Interspecies interactions promote dual-species biofilm formation by Lactiplantibacillus plantarum and Limosilactobacillus fermentum: Phenotypic and metabolomic insights

Probiotics are live microorganisms offering various health benefits to hosts, but exposure to adverse conditions can compromise their viability during gastrointestinal transit. Probiotics in the biofilm state have been proven as an alternative way to the probiotic survival challenge; however, knowledge of mixed-species biofilms by probiotics is limited. This study aimed to examine the ecological interactions between Lactiplantibacillus plantarum LP-52 and Limosilactobacillus fermentum LF-56 from a phenotypic and metabolomics perspective during their mixed-species biofilm development. In specific, we investigated how their interaction changes bacterial growth, biofilm-forming capacity, biofilm structure, biofilm metabolic activity, EPS production, and biofilm tolerance under gastrointestinal conditions. Moreover, a comprehensive metabolomics analysis was conducted to identify different metabolic profiles and elucidate the underlying mechanisms during the development of mixed-species biofilm. Results showed that their cooperative interaction significantly promoted the planktonic cell growth of L. fermentum LF-56 and L. plantarum LP-52 during their co-cultivation. The synergistic effect also markedly improved the biofilm formation, with increased cell counts in biofilms and higher metabolic activity when compared to each single-species biofilm. Confocal laser scanning microscopy imaging showed denser and more diverse structures of mixed-species biofilm with higher coverage and thickness. In addition, dual-species biofilms were best tolerated under simulated gastric and intestinal conditions. Untargeted metabolomics assay identified 852 differential metabolites, primarily associated with seven pathways: two pathways of nucleotide metabolism (purine metabolism, pyrimidine metabolism), two pathways of carbohydrate metabolism (TCA cycle, glycolysis), alanine, aspartate, and glutamate metabolism, riboflavin metabolism, and ABC transporters, which an enhanced energy metabolism, stress adaptation, and potential biofunctional benefits. With this respect, this investigation underscores the benefits of mixed probiotics biofilms and contributes to further application of probiotics in the food and biotechnology industry.