR using particular groups of universal primers depending on the nature of the sample (bacteria, Saccharomyces or non-Saccharomyces yeasts).

Saccharomyces sensu stricto yeasts were amplified with ShafGC and Shar specific primers. This yeast was found in the Winery and in the Picolit wine. The DGGE study showed that all samples corresponded to the Saccharomyces cerevisiae species, being identical to the commercial yeast used as a starter. Non-Saccharomyces 26S ribosomal rDNA genes were amplified by Nested PCR using the primers NL1-NL4 for step 1 and NL1GC-LS2 for step 2. In the vineyard, the DGGE analysis allowed the identification of several yeasts like Kloeckera sp., Metchnikowia sp., Pichia sp., Hansenula sp. and Schizosaccharomyces sp. Other than Saccharomyces cerevisiae, no other yeast was found in the wine, indicating the starter’s ability to reduce the variability of the yeasts during the fermentation process. Brettanomyces sp. and Candida sp. were absent in the yeast analyzed.
Samples that didn’t correspond to a reference strain used were sequenced allowing the identification of Torulaspora delbrueckii and Debaryomyces hansenii.

Finally, bacteria samples were analyzed by employing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified with 338fGC and P4V3 primers. Several
microorganisms were found in the vineyard and the winery but only the strains of Lactobacillus casei/ Lactobacillus paracasei and Leuconostoc mesenteroides were found in the Picolit wine, indicating the selection due to the yeast fermentation process. Only 2 samples of the vineyard and one contact plate of the cellar were identified as Oenococcus oeni.
Samples that didn’t correspond to a reference strain used were sequenced allowing the identification of Lactobacillus hilgardii, Lactobacillus mali, Pediococcus parvulus.
In this work we demonstrate that PCR-DGGE is a viable alternative to standard plating methods for a qualitative assessment of the microbial constituents from de vineyard to the wine.

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