RESEARCH PAPER

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

  • LIU Aiyu ,
  • TIAN Jun ,
  • ZHAO Xinnian ,
  • LI Yongliang ,
  • LI Xiaoyu ,
  • ZHAO Lichen ,
  • GAO Yuhong ,
  • KANG Xinyang ,
  • ZHANG Huiwen
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  • 1. College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China;
    2. Fengning County Agriculture and Rural Affairs Bureau, Chengde 067000, China;
    3. Technology and Education Station of Chengde Municipal Bureau of Agriculture and Rural Affairs, Chengde 067000, China

Received date: 2022-05-13

  Online published: 2022-12-15

Abstract

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 CaCO3-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.

Cite this article

LIU Aiyu , TIAN Jun , ZHAO Xinnian , LI Yongliang , LI Xiaoyu , ZHAO Lichen , GAO Yuhong , KANG Xinyang , ZHANG Huiwen . Isolation, Identification and Partial in Vitro Probiotic Characteristics Evaluation of Lactic Acid Bacteria in Gastrointestinal Tract of Fattening Sheep[J]. Chinese Journal of Animal Nutrition, 2022 , 34(12) : 8149 -8160 . DOI: 10.3969/j.issn.1006-267x.2022.12.061

References

[1] BARBOSA T M, SERRA C R, LA RAGIONE R M, et al.Screening for Bacillus isolates in the broiler gastrointestinal tract[J].Applied and Environmental Microbiology, 2005, 71(2):968-978.  
[2] SALZMAN N H, DE JONG H, PATERSON Y, et al.Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria[J].Microbiology, 2002, 148(Pt 11):3651-3660.
[3] 夏雪茹.兔源乳酸菌的筛选及其益生潜能评价[D].硕士学位论文.保定:河北农业大学, 2021. XIA X R.Screening of rabbit-derived lactic acid bacteria and evaluation of its probiotic potential[D].Master's Thesis.Baoding:Hebei Agricultural University, 2021.(in Chinese)
[4] 王银梦, 夏洪泽, 张晓涵, 等.牦牛犊牛肠源益生性抑菌乳酸菌的筛选与鉴定[J].饲料研究, 2020, 43(12):92-95. WANG Y M, XIA H Z, ZHANG X H, et al.Isolation and screening of probiotic lactic acid bacteria from the intestine of yak calves[J].Feed Research, 2020, 43(12):92-95.(in Chinese)
[5] 谢璐娜.番鸭肠道乳酸菌筛选鉴定、抑菌作用及对Caco-2细胞粘附的影响[D].硕士学位论文.福州:福建农林大学, 2015. XIE L N.Study on screening, identification, bacteriostatic efficiency and effects on Caco-2 cell adhesion of Lactobacillus from muscovy duck intestine[D].Master's Thesis.Fuzhou:Fujian Agriculture and Forestry University, 2015.(in Chinese)
[6] 汤凯, 杨劲松, 杨天澍, 等.海南黑山羊肠道源乳酸菌的分离鉴定与生物学特性分析[J].中国酿造, 2021, 40(11):149-153. TANG K, YANG J S, YANG T S, et al.Isolation, identification and biological characteristics of lactic acid bacteria from Hainan black goat intestinal tract[J].China Brewing, 2021, 40(11):149-153.(in Chinese)
[7] 郝贺, 朱原, 阿合玛尔列·海依拉提, 等.羊瘤胃乳酸菌的分离鉴定及抗逆性筛选[J].中国草食动物科学, 2021, 41(2):44-48, 54. HAO H, ZHU Y, HAIYILATI A, et al.Isolation, identification and stress resistance screening of rumen lactic acid bacteria in goat[J].China Herbivore Science, 2021, 41(2):44-48, 54.(in Chinese)
[8] 何江波.羊瘤胃源乳酸菌的分离筛选及其菌剂制备方法研究[D].硕士学位论文.杨凌:西北农林科技大学, 2021. HE J B.Isolation and screening of lactic acid bacteria from rumen and study on the preparation method of bacteria agent[D].Master's Thesis.Yangling:Northwest A & F University, 2021.(in Chinese)
[9] 宗学阳, 张华文, 史怀平.羔羊消化生理及消化道微生物研究进展[J].畜牧与兽医, 2022, 54(4):124-129. ZONG X Y, ZHANG H W, SHI H P.Advance in research on digestive physiology and gastrointestinal microflora of lambs[J].Animal Husbandry & Veterinary Medicine, 2022, 54(4):124-129.(in Chinese)
[10] 徐凤, 张焕容, 张兴民, 等.不同年龄山羊胃肠道乳酸菌的分离鉴定[J].黑龙江畜牧兽医, 2017(5):108-110. XU F, ZHANG H R, ZHANG X M, et al.Isolation and isolation of lactic acid bacteria in the gastrointestinal tracts of goats at different ages[J].Heilongjiang Animal Science and Veterinary Medicine, 2017(5):108-110.(in Chinese)
[11] 韩佳临.种猪肠道微生物分析、益生菌筛选及发酵豆粕的初步研究[D].硕士学位论文.南宁:广西大学, 2020. HAN J L.Preliminary study on the analysis of intestinal microbes in breeding pigs, screening of probiotics and fermented soybean meal[D].Master's Thesis.Nanning:Guangxi University, 2020.
[12] NCCLS.Performance standards for antimicrobial susceptibility testing[J].Twelfth Informational Supplement, 2002, 22(1):48-49.
[13] CHARTERIS W P, KELLY P M, MORELLI L, et al.Antibiotic susceptibility of potentially probiotic Lactobacillus species[J].Journal of food protection, 1998, 61(12):1636-1643.  
[14] 蔺豆豆, 琚泽亮, 柴继宽, 等.青藏高原燕麦附着耐低温乳酸菌的筛选与鉴定[J].草业学报, 2022, 31(5):103-114. LIN D D, JU Z L, CHAI J K, et al.Screening and identification of low temperature tolerant lactic acid bacterial epiphytes from oats on the Qinghai-Tibetan Plateau[J].Acta Prataculturae Sinica, 2022, 31(5):103-114.(in Chinese)
[15] 丁宁, 陆兆新, 别小妹, 等.谷物中具有抑制霉菌活性乳酸菌的分离筛选及鉴定[J].南京农业大学学报, 2021, 44(6):1187-1196. DING N, LU Z X, BIE X M, et al.Isolation and identification of antifungal lactic acid bacteria from different grains[J].Journal of Nanjing Agricultural University, 2021, 44(6):1187-1196.(in Chinese)
[16] GUO X H, KIM J M, NAM H M, et al.Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties[J].Anaerobe, 2010, 16(4):321-326.  
[17] GUO X H, LI D F, LU W Q, et al.Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs[J].Antonie Van Leeuwenhoek, 2006, 90(2):139-146.  
[18] MISHRA V, PRASAD D N.Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics[J].International Journal of Food Microbiology, 2005, 103(1):109-115.  
[19] NESS I F, DIEP D B, IKE Y.Enterococcal bacteriocins and antimicrobial proteins that contribute to niche control[M]//GILMORE M S, CLEWELL D B, IKE Y, et al.Enterococci:from commensals to leading causes of drug resistant infection.Boston:Massachusetts Eye and Ear Infirmary, 2014.
[20] GUPTA A, TIWARI S K.Probiotic potential of bacteriocin-producing Enterococcus hirae strain LD3 isolated from dosa batter[J].Annals of Microbiology, 2015, 65(4):2333-2342.  
[21] BIRRI D J, BREDE D A, FORBERG T, et al.Molecular and genetic characterization of a novel bacteriocin locus in Enterococcus avium isolates from infants[J].Applied and Environmental Microbiology, 2010, 76(2):483-492.  
[22] 余琴, 马现永, 邓盾, 等.海氏肠球菌IDO5对猪粪废水中吲哚降解条件优化及降解途径分析[J].生物技术通报, 2021, 37(12):113-123. YU Q, MA X Y, DENG D, et al.Optimization of indole-degrading conditions in pig manure waste water by Enteroccus hirae IDO5 and analysis of its corresponding degradation pathway[J].Biotechnology Bulletin, 2021, 37(12):113-123.(in Chinese)
[23] 董翎逸, 刘正奇, 裘梁, 等.屎肠球菌WEFA23和海氏肠球菌WEHI01 发酵荷叶上清的抑菌及抗氧化评价[J].南昌大学学报(理科版), 2018, 42(4):375-381. DONG L Y, LIU Z Q, QIU L, et al.Antibacterial and antioxidant evaluation of supernatant of the fermented lotus leaf by Enterococcus faecium WEFA23 and Enterococcus hirae WHEHI01[J].Journal of Nanchang University (Natural Science), 2018, 42(4):375-381.(in Chinese)
[24] 周文文, 高慧, 孙亚芳, 等.海氏肠球菌的特殊功能——解酒和抗衰老[C]//2017中国食品科学技术学会第十四届年会暨第九届中美食品业高层论坛论文摘要集.无锡:中国食品科学技术学会, 2017:346-347. ZHOU W W, GAO H, SUN Y F, et al.The special function of Enterococcus hirae-hangover and anti-aging[C]//2017 The 14th Annual Conference of China Food Science and Technology Society and the 9th China-Us Food Industry High-Level Forum Paper Abstract Collection.Wuxi:Chinese Institute of Food Science and Technology, 2017:346-347.(in Chinese)
[25] 胡颖, 杨琴, 甘盛力, 等.海氏肠球菌(Enterococcus hirae)发酵特性及应用[J].乳业科学与技术, 2012, 35(1):15-19. HU Y, YANG Q, GAN S L, et al.Fermentation characteristics of Enterococcus hirae and application in yogurt starter[J].Journal of Dairy Science and Technology, 2012, 35(1):15-19.(in Chinese)
[26] 张旭, 李祥, 曾祥伟.一株雀形目野鸟源海氏肠球菌的分离、鉴定[J].黑龙江畜牧兽医, 2015(18):170-172. ZHANG X, LI X, ZENG X W.Isolation and identification of a strain of Enterococcus hirae from wild birds[J].Heilongjiang Animal Science and Veterinary Medicine, 2015(18):170-172.(in Chinese)
[27] 王佳慧, 李禤, 贾丹, 等.牛源性益生菌的分离鉴定及抑菌性研究[C]//第十三届(2018)中国牛业发展大会论文集.晋中:中国畜牧业协会, 2018:225-232. WANG J H, LI X, JIA D, et al.Isolation, identification and antibacterial activity of bovine probiotics[C]//Proceedings of the 13th (2018) China Cattle Industry Development Conference.Jinzhong:China Animal Agriculture Association, 2018:225-232.(in Chinese)
[28] 石磊, 李禤, 李有全, 等.牦牛粪便中乳酸菌的分离与鉴定[J].中国兽医杂志, 2019, 55(8):21-23, 130. SHI L, LI X, LI Y Q, et al.Isolation and identification of Lactobacillus from yak dung in Gonghe county, Qinghai province[J].Chinese Journal of Veterinary Medicine, 2019, 55(8):21-23, 130.(in Chinese)
[29] CHIU C C, LIN T C, WU R X, et al.Etiologies of community-onset urinary tract infections requiring hospitalization and antimicrobial susceptibilities of causative microorganisms[J].Journal of Microbiology, Immunology, and Infection, 2017, 50(6):879-885.  
[30] 张芬.婴儿源肠球菌的安全性评价及其体内外降胆固醇的益生功能和机制探究[D].硕士学位论文.南昌:南昌大学, 2017. ZHANG F.Safety evaluation of enterococci from healthy Chinese infants as well as their cholesterol-lowering effect in vitro and in vivo[D].Master's Thesis.Nanchang:Nanchang University, 2017.(in Chinese)
[31] 张浩.年龄和腹泻因素对波尔山羊肠道菌群的影响及肠道益生菌的筛选[D].硕士学位论文.泰安:山东农业大学, 2018. ZHANG H.Effects of age and diarrhea on intestinal flora of Boer goats and screening of intestinal probiotics[D].Master's Thesis.Tai'an:Shandong Agricultural University, 2018.(in Chinese)
[32] ZHANG H, SHAO M X, HUANG H, et al.The dynamic distribution of small-tail Han sheep microbiota across different intestinal segments[J].Frontiers in Microbiology, 2018, 9:32.
[33] 贺曦, 徐淑琴, 马祥兆, 等.藏羊源肠道海氏肠球菌的分离鉴定及益生特性研究[J].黑龙江畜牧兽医, 2021(22):63-68, 150. HE X, XU S Q, MA X Z, et al.Isolation and identification of Enterococcus hirae from intestine of Tibetan sheep and its probiotic properties[J].Heilongjiang Animal Science and Veterinary Medicine, 2021(22):63-68, 150.(in Chinese)
[34] AROKIYARAJ S, HAIRUL ISLAM V I, BHARANIDHARAN R, et al.Antibacterial, anti-inflammatory and probiotic potential of Enterococcus hirae isolated from the rumen of Bos primigenius[J].World Journal of Microbiology and Biotechnology, 2014, 30(7):2111-2118.  
[35] VALLEJO M, PARADA R B, MARGUET E R.Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis)[J].Revista Argentina de Microbiología, 2020, 52(2):136-144.  
[36] 朱惠.牦牛源海氏肠球菌Swun3株对小鼠生长性能和肠黏膜免疫的影响[D].硕士学位论文.成都:西南民族大学, 2016. ZHU H.Effect of Enterococcus hirae Swun3 isolated from yak on the growth performance and intestinal mucosal immunity in mice[D].Master's Thesis.Chengdu:Southwest Minzu University, 2016.(in Chinese)
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