牡蛎中产广谱细菌素芽孢杆菌的筛选及鉴定  

Screening and Identification of Broad-spectrum Bacteriocins Produced by Bacillus sp.Isolated from Oysters

作　　者：王伟[1,2] 陈渊戈[1] 迟海[1,2] 陶乐仁[2] Wang Wei;Chen Yuange;Chi Hai;Tao Leren(East China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Shanghai 200090;School of Medical Instrument and Food Engineering,University of Shanghai for Science and Technology,Shanghai 200093)

机构地区：[1]中国水产科学研究院东海水产研究所,上海200090 [2]上海理工大学医疗器械与食品学院,上海200093

出　　处：《中国食品学报》2021年第3期282-290,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31801663);上海市自然科学基金项目(18ZR1449900);中央级公益性科研院所基本科研业务费专项资金(2016M02,2019ZD0802);福建省属公益类科研院所基本科研专项(2018R1037-2)。

摘　　要：以不同革兰氏阳性菌(金黄色葡萄球菌、蜡样芽孢杆菌、植物乳杆菌)和革兰氏阴性菌(弗氏柠檬酸杆菌、副溶血性弧菌、大肠杆菌、肠道沙门氏菌、福氏志贺氏菌)为指示菌,利用从福建霞浦海域牡蛎中分离的细菌为指标菌,筛选具有产广谱细菌素的细菌。其中,14株具有广谱抑制作用的细菌经形态特征和16s rDNA分析,初步鉴定均为芽孢杆菌属。这些芽孢杆菌产的细菌素具有热稳定性强,易被蛋白酶分解等细菌素基本特征。通过抑菌谱的范围和重复片段基因指纹分析(rep-PCR),结果显示:这14株芽孢杆菌可进一步分为4株基因型的芽孢杆菌,做后续试验。通过质粒提取结果判断这4株芽孢杆菌细菌素是由染色体编码合成。经硫酸铵盐析法及凝胶过滤层析得到的细菌素粗提物质仍具有较强的抑菌作用。将细菌素的粗提物通过Tricine-SDS-PAGE电泳试验,结果显示:这4株具有广谱细菌素的芽孢杆菌分子质量均小于20 ku,应归属于Class Ⅱ类细菌素。结合LC-MS/MS比对鉴定,新发现的细菌素未列入现有蛋白质数据库中,这说明新发现的4株芽孢杆菌可能产生新型广谱细菌素。By using different Gram-positive bacteria(Staphylococcus aureus,Bacillus cereus,Lactobacillus plantanrum)and Gram-negative bacteria(Citrobacter freundii,Vibrio parahemolyticus,Escherichia coli,Salmonella enterica,Shigella flexneri)as indicator strains,some collection of bacterial strains isolated from the oysters of Xiapu Sea in Fujian province were screened for bacteriocin producers with broad inhibition spectra.All the fourteen strains with broad-spectrum inhibitory effect were initially identified as Bacillus sp.by morphological characteristics and 16s rDNA analysis.The bacteriocins produced by the Bacillus sp.are characterized by strong thermal stability and easy-decomposition by protease.The range of inhibition spectrum and the results of repeated fragment gene fingerprinting(rep-PCR)showed that the 14 strains of Bacillus could be further divided into four Bacillus strains with different genotypes for subsequent experiments.It was found from the plasmid extraction results that the bacteriocins producing by four strains of Bacillus sp.were synthesized by chromosome coding.The crude bacteriocins extracted by ammonium sulfate method and gel filtration chromatography still showed strong antibacterial activity.The Tricine-SDS-PAGE electrophoresis results showed that the molecular weights of bacteriocins producing by the four Bacillus strains were all less than 20 ku,which can be classified as Class Ⅱ bacteriocins from lactic acid bacteria.Combined with the comparisons and identification of LC-MS/MS,no related well-known bacteriocins were found in the database,indicating that the four strains of Bacillus may produce novel broad-spectrum bacteriocins.

关 键 词：芽孢杆菌 细菌素 抑菌活性 蛋白质鉴定 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]