一株丁酸梭菌的分离鉴定及其益生特性研究

doi:10.16431/j.cnki.1671-7236.2020.01.030

摘要/Abstract

摘要： 本研究探索了丁酸梭菌分离株的益生特性，旨在为其在仔猪日粮中的应用提供理论依据。利用常规方法分离丁酸梭菌并进行纯培养，经生化检测和16S rRNA基因测序，对分离菌株进行鉴定；采用活菌计数法和牛津杯法研究分离株培养上清对3种致病菌的抑制作用、分离株黏附猪小肠上皮细胞（IPEC-J2）的特性及其对致病菌黏附细胞的抑制作用；采用细胞计数法研究分离株对IPEC-J2生长的影响；采用ELISA检测分离株处理细胞后培养上清液中细胞因子水平。结果表明，本研究成功分离到一株丁酸梭菌。与对照组相比，丁酸梭菌培养上清对3种致病菌生长抑制效果均显著（P<0.05）；丁酸梭菌可黏附于IPEC-J2，并且黏附效果最佳的感染复数（MOI）为50，最适处理时长为3 h；丁酸梭菌对3种致病菌黏附IPEC-J2均具有显著抑制作用（P<0.05）；丁酸梭菌MOI为1、10和50时细胞生长正常、形态完好，MOI为100时IPEC-J2生长受到显著抑制，同时出现部分细胞死亡；MOI为1时细胞因子水平无差异，MOI为10、50和100时细胞因子水平均显著增高（P<0.05）。综上所述，本研究分离的一株丁酸梭菌具有较好的益生特性，为其在生产中的应用提供了理论依据。

关键词: 丁酸梭菌; 分离鉴定; 猪小肠上皮细胞; 益生特性

Abstract: This study explored the probiotic characteristics of Clostridium butyricum isolates and aimed to provide a theoretical basis for their application in piglet diets.Clostridium butyricum was isolated by routine methods and cultured in pure culture.The isolates were identified by biochemical detection and 16S rRNA gene sequencing;The viable cell count method and the Oxford cup method were used to study the inhibitory effect of the culture supernatant of the isolate on the three pathogenic bacteria,the adhesion of the isolated pig intestinal epithelial cells (IPEC-J2) and its inhibitory effect on the adherent cells of pathogenic bacteria;The effect of the isolate on the growth of IPEC-J2 was studied by cell counting method;The cytokine level in the culture supernatant was determined by ELISA after treatment of the isolate.The results showed that a strain of Clostridium butyricum was successfully isolated in this study.Compared with the control group,the antibacterial effect of Clostridium butyricum culture supernatant on the three pathogenic bacteria was significant (P<0.05).Clostridium butyricum could adhere to IPEC-J2,and the optimal multiplicity of infection(MOI) was 50,and the optimal treatment time was 3 h;Clostridium butyricum had inhibitory effect on three pathogenic adhesion IPEC-J2 cells (P<0.05).When the MOI of Clostridium butyricum was 1,10 and 50,the cell growth was normal and the morphology was intact.When MOI was 100,the growth of IPEC-J2 was significantly inhibited,and some cell death occurred at the same time.There was no difference in cytokine production levels when MOI was 1,and cytokine production levels were significantly increased at MOI of 10,50,and 100 (P<0.05).In conclusion,a Clostridium butyricum strain was successfully isolated in this study,which has good probiotic characteristics.The results provided a theoretical basis for the application of the isolated strain in production.

Key words: Clostridium butyricum; isolation and identification; pig intestinal epithelial cells; probiotic characteristics

中图分类号:

S816.3

王倩, 李海花, 窦彩霞, 刘丽丽, 乔家运. 一株丁酸梭菌的分离鉴定及其益生特性研究[J]. 中国畜牧兽医, 2020, 47(1): 258-266.

WANG Qian, LI Haihua, DOU Caixia, LIU Lili, QIAO Jiayun. Isolation,Identification and Probiotic Characteristics of a Clostridium butyricum[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 258-266.

参考文献

[1] 朱翠,蒋宗勇.谷氨酰胺对早期断奶仔猪肠黏膜免疫的调节作用[J].中国畜牧兽医,2011,38(8):10-16. ZHU C,JIANG Z Y.Regulative function of glutamine on intestinal mucosal immunity in early-weaning piglets[J].China Animal Husbandry & Veterinary Medicine,2011,38(8):10-16.(in Chinese)
[2] QI M,HUAN S Y,YU L Y,et al.Amino acids influencing intestinal development and health of the piglets[J].Animal,2019,9(6):E302.
[3] 马树薇,杨贵燕,朱要宏,等.断奶腹泻仔猪肠道沙门菌与奇异变形杆菌的分离鉴定[J].中国兽医学报,2017,37(7):1288-1291. MA S W,YANG G Y,ZHU Y H,et al.Isolation and identification of Salmonella and Proteus mirabilis in weaned pigs with diarrhea[J].Chinese Journal of Veterinary Science,2017,37(7):1288-1291.(in Chinese)
[4] 赵文艳.铜－壳聚糖/蒙脱土复合材料的抗菌性能及其对肉鸡的应用效果[D].杭州:浙江大学,2007. ZHAO W Y.The antibacterial activity of Cu²⁺-chitosan/montmorillonite composite and its applied effects in broilers[D].Hangzhou:Zhejiang University,2007.(in Chinese)
[5] 贾鹏,马涛,万凡,等.益生菌在反刍动物饲粮中的应用[J].饲料工业,2019,40(1):59-64. JIA P,MA T,WAN F,et al.Application of probiotics in diet of ruminants[J].Feed Industry Magazine,2019,40(1):59-64.(in Chinese)
[6] 肖宏德,肖运才,何熙贞,等.用益生菌制剂代替断奶仔猪日粮中抗生素的研究[J].动物医学进展,2014,35(3):53-58. XIAO H D,XIAO Y C,HE X Z,et al.Study on probiotic agents substituting for antibiotics in weaned piglet diets[J].Progress in Veterinary Medicine,2014,35(3):53-58.(in Chinese)
[7] WYDAU-DEMATTEIS S,LOUIS M,ZAHR N,et al.The functional dlt operon of Clostridium butyricum controls the D-alanylation of cell wall components and influences cell septation and vancomycin-induced lysis[J].Anaerobe,2015,35:105-114.
[8] 谢丽静,王伟华,王海宽,等.丁酸梭菌的应用及对食品安全的影响[J].中国食物与营养,2017,23(7):5-9. XIE L J,WANG W H,WANG H K,et al.Application of Clostridium butyricum and its effect on food safety[J].Food and Nutrition in China,2017,23(7):5-9.(in Chinese)
[9] 赵敏孟,单昊书,沈永华,等.丁酸梭菌在畜禽生产中的应用研究进展[J].动物营养学报,2018,30(7):1-5. ZHAO M M,SHAN H S,SHEN Y H,et al.Research progress on application of Clostridium butyricum in livestock and poultry production[J].Chinese Journal of Animal Nutrition,2018,30(7):1-5.(in Chinese)
[10] 布坎南R E,吉本斯N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984. BUKANMAN R E,JIBENSI N E.Berger Bacterial Identification Manual[M].8th Edition.Beijing:Science Press,1984.(in Chinese)
[11] 李圣杰,丁轲,姜芳芳,等.鸡源高产淀粉酶丁酸梭菌的分离鉴定[J].中国畜牧兽医,2015,42(11):3073-3079. LI S J,DING K,JIANG F F,et al.Isolation and identification of producing High-yielding amylase Clostridium butyricum from chicken[J].China Animal Husbandry & Veterinary Medicine, 2015,42(11):3073-3079.(in Chinese)
[12] 袁华伟,赵猛,蒋君,等.窖泥中产丁酸菌的筛选、鉴定及生长性能研究[J].酿酒科技,2017,5:35-40. YUAN H W,ZHAO M,JIANG J,et al.Isolation and identification of a butyric acid-producing strain from pit mud and study on its growth performance[J].Liquor-Making Science and Technology,2017,5:35-40.(in Chinese)
[13] 梁东梅,李玉鹏,杜艳芬,等.三株乳酸杆菌的分离鉴定与益生特性研究[J].中国饲料,2018,11:24-29. LIANG D M,LI Y P,DU Y F,et al.Study on the isolation,identification of three Lactobacilli and their probiotic properties[J].China Feed,2018,11:24-29.(in Chinese)
[14] 王腾浩,宗鑫,宋德广,等.产抑菌蛋白的丁酸梭菌的筛选和鉴定及体外益生功能研究[J].动物生产,2015,51(13):75-81. WANG T H,ZONG X,SONG D G,et al.Screening,identification and in vitro functional study of Clostridium butyricum which produce antibacterial protein[J].Animal Production,2015,51(13):75-81.(in Chinese)
[15] 张晓阳,卢忆,马艳莉,等.丁酸梭菌生理功能及应用研究进展[J].中国食物与营养,2012,18(12):31-35. ZHANG X Y,LU Y,MA Y L,et al.Research progress of physiological function and application of Clostridium butyricum[J].Food and Nutrition in China,2012,18(12):31-35.(in Chinese)
[16] 贾丽楠,崔嘉,李占一,等.丁酸梭菌对肉仔鸡肠道致病菌抑菌作用研究[J].中国家禽,2017,23:22-25. JIA L N,CUI J,LI Z Y,et al.Study on mechanism and inhibitory effect of Clostridium butyricum on bacteria in intestinal of broilers[J].China Poultry,2017,23:22-25.(in Chinese)
[17] 杨静.丁酸梭菌及其磷壁酸与肠道细胞互作的分子机制[D].泰安:山东农业大学,2014. YANG J.The molecular mechanism of Clostridium butyricum and Clostridium butyricum teichoic acid acting on intestinal epithelial cells[D].Tai’an:Shandong Agricultural University,2014.(in Chinese)
[18] 赵静,李向楠,王军.益生乳酸菌对奶牛阴道上皮细胞黏附作用的研究[J].中国预防兽医学报,2014,36(9):701-703. ZHAO J,LI X N,WANG J.Adherence probioties of Lactobacillus to the vaginal epithelial cell of cows[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(9):701-703.(in Chinese)
[19] 刘海鸿,陶柯衡,张国荣,等.卷曲乳酸杆菌对Hela细胞的黏附研究[J].中国微生态学杂志,2017,29(1):9-21. LIU H H,TAO K H,ZHANG G R,et al.The adhesion of Lactobacillus crispatus on Hela cell[J].Chinese Journal of Microecology,2017,29(1):9-21.(in Chinese)
[20] 向鑫玲,张英春,马放.乳酸杆菌的表面特性及其黏附能力的研究[J].食品工业科技,2016,37(7):126-130. XIANG X L,ZHANG Y C,MA F.Surface properties and adhesion ability of Lactobacillus[J].Science and Technology of Food Industry,2016,37(7):126-130.(in Chinese)
[21] 贾聪慧,杨彩梅,曾新福,等.丁酸梭菌对肉鸡生长性能、抗氧化能力、免疫功能和血清生化指标的影响[J].动物营养学报,2016,28(3):908-915. JIA C H,YANG C M,ZENG X F,et al.Effects of Clostridium butyricum on growth performance,antioxidant capacity,immune function and serum biochemical parameters of broilers[J].Chinese Journal of Animal Nutrition,2016,28(3):908-915.(in Chinese)
[22] 张传健.抗生素干预对猪肠道微生物和肠道黏膜免疫的影响[D].南京:南京农业大学,2013. ZHANG C J.Influence of antibiotic intervention on gut microbiota anf intestinal mucosal immunity in swine[D].Nanjing:Nanjing Agricultural University,2013.(in Chinese)