发酵乳杆菌CSC-19胞外粗多糖提取物抑制单核细胞增生李斯特菌生物被膜形成能力的研究

【目的】筛选能有效抑制单核细胞增生李斯特菌(Listeria monocytogenes,LM)形成生物被膜的乳酸菌，分析其活性成分并进行功能表征。【方法】采用结晶紫染色法筛选抑制LM形成生物被膜的不同乳酸菌提取物；通过酸中和、蛋白酶处理及热处理，推测抑制生物被膜活性物质以胞外多糖(extracellular crude polysaccharide,ECP)为主；乙醇沉淀法提取目标乳酸菌分离株胞外粗多糖，分析其抑制生物被膜形成活性和对LM生长的影响；运用激光共聚焦扫描显微镜(laser confocal scanning microscopy,LCSM)和扫描电子显微镜(scanning electron microscopy,SEM)观察胞外粗多糖对生物被膜细胞形态和结构的影响。【结果】发酵乳杆菌CSC-19发酵上清液对1516-2LM生物被膜的抑制率为81.7%；经热和蛋白酶处理后，发酵上清抑制生物被膜形成的活性未发生显著变化(P>0.05)，表明发酵上清液中抑制生物被膜形成的物质可能为胞外多糖；在不抑制LM生长的条件下所提取的胞外粗多糖抑制生物被膜形成能力具有浓度依赖性。激光共聚焦扫描显微镜和扫描电子显微镜结果显示，胞外粗多糖显著抑制了生物被膜的形成能力，生物被膜三维、有组织的蜂窝状结构被破坏，仅有少量的粘附细胞分散于细胞爬片表面。【结论】发酵乳杆菌CSC-19胞外粗多糖能有效抑制LM生物被膜的形成，有望应用于高效防控该菌污染食品。

Extracellular crude polysaccharide of Limosilactobacillus fermentum CSC-19 inhibits the biofilm formation of Listeria monocytogenes

Objective] To screen out the lactic acid bacteria that can inhibit the biofilm formation of Listeria monocytogenes (LM), identify the active component of the bacteria, and analyze the function of the active component. Methods] We employed crystal violet staining to screen out the lactic acid bacteria of which the extract could inhibit the biofilm formation of L. monocytogenes. We analyzed the potential active component that inhibited biofilm formation by acid neutralization, protease treatment, and heat treatment and hypothesized that the main active component was extracellular crude polysaccharide (ECP). We then employed ethanol precipitation to extract the ECP from the lactic acid bacteria and analyzed the inhibitory activity of ECP on the biofilm formation and growth of L. monocytogenes. The morphology and structure of the biofilm treated with ECP were characterized by laser confocal scanning microscopy and scanning electron microscopy. Results] The inhibition rate of Limosilactobacillus fermentum CSC-19 fermentation supernatant on the biofilm formation of L. monocytogenes 1516-2LM was 81.7%. After heat treatment and protease treatment, the activity of the fermentation supernatant did not significantly change (P>0.05), which indicated that the active component in the fermentation supernatant may be ECP. ECP inhibited the biofilm formation of L. monocytogenes in a concentration-dependent manner, while it did not inhibit the growth of L. monocytogenes. The results of confocal laser scanning microscopy and scanning electron microscopy showed that ECP destroyed the three-dimensional and honeycomb-structured biofilm and left only a few scattered adherent cells on the surface of the glass slide. Conclusion] The ECP of L. fermentum CSC-19 inhibits the biofilm formation of L. monocytogenes, which is expected to be applied to the prevention and control of food contamination.