Comparative metatranscriptome analysis of Brazilian milk and water kefir beverages.

The present study compared bacterial and fungal diversity of kefir beverages produced using milk (MK) or sugared water (WK) as propagation matrices and grains from the cities of Curitiba (CU) or Salvador (SA), Brazil, by sequencing the complete set of RNA transcripts produced in four products. In Brazil, milk and sugared water are used as matrices to propagate kefir grains. In all beverages, the bacterial community was composed of Lactobacillaceae and Acetobacteraceae. Saccharomycetaceae was the yeast family more abundant in WK, and Dipodascaceae and Pichiaceae in MK. Regarding KEGG mapping of functional orthologs, the four kefir samples shared 70% of KO entries of yeast genes but only 36% of bacterial genes. Concerning main metabolic processes, the relative abundance of transcripts associated with metabolism (energy metabolism) and environmental information processing (membrane transport) had the highest water/milk kefir ratio observed in Firmicutes. In contrast, transcripts associated with genetic information processing (protein translation, folding, sorting, and degradation) oppositely had the lowest water/milk ratios. Concluding, milk and water kefir have quite different communities of microorganisms. Still, the main mapped functional processes are similar, with only quantitative variation in membrane transport and energy acquisition in the water kefir and protein synthesis and turnover in the milk kefir.

Kefiran antagonizes cytopathic effects of Bacillus cereus extracellular factors.

Kefiran, the polysaccharide produced by microorganisms present in kefir grains, is a water-soluble branched glucogalactan containing equal amounts of D-glucose and D-galactose. In this study, the effect of kefiran on the biological activity of Bacillus cereus strain B10502 extracellular factors was assessed by using cultured human enterocytes (Caco-2 cells) and human erythrocytes. In the presence of kefiran concentrations ranging from 300 to 1000 mg/L, the ability of B. cereus B10502 spent culture supernatants to detach and damage cultured human enterocytes was significantly abrogated. In addition, mitochondrial dehydrogenase activity was higher when kefiran was present during the cell toxicity assays. Protection was also demonstrated in hemolysis and apoptosis/necrosis assays. Scanning electron microscopy showed the protective effect of kefiran against structural cell damages produced by factors synthesized by B. cereus strain B10502. Protective effect of kefiran depended on strain of B. cereus. Our findings demonstrate the ability of kefiran to antagonize key events of B. cereus B10502 virulence. This property, although strain-specific, gives new perspectives for the role of bacterial exopolysaccharides in functional foods.

Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.

Gastrointestinal conditions along the digestive tract are the main stress to which probiotics administrated orally are exposed because they must survive these adverse conditions and arrive alive to the intestine. Adhesion to epithelium has been considered one of the key criteria for the characterization of probiotics because it extends their residence time in the intestine and as a consequence, can influence the health of the host by modifying the local microbiota or modulating the immune response. Nevertheless, there are very few reports on the adhesion properties to epithelium and mucus of microorganisms after passing through the gastrointestinal tract. In the present work, we evaluate the adhesion ability in vitro of L. paracasei strains isolated from kefir grains after acid and bile stress and we observed that they survive simulated gastrointestinal passage in different levels depending on the strain. L. paracasei CIDCA 8339, 83120 and 83123 were more resistant than L. paracasei CIDCA 83121 and 83124, with a higher susceptibility to simulated gastric conditions. Proteomic analysis of L. paracasei subjected to acid and bile stress revealed that most of the proteins that were positively regulated correspond to the glycolytic pathway enzymes, with an overall effect of stress on the activation of the energy source. Moreover, it is worth to remark that after gastrointestinal passage, L. paracasei strains have increased their ability to adhere to mucin and epithelial cells in vitro being this factor of relevance for maintenance of the strain in the gut environment to exert its probiotic action.

Shear and extensional properties of kefiran.

Kefiran is a neutral polysaccharide constituted by glucose and galactose produced by Lactobacillus kefiranofaciens. It is included into kefir grains and has several health promoting properties. In the present work, shear and extensional properties of different kefiran aqueous dispersions (0.5, 1 and 2% wt.) were assessed and compared to other neutral gums commonly used in food, cosmetic and pharmaceutics industries (methylcellulose, locust bean gum and guar gum). Kefiran showed shear flow characteristics similar to that displayed by other representative neutral gums, although it always yielded lower viscosities at a given concentration. For each gum system it was possible to find a correlation between dynamic and steady shear properties by a master curve including both the apparent and complex viscosities. When studying extensional properties of selected gums at 2% wt. by means of a capillary break-up rheometer, kefiran solutions did not show important extensional properties, displaying a behaviour close the Newtonian.

Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins.

Several microbial interactions involving yeast and lactobacilli have been suggested in fermented products. Co-aggregation between Lactobacillus kefir and yeast Saccharomyces lipolytica isolated from kefir grains was studied by scanning electron microscopy and aggregation assays. Six out of twenty Lb. kefir strains were able to co-aggregate with Sacch. lipolytica CIDCA 812 and showed thermolabile non-covalently bound surface molecules involved in this interaction. Co-aggregation inhibition after Lb. kefir pre-treatment with 5 m-LiCl or 20 g SDS/l showed that bacterial S-layer proteins play an important role in this interaction. Presence of different sugar (mannose, sucrose and fructose) or yeast pre-treatment with sodium periodate inhibited co-aggregation between Lb. kefir and Sacch. lipolytica. Co-aggregating Lb. kefir strains were also able to agglutinate with human red blood cells and they lost this ability after treatment with 5 m-LiCl. These results and the capacity of purified S-layer proteins of Lb. kefir to haemagglutinate, strongly suggest that a lectin-like activity of bacterial surface proteins (S-layer) mediates the aggregation with yeast cells.

Lactobacilli isolated from kefir grains: evidence of the presence of S-layer proteins.

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66-71 kDa as measured by SDS-PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.