Screening and characterisation of wine related enzymes produced by wine associated lactic acid bacteria

Malolactic fermentation (MLF) is the secondary fermentation process which is normally conducted by lactic acid bacteria (LAB) present in wine after alcoholic fermentation. The former fermentation process involves the conversion of a dicarboxylic acid (malate) to a monocarboxylic acid (lactate) and carbon dioxide (CO2). MLF is beneficial to wine due to its contribution to deacidification, microbial stabilisation and wine aroma formation, with the latter being an important part of research in this study. LAB isolated from South African wines during spontaneous MLF were screened for different enzymes of interest in winemaking using both classical and molecular techniques. The results obtained from screening have shown that LAB possess different combinations of enzymes such as β- glucosidase, protease, esterase, glucanase and lipase. Isolates were screened for β- glucosidase and glucanase with plate assays. Results obtained showed that 40% were positive for β-glucosidase, 80% for glucanase tested with carboxymethylcellulose, and 65% for glucanase tested with lichenan. Isolates were also screened using molecular techniques. The enzyme-specific primers used for PCR amplifications were deduced from nucleotide sequences previously identified in putative genes from Lactobacillus plantarum WCFS1 strain. These primers amplified genes with sizes corresponding to 1392 bp (β- glucosidase), 1263 bp (protease), 1020 bp (esterase) and 1644 bp (malolactic enzyme). Of all the isolates tested, 40% were positive for β-glucosidase, 35% for serine protease, 42% for esterase and 36% for malolactic enzyme. Among the isolates that possessed all four enzyme genes, 11 were selected from which genomic DNA was extracted and used as template to amplify the coding regions of the respective genes. Purified amplicons generated with enzyme-specific primers were further cloned into pGEM-T easy vector and sequenced. Analysis of sequences revealed that gene sequences were highly conserved between species, and these sequences exhibited significant homology with nucleotide sequences available from GenBank database. These results suggest that wine LAB can be the potential source of enzymes for use in vinification.