EFECT OF GASTROINTESTINAL CONDITIONS ON THE VIABILITY AND ANTIMICROBIAL ACTIVITY OF Enterococcus gallinarum CRL 1826

ACEVEDO, M.A.; ALE, C.E.; NIEDERLE, M.V.; NADER-MACÍAS, M.E.F; PASTERIS, S.E.

Tucumán

Simposio; V SIMPOSIO INTERNACIONAL DE BACTERIAS LACTICAS; 2016

CERELA-CONICET

In previous work, our research group have shown that Enterococcus gallinarum CRL 1826 is a probiotic candidate to control Red Leg Syndrome (RLS)-related pathogens outbreaks during American Bullfrog culture. Autochthonous lactic acid bacteria (LAB) were selected according to its beneficial properties (production of antimicrobial metabolites, autoaggregation and hydrophobic characteristics) and absence of adverse effects when fresh bacterial suspensions were administered to bullfrog embryo up to the tadpole stage (30 days). However, during the metamorphosis, physiological and anatomical modifications were observed by changes in the feed habits. Therefore, the viability and antimicrobial properties of fresh and lyophilized LAB strain under the adults? gastrointestinal conditions was studied. In order to determine the growth and antimicrobial activity of E. gallinarum CRL 1826, cells (fresh cultures incubated in LAPTg broth or freeze dried in 5% sucrose + 5% skim milk) were collected and/or resuspended (109 CFU/mL) in physiological solution (10% w/v) and individually inoculated in: a- LAPTg broth pH 2, 3, 4, 5, 6, 7 and 8; b- LAPTg + 0.1, 0.3, 0.5, 1, 1.5, 3, 6 and 10% bile, pH 6.8; c- LAPTg + 0.05; 0.15; 0.3 y 0.6% pepsin, pH 2 and d- LAPTg + 0.01, 0.05, 0.1, 0.15 and 0.2% pancreatine, pH 6.8. Total biomass and antimicrobial activity against L. monocytogenes Scott A by using standardized protocols were performed after 20 h of each treatment. In those cases where microbial growth was not detected, viable cell counts after fixed times for each treatment were determined. Our results show that fresh cultures of E. gallinarum grew between pH 5 to pH 8, in all pancreatine concentrations and only with 0.1% bile up to 20 h. However, viable cells around 107 CFU/mL were detected in bile up to 0.6% after 90 min of incubation, while a decrease in 2 log units was observed when LAB were treated for 10 min with higher bile concentrations. Also, when cells were incubated during 18 min at pH 2-4, 107 CFU/mL were recovered. Lyophilized and rehydrated cells grew at pH 4 to 8, 0.1 to 3% bile, and in all pancreatine concentrations. Treatments with 6 and 10% bile during 10 min reduced the viable LAB numbers to 107 CFU/mL. Both, fresh and lyophilized LAB were resistant 18 min to pepsin treatment (~105-106 CFU/mL). At pH 2, 1.5% bile and 0.2% pancreatine, 1% of residual bacteriocin activity was observed. The results obtained represents the first step in the design of a simulated gastrointestinal digestion model to evaluate the E. gallinarum CRL 1826 viability and antimicrobial properties to apply during the bullfrog intensive culture and thus to improve this commercial activity in Argentina