Effect of yeast culture supernatant on the probiotic properties and biochemical characteristics of three probiotics, in vitro and in vivo evaluation of yeast-probiotic combination against the pathogen V. anguillarum
Research hypothesis: If yeast and probiotics enhance each other they might provide improved protection against pathogens. Growth, aggregation, biofilm formation and hydrophobicity as well as evaluation, of pH lactic acid, free amino nitrogen and reducing sugars in the growth media were estimated for probiotics in the presence of three ratios of yeast cell free supernatant (CFS). The effect of the best working ratio of each probiotic with yeast in terms of the characteristics examined, on the fish pathogen Vibrio anguillarum properties, namely aggregation, biofilm formation and hydrophobicity, was studied in vitro. Then an in vivo examination on Artemia franciscana nauplii was performed following administration of yeast and probiotic bacteria at the selected ratios and a challenge test with the pathogen to examine any possible protection offered in terms of Artemia survival.
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The bacterial strains that were used as probiotics were Bacillus subtilis NCIMB 3610, Lactococcus lactis ATCC 11454 and Lactobacillus plantarum CECT 220 and were grown in NB, BHI and MRS respectively, at 28°C, in a rotary shaker, while the Greek strain of the pathogen Vibrio (Listonella) anguillarum, 332A (WDCM 908, identification Nr 1.1) used for challenge experiments was grown in TSB agar containing 2% NaCl, at 22-24 °C, for 48 h .S. cerevisiae wild type strain JD47–13C was used. After incubation the bacterial and the yeast cultures were centrifuged at 4°C, 4200 x g for 30 min. The resulting supernatants were filtered through glassfiber filters followed by 0.20 μm, 47 mm membranes, and stored in sterile flasks at 4 °C until used in further experiments.Liquid cultures of each probiotic were cultured in the appropriate medium for each bacterium containing yeast CFS at a ratio of 1:1, 1:2 and 2:1. Monitoring of probiotics and yeast growth was performed by absorbance measurements (600 nm) as well as with plating at timed intervals Auto-aggregation of cells was estimated according to the method of Del Re et al. (2000) ,hydrophobicity of cells was estimated as the ability of microbial adhesion to hydrocarbons (MATH test), Biofilm formation capacity was performed by the method of O’Toole and colleagues Lactic acid was determined enzymatically, Free amino nitrogen concentration (FAN) was determined using the EBC- ninhydrin method, total reducing sugars (TRS) was performed using the dinitrosalicylic method CFS from uninterrupted cultures of yeast at 48 h and of the probiotics at 24 h of incubation, mixed at the specific yeast: probiotic ratio determined on the basis of the results of the aforementioned experiments, were added in a 24 h V. anguillarum culture. Growth at 24 h (OD 600 nm at 24 h), auto-aggregation, hydrophobicity and biofilm formation capacity of the pathogen were determined at the end of a 24 h incubation period. Experiments regarding the in vivo evaluation of yeast-probiotics combinations were performed on axenic cultures of nauplii The probiotics and/or yeast, were administered at 48 h, for two consecutive days, two doses per day. Then the challenge with Vibrio anguillarum was performed