Lactobacillus spp. isolated from Brazilian water kefir grains and their exopolysaccharides inhibit adhesion and/or invasion of Salmonella enterica serovar Enteritidis in CACO-2/TC7 cell

NEUMANN E ; ABRAHAM A.G.; GARROTE G L; VILLANOEVA C.N.; NICOLI J.R.

Rosario

Congreso; XXIIII Congreso Latinoamericano de Microbiología. XIV Congreso Argentino Microbiología. IV Congreso Latinoamericano de Microbiología de Medicamentos y Cosméticos; 2016

AAM

Lactobacillus spp. from kefir grains have been reported as good exopolysaccharide (EPS) producers. These EPS constitute the grain matrix and are involved in some human benefits atributed to kefir. The molecular mass (MM) is one of the factors that determine the functional properties of the EPS, and depending on their chemical structure, they are not hydrolyzed by the digestive enzymes and, therefore, can reach the small intestine where can exert the biological effect. In face of that, EPS produced by Lactobacillus spp. isolated from Brazilian water kefir grains were chemically characterised and their protective action on adhesion and invasion of Salmonella enterica serovar Enteritidis in Caco-2/TC7 cells was tested. Lactobacillus satsumensis 10P; L. satsumensis 10P2, L. satsumensis 18P, L. perolens 11P3, L. perolens 17P2 and L. mali 19U were inoculated in MRS broth containing 8% sucrose and incubated at 25°C/48h, and then the cells were removed by centrifugation. The EPS were extracted by alcohol precipitation (-20°C /12 hours), followed by centrifugation and lyophilization. The MM was determined by high-performance liquid chromatography of molecular exclusion. The Structural characterization was done by magnetic resonance spectroscopy. Caco-2/TC7 cells were inoculated with Lactobacillus spp. (107-8 UFC/mL) or EPS (1,0g/L) for 1h//37° and inoculated with S. Enteritidis (1.5 × 106cfu/mL, 1h/37°C). After that, the cells were washed with PBS, disrupted with Milli-Q water and the number of adhered cells was checked in nutrient agar. To verify the invasion of S. Enteritidis the cells were treated with 0,5 ml of gentamicin (100 µ g/mL in PBS, 1h/37°C) prior to lysis and S. Enteritidis enumeration. All strains produced EPS in different growing conditions. The EPS molecular mass showed great diversity: 10P (1,44x106Da/38,42% and 2,13x104Da/61,58%); 10P2 (2,25x104Da/100%); 18P (7,09x107Da/79,63%); 11P3 (7,27x107Da/43,45% and 2,40x104Da/56,55%); 17P2 (7,46x107Da/27,08% and 2,34x104Da/72,92%); and 19U (5,96x107Da/100%). The EPS were composed mostly by α-glucose and in a smaller proportion by α-rhamnose and α-galactose. The EPS tested were able to inhibit adhesion and invasion of S. Enteritidis in Caco-2/TC-7 cells, although the protective mechanism is not yet understood. Some strains were capable of antagonizing the invasion of S. Enteritidis in vitro, which shows that we can get a kefir with probiotic properties if we use these strains as starter cultures.