摘要
为筛选能够抑制猪源致病性大肠杆菌(Escherichia coli)的乳酸菌(LAB)并分析LAB的主要抑菌成分,本研究利用牛津杯法测定了21株从健康猪体内分离到的LAB对4株不同血清型猪源致病性E. coli的抑菌活性,并采用微量稀释法测定了其最小抑菌浓度(MIC)。结果显示,筛选到6株LAB,分别为唾液乳杆菌和粪肠球菌各2株、黏膜乳杆菌和约氏乳杆菌各1株,对4株致病性E. coli均具有明显的抑制作用,其中唾液乳杆菌L1-5的抑制效果最明显,MIC值最低,而约氏乳杆菌L4-3的抑菌活性最低,MIC值相对较高。通过测定生长曲线分析筛选到的LAB对致病性E. coli增殖的影响,采用有机酸排除、过氧化氢酶处理和蛋白酶敏感性检测等方法分析LAB发挥抑菌作用的主要成分,并进一步分析温度、紫外线和表面活性剂对LAB抑菌成分的影响。生长曲线测定结果显示,与LAB共培养后,4株E. coli的增殖速度均降低,且在各时间点的OD_(600nm)值均低于不加LAB的对照组。对LAB抑菌成分的分析结果显示,pH值调整为6.5后4株乳杆菌的抑菌活性均消失,而不同蛋白酶处理后2株粪肠球菌的抑菌活性极显著降低(P<0.01、P<0.001),分析可知其中4株乳杆菌的主要抑菌成分为有机酸,而2株粪肠球菌的主要抑菌成分推测为细菌素。抑菌作用稳定性分析结果显示,6株LAB在不同温度、紫外线照射和表面活性剂处理后仍具有较强的抑菌活性。上述结果表明,6株分离自健康猪体内的LAB对4株猪源致病性E.coli均具有明显的抑菌活性。本研究为防治猪大肠杆菌病的新型微生态制剂开发奠定了基础。
In order to screen the lactic acid bacteria(LAB) for inhibiting porcine pathogenic Escherichia coli and analyze its main antibacterial ingredient, the Oxford cup method was used to determine the antimicrobial activity of 21 strains of LAB isolated from healthy pigs against 4 strains of pathogenic E. coli with different serotypes. And the minimum inhibitory concentration(MIC)of LAB against pathogenic E. coli was determined by the microdilution method. The results showed that 6 strains of LAB, which including 2 strains of Lactobacillus salivarius, 1 strain of Lactobacillus mucosus, 1 strain of Lactobacillus johnsonii, and 2 strains of Enterococcus faecalis had significant antimicrobial activities against 4 pathogenic E. coli strains, among which L. salivarius L1-5had the most obvious inhibitory effect and the lowest MIC value, while L. johnsonii L4-3 had the lowest antimicrobial activity and a relatively high MIC value. The effect of LAB on the proliferation of pathogenic E. coli was determined by measuring the growth curve, and the antibacterial components were analyzed by eliminating interference of organic acid, catalase treatment and protease sensitivity test. Then the influence of temperature, ultraviolet light and surfactant on the antimicrobial activity of LAB was further analyzed. The results of growth curve measurement showed that the proliferation rate of four strains of pathogenic E. coli decreased very slow after co-culture with LAB and the OD_(600nm)value was lower than that of the control group at all time points. The analysis of the antimicrobial substance of LAB showed that the antibacterial activities of 4 strains of Lactobacillus disappeared when p H value was adjusted to 6.5, while the antimicrobial activities of 2 strains of E. faecalis significantly decreased after treatment with different proteases(P<0.01, P<0.001). The main antibacterial component of four strains of LAB was organic acid and the other two stains of Enterococcus faecalis was bacteriocin. The stability of antibacterial activity analysis showed that the 6 LAB strains still had high bacteriostatic activity under different temperatures, ultraviolet irradiation and surfactant treatment. The results indicated that 6 strains of LAB isolated from healthy pigs had obvious antibacterial activities against the four swine pathogenic E. coli strains.This study has important reference value for replacing antibiotics in the prevention and treatment of swine E. coli disease and developing new probiotics.
作者
冯瑜菲
刘文鑫
袁超文
胡清泉
FENG Yu-fei;LIU Wen-xin;YUAN Chao-wen;HU Qing-quan(College of Life Science and Technology,LingNan Normal University,Zhanjiang 524048,China;Central People's Hospital of Zhanjiang,Zhanjiang 524045,China;College of Life Sciences and Health,Notheastem University,Shenyang 10006,China;College of Animal Science and Medicine,Shenyang Agricultural University,Shenyang 110866,Chin)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第12期1277-1282,1297,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
广东省基础与应用基础研究基金企业联合基金(公共卫生与医药健康领域)项目(2021A1515220027)
广东省自然科学基金-面上项目(2020A1515010639)
湛江市科技发展专项资金竞争性分配项目(2020A01045、2021A05147)
湛江中心人民医院院级科研项目-博士启动项目(2020A07)。
关键词
致病性大肠杆菌
乳酸菌
抑菌活性
有机酸
细菌素
pathogenic Escherichia coli
lactic acid bacteria
antimicrobial activity
organic acid
bacteriocin
