BF25-IN VITRO AND IN SILICO STUDY OF PREBIOTIC OLIGOSACHARIDES FERMENTATION PATHWAY BY A PROBIOTIC Limosilactobacillus reuteri STRAIN

GOMEZ, JORGE; LEDESMA, ANA E.; TARANTO, MARÍA PÍA; BUSTOS, ANA Y.

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Research on the ecology of the intestinal microbiota, as well as the commercial application ofprebiotics, has awakened the interest of the scientific community in the metabolic pathways of nondigestible oligosaccharides. Although oligosaccharide metabolism is essential for the ecological fitness of lactic acid bacteria, few data are currently available.The objective of the present work was to evaluate the ability of the probiotic strain Limosilactobacillus (L.) reuteri to ferment commercial oligosaccharides using in vivo assays, bioinformatic analysis and computational studies.For this purpose, the commercial prebiotics lactulose and commercial insulins of different degree of polymerization (DP) and purity (Orafti® GR, HP, HSI and Raftilose) were added to the MRS broth as the sole carbon source. The cultures were incubated for 24 h at 37°C and growth was evaluated by plate count. In addition, the metabolic pathways of prebiotic oligosaccharides in L. reuteri CRL 1098 were evaluated by bioinformatics analysis. Data were obtained from NCBI GenBank and Blast and MUSCLE algorithms were used to analyze and align the sequences. Finally, the 3D structures of the main enzymes involved in the metabolic pathways were obtained by homology modeling usingAlphaFold Colab. The results show that the strain was not able to grow in medium with pure inulin (HP) as the sole carbon source. However, in the presence of lower purity and low GP insulins, growth of 1.5 Log CFU/mL on average was observed. Notably, in the presence of lactulose, growths of 2.4 Log CFU/mL were observed, comparable to that of the glucose control. Our bioinformatics search indicates that strain CRL 1098 lacks endo- or exoinulinase enzymes, responsible for inulin hydrolysis, as well as transport systems described in other related genera and species. On the other hand, the LacLM system responsible of β-galacto-oligosaccharides such as lactulose metabolism was identified in the genome of L. reuteri CRL 1098. The enzyme consists of heterodimers encoded by two partially overlapping chromosomal genes, LacL (long subunit), where the active site is located, and LacM (small subunit). Remarkably, two LacL encoding proteins of 672 and 628 amino acids were found in CRL 1098 genome, (genBank: OAV47989.1, 672, OAV47785.1, 628 , respectively) and one lacM encoding a 319 aa protein (genBank: OAV47784.1). The output of the local alignment performed with LacL subunit shows repetitive patterns where residues such as ASP197, GLU303, GLU460 and GLU546 were partially conserved. Finally, the 3D structures of the beta-galactosidase enzymes were modeled and the secondary structures were calculated. A high percentage of disordered structure was observed (range of 47-53 %), followed by alpha helix (21-35 %), beta sheets (11-21 %) and turn (δ) (5-7 %). These results allow us to further study the metabolism of prebiotics by probiotic strains and represent the basis for the design of symbiotic foods.