Ecology and technological capability of lactic acid bacteria associated with Grillo grapevine used as base wine for Marsala production.

Lactic acid bacteria (LAB) have a defining role in winemaking process since their activities determine an important contribute to wine quality. Besides sulphur dioxide, lysozyme is becoming a common supplement in wine for bacterial growth inhibition (Sonni et al, 2009). It is a natural enzyme with muramidase activity working against a wide range of LAB, including Oenococcus spp., Pediococcus spp., Lactococcus spp. and Lactobacillus spp. (Cunningham et al, 1991). To obtain a first mapping of LAB inhabiting Marsala wine production area, grapes of “Grillo” variety were harvested from five vineyards different for climatic and agronomic parameters. A Marsala base wine large-scale process was followed and samples were collected from must to bottling. The influence of lysozyme and SO2 on LAB was also evaluated through two experimental micro-vinification processes. Microbial communities and conventional chemical parameters were periodically analysed. Total microflora on grapes was barely around 10 CFU mL-1; while must from large-scale vinification hosted a higher concentration, around 103 CFU mL-1, that decreased during wine process. No difference in terms of microbial load was detected between the two experimental micro-vinifications (both containing around 10 CFU mL-1) and the chemical parameters were those commonly reported in literature. A total of 146 bacterial isolates were analyzed: only 35 cultures were presumptively identified as LAB (Gram positive, catalase and oxidase negative). On the basis of isolation source and cell morphology, 16 isolates were genetically identified. 16S rRNA gene sequencing revealed the presence of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Enterococcus lactis, Leuconostoc fallax and Sporalactobacillus nakayamae subsp. nakayamae. Subsequently, strains were characterized for lysozyme and SO2 resistance. Lactococcus lactis subsp. lactis strains, most frequently isolated during winemaking, showed the highest resistance to SO2 and to Lysozyme: up to 1600 mg L-1, namely a concentration higher than the one usually employed in commercial vinification processes. Sporalactobacillus nakayamae subsp. nakayamae strains, isolated during the first stage of large-scale wine aging, were inhibited by 100 mg L-1 lysozyme, but were resistant to 800 mg L-1 SO2. The other species, collected from different steps of winemakings, showed a medium-high resistance to the tested inhibitory concentrations. In conclusion, this study, in agreement with previous works (Delfini et al, 2004), underlines the lack of antimicrobial activity of lysozyme and SO2 against Lactococcus lactis subsp. lactis strains. Furthermore, the presence of Sporalactobacillus nakayamae subsp. nakayamae in wine has been reported for the first time. However, its presence might be associated to the LAB contamination of commercial yeast starter culture used in winemaking.