Derived Postbiotics of a Multi‐strain Probiotic Formula Clinically Validated for the Treatment of Irritable Bowel Syndrome

Introduction The pathophysiology of Irritable Bowel Syndrome (IBS) involves increased intestinal permeability, micro-inflammation and gut dysbiosis with associated visceral hypersensitivity. The probiotic mix of Lactobacillus plantarum strains CECT7484 (KABP022) and CECT7485 (KABP023) and Pediococcus acidilactici CECT7483 (KABP021) has shown positive clinical outcomes in IBS. However, their exact mechanisms of action have not been elucidated. Postbiotics are probiotic-derived metabolites or cell components that can improve host health. In this work we sought to characterize said strains about the production of specific postbiotics known to influence gut permeability and inflammation, as well as their inhibitory activity against bacteria known to be increased in IBS. Methods We measured the capacity to biosynthesize acetate from 3 carbon sources (glucose, xylose and arabinose) by colorimetric assay, acetylcholine (ACh) by HPLC-ESI-MS/MS and polyphosphate (polyP) granules by fluorescence, using Lactobacillus rhamnosus ATCC53103 as control. Inhibitory activity was tested on 6 IBS-associated bacteria (2 enterobacteria, 2 Actinomyces spp. and 2 Streptococcus spp. strains). Supernatants from co-cultures of probiotic and target bacteria were obtained and adjusted to pH 6 to only detect organic-acid independent inhibition. Target bacteria were cultured on the supernatants for 16 h. Genomes were sequenced by Hiseq 2500–3000 and key genes involved in the biosynthesis of these postbiotics were searched. Additionally, susceptibility of the 3 strains to 8 antibiotics were determined according to ISO10932. Results Results are summarized in Table 1. Production of acetate was dependent on carbon source. The production from glucose was low (< 1 mM). The fermentation of xylose increased the acetate production by all the strains (3 mM approx.) while arabinose yielded the greatest concentrations (up to 13.5 mM for L. plantarum CECT7484). L. plantarum strains synthesized the largest amounts of ACh (12.5 mg/L for CECT7484 and 13.8 mg/L for CECT7485) whereas P. acidilactici CECT7483 produced 1.9 mg/L and control strain produced none. After 6 h of growth, accumulation of PolyP granules was observed for the L. plantarum and control strains but not for Pediococcus. The 3 strains produced antimicrobials against all 6 target bacteria. Consistent with phenotypic analysis, in silico study confirmed the presence of phosphofructokinase (pfk) gene (responsible for acetate synthesis) in the 3 genomes and polyphosphate kinase (ppk) gene (polyP synthesis) in the 2 L. plantarum genomes. Bacteriocin (antimicrobial compound) genes were found in the genomes of L. plantarum CECT7484 and CECT7485. Moreover, the 3 strains were susceptible to all the antibiotics tested. Conclusion The probiotic strains were proven to produce postbiotic molecules including acetate, polyP, ACh and antimicrobial compounds (potentially bacteriocins) against IBS-associated microorganisms. This unique combination of postbiotics could explain the clinically beneficial effects of the probiotic mix in IBS subjects. Support or Funding Information AB-Biotics -KANEKA Table 1. Postbiotics produced by the 3 probiotic strains and the control strain. Acetate production was tested from three carbon sources. Production of antimicrobials was determined by testing the inhibition of growth of 6 IBS-related bacteria. ACh, acetylcholine; PolyP, polyphosphate (−/+, traces; +, production). Strains Acetate ACh PolyP No. bacteria inhibited Glucose Xylose Arabinose L. plantarum CECT7484 (KABP022) −/+ ++ ++++ ++++ +++ 6 out of 6 L. plantarum CECT7485 (KABP023) −/+ ++ +++ ++++ ++ 6 out of 6 P. acidilactici CECT7483 (KABP021) −/+ ++ +++ + − 6 out of 6 L. rhamnosus ATCC53103 (Control) −/+ ++ + − + NA