育肥绵羊胃肠道乳酸菌的分离鉴定及部分体外益生特性评价

Isolation,Identification and Partial in Vitro Probiotic Characteristics Evaluation of Lactic Acid Bacteria in Gastrointestinal Tract of Fattening Sheep

本研究旨在探寻益生特性优良、可作为抗生素替代品的羊源乳酸菌,为后续饲用益生菌的开发与利用提供依据。以育肥羊不同肠段和瘤胃内容物为菌株分离的来源,采用碳酸钙-MRS固体培养基分离纯化乳酸菌,通过初筛、溶血试验复筛获取安全菌株,对安全菌株进行形态学、生化鉴定和16S rRNA测序鉴定,并评价其抑菌能力、抗生素敏感性和耐受性。结果显示:共获取12株同型乳酸菌——海氏肠球菌的安全菌株,分别来源于空肠(K1、K2、K3、K4)、回肠(H1、H2、H3、H4、H5、H6)、直肠(Z1)和瘤胃(L1)。在抑菌能力方面,12株海氏肠球菌对2种典型致病菌金黄色葡萄球菌和大肠杆菌均具有一定的抑制性,且对金黄色葡萄球菌的抑菌圈直径极大于大肠杆菌,对这2种指示菌的抑菌圈直径分别为7.30-12.30 mm(平均为9.90 mm)和6.90-9.47 mm(平均为8.43 mm),尤其是来自空肠的K2菌株对这2种指示菌的抑菌圈最大;从抗生素敏感性分析,K2和H5菌株具广泛的抗生素敏感性,对6种抗生素敏感,其余菌株(L1除外)对5种抗生素敏感;从耐受性上看,这12株海氏肠球菌对胃肠液均具有一定的耐受性,L1和K2菌株在胃液中存活率较高,分别达175.61%和173.72%,极显著高于其他菌株(P<0.01),K2菌株在肠液中存活率最高,达到143.68%,极显著高于其他菌株(P<0.01);另外,从细菌生长规律可以看出,这12株海氏肠球菌的生长对数期均为2-14 h。综上可知,本试验筛选出的12株海氏肠球菌抑菌能力强、具有一定抗生素敏感性且能耐受胃肠液,尤其是分离自空肠的K2菌株,可为饲用益生菌剂的开发提供参考菌种。

The purpose of this study was to explore sheep-derived lactic acid bacteria with excellent probiotic properties as a substitute for antibiotics,which would provide a basis for exploitation and utilization of subsequent feeding probiotics.The contents from different intestinal segments and rumen of fattening sheep were used to isolate lactic acid bacteria.The strains of bacteria were isolated and purified by solid medium of CaCO_(3)-MRS,and the safe strains were obtained via primary screening and re-screening in hemolysis test.The safe strains were identified by three methods of morphological,biochemical identification,and 16S rRNA sequencing,and their bacteriostatic ability,antibiotic susceptibility and tolerance were also evaluated.The results were as follows:twelve safe strains of homotypic lactic acid bacteria—Enterococcus hirae were obtained,which were derived from jejunum(K1,K2,K3 and K4),ileum(H1,H2,H3,H4,H5 and H6),rectum(Z1)and rumen(L1).In terms of bacteriostatic activity,the 12 strains of Enterococcus hirae had inhibitory abilities against two typical pathogenic bacteria—Staphylococcus aureus and Escherichia coli,suggesting that the inhibition zone diameter against Staphylococcus aureus was bigger than that against Escherichia coli.The diameters of inhibition zones ranged from 7.30 to 12.30 mm(averaging 9.90 mm)in Staphylococcus aureus and 6.90 to 9.47 mm(averaging 8.43 mm)in Escherichia coli,Particularly,the K2 strain from jejunum demonstrated the biggest inhibition zone against both above indicative bacteria among all isolated strains.From antibiotic susceptibility,the K2 and H5 strains had a wide range of antibiotic susceptibility,sensitive to 6 kinds of antibiotics,and other isolated strains were only sensitive to 5 kinds of antibiotics,except for L1.From perspective of tolerance,all isolated strains showed the tolerance to gastrointestinal fluid.The L1 and K2 strains demonstrated the higher survival rate in gastric fluid among all strains,reaching 175.61%and 173.72%,respectively,and were significantly higher than the other strains(P<0.01),while K2 strain had the highest survival rate in intestinal fluid among all strains,reaching up to 143.68%,and was significantly higher than the other strains(P<0.01).In addition,from bacteria growth curve,the growth logarithmic phase of 12 strains of Enterococcus hirae lasted for 2 to 14 h.In conclusion,the 12 strains of Enterococcus hirae isolated from gastrointestinal tract of fattening sheep in this experiment has stronger bacteriostatic activity,a certain antibiotic sensitivity,and can tolerate gastrointestinal fluid,particularly K2 strain which isolated from jejunum,can provide reference stains for exploitation of feeding probiotics.