Phenotypic and genotypic identificaiton of probiotic bacteria and their application in the development of probiotic dairy products

This study was conducted to isolate, identify, preserve, and use the probiotic bacteria isolated from probiotic dairy products sold locally, in the development of probiotic 'kesong puti' and 'pastillas de leche'. A total of 15 probiotic bacteria were isolated from Chamyto Plain and Yakult. Fourteen isolates were found Gram positive, catalase negative, rods and one isolate was Gram positive, catalase negative coccus. The biochemical reaction profile showed that all isolates were Lb. paracasei. Their ITS-PCK products showed 65 percent homology to the indicator strains which are Lb. paracasei-paracasei. The DNA fingerprint results however, indicated that identities of the three isolates (1C10, 2Y9, and 3C10) showed complete (100 percent) similarity with the 16S rRNA genes of Lb. paracasei and one isolate (1Y9) had 97 percent similarity with the A6S rRNA genes of Enterococcus faecalis. The other 2 isolates were not subjected to 16S rRNA sequencing. Bacteriocin activity assay showed that all the isolates were inhibitory to Bacillus subtilis. Isolates 1C10, 2C10 and 2Y9 (all Lb. paracasei) inhibited Stophylococcus aureus. Isolate 3C10 which was identified as Lb. paracasei was the most acid and bile salt tolerant with mean viable count of 2.30 x 10 squareCFU/mL after 3 h in the artificial gastric juice (pH 2.) which increased to 2.10 x 10 sup 7 (FU/mL after 3 h in the simulated interstinal fluid. Lb. paracasei (isolate 3C10) was used in the development of probiotic 'kesong puti' and 'pastillas de leche'. Lb. paracasei survived the conditions in the 'kesong puti' and 'pastillas de leche' with mean viable counts of 3.20 x 10 sup 9 CFU/g and 4.90 x 10 sup 6 CFU/g, respectively. Increasing the level of Lb. paracasei caused a highly significant increase in yield, protein, fat, and ash contents of the 'kesong puti'. The response surface plot showed that viable counts increased with an increase in culture level up to one week refrigerated storage, after which, viable counts decreased. Increasing the cell concentration of Lb. paracasei caused a highly significant increase in the yield, protein, and ash contents of 'pastillas de leche'. Response surface plot of viable count vs. Lb. paracasei cell concentration in the 'pastillas de leche' and storage period showed that both 10 sup 8 and 10 sup 10 CFU/g and one week storage at room temperature will guarantee the minimum therapeutic daily dose of 10 sup 8 to 10 sup 9 viable cells. For the consumer to benefit from the probiotic effect minimum daily consumption should be at least 1 g for 'kesong puti' and 10 pieces of 10 to 12 g per piece for 'pastillas de leche'.