产细菌素屎肠球菌的筛选鉴定及其抑菌特性

Screening for and Identification for Bacteriocin-producing Enterococcus faecium and Its Antibacterial Properties

从采自四川红原的传统发酵酸乳中分离得到308株疑似乳酸菌,通过96孔板法初筛和平板打孔法复筛,筛选出1株具有明显抑菌作用的菌株SC-Y112。通过形态学观察、16S rDNA序列同源性分析和ropA管家基因序列同源性分析结合生理生化实验,鉴定该菌株为屎肠球菌(Enterococcus faecium)。通过排酸及排除H2O2影响后,该菌株的发酵上清液仍具有明显抑菌活性,经蛋白酶处理后,抑菌效果明显消失或下降,说明发酵上清液中的抑菌成分具有蛋白质性质。进一步探究该菌株所产细菌素的生物学特性、遗传稳定性及抑菌特性表明,该细菌素在菌株接种后6 h产生,12 h达到最高;菌株在连续传代86代内,产细菌素能力较为稳定;细菌素在pH 3.0~7.0的条件下稳定,在100℃处理30 min后仍具有抑菌活性;SC-Y112所产细菌素具有广谱抗菌性,对单核细胞性李斯特菌有明显的抑菌活性。

A total of 308 strains suspected of being Lactobacillus were isolated from traditional fermented yoghurt collected from Hongyuan,Sichuan province,and were screened by 96-well plate method and agar well diffusion method.Among these strains,SC-Y112 was selected for its obvious antibacterial effect.It was identified as Enterococcus faecium by morphological observation,16S rDNA and ropA housekeeping gene sequencing analysis,and physiological and biochemical tests.After removing the influence of organic acid and hydrogen peroxide,the fermentation supernatant of the strain still had considerable antibacterial activity.The antibacterial effect decreased and even disappeared after being treated with protease,which indicated that the antibacterial components in the fermentation supernatant were proteins.Further,the biological characteristics,genetic stability and bacteriostatic characteristics of the bacteriocin produced by the strain were investigated,which showed that the bacteriocin was produced at 6 h and reached the maximum at 12 h after inoculation;the bacteriocin production ability was relatively stable within 86 consecutive generations;the bacteriocin was stable under the condition of pH 3.0-7.0 and heat-treatment at 100℃for 30 min.The bacteriocin had a wide antibacterial spectrum,especially against Listeria monocytogenes.