宋朋杰 武小虎 王东升 杨洁 李亚娟 蒋威 沈文祥 邵丹 严作廷

摘要：【目的】評价健康奶牛生殖道分离乳酸菌益生特性，发掘其作为益生菌制剂细菌成分的可能性，为开发防治奶牛子宫内膜炎的微生态制剂提供科学依据。【方法】以直肠把握法无菌采集产后40～60 d健康奶牛子宫分泌物，采用MRS培养基在厌氧条件（氧气浓度<1.2%，37 ℃）下分离乳酸菌，通过扩增16S rDNA序列进行鉴定，并进行抑菌活性、耐药性、黏附子宫内膜上皮细胞特性及安全性评估，分析其开发成益生菌制剂的可能性。【结果】经16S rDNA测序分析共获得8株乳酸菌，分别为植物乳杆菌、副干酪乳杆菌、蜡样芽孢杆菌、鼠李糖杆菌、蒙氏肠球菌、德氏乳杆菌、戊糖乳杆菌和嗜热链球菌，根据预试验结果选取其中3株（植物乳杆菌、副干酪乳杆菌和鼠李糖杆菌）进行益生特性评价。3株奶牛生殖道乳酸菌培养上清液对大肠杆菌、无乳链球菌及金黄色葡萄球菌均表现出良好的抑制效果，约在接种 3 h后进入典型的指数生长期，生长至平台期后植物乳杆菌OD620 nm为1.375、鼠李糖杆菌OD620 nm为1.314、副干酪乳杆菌OD620 nm为1.250。副干酪乳杆菌的产酸能力最强，植物乳杆菌和鼠李糖杆菌的产酸能力几乎相同，但均不产H2O2。3株奶牛生殖道乳酸菌对青霉素G、氯霉素、阿莫西林、氨苄西林、头孢噻肟、头孢唑林和四环素等7种抗菌药物高度敏感;能同时检测到磺胺类耐药基因sul2，在副干酪乳杆菌和鼠李糖杆菌中还同时检测到氨基糖苷类耐药基因aphA-2和β-内酰胺类耐药基因blaIMP。副干酪乳杆菌能很好地黏附于奶牛子宫内膜上皮细胞，且黏附效率与副干酪乳杆菌浓度呈正相关。3株奶牛生殖道乳酸菌的混合菌液经腹腔注射小鼠后未引起脏器病变，且对小鼠的行为活动、采食饮水亦无明显影响。【结论】从奶牛生殖道分离获得的植物乳杆菌、副干酪乳杆菌和鼠李糖杆菌可作为潜在的益生菌，用于开发防治奶牛子宫内膜炎的微生态制剂。

关键词： 奶牛;生殖道;乳酸菌;子宫内膜炎;益生特性;微生态制剂

中图分类号： S852.6                          文献标志码： A 文章编号：2095-1191（2020）09-2278-09

Probiotic evaluation of three strains of lactic acid

bacteria isolated from cow genital tract

SONG Peng-jie1， WU Xiao-hu1*， WANG Dong-sheng1， YANG Jie1， LI Ya-juan1，2，

JIANG Wei1， SHEN Wen-xiang1， SHAO Dan1， YAN Zuo-ting1*

（1Lanzhou Institude of Husbandry and Pharmaceutical Sciences， Chinese Academic of Agricultural Sciences/Key Laboratory of Veterinary Pharmaceutical Development of Ministry of Agriculture and Rural Affairs/Engineering & Technology Research Center of  Traditional Chinese Veterinary Medicine of Gansu Province， Chinese Academic of Agricultural Sciences， Lanzhou  730050， China; 2College of Animal Science and Technology，

Guangxi University， Nanning  530004， China）

Abstract：【Objective】To evaluate the probiotic characteristics of lactic acid bacteria isolated from the reproductive tract of dairy cows，  explore its potential to be bacterial component to provide raw materials for microecological agents， and offer theoretical basis for the development of microecological agents for prevention and treatment of endometritis in cows. 【Method】The vaginal secretions of healthy dairy cows from 40 to 60 d after parturition were collected aseptically by rectal palpation method. Lactic acid bacteria were isolated by MRS medium under anaerobic conditions（oxygen concentration <1.2%， 37 ℃）. The antimicrobial activity， drug resistance， adhesion to endometrial epithelial cells and safety were evaluated to analyze the possibility of its development as probiotics by 16S rDNA sequence amplification. 【Result】A total of 8 lactic acid bacteria were obtained by 16S rDNA sequencing， including Lactobacillus plantarum， L. paracasei， Bacillus cereus， Rhamnose lactobacillus， Enterococcus mongholicus， L. delbrueckii， L. pentosus and Streptococcus thermophilus. According to the preliminary experimental results， three of them（L. plantarum， L. paracasei and Rhamnose lactobacillus） were selected for probiotic evaluation. About 3 h after inoculation， all three strains entered the typical exponential growth phase，and the supernatant of genital tractlactic acid bacteria showed fine inhibition to Escherichia coli，  Streptococcus agalactiae and Staphylococcus aureus， and the OD620nm of L. plantarum was 1.375， R. lactobacillus was 1.314， L. paracasei was 1.250 at platform stage. L. paracasei had the strongest acid producing ability， while L. plantarum and R. lactobacillus had almost the same acid production capacity， but they did not produce H2O2. Three strains of genital tract lactic acid bacteria were sensitive to 7 antibiotics such as penicillin G， chloramphenicol， amoxicillin， ampicillin， cefotaxime， cefazolin and tetracycline， and sulfa resistance genes sul2 were detected. In L. paracasei and L. rhamnosus， amino glycoside  resistance gene aphA-2 and β-lactam resistance gene blaIMP were detected. L. paracasei could adhere to epithelial cells of genital tract， and adhesion rate was positively correlated with L. paracasei concentration. The mixed bacteria liquid of the three strains of lactic acid bacteria isolated from the reproductive tract of cows were injected into rat， and did not cause visceral diseases， and had no obvious effects on behavior and ingestion of rat. 【Conclusion】L. plantarum，L. paracasei and L. rhamnosus have the potential as probiotics to further develop lactic acid bacteria microecological preparations.

Key words： cows; genital tract; lactic acid bacteria; endometritis; probiotic characters; microecological preparations

Foundation item：National Key Research and Development Program（2017YFD0502201）; Central Public-interest Scientific Institution Basal Research Fund（1610322018003）; Lanzhou Science and Technology Planning Project（2018-1-100）

0 引言

【研究意义】子宫内膜炎作为奶牛产后的一种多发病，是导致奶牛不孕、受孕率低下及空怀期延长的主要病因。据统计，在不孕奶牛中患子宫内膜炎的比例高达95%（Zobel，2013;马帅等，2018;邵丹等，2019）。患子宫内膜炎的奶牛因其子宫局部存在炎症，而阻碍精子运行及受精卵着床，严重影响生殖激素水平，致使其繁殖效率明显降低，给整个奶牛业带来巨大经济损失。益生菌（Probiotics）是指适当摄入后对宿主产生各种有益影响的可传代培养微生物（Prado et al.，2015;杨虹等，2019），具有抵抗机体各种不利条件（唾液酶、低pH、胰液和胆汁等）的能力，且能在胃肠道黏膜、呼吸道黏膜、乳腺上皮及子宫内膜上皮等部位定殖而发挥生物学功能，有利于宿主微生物环境的调节（Zoumpopoulou et al.，2017）。乳酸菌是最常见的一种益生菌，具有绿色安全的特点，在维持奶牛胃肠道菌群平衡上已得到广泛应用。Peter等（2018）研究表明，将布氏乳杆菌投入患子宫内膜炎奶牛子宫中能显著降低IL-1β和IL-8等炎性细胞因子的释放，对奶牛子宫内膜炎症反应具有明显抑制作用。……