CONICET | Buscador de Institutos y Recursos Humanos

The development of functional foods containing lactic acid bacteria (LAB) with beneficial effects on human health has increased in the last few years. Probiotic LAB of oral administration should be able to survive gastrointestinal passage and to overcome toxic detergent-like compounds such as bile salts in the duodenal loop. Thus, the tolerance of the cells to bile stress is an important criteria for selecting probiotics as dietary supplements.Previous studies have evinced differences in the bile tolerance of different Lactobacillus strains . Assessment of viable microorganisms within a population is often difficult, since no single sensitive method is able to identify viable cells under certain conditions.Although the plate count approach is employed as the standard method for determining bacterial viability, it is less sensitive, laborious and requires long incubation times.The subject of this study was to find a rapid and effective method to evaluate the survival of probiotic lactobacilli to bile acids Plate counts were performed in order to compare the flow cytometry (FC) viability assessment and subsequently, determine the correlation between methods.Eight probiotic lactobacilli strains (from CERELA culture collection, Tucumán, Argentina) were selected for the study. Cells in acetate buffer pH 5.2, supplemented with 0,5% w/v glucose and 1 mM TDCA or GDCA, were incubated at 37ºC. Samples for FC and viability assays were taken at 0 and after 30 min incubation. The cells for FC were incubated with a fluorescent dye combination of BDTM Cell Viability Kit containing thiazole orange and propidium iodide According to the manufacturers recommendation.To achieve the unit forming colony (ufc/ml) by plate counts, serial dilutions of each sample were pour plated (MRS agar) and incubated at 37ºC for 48 h.Results showed that the bile tolerance was strain-dependent. A high correlation between both methods (FC and plate counts) was observed, which validates the FC assessment. It is known the presence of Viable-nonculturable-cells within a bacterial population when subjected to different kind of environmental stress e.g., acid or bile. In this particular state, microorganisms maintain metabolic activity but loose the ability to form colonies on agar media. These particular cells can not be detected through conventional microbiological methods, which led to underestimation of the real number of functional probiotics.The application of sensitive methods to assure the correct selection of bile resistant probiotic strains is a critical step for novel functional foods with proved beneficial characteristics. The FC technique, although being semi-quantitative, has the advantage of a high sensitivity and it isperformed within minutes by using small volumes of sample. Studies are being conducted to improve this methodology