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L-citrulline-producing bacterium BH-01 was isolated from raw buffalo milk. The isolate was tested for probiotic activities such as tolerance to simulated gastric and intestinal juices, antagonistic activity against six antibiotic-resistant bacteria, and temperature tolerance. L-citrulline production and arginine deiminase (ADI) activity were optimized using statistical designs. The bacterial isolate was molecularly identified as <jats:italic>Bacillus subtilis<\/jats:italic> strain AUMC B-498 (accession number PP574248.1). The strain exhibited resistance at pH 2.0 and bile salt 0.5% for a two-hour exposure period. It could inhibit the growth of <jats:italic>Escherichia coli<\/jats:italic>, <jats:italic>Klebsiella pneumonia<\/jats:italic>, <jats:italic>Serratia<\/jats:italic> sp., <jats:italic>Staphylococcus aureus<\/jats:italic>, methicillin<jats:italic>-<\/jats:italic>resistant <jats:italic>Staphylococcus aureus<\/jats:italic> (MRSA), and <jats:italic>Streptococcus pneumoniae.<\/jats:italic> From the results of statistical optimization, the Plackett-Burman design identified temperature, L-arginine, incubation period, and peptone as the most effective factors among the eight selected variables. Based on these, the Box-Behnken design was used to optimize the factors required to maximize citrulline production. The maximum L-citrulline was 632.5\u00a0\u00b5g\/L, and ADI activity was 1.42 U\/mL. 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