滇池金线鲃肠道产细菌素细菌的筛选鉴定及细菌素LSP01的抑菌作用

Screening and identification of bacteriocin-producing bacteria in the intestines of Sinocyclocheilus grahami in Dianchi and the antibacterial effect of bacteriocin LSP01

摘要:

[背景] 细菌素是微生物在生长过程中产生的一类具有抑菌作用的蛋白质或多肽类物质，可有效抑制或杀灭多种病原微生物。滇池金线鲃（Sinocyclocheilus grahami）是云南滇池特有鱼种，长期生存在滇池恶劣的生态环境中，其肠道内可能存在着大量产细菌素的微生物资源。[目的] 从滇池金线鲃肠道内筛选产细菌素菌株，并对其所产细菌素的抑菌特性及机制进行探究。[方法] 对滇池金线鲃肠道细菌进行分离鉴定，利用牛津杯双层平板法筛选具有抑菌活性的产细菌素菌株，测定抑菌活性最佳菌株的细菌素酶敏感性、酸碱与高温耐受性、最小抑菌浓度（minimum inhibitory concentration，MIC）与抑菌广谱性等抑菌特性，并借助细胞膜通透性、2，3-bis （2-methoxy-4-nitro-5-sulfonyl）-2h-tetrazolium-5-carboxanilide （XTT）实验及扫描电子显微镜（scanning electron microscope，SEM）观察等实验探究细菌素的抑菌机制。[结果] 从滇池金线鲃肠道中共筛选得到5株产细菌素细菌，隶属于芽孢杆菌属（Bacillus）和乳杆菌属（Lactobacillus）。其中，菌株S01所产细菌素（LSP01）的抑菌活性最佳，抑菌圈直径可达到（29.21±0.25） mm，对多种蛋白酶敏感，在100 ℃和pH 10.0处理后抑菌活性仍维持在60%以上，并对大肠杆菌（Escherichia coli）、福氏志贺氏菌（Shigella flexneri）和金黄色葡萄球菌（Staphylococcus aureus）等12株不同类型的革兰氏阳性及阴性致病菌具有良好的抑制效果。此外，LSP01对嗜水气单胞菌（Aeromonas hydrophila）的MIC仅为11.06 μg/mL，经LSP01处理后的嗜水气单胞菌细胞膜通透性上升、细胞代谢活性下降、细胞结构出现破裂，致其死亡。[结论] 滇池金线鲃肠道产细菌素细菌资源较为丰富，尤其是菌株S01所产生细菌素（LSP01）具有抑菌活性高、稳定性好、抑菌谱广等特性，能够明显破坏嗜水气单胞菌的细胞结构、降低代谢活性及细胞膜通透性，显示出了良好的替代抗生素应用潜能。

Abstract:

[Background] Bacteriocin is a kind of protein or polypeptide substances with antibacterial effect produced during the growth of microorganisms, which can effectively inhibit or kill for various foodborne pathogens. Sinocyclocheilus grahami is one of specific fish species in Dianchi, Yunnan. S. grahami survive in harsh ecological environment with long-term in Dianchi, intestines of which might contain a large amount of bacteriocin-producing microorganism resources. [Objective] The bacteriocin-producing strains were screened from the intestinal tract of the S. grahami in Dianchi and the antibacterial properties as well as mechanisms of the bacteriocin-producing strains were explored. [Methods] The intestinal bacteria of S. grahami in Dianchi were isolated and identified. The antibacterial effect of the bacteriocin-producing strains was screened by the Oxford cup double-layer method. The antibacterial properties including enzyme sensitivity, acid-base and high temperature tolerance, determination of the minimum inhibitory concentration (MIC) and antibacterial spectrum of the best strains were determined. The antibacterial mechanism of the bacteriocin was explored through cell membrane permeability, XTT (2,3-bis(2-methoxy-4-nitro-5-sulfonyl)-2h-tetrazolium-5-carboxanilide) experiments, and scanning electron microscopy analysis. [Results] A total of 5 strains of bacteriocin-producing bacteria were screened from the intestinal tract of the S. grahami in Dianchi, and they all belonged to the Bacillus and Lactobacillus. Among them, the antibacterial activity of bacteriocin (LSP01) produced by strain S01 was the best, inhibition zone of which reached to (29.21±0.25) mm. The produced bacteriocin was sensitive to a variety of proteases and maintained more than 60% antibacterial activity after treatment at 100℃ and pH 10.0. LSP01 was also presented a good antibacterial effect against other 12 Gram-positive and Gram-negative pathogenic bacteria, e.g. Escherichia coli, Shigella flexneri, and Staphylococcus aureus etc. In addition, the MIC of LSP01 against Aeromonas hydrophila was 11.06 μg/mL. After treatment with LSP01, the permeability of the cell membrane of A. hydrophila was increased, the metabolic activity of the cells was decreased, and the cell structure was ruptured, resulting in the death of A. hydrophila. [Conclusion] The bacteriocin resources in the intestines of S. graham in Dianchi are abundant, especially the bacteriocin (LSP01) produced by strain S01 has the characteristics of high antibacterial activity, good stability, and broad antibacterial spectrum, which can destroy the cell structure of A. hydrophila, reduce metabolic activity and cell membrane permeability, suggesting potential for alternative antibiotics.