HIGH-THROUGHPUT SCREENING OF NOVEL BACTERIOCINS FROM LACTIC ACID BACTERIA AND CONSTRUCTION OF THE FERMENTATION SYSTEM

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High-throughput screening strategy for rapid evaluation prior to purification and structural analysis is an urgent necessity to obtain diverse bacteriocins. Firstly, we developed a new high-throughput screening system involving two methods ; antimicrobial spectrum analysis by statistical procedure and molecular mass determination by liquid chromatography/mass spectrometry (LC/MS).Antimicrobial spectra of bacteriocins were analyzed by spot-on-lawn method against 12 selected indicator strains. The results obtained were converted to numeric values and then analyzed by principal component analysis (PCA). PCA enabled not only grouping of bacteriocins but also exploration of new ones.LC/MS permitted direct detection of bacteriocins from culture supernatants, simultaneous removal of impurities, and high sensitivity and resolution with accurate molecular masses. Till now, nisin variants and some bacteriocins could be detected and identified from 25 μl culture supernatants. Moreover, statistica … More l analysis of antibacterial spectra against selected indicator strains enabled classification of bacteriocins and exploration of new ones. Combination of these methods could save the time and provide a high probability for discovering novel bacteriocins.Some of the potential bacteriocins have been further characterized, which resulted in novel compounds such as nisin Q, lactococcin Q and lacticin Q. Especially, lacticin Q, produced by a new corn isolate Lc.lactis QU 5,showed a wide antibacterial spectrum comparable to nisin A, and unique characteristics compared to general LAB bacteriocins. Activity of lacticin Q was stable against heat treatment and pH change, particularly in alkaline pH, whereas most of LAB bacteriocins were not stable in alkaline conditions. In addition, lacticin Q induced ATP leakage in shorter time and lower concentration than that of nisin A. Lacticin Q would thus be a potential candidate to be a safe natural antimicrobial satisfying our diversifying demands.Finally we developed the fermentation systems of novel bacteriocins for practical uses as biopreservatives and antimicrobials. Less