Deciphering the effects of co-culturing on the metabolite profiles of Ligilactobacillus reuteri ZJ625 and Limosilactobacillus salivarius ZJ614 using untargeted metabolomics

Abstract Ligilactobacillus reuteri ZJ625 (ZJ625) and Limosilactobacillus salivarius ZJ614 (ZJ614) are important lactic acid bacteria with the potential for use as probiotics. To better understand the mechanisms of cell-to-cell interactions between ZJ625 and ZJ614 and elucidate their probiotics potentials, we studied the effects of co-culturing in defined media on the metabolomes. The strains were cultured singly (L. reuteri ZJ625 or L. salivarius ZJ614) and in co-cultures (L. reuteri ZJ625 plus L. salivarius ZJ614) and harvested at the mid-exponential phase. Single-phase extraction method was used to extract the metabolites from pre-weighed cells for the two-dimensional gas-column time-of-flight-mass spectrometry (GCxGC- TOFMS) analysis. The data was obtained using a Leco corporation ChromaTOF software and exported for statistical, pathway enrichment, and impact analyses using MetaboAnalyst 5.0. Principal component analysis (PCA) indicated an overlap of the samples from the two batches, indicating no significant batch effect. The data was then analyzed using the group allocations. This study revealed 1388 metabolites from L. reuteri ZJ625 and L. salivarius ZJ614 single and co-cultures in defined media, of which 18.87% were significantly expressed (p < 0.05). Of the significantly expressed metabolites, 28% DEMs were identified (p < 0.05, VIP > 1), of which 51.3% were annotated while 48.6% are currently not annotated in the databases. The whole metabolome of the strains in single and co-cultures revealed the expression of several important metabolites, including arabinofuranose, methyl-galactoside, N-acetyl-glutamic acid, phosphoric acid, decanoic acid, metyrosine, and deoxypentafuranose which play roles in different metabolic and biosynthetic pathways, hence confirming the probiotic potential as single- or multi-strain cultures. The co-culturing of the strains influenced the metabolites profiles of the strains showing additive and regulatory differential metabolites expressions. Pathway enrichment and impact analyses revealed the association of the differentially expressed metabolites with functional effects such as antihypertensive, antimicrobial, and microbiome stimulation.