Abstract:
Wine is the product of microbial activity for it is microorganisms (predominantly yeasts) that turn grape juice into wine. Somewhat surprisingly, there have been relatively few studies of wine-associated microorganisms in New Zealand, and globally, wine-associated bacteria are poorly studied in comparison with yeasts. Most likely this is because culturebased techniques are limited in their ability to accurately identify species within microbial communities and also because industrially important or dominant species are prioritised. The primary aim of this study was to develop a culture-independent molecular method for co-extracting DNA and RNA from a spontaneous Pinot Gris ferment. A secondary aim was to use PCR and RT-PCR amplicons of the extracted DNA and RNA, respectively, for molecular fingerprinting via denaturing gradient electrophoresis (DGGE). This approach was chosen to enable rapid determination of patterns of biodiversity and community dynamics of the yeast and bacteria present throughout vinification at a commercial winery. Simultaneous extraction of DNA and RNA from the same sample enables identification of species composition and a 'snapshot' of which species are metabolically active at each timepoint. The protocols trialed were a CTAB bead-beating protocol + DNase digestion, Griffiths' co-extraction CTAB + phenol:chloroform:isoamyl protocol, McIlroy's co-extraction protocol based on NaTCA and a Triton X-100 + DNase digestion protocol. The Triton-X protocol proved to be the most suitable due to its very high yield of quality RNA amplifiable by RT-PCR, high yield of quality DNA suitable for subsequent PCR amplification and ease of use. Attempts to optimise DGGE molecular fingerprinting of yeast PCR products from wine ferment samples using species-level (NL1-GC, NL4) as opposed to the common genus-level (NL1-GC, LS2) primers were hampered by excessive smearing that may have been a result of the PCR fragment being too large for adequate resolution on a DGGE gel. It is suggested that future studies aim at improving the efficiencies of the Triton-X co-extraction protocol and perhaps try another molecular fingerprinting method.