可抑制致病菌的益生菌筛选及抑菌物质的初步确定

doi:10.13560/j.cnki.biotech.bull.1985.2017-0411

摘要/Abstract

摘要： 为了得到可抑制致病菌的益生菌并初步确定其抑菌物质。从土壤、饲料添加剂和肥料添加剂3种样品中初筛能产生抑菌圈的菌株。用牛津杯法,对初筛得到的菌株和实验室保存的菌株进行复筛。对复筛的未知菌株进行形态学、16S rRNA基因序列和生理生化特征鉴定。对复筛菌株的发酵上清液进行酸碱作用验证、高温处理、蛋白酶处理、盐析处理和透析处理,再进行抑菌活力测定。结果显示复筛中有8株对大肠杆菌、金黄色葡萄球菌和藤黄微球菌都有较强抑制作用的菌株。8株菌中有4株已知菌,4株未知菌。已知菌株分别为保加利亚乳杆菌、干酪乳杆菌、鼠李糖乳杆菌和植物乳杆菌。4株未知菌中3株被鉴定为地衣芽孢杆菌,1株被鉴定为乳酸片球菌。地衣芽孢杆菌能产生多种耐高温的抑菌物质,其中包含能透过8-14 kD透析袋的小分子和不能透过8-14 kD透析袋的大分子,初步判定为多肽类物质。5株产酸菌株产生的抑菌物质主要是酸性物质。

关键词: 益生菌, 牛津杯法, 16S rRNA, 抑菌物质

Abstract: In order to obtain probiotics that inhibit pathogens and to determine their antimicrobial substances,strains that can produce inhibitory zones were isolated from the soil,feed additives and fertilizer additives. Strains isolated and preserved in the laboratory were re-screened with the Oxford Cup method. Then the unknown strains were identified with morphological observation,16S rRNA -based phylogeny and physiological and biochemical reaction. The antimicrobial activity of fermentation supernatant was determined after treated by acid-base reaction,high temperature,protease decomposition,salting-out and dialysis. Results showed that 8 strains presented the strong inhibitory effect on Escherichia coli,Staphylococcus aureus and Micrococcus luteus,and 4 strains were known as Lactobacillus bulgaricus,Lactobacillus casei,Lactobacillus rhamnosus and Lactobacillus plantarum. Three of the 4 unknown strains were identified as Bacillus licheniformis and one was identified as Pediococcus lactis. Bacillus licheniformis can produce a variety of antimicrobial substances with different molecular weights and high temperature resistance,which can or cannot pass through 8-14 kD dialysis bags,and the antimicrobial substances produced by Bacillus licheniformis are preliminarily determined as polypeptide,and antimicrobial substances from 5 acid-producing strains are mainly acidic.

Key words: probiotics, Oxford Cup method, 16S rRNA, antimicrobial substances

王利杰, 余晓斌, 方银兵, 顾秋亚. 可抑制致病菌的益生菌筛选及抑菌物质的初步确定[J]. 生物技术通报, 2017, 33(11): 123-129.

WANG Li-jie, YU Xiao-bin, FANG Yin-bing, GU Qiu-ya. Screening of Probiotics for Inhibiting Pathogens and Preliminary Determination of Antimicrobial Substances[J]. Biotechnology Bulletin, 2017, 33(11): 123-129.

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