Control of single and multi-species biofilms in food processing by environmental bacteria metabolites

Biofilm formation by food spoilage bacteria on surfaces in food processing environments remains a major challenge in food safety due to increased resistance to conventional disinfectants. Food is susceptible to spoilage or deterioration caused by the growth of food-spoilage pathogenic bacteria such as Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens. These contaminants can affect food quality, pose a significant risk of food poisoning, and cause significant economic losses. These pathogenic bacteria can form biofilms and show resistance to antimicrobial compounds. Traditional approach to handling food spoilage usually using chemical preservative or physical approach. But this approach may not be sufficient to control the biofilm produced by the bacteria; it also may have side effects for human health. Naturally, multispecies biofilm formation is more commonly observed in natural environments. Therefore, this study aims to evaluate the potential of bacterial metabolites extracted from environmental sources to inhibit and destroy single-species and multi-species biofilms by identifying their inhibition and disruption times. This study evaluated how environmental metabolites affect biofilm. The potential of metabolites produced by environmental Enterobacter sp. isolates B10 and J70 against single and multispecies biofilms of Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens. Metabolites were extracted using ethyl acetate and tested for biofilm inhibition and disruption using a crystal violet assay. Chemical profiling was performed by GC-MS and LC-MS, toxicity was assessed using the Brine Shrimp Lethality Assay (BSLA), and biofilm morphology was observed by scanning electron microscopy. Both extracts exhibited significant antibiofilm activity. The J70 extract achieved the highest disruption of B. cereus biofilm (≈ 60%), while multispecies B. cereus & others biofilms were disrupted by 26-60%. Meanwhile the B10 achieved the highest disruption of B. cereus biofilm (≈ 55%), while multispecies biofilms were disrupted by 24-63%. GC-MS and LC-MS analyses revealed unsaturated fatty acids, cyclic dipeptides, and phenolic-related compounds as putative active metabolites. Toxicity evaluation showed LC₅₀ values > 1000 ppm, indicating low acute toxicity. These findings demonstrate that metabolites from environmental bacteria represent a promising natural and safe alternative for controlling single- and multispecies biofilms associated with food spoilage and for supporting improved food safety management.