具有拮抗无乳链球菌能力乳杆菌的筛选及抑菌特性

Screening of Lactobacillus with the ability of antagonizing Streptococcus agalactiae and analysis of its bacteriostatic characteristics

目的从罗非鱼(Nile tilapia)肠道中筛选出具有抑菌作用的乳杆菌,测定其对无乳链球菌(Streptococcus agalactiae)的抑菌效果,分析乳杆菌抑制无乳链球菌的有效成分,并利用分子生物学手段对筛选的乳杆菌菌种进行鉴定。方法采用双层平板法对具有抑制无乳链球菌的乳杆菌进行筛选,牛津杯法对抑菌效果进行测定,酶蛋白敏感性测定、热处理、有机酸处理等方法分析抑菌活性物质有效成分,16S rDNA分子标记对乳杆菌进行鉴定。结果从罗非鱼肠道中筛选出14株乳杆菌,其中菌株RS2对无乳链球菌具有明显的抑菌效果;不同蛋白酶种类、pH处理对乳杆菌无细胞培养液均有不同的影响,经80℃处理的乳杆菌无细胞培养液,其抑菌效果未显著改变(t=0.169 2,P=0.873 8)。此外,此株乳杆菌对猪霍乱沙门菌(Salmonella choleraesuis)、大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和志贺菌(Shigella sp.)等病原菌具有良好的抑制作用。经鉴定,该乳杆菌为植物乳杆菌(Lactobacillus plantarum)。结论从罗非鱼肠道中分离得到的植物乳杆菌菌株RS2对无乳链球菌等致病菌具有一定的抑制作用,推断其抑菌有效成分为细菌素类物质。此项研究对开发抗生素替代产品,提高食品的品质具有一定价值。

Objective To screen Lactobacillus with bacteriostatic effect from tilapia(Nile tilapia) intestine, determine its bacteriostatic effect on Streptococcus agalactiae, analyze its effective components against S. agalactiae, and identity the species of the Lactobacillus using molecular biology. Methods Lactobacillus strains inhibiting S. agalactiae were screened with double-layer plate methods. The antimicrobial effect of the Lactobacillus was determined using Oxford cup methods. The active ingredients of antimicrobial substances were analyzed with enzyme-protein sensitivity test, heat treatment and organic acid treatment. The Lactobacillus species was identified using 16 S rDNA gene molecular marker. Results A total of 14 strains of Lactobacillus were screened from tilapia intestines. Among them, strain RS2 had obvious bacteriostatic effect on S. agalactiae. Different protease types and pH treatments exhibited different effects on the fermentation broth of the Lactobacillus, and the antibacterial effect of Lactobacillus cell-free culture was not significantly changed after treating under 80 ℃(t=0.169 2, P=0.873 8). In addition, this strain of Lactobacillus presented good inhibitory effects on Salmonella choleraesuis, Escherichia coli, Staphylococcus aureus and Shigella sp.. The Lactobacillus was identified as Lactobacillus plantarum. Conclusion L.plantarum strain RS2 isolated from tilapia intestine presented inhibitory effects on pathogenic bacteria of fish, such as S. agalactiae. It was speculated that the active bacteriostatic ingredient is bacteriocins. This result may contribute to the development of alternative antibiotics and improvement of food quality.