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