有抑菌能力乳杆菌的筛选及其抑菌物质的初步探究

Screening of Lactobacillus with antibacterial ability and preliminary study on its antibacterial substances

目的筛选具有抑菌能力的乳杆菌菌株并对其抑菌物质进行初步探究。方法采用琼脂扩散法进行抑菌试验,筛选出具有抑菌能力的乳杆菌菌株;通过热稳定性试验检测抑菌物质耐高温的能力;通过有机酸排除与过氧化氢排除试验检测这两种物质对抑菌作用是否有影响;用胃蛋白酶、胰蛋白酶和蛋白酶K消化处理各株乳杆菌无细胞发酵液后进行抑菌试验,判断抑菌物质是否为蛋白多肽类物质。结果 2株副干酪乳杆菌与1株保加利亚乳杆菌对大肠埃希菌、铜绿假单胞菌、伤寒沙门菌和痢疾志贺菌有抑菌效果。这3株乳杆菌无细胞发酵液中的抑菌物质经过高温处理后仍具有抑菌能力,但抑菌能力与处理前相比显著降低(P<0.05);有机酸对照组未产生明显的抑菌圈;过氧化氢排除后的无细胞发酵液的抑菌能力未受影响;经过蛋白酶作用3株乳杆菌无细胞发酵液的抑菌能力显著降低(P<0.05)或消失。生物被膜态副干酪乳杆菌2的抑菌能力与浮游态相近,其无细胞发酵液中的抑菌物质可以完全耐受100℃处理与胃蛋白酶的消化作用,可部分耐受胰蛋白酶的消化作用。结论副干酪乳杆菌1、副干酪乳杆菌2和保加利亚乳杆菌具有良好的抑菌能力,它们产生的主要抑菌物质为蛋白多肽类,此物质具有较好的耐高温能力与耐蛋白酶能力;被膜态副干酪乳杆菌产生的抑菌物质表现出了更强的抗胁迫能力与稳定性。

Objective To screen Lactobacillus strains with antibacterial ability and preliminarily investigate their antibacterial substances.Methods Agar diffusion method was used to screen the Lactobacillus strains with antibacterial ability.The ability of the bacteriostatic substances to tolerate high temperature was tested with thermal stability test.The antibacterial effects of organic acids and hydrogen peroxide were detected through bacteriostatic test after eliminating these substances.Bacteriostasis tests were also carried out after pepsin,trypsin and protease K were used to digest the cell-free fermentation broth of each Lactobacillus strain.Results Two strains of Lactobacillus paracasei and one strain of Lactobacillus bulgaricus had antibacterial activity against Escherichia coli,Pseudomonas aeruginosa,Salmonella typhi and Shigella dysenteriae.The bacteriostatic substances in cell-free fermentation broths of these three strains still had bacteriostatic ability after high-temperature treatment at 100℃.However,the bacteriostatic effect was significantly decreased compared with that before treatment(P<0.05).In the organic acid control group,there was no obvious bacteriostatic zone.After the removal of hydrogen peroxide,the bacteriostatic ability of cell-free fermentation broth was not affected.The antibacterial ability of the cell-free fermentation broths of these three strains of Lactobacillus significantly decreased(P<0.05)or disappeared after the treatment with protease.The antibacterial ability of the biofilm cells of Lactobacillus paracasei 2 was similar to that in planktonic state,and the antibacterial substances in the cell-free fermentation broth of the biofilm cells completely tolerated the treatment with 100℃ temperature and pepsin,and partially tolerated the digestion by trypsin.Conclusion Lactobacillus paracasei 1,Lactobacillus paracasei 2 and Lactobacillus bulgaricus have excellent antibacterial ability.The main bacteriostatic substances were protein peptides which were heat-and protease-tolerant.The bacteriostatic substances produced by Lactobacillus in biofilm state showed a stronger tolerance and stability.