具有优良抑菌特性乳酸菌的筛选鉴定及活性物质检测

Screening and identification of lactic acid bacteria with excellent antibacterial properties and detection of active substances

【背景】有益性乳酸菌在人体和动物体内分布极为广泛,是维持胃肠道菌群平衡、提高机体免疫力的主力军。近年来,为了解决禁用抗生素而导致动物发病率不断增高的问题,分析和研究乳酸菌及其所产活性物质的益生特性并开发新型饲料添加剂成为一个重要手段。【目的】本实验旨在从土壤中分离筛选出具有优良抑菌特性的乳酸菌,并对其所产活性物质的特性进行分析评价。【方法】采用溴甲酚紫平板法筛选并结合抑菌能力检测,得到2株具有优良抑菌特性的产酸菌株,分别命名为H-3和H-4。经形态学鉴定及16S rRNA基因序列测定后,对2株菌分别进行生长曲线和产酸量检测;通过排除酸处理、蛋白酶处理和热处理的方法分析2株菌所产抑菌物质的有效成分。【结果】H-3和H-4菌株经初步鉴定为乳酸片球菌(Pediococcus acidilactici),2株菌均具有良好的生长性能及产酸性能。菌株发酵上清液对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、猪霍乱沙门氏菌(Salmonella choleraesuis)、福氏志贺氏菌(Shigella flexneri)均表现出明显的抑制作用。pH值调至6.0时,菌株发酵上清液仍保持较好的抑菌活性;pH值调至7.4时,H-4菌株上清液抑菌活性消失。经蛋白酶处理后上清液的抑菌活性减弱甚至消失,但高温处理对其抑菌活性几乎无影响,说明抑菌物质中存在一种蛋白质且具有良好的热稳定性。【结论】本实验从土壤中分离得到的2株乳酸片球菌具有广谱抑菌特性,而且所产抑菌物质中除有机酸外还存在一种蛋白质。该2株菌的发现及对它们所产抑菌物质的研究为新型绿色饲料添加剂及替抗产品的研发提供了材料并奠定了理论基础。

[Background]Lactic acid bacteria are widely distributed in human and animals.They are the main force to maintain the balance of gastrointestinal flora and improve immunity.In recent years,as antibiotics are banned to be used as feed supplements for animals,the incidence of animal diseases has been on the rise.An essential solution is to develop new feed supplements based on the characteristics of lactic acid bacteria and their active metabolites.[Objective]To isolate and screen lactic acid bacteria with excellent antibacterial properties from soil and to analyze and evaluate the characteristics of their active metabolites.[Methods]Two acid-producing strains with excellent antibacterial activity were screened with bromocresol purple plate method and Oxford cup method,which were named H-3 and H-4,respectively.After morphological identification and 16S rRNA gene sequencing,the growth curves and acid production of the two strains were detected,respectively.The effective components of antibacterial substances produced by the two strains were analyzed based on acid exclusion,protease treatment,and heat treatment.[Results]H-3 and H-4 were preliminarily identified as Pediococcus acidilactici,which demonstrated good growth performance and acid production capacity.The supernatant of the fermentation broth of the two strains showed obvious inhibitory effect on Escherichia coli,Staphylococcus aureus,Salmonella choleraesuis and Shigella flexneri.At pH 6.0,the supernatant maintained strong antibacterial activity,while at pH 7.4,the antibacterial activity of the supernatant of H-4 disappeared.The antibacterial activity of the supernatant treated with protease decreased or even disappeared,but the high-temperature treatment had little influence on the activity,suggesting the existence of a protein in the antibacterial substances with thermal stability.[Conclusion]H-3 and H-4 have broad-spectrum antibacterial activity,and in addition to organic acids,there is also a protein in the produced antibacterial substances.The result in this study lays a theoretical foundation for the research and development of new green feed supplements and antibiotic alternatives.