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

GOMEZ, J. N; LEDESMA, ANA ESTELA; TARANTO, MARIA PIA; BUSTOS, ANA YANINA

Los Cocos, Córdoba

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Sociedad Argentina de Microbiología General (SAMIGE)

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 fitnessof 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 andcomputational studies.For this purpose, the commercial prebiotics lactulose and commercial insulinsof different degree of polymerization (DP) and purity (Orafti® GR, HP, HSI and Raftilose) were addedto the MRS broth as the sole carbon source. The cultures were incubated for 24 h at 37°C and growthwas 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 GenBankand Blast and MUSCLE algorithms were used to analyze and align the sequences. Finally, the 3Dstructures of the main enzymes involved in the metabolic pathways were obtained by homologymodeling usingAlphaFold Colab. The results show that the strain was not able to grow in medium withpure 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. Ourbioinformatics search indicates that strain CRL 1098 lacks endo- or exoinulinase enzymes, responsiblefor inulin hydrolysis, as well as transport systems described in other related genera and species. Onthe other hand, the LacLM system responsible of β-galacto-oligosaccharides such as lactulosemetabolism was identified in the genome of L. reuteri CRL 1098. The enzyme consists of heterodimersencoded by two partially overlapping chromosomal genes, LacL (long subunit), where the active siteis located, and LacM (small subunit). Remarkably, two LacL encoding proteins of 672 and 628 aminoacids 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 alignmentperformed 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-galactosidaseenzymes were modeled and the secondary structures were calculated. A high percentage ofdisordered 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 byprobiotic strains and represent the basis for the design of symbiotic foods.