一株可拮抗致病性肠球菌的益生菌D-19发酵条件和拮抗稳定性  

Fermentation conditions and antagonistic stability of a probiotic strain D-19 against pathogenic Enterococcus

作　　者：车雨辰 孙冶[2,3] 李舒音[2,4] 肖纯凌 CHE Yuchen;SUN Ye;LI Shuyin;XIAO Chunling(Department of Immunology,Shenyang Medical College,Shenyang,Liaoning 110034,China;不详)

机构地区：[1]沈阳医学院免疫学教研室,辽宁沈阳110034 [2]沈阳医学院,辽宁省环境污染与微生态重点实验室,沈阳市肖纯凌劳模创新工作室,辽宁沈阳110034 [3]沈阳医学院病原生物学教研室,辽宁沈阳110034 [4]沈阳医学院人事处,辽宁沈阳110034

出　　处：《中国微生态学杂志》2024年第8期915-921,共7页Chinese Journal of Microecology

基　　金：中国医药教育协会重大科学攻关问题和医药技术难题课题项目(2022KTM026)。

摘　　要：目的研究益生菌D-19发酵液对致病性肠球菌的拮抗能力,多方面评价其拮抗稳定性,为挖掘其作为微生态制剂的潜在益生特性和医疗应用提供理论基础。方法采用纸片扩散法探讨益生菌D-19发酵液对粪链球菌和屎肠球菌的拮抗能力,优化其发酵条件,从理化因素和保存条件等方面探讨其拮抗稳定性,通过生物膜形成试验研究益生菌D-19的抑菌机制。结果益生菌D-19发酵液原液、无菌体上清液可以抑制粪链球菌和屎肠球菌的生长,菌体本身无明显抑菌作用;在培养液接种量1%、初始液pH 7、37℃培养24 h的优化条件下,益生菌D-19发酵液对粪链球菌和屎肠球菌表现出较强的拮抗能力,抑菌圈直径分别为(24.70±0.71)mm和(22.57±0.43)mm;在40~50℃时,益生菌D-19发酵液拮抗粪链球菌和屎肠球菌的能力无显著变化,随着温度的升高、处理时间的增加,其拮抗能力逐渐降低直至失去;同样,在pH5~8时,益生菌D-19发酵液表现出稳定的拮抗能力,低于或高于这个范围,其将逐渐失去拮抗粪链球菌和屎肠球菌的能力;当保存温度为-20℃、保存月数m=5时,益生菌D-19发酵液对粪链球菌和屎肠球菌的抑菌圈直径分别降至(7.56±8.20)mm和(5.64±0.32)mm;当保存温度为4℃、保存天数d=75时,对粪链球菌和屎肠球菌的抑菌圈直径分别降至(10.02±0.91)mm和(6.62±0.55)mm;当保存温度为25℃、保存天数d=15时,对粪链球菌和屎肠球菌的抑菌圈直径分别降至(8.26±0.42)mm和(5.99±0.35)mm;益生菌D-19发酵液分别与2种肠球菌共培养可以显著抑制其生物膜的形成。结论益生菌D-19发酵液在最佳发酵条件下对粪链球菌和屎肠球菌具有较强的拮抗能力,其中主要抑菌活性成分为无菌体上清液,其无菌体上清液具有一定的酸碱稳定性和热稳定性,在pH 5~8、40~50℃的环境下能保持稳定的拮抗能力,对强酸强碱、高热敏感,可在-20℃的条件下保持拮抗活性1~5个月Objective To explore the antagonistic ability of probiotic D-19 fermentation broth against pathogenic Enterococcus,evaluate its antagonistic stability from multiple aspects,and provide a theoretical basis for exploring its potential probiotic properties and medical applications as microecologics.Methods Kirby-Bauer method was used to explore the antagonistic ability of probiotic D-19 fermentation broth against Enterococcus faecalis and Enterococcus faecium.The fermentation conditions of it were optimized and the antagonistic stability was observed from viewpoints of physicochemical factors and preservation conditions.The antibacterial mechanism of probiotic D-19 was studied through biofilm formation experiments.Results The fermentation broth of probiotic D-19 and the supernatant of sterile bodies could inhibit the growth of E.faecalis and E.faecium,while the bacterial bodies themselves had no significant antibacterial effect.Under the optimized conditions of 1%inoculation in the culture medium,initial pH 7,and 24 hours of cultivation at 37℃,the fermentation broth of probiotic D-19 showed strong antagonistic ability against the two Enterococcus,with inhibition zone diameter of(24.70±0.71)mm and(22.57±0.43)mm against E.faecalis and E.faecium,respectively.At 40-50℃,there was no significant change in the ability of probiotic D-19 fermentation broth to antagonize E.faecalis and E.faecium.As the temperature and treatment time increased,its antagonism gradually decreased until finally lost.Similarly,probiotic D-19 fermentation broth exhibited stable antagonistic ability at pH 5-8.If the pH falls below or exceeds this range,it will gradually lose its antagonistic ability to E.faecalis and E.faecium;When the storage temperature was‒20℃and m=5,the antibacterial zone diameter of probiotic D-19 fermentation broth against E.faecalis and E.faecium decreased to(7.56±8.20)mm and(5.64±0.32)mm,respectively;When stored at a temperature of 4℃and d=75,the size of inhibition zone against E.faecalis and E.faecium decrease

关 键 词：益生菌 肠球菌 抑菌 拮抗作用 稳定性 

分 类 号：R378.99[医药卫生—病原生物学]