Probiotic potential of Lactobacillus helveticus CMI1 isolated from Iranian Khiki cheese: antimicrobial, anti-biofilm, and antioxidant properties for food preservatives

BACKGROUND AND OBJECTIVE: Lactic acid bacteria (LAB) are widely recognized for their probiotic and antimicrobial potential in food and health-related applications. This study aimed to isolate and characterize Lactobacillus helveticus strain CMI1 from traditional Iranian Khiki cheese and to evaluate its probiotic-related properties, including antimicrobial, anti-biofilm, antioxidant, and cytotoxic activities. METHODS: The isolate was identified by 16S rRNA gene sequencing. Antimicrobial activity of acidic and neutralized cell-free supernatants (CFS) was evaluated against major Gram-positive and Gram-negative foodborne pathogens using disk diffusion, well diffusion, modified double-layer, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. Anti-biofilm activity against Staphylococcus aureus was assessed using crystal violet staining and quantitative real-time PCR analysis of biofilm-related genes. Antioxidant activity was determined using DPPH, ABTS, and β-carotene/linoleic acid assays. Cytotoxic effects were evaluated against HT-29, HeLa, and MCF-7 cancer cell lines using the MTT assay. Antibiotic susceptibility was also examined. RESULTS: L. helveticus CMI1 exhibited strong antimicrobial activity, with higher efficacy observed for acidic CFS compared to neutralized CFS, particularly against S. aureus. The strain significantly inhibited S. aureus biofilm formation and downregulated key biofilm- and virulence-related genes (mecA, agr, spa, icaA, and hla). Additionally, CMI1 demonstrated notable antioxidant activity and dose-dependent cytotoxic effects against cancer cell lines, while remaining sensitive to clinically relevant antibiotics. CONCLUSION: These findings indicate that L. helveticus CMI1 possesses promising probiotic and functional properties, supporting its potential application in food preservation and functional food development.