Isolation, Identification and Characterization of Probiotic Bacteria, Development of Bio-Yogurt and Its Effects of Feeding on Mice

Abstract

Widespread interest in probiotic bacteria that can be observed nowadays results from their medicinal properties reported both for human and animal subjects. Probiotics are generally recognized as preparation of or a product containing viable single or mixed cultures of safe, defined microorganisms in sufficient numbers that are used as functional food components with claimed health-promoting effects. Yogurt is one of the potential source of probiotic bacteria as well as an ancient famous nutritious food which have variety of beneficial effect on human. With this perspective, the aim of the present study was the isolation of probiotic bacteria from regional yogurt, their physiological and biochemical identification, study of their probiotic properties along with development of yogurt using the isolates and in vivo mice trial regarding body weight gain, blood cholesterol level and antibiotic resistance of gut microflora. In the present study, a total of nine isolates of which three isolates of each Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus and Bifidobacterium spp. were obtained from three different yoghurt samples of Khulna City Corporation area, Khulna, Bangladesh. According to their morphological, physiological and biochemical assay, it was observed that all the nine isolates of three species were gram positive, endospore negative, catalase negative which are the characteristic of typical probiotic bacteria. Carbohydrate/sugar fermentation profiles experiment using eight important sugars ensured the presumptive identification of these three species. The NaCl tolerance test results have shown that all the isolates had excellent tolerance against 1-5% NaCl, moderate in 6-8% and in 9%-10% NaCl, low or no growth were observed after 24 hours of incubation at 37° C. They showed very good tolerance against 0.1% and 0.3% phenol and moderate or low tolerance against at 0.4% phenol after 24 hours of incubation at 37° C, respectively. All the nine isolates were passed to bile salt tolerance test at 0.05%, 0.1%, 0.15%, 0.2% and 0.3% during 4, 8 and 24 hours of incubation. They showed resistance to artificial gastric juice environment at pH 2.2 and 6.6. These nine isolates showed antimicrobial activity against Shigella flexeri, Shigella dysenteriae, Vibrio cholerae and Salmonella typhi. Lactobacillus delbrueckii ssp bulgaricus and Bifidobacterium spp. also showed antimicrobial activity against Staphylococcus epidermis, Pseudomonas spp. and E. coli. After confirming their probiotic properties, yogurt was developed using all the nine isolates as inoculums. Developed yogurt was tested for pH and their probiotic dose and subjected to mouse trial for conducting efficacy and safety study. After 28 days in vivo mice trial, all three treatment groups showed significant body weight gain and decreased blood cholesterol level along with increased antibiotic resistant gut microflora in comparison to control group. All these result showed that these nine isolates had probiotic properties and the probiotic yogurt produced using the isolates had a beneficial effects on mice. However, most of the results from the experiments have shown that there were variations in probiotic properties of the nine isolates belonging to three species. Molecular identification of isolated Probiotic bacteria is needed for technological interest and probiotic product development both for human and poultry.