No abstract

The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of antiapoptotic genes and over-expression of cell cycle-related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time-and concentrationdependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain, isolated recently from feta-type cheese. Its probiotic potential has been demonstrated in a series of established in vitro tests. Moreover, incorporation of L. paracasei K5 as starter culture offered organoleptic and technological advantages to novel fermented food products. In the present study, further investigation of the potential probiotic activity of L. paracasei K5 was performed and its mechanisms of action were investigated. Employing quantitative analysis and confocal, fluorescent microscopy the adhesion properties of the above strain were studied. L. paracasei K5 displayed efficient adherence capacity to Caco-2 colon cancer cells, similarly to the reference strains Lactobacillus casei ATCC 393 and Lactobacillus rhamnosus GG. Moreover, treatment of Caco-2 cells with L. paracasei K5 inhibited cell proliferation in a time-and dose-dependent manner. The anti-proliferative effects appear to be mediated through induction of apoptosis via modulation of expression of specific Bcl-2 family proteins. These results elucidate the mechanisms of action of L. paracasei K5 and enhance its potential probiotic activity.

Lactobacillus pentosus B281 and Lactobacillus plantarum B282 are two Lactobacillus strains previously isolated from fermented table olives. Both strains were found to possess probiotic properties and displayed desirable technological characteristics for application as starters in novel functional food production. In the present study the anti-proliferative and immunostimulatory activities of the two strains were investigated. Firstly, we demonstrated that live L. pentosus B281 and L. plantarum B282 significantly inhibited the growth of human colon cancer cells (Caco-2) in a time- and dose-dependent manner. By employing the air pouch system in mice, we showed that administration of both strains led to a rapid and statistically significant infiltration of leukocytes in the air pouch exudates. The phenotypical characterisation of the recruited immune cells was performed by flow cytometry analysis. We demonstrated that the majority of the infiltrated leukocytes were neutrophils. Finally by using the Mouse Cytokine Array Panel A Detection Antibody cocktail, we showed that both strains induced the expression of granulocyte-colony stimulating factor, interleukin (IL)-1α, IL-1β, IL-6, chemokine (C-X-C motif) ligand (CXCL)-1, chemokine (C-C motif) ligand (CCL)-3, CCL-4, and CXCL-2 and diminished the expression levels of soluble intercellular adhesion molecule, macrophage colony-stimulating factor and metallopeptidase inhibitor 1. Our results showed that both strains display anti-proliferative and immunostimulatory properties equal or even better in some cases than those of established and commonly used probiotic strains. These findings further support the probiotic character of the two strains.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1α (IL-1α), ΙL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character.