奶牛场明串珠菌的分离鉴定及其生物学特性分析

doi:10.16431/j.cnki.1671-7236.2025.06.039

摘要/Abstract

摘要： 【目的】从奶牛场环境和牛体样本中分离明串珠菌(Leuconostoc)并深入探究其生物学特性，为防治奶牛乳房炎提供益生菌菌株。【方法】通过菌体形态学观察及分子生物学手段对分离菌进行菌种鉴定，通过双层平板法分析菌株对奶牛乳房炎常见病原菌的抗菌活性，并分析其主要抑菌物质成分，建立生长曲线及产酸曲线，探究其消化道环境耐受特性，并通过药敏试验和溶血试验评估菌株的安全性。【结果】分离得到3株益生菌，在MRS琼脂培养基上均呈乳白色单菌落，镜检为革兰阳性球菌,分别命名为11、14和18号菌株。16S rDNA测序结果显示，11和14号菌株与NCBI登录的肠膜明串珠菌相似性均在99%以上，18号菌株与乳明串珠菌相似性在99%以上，确定3株分离株均为明串珠菌。3株明串珠菌对大肠杆菌和肺炎克雷伯菌的抑菌圈直径均＞19 mm，具有良好的抑菌活性。3株明串珠菌对大肠杆菌的抑菌物质主要包括有机酸、细菌素和过氧化氢；而11、14号菌株对肺炎克雷伯菌的主要抗菌物质包括有机酸和过氧化氢，18号菌株主要包括有机酸。生长曲线显示，3株明串珠菌生长规律基本一致，其中0～2 h为迟缓期，2～12 h为对数期，12 h后为稳定期，菌液产酸pH均稳定为4.0左右,具有较好的产酸性能。3株明串珠菌在人工胃液、人工肠液中培养4 h后的存活率均在56%以上，胆盐浓度为0.3%时，3株明串珠菌存活率均在26%以上，说明菌株具有良好的耐受性。药敏试验结果显示，3株明串珠菌对红霉素、米诺霉素、妥布霉素等高度敏感；对青霉素、氨苄西林和万古霉素耐受；对头孢呋辛、头孢曲松、左氧氟沙星等中度敏感。3株明串珠菌均不具有溶血性。【结论】本研究分离得到的3株明串珠菌均具有较好的抑菌特性和安全性，为后续开展益生菌防治细菌性奶牛乳房炎提供了研究基础。

关键词: 奶牛乳房炎; 明串珠菌; 抑菌活性

Abstract: 【Objective】 This study was conducted to isolate Leuconostoc from dairy farm environment and bovine body samples and further explore its biological characteristics,so as to provide probiotic strains for the future prevention and treatment of dairy mastitis.【Method】 The isolates were identified by means of morphological observation and molecular biology,and the antibacterial activity of the isolates against common pathogens of dairy cow mastitis was analyzed by double-layer plate method,the main bacteriostatic substances were analyzed.Growth and acid production curves were established.The digestive tract environmental tolerance characteristics were investigated,and the safety of the strain was evaluated by drug sensitivity test and hemolysis test.【Result】 Three strains of probiotics were isolated.The three isolated probiotics showed milky white single colonies on MRS agar medium,and were identified as Gram-positive cocci by microscopy.They were named as strains 11,14 and 18,respectively.16S rDNA sequencing revealed that the strains 11 and 14 shared over 99% similarity with Leuconostoc mesenteroides identified by NCBI,while strain 18 exhibited over 99% similarity with Leuconostoc lactis.Consequently,the three isolates were classified as Leuconostoc.The inhibitory zone diameter of the three strains against Escherichia coli and Klebsiella pneumoniae was greater than 19 mm,which showed good inhibitory activity.The antibacterial substances produced by the three Leuconostoc strains against Escherichia coli mainly consist of organic acids,bacteriocins and hydrogen peroxide.For Klebsiella pneumoniae,the primary antibacterial substances of strains 11 and 14 included organic acids and hydrogen peroxide,while strain 18 mainly produced organic acids.The growth curve showed that the growth patterns of the three Leuconostoc strains were basically consistent,with 0 to 2 h being the delayed phase,2 to 12 h being the logarithmic phase,and after 12 h being the stable phase.The pH of the bacterial solution was stable at about 4.0,showing good acid production ability.The survival rate of the three Leuconostoc strains was above 56% after 4 h culture in artificial gastric juice and artificial intestinal juice.When the bile salt concentration was 0.3%,the survival rates of three Leuconostoc strains were all more than 26%,indicating that the strains had good tolerance.The results of the drug sensitivity test showed that three strains of Leuconostoc were highly sensitive to erythromycin,minomycin and tobramycin,resistant to penicillin,ampicillin and vancomycin,and moderately sensitive to cefuroxime,ceftriaxone and levofloxacin.And all of the three strains of Leuconostoc did not exhibit hemolytic activity.【Conclusion】 The three isolated Leuconostoc strains exhibited favorable antibacterial properties and safety profiles,laying a solid foundation for future research on probiotics for the prevention and treatment of bacterial bovine mastitis.

Key words: bovine mastitis; Leuconostoc; antibacterial activity

中图分类号:

仝静迪, 李霄韩, 任美奕, 宋德源, 赵岩, 宋金尚, 苏娅澜, 温向福, 程佳, 刘明超. 奶牛场明串珠菌的分离鉴定及其生物学特性分析[J]. 中国畜牧兽医, 2025, 52(6): 2874-2883.

TONG Jingdi, LI Xiaohan, REN Meiyi, SONG Deyuan, ZHAO Yan, SONG Jinshang, SU Yalan, WEN Xiangfu, CHENG Jia, LIU Mingchao. Isolation and Identification of Leuconostoc from Dairy Farms and Analysis of Their Biological Characteristics[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2874-2883.

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