Screening, identification and characterisation of bacteriocins produced by the wine isolated LAB

[摘要] ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to itsaroma and flavour. However, they can also be responsible for many wine spoilage problems thatcompromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics tothe sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect thewholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethylcarbamate.Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilagemicro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains thebenefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand toreduce SO2 levels due to the increasing health related risks and other factors.All these considerations have increased the interest in research to look for new preservationstrategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in thelast decade. Various types of bacteriocins have been identified and characterized. However, there are fewreports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finishedwine.The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocinproduction. The isolates originated from South African red wines undergoing spontenous malolacticfermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibitionzones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum ofantimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeriamonocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important inwinemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocinsthey produce could possibly find application in the food fermentation industry.The physiological results, biochemical tests and sugar fermentation profiles all gave the sameresults for the seven isolates, which were indicative of enterococci. The identification through 16S rRNAgene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCRfingerprinting gave the same profile for the seven strains confirming that they were all identical ongenetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search resultsconfirmed that all eight strains contained the enterocin P gene from Ent. faecium.The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enableenterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococciproducing bacteriocins have potential applications in various foods and fermented products. The pH testsshowed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH rangemake them potentially more suitable as natural preservatives of foods and fermented products whereproducts are acidified or pH decreases due to natural LAB present. They also have potential applicationsin oenological process where pH levels are as low as 3 and 4.Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibitenterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydratemoieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicatedthat the compound was proteinaceous in nature.Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with theexponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication ofprimary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours andwas maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell freesupernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314was clearly demonstrated by complete inhibition of growth for most of the experimental period, while thecontrol increased exponentially throughout the experiment.In conclusion, this study has confirmed the isolation and identification of Ent. faecium strainsfrom wine, a genus that is rarely found in the wine environment. Although one can speculate on the originof this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolatesproduced enterocin P which have antimicrobial action against wine-related LAB genera and thereforehave a potential role in wine spoilage control.

[发布机构] Stellenbosch University