Biodiversity and VBNC state in Brettanomyces bruxellensis isolated from Apulian wineries

wines and identified. The identification was performed amplifying using the primers ITS1/ITS4, with a successive restriction analysis (Hae III, Hinf I e Cfo I). In addition, another specific PCR (primers DB90/DB394), that form amplification products only with the species B. bruxellensis and B. anomalus was used and the differences between these two species was assessed after restriction enzyme analysis (DdeI) of the amplified products. The strains identified as B. bruxellensis (50) were characterized genotypically through the technique of SAU-PCR, amethod previously used to characterize lactic acid bacteria populations (Corich et al., 2005), to define the intra-species diversity of Saccharomyces cerevisiae isolates obtained from former and modern wineries in Italy (Cocolin et al., 2004b), and to study the biodiversity of B. bruxellensis in north-western wine (Campolongo et al., 2010). The SAU-PCR fingerprinting was used to investigate B. bruxellensis diversity at the strain level, highlighting differences related to geographical origin and to sample type. With this method, genomic DNA is first subjected to enzymatic cleavage using restriction enzyme Sau3AI and then amplified using a specific primer constructed on the basis of the restriction site of the enzyme, so that the obtained profiles reflect the presence or the absence of Sau3AI restriction site within the genome of the strain under investigation. One representative strain from each of the 7 different clusters (SAU-PCR) were subjected to analysis of volatile phenol production in synthetic media and in wine with chemical analysis by GC-MS. The strains were also tested to assess their VBNC behaviour using different concentration of SO2 as recently reported (Serpaggi et al., 2012). The VBNC condition is a survival strategy adopted by many microorganisms when exposed to harsh environmental stresses. The VBNC is a reversible state, indeed under appropriate conditions the yeasts can resuscitate to culturable state. The comprehension of strain behaviour in relationship of the VBNC state is crucial, giving that VBNC state of Brettanomyces can be responsible of significant spoilage risks.