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

在细菌素纯化和结构分析之前进行快速评价的高通量筛选策略是获得多样化细菌素的迫切需要。首先，建立了一种新的细菌素高通量筛选体系，采用统计学方法分析抗菌谱和液相色谱/质谱（LC/MS）测定分子量，并对筛选出的12株指示菌进行了抗菌谱分析。将所得结果转换为数值，然后通过主成分分析（PCA）进行分析。PCA不仅可以对细菌素进行分类，还可以探索新的细菌素。LC/MS可以直接从培养上清液中检测细菌素，同时去除杂质，具有高灵敏度和高分辨率，分子量准确。到目前为止，从25 μl培养上清液中可以检测到nisin变体和一些细菌素。此外，南极洲 ...更多信息 针对选定的指示菌株的抗菌谱分析使得能够对细菌素进行分类并探索新的细菌素。这些方法的结合可以节省大量的时间，并为发现新的细菌素提供了很大的可能性，一些潜在的细菌素已经被进一步鉴定，产生了新的化合物，如nisin Q，lactococcin Q和lacticin Q。特别是由一种新的玉米分离物Lc.lactis QU 5产生的乳酸菌素Q，显示出与乳链菌肽A相当的广泛的抗菌谱，并且与一般的LAB细菌素相比具有独特的特性。乳酸菌素Q的活性对热处理和pH变化是稳定的，特别是在碱性pH下，而大多数LAB细菌素在碱性条件下不稳定。此外，乳酸菌素Q诱导ATP渗漏的时间短，浓度低，比乳酸链球菌素A。因此，乳酸菌素Q有望成为一种安全的天然抗菌剂，以满足人们多样化的需求。最后，我们开发了新型细菌素的发酵系统，并将其作为生物防腐剂和抗菌剂应用于实际。少

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