Caracterização de Lactobacillus spp. e desenvolvimento de um sistema de simulação de sobrevivência bacteriana no trato gstroentestinal

Abstract

This work aimed to study the probiotic characteristics of lactic acid bacteria (LAB) isolated from three food products / processes (fresh sausage, cocoa fermentation and cauim). A model of the gastrointestinal (GI tract to evaluate the physiological conditions of cells by intracellular pH (pHi) analysis was developed. The model was used to evaluate two selected potential probiotic Lactobacillus plantarum UFLA CH3 and Lb. brevis UFLA FFC199. Initially, 234 LAB were subject to pH 2.0 test, and then 51 were selected. They were identified as Lb. fermentum (34 isolates), Lb. plantarum (10) and Lb. brevis (7). Based on the results of bile tolerance (0.3%), ability to auto-aggregation and hydrophobic properties, the isolate Lb. fermentum (UFLA CH58), Lb. plantarum (UFLA CH3, UFLA CH41 and UFLA SAU96) and Lb. brevis (UFLA SAU105 and UFLA FFC199) were selected. Lb. plantarum UFLA CH41 showed the highest ability to co-aggregate with Escherichia coli, while Lb.brevis (UFLA SAU105 and UFLA FFC199) and Lb. fermentum UFLA CH58 exhibited antagonistic activity against the pathogens Listeria monocytogenes and Staphylococcus aureus. Regarding adherence to Caco-2 cells, Lb. plantarum UFLA CH3 and UFLA CH41 and Lb. brevis UFLA FFC199 showed similar results (1.6, 1.1 and 0.9%, respectively) to the commercial probiotic Lb. rhamnosus GG (1.5%). These isolates were also able to significantly increase the transepithelial electrical resistance (TEER) of Caco-2 cells over 24 h (p <0.05). Therefore, due to their potential probiotic characteristics, Lb. plantarum UFLA CH3 (cocoa) and Lb. brevis UFLA FFC199 (cauim) were selected and applied in the GI tract model developed in the present study. Analyses of pHi were determined employing Fluorescence Ratio Imaging Microscopy (FRIM). We observed heterogenous populations, with pHi values ranging from 6.5-7.5 and 6.5-8.0 or higher during passage of saliva (pH 6.4) and intestinal juice (pH 6.4), respectively. Exposure to gastric juice (pH 3.5) showed two sub-populations with pHi ranging from 3.5-4.5 and 4.5-5.6. The isolate Lb. brevis UFLA FFC199 showed pHi values closest to pHex when a nutrient solution was added to gastric juice, which was not observed for Lb. plantarum UFLA CH3. The GI model developed employing FRIM, enabled a real-time monitoring of pH homeostasis of single cells. The isolates Lb. plantarum UFLA CH3 and Lb. brevis UFLA FFC199 showed characteristics for potential probiotic use, being able to survive the conditions of the GI tract maintaining viable and vital.