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Background and Objectives: Probiotic bacteria is added directly to food components and it has beneficial effect on function and the health of organisms. The bifidogenic factors enter the colon where they contribute to an increase lactic acid bacteria population including Lactobacilli and Bifidobacteria and they inhibit enteric pathogenic bacterial growth. The aim of this study is to investigate the effect of culture medium on metabolic and antibacterial of probiotic bacteria.Methods: In this study, the probiotics bacterial and intestine pathogenic are to be used. Lactobacilli and Bifidobacterium were identified by plating samples on MRS medium, Gram Staining and standard biochemical methods. The effect of antagonistic probiotics was investigated in the presence of growth factor in the method well diffusion Ager on the Shigella flexneri (PTCC 1234), Escherichia coli (PTCC 1552), Salmonella typhi ( PTCC 1609) and the culture medium pH was measured.Results: The probiotics bacterial growth in MRS and lactose1%, sorbitol, raffinose, riboflavin were shown the effect antibacterial. The results of the study show the most antagonistic activity in commercial strain Lactobacillus acidophilus on Shigella flexneri and lower activity was in Lactobacillus casei (PTCC 1608), and Salmonella typhimurium (PTCC 1609), and also in Bbifidobacterium bifidum, it showed the most decrease pH value.Conclusion: According to the result of the study, adding growth factors to MRS medium base and lactose 1%, probiotic growth was increased and which also increased antagonistic activity.