散养云南地方猪源乳酸菌分离、鉴定及其益生特性的研究  

Isolation,Identification and Probiotic Characteristics of Lactic Acid Bacteria Derived from Free Range Local Pigs in Yunnan Province

作　　者：鲍国樱 余行 王孝义[1] 叶朋飞 殷桦娟 胡天念 曹振辉[1] BAO Guoying;YU Hang;WANG Xiaoyi;YE Pengfei;YIN Huajuan;HU Tiannian;CAO Zhenhui(College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学动物科学技术学院,昆明650201

出　　处：《动物营养学报》2023年第8期5418-5429,共12页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金(32160809);云南省中青年学术和技术带头人后备人才(202305AC160040)。

摘　　要：本试验旨在筛选优质猪源乳酸菌,为猪用益生菌制剂研发奠定基础。以散养云南地方猪新鲜粪便为菌株分离来源,采用MRS+碳酸钙固体培养基分离纯化乳酸菌,经生理生化和16S rRNA测序鉴定得到9株乳酸菌,测定并评价其抗生素敏感性、抑菌活性、胃肠道耐受性、黏附能力和抗炎能力,综合比较不同菌株之间的益生特性。结果表明:从散养云南地方猪新鲜粪便中分离筛选出9株对氨苄青霉素、头孢噻吩和青霉素G等6种抗生素敏感或中度敏感且抑菌活性较强的菌株,经过16S rRNA基因序列分析和生理生化鉴定,DBS4-3、DBS5-5、DBS2-3、SBC4-9、SBC5-3和ZK3-1为罗伊氏乳杆菌,SBC1-6和ZK7-2为发酵乳杆菌,ZK2-2为黏膜乳杆菌。DBS2-3、SBC5-3和ZK7-2可耐受低pH和高胆盐环境;SBC5-3和ZK7-2对肠上皮细胞HT-29具有较强的黏附能力;SBC5-3可显著降低肿瘤坏死因子-α(TNF-α)诱导HT-29细胞白细胞介素-8(IL-8)基因相对表达量(P<0.05)。综上所述,SBC5-3是一株具有良好的黏附和抗炎效果、抑菌能力强、对抗生素敏感且能耐受胃肠道环境的猪源乳酸菌,可用作微生态制剂候选菌株进一步开展深层次研究。This study aimed to isolate pig⁃derived lactic acid bacteria with excellent probiotic properties,which would provide a basis for exploitation and utilization of potential probiotics in pig production.Fresh feces of free⁃range Yunnan local pigs were used as the source of strain isolation,and lactobacilli were isolated and puri⁃fied using MRS agar supplemented with calcium carbonate.Nine strains of lactobacilli were identified based on physiological and biochemical tests and 16S rRNA sequencing.Their antibiotic sensitivity,antagonistic activi⁃ty,gastrointestinal tolerance,adhesion ability and anti⁃inflammatory ability were determined and evaluated.The probiotic characteristics between different strains were compared comprehensively.The results showed that nine strains which were sensitive or moderately sensitive to six used antibiotics such as ampicillin,cephalothin,penicillin G and exhibited greater antagonistic activity were isolated from fresh feces of free⁃range local pigs in Yunnan Province.According to data obtained from 16S rRNA gene sequence analysis and physiological and bi⁃ochemical identification,DBS4⁃3,DBS5⁃5,DBS2⁃3,SBC4⁃9,SBC5⁃3 and ZK3⁃1 were Lactobacillus reu⁃teri,SBC1⁃6 and ZK7⁃2 were Lactobacillus fermentum,and ZK2⁃2 was Limosilactobacillus mucosae.DBS2⁃3,SBC5⁃3 and ZK7⁃2 could tolerate low pH and high bile salt conditions.SBC5⁃3 and ZK7⁃2 showed greater adhesion ability to HT⁃29 intestinal epithelial cells.SBC5⁃3 could significantly decrease the relative ex⁃pression level of proinflammatory gene interleukin⁃8(IL⁃8)in tumor necrosis factor⁃alpha(TNF⁃α)⁃induced HT⁃29 cells(P<0.05).In conclusion,SBC5⁃3 is a porcine lactic acid bacteria with excellent adhesion and an⁃ti⁃inflammatory effect,strong antagonistic ability,antibiotics susceptibility and tolerance to gastrointestinal en⁃vironment.It can be used as a candidate strain for microecological preparations for further in⁃depth research.

关 键 词：云南地方猪 乳酸菌 益生菌 胃肠道耐受性 抑菌活性 抗生素敏感性 

分 类 号：S828[农业科学—畜牧学]