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With respect to the food safety and quality, it is necessary to isolate and apply protective microbes for food preservation. Lactic acid bacteria can produce some antimicrobial substances by metabolism, which are related with biological preservation of foods. Lactobacillus brevis P-319 is a strain isolated from sauerkraut. Antimicrobial activity of Lactobacillus brevis P-319 cell-free broth was studied under some conditions of food processing and preservation. Incubating the cell-free broth in the presence of trypsin and papain resulted in loss of antimicrobial activity of the cell-free broth, which demonstrated the substance with antimicrobial activity might have the molecular structure of protein or peptide. The cell-free broth kept at 100°C or at pH 2.0~6.0 for 2 h or under natural conditions for four weeks still displayed antimicrobial activity. No loss in antimicrobial activity of the cell-free broth was observed after treatment with ethanol below 8% and NaCl below 6%, but significant reduction after incubation in the presence of 8% NaCl. Lactobacillus brevis P-319 can be considered to have commercial potential, applied perspectives for food manufacture and chemical preservatives in foods.