Exopolysaccharides From Lactobacillus paracasei Isolated From Kefir as Potential Bioactive Compounds for Microbiota Modulation

Abstract

Microbiota coexists in true symbiosis with the host playing pivotal roles as a key element for well-being and health. Exopolysaccharides from lactic acid bacteria are an alternative as novel potential prebiotics that increase microbiota diversity. Considering this, the aim of the present work was to evaluate the capacity of the EPS produced by two L. paracasei strains isolated from kefir grains, to be metabolized in vitro by fecal microbiota producing short chain fatty acids. For this purpose, fecal samples from healthy children were inoculated in a basal medium with EPS and incubated in anaerobiosis at 37°C for 24, 48, and 72 h. DGGE profiles and the production of SCFA after fermentation were analyzed. Additionally, three selected samples were sequenced by mass sequencing analysis using Ion Torrent PGM. EPS produced by L. paracasei CIDCA 8339 (EPS₈₃₃₉) and CIDCA 83124 (EPS₈₃₁₂₄) are metabolized by fecal microbiota producing a significant increase in SCFA. EPS₈₃₃₉ fermentation led to an increment of propionate and butyrate, while fermentation of EPS₈₃₁₂₄ increased butyrate levels. Both EPS led to a profile of SCFA different from the ones obtained with inulin or glucose fermentation. DGGE profiles of 72 h fermentation demonstrated that both EPS showed a different band profile when compared to the controls; EPS profiles grouped in a cluster that have only 65% similarity with glucose or inulin profiles. Mass sequencing analysis demonstrated that the fermentation of EPS₈₃₃₉ leads to an increase in the proportion of the genera Victivallis, Acidaminococcus and Comamonas and a significant drop in the proportion of enterobacteria. In the same direction, the fermentation of EPS₈₃₁₂₄ also resulted in a marked reduction of Enterobacteriaceae with a significant increase in the genus Comamonas. It was observed that the changes in fecal microbiota and SCFA profile exerted by both polymers are different probably due to differences in their structural characteristics. It can be concluded that EPS synthesized by both L. paracasei strains, could be potentially used as bioactive compound that modify the microbiota increasing the production of propionic and butyric acid, two metabolites highly associated with beneficial effects both at the gastrointestinal and extra-intestinal level.

Keywords: exopolysaccharide; lactic acid bacteria; microbiota; prebiotics; probiotics; short chain fatty acids.

FIGURE 1

PCR-based DGGE profiles (A) and the respective dendrograms (B) of fecal homogenates after 24, 48, and 72 h fermentation with EPS₈₃₃₉ (E 8339), EPS₈₃₁₂₄ (E 83124), glucose (Glu), inulin (Inu) or without extra sugar added (BM). UF profile corresponds to the one obtained with the unfermented fecal homogenate and Rs corresponds to reference strains. Lp, Lactobacillus. plantarum; Lk, Lactobacillus kefiri; Lc, Lactobacillus casei; Ba, Bifidobacterium adolescentis. Dendrograms were obtained by similarity analysis of DGGE profiles using Dice similarity coefficient and UPGMA cluster analysis.

FIGURE 2

UPGMA dendrogram of DGGE profiles of fecal homogenates microbiota after different fermentation times with EPS₈₃₃₉ (E 8339), EPS₈₃₁₂₄ (E 83124), glucose (GLU), inulin (INU) or without sugar source added (BM). UF corresponds to the unfermented fecal homogenate. Dendrogram was obtained by similarity analysis of DGGE profiles using Dice similarity coefficient and UPGMA cluster analysis.

FIGURE 3

Relative abundance at the phylum level of fecal homogenates fermented for 72 h in the presence EPS₈₃₃₉, EPS₈₃₁₂₄ or without extra sugar added (BM). Only phyla with relative abundance higher than 1% in at least one sample were included (A). Differences in bacterial relative abundance at the genus level in fecal homogenates fermented for 72 h with EPS₈₃₃₉ and EPS₈₃₁₂₄ in comparison with homogenates fermented 72 h without extra sugar added (BM). Only genera that showed differences higher than 1% in at least one sample were included (B).

FIGURE 4

Total SCFA (A) and individual acetate (B), propionate (C) and butyrate (D) levels of fecal homogenates fermented during 24, 48, and 72 h with and without sugar source added. # indicates significant differences against carbohydrate-free basal medium at the same fermentation time. *indicates significant differences between samples with a specific sugar at different fermentation time. (*/#p < 0.05, **/##p < 0.01, ***/###p < 0.001, Tukey’s multiple comparison test).