CONICET | Buscador de Institutos y Recursos Humanos

Milk constitutes an excellent carrier for probiotics wherein specific bacterial-fermentation processes may lead to functional products with new beneficial health effects beyond the normal nutritional function of the dairy product itself. These health benefits associated to fermented milk consumption may be originated by the presence of the microorganisms themselves or other bioactive components produced during fermentation such as organic acids, peptides and/or exopolysaccharides (EPS). It has been well documented that certain fermented milks with lactic acid bacteria such as kefir, present the capacity to modulate the host?s immune response resulting in an interesting alternative to alleviates symptoms of gastrointestinal inflammatory diseases (1). Considering this, the aim of this work was to evaluate the bioactive properties of the nonbacterial fraction of Lactobacillus paracasei fermented milks with a focus on their ability to modulate the innate immune response on gastric and intestinal epithelial cells growing in culture.L. paracasei CIDCA 8339 and CIDCA 83124, isolated from kefir grains were characterized as EPS-producing (2) and potentially probiotics strains. These strains are able to resist the gastrointestinal conditions (3, 4), adhere to intestinal epithelial cells (3), downregulate the innate immune response on intestinal epithelial cells (4) and exert a protective effect against Salmonella infection (5). Moreover, it was evidenced that polysaccharide production is influenced by growth temperature without affecting probiotic properties being this aspect of relevance for industrial application (4).Lactobacilli were inoculated in UHT low fat milk at 5% v/v and incubated at 30°C for 24h. The nonbacterial fractions (NBF) of fermented milks were obtained by centrifugation (5min at 5000g); the supernatants were then neutralized with 5N NaOH and filtered through a 0.45 μm of pore diameter. The lactic and acetic acid concentration in the NBF were determined by HPLC with an AMINEX HPX-87H ion exchange column. To evaluate the immunomodulatory properties of the NBF of fermented products, gastric-epithelial-carcinoma cells (AGS) and reporter intestinal epithelial cells Caco-2-ccl20:luc were treated for 30 min with the NBF and then exposed to stimulation by 1µg/ml Flagellin (FliC) during 8h at 37 °C in an atmosphere of 5% CO2?95% air. Artificially acidified milk (AAM) with lactic and acetic acid, as well as aqueous solutions of lactic acid, acetic acid and EPS isolated from fermented milks were also tested in these systems. A basal condition without any treatment was included as a control; and a FliC stimulated condition was used to define the 100% induction of the proinflammatory response. The innate immune response was define by determination of IL-8 levels in AGS supernatants by ELISA (BD Bioscience OptEIATM human IL-8 ELISA Kit USA) and by measuring luciferase activity in Caco-2-ccl20:luc in a Labsystems Luminoskan TL Plus luminometer (Thermo Scientific, USA) using a luciferase assay system (Promega, USA). IL-8 levels and luminescence were normalized to the stimulated control cells and expressed as a percentage of the normalized average ± standard deviation (SD). In order to evaluate the treatment-induced cytotoxicity, the mitochondrial activity of the cells was evaluated by measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co) to a purple formazan at 550 nm.The organic acids concentration in the NBF of fermented milks with L. paracasei CIDCA 8339 and CIDCA 83124 were similar, with a value near 100 mM for lactic acid and 12 mM for acetic acid. Both NBF were able to significantly modulate the epithelial innate immune response in vitro. The pre-incubation of the cells with the NBF led to a reduction of an 85-90% of luciferase activity in intestinal cells and a 70% modulation of IL-8 expression in gastric cells (Figure 1, p