BETA-GALACTOSIDASE PRODUCTION BY Bifidobacterium animalis SUBSP. lactis BB-12 IN WHEY MEDIUM

Probiotics are live microorganisms that provide health benefits to the host. In the presence of lactose, the probiotic Bifidobacterium animalis subsp. lactis produce β-galactosidase, also known as lactase, responsible of lactose hydrolysis. This enzyme is present in the human gut and its absence can cause lactose intolerance. Thus, this study objective was to produce β-galactosidase from Bifidobacteirum animalis subsp. lactis BB-12 cultivated in whey medium. The fermentation process was performed at 35ºC ± 2 with a 150 rpm agitation in a whey medium standardized for pH 5. The enzymatic activity (U/ mL) was analyzed through a Box-Benhken statistical experiment with three independent factors. Being those the fermentation time of the probiotic in the whey medium (24, 48 and 72h); the whey powder concentration (5, 10 and 15% - p/ v) dissolved in distilled water to produce the fermentation medium and the ethanol (35, 50 and 85% - v/ v) diluted in phosphate buffer used in the permeation process necessary to measure the enzymatic activity, thereby 15 runs were performed. The enzymatic activity was observe in the probiotic biomass by centrifuging 5mL of the whey medium at 5.000rpm for 30 minutes, the supernatant was discarded and biomass permeated with the different ethanol percentages among with phosphate buffer (pH 6.8) per 20 minutes at 20ºC. The permeated biomass was centrifuged (5.000rpm, 10 minutes), and resuspended in phosphate buffer. The Beta-galactosidase activity was evaluate through ONPG method by adding 50µL of the sample into 2mL of ONPG (1.25mM diluted in phosphate buffer 6.6 with manganese), the reaction was incubated for 5 minutes at 37ºC and stopped by 0.5mL of sodium carbonate addition. The released ONPG was measured in a spectrophotometer at 420nm. Using response surface applied to the Box-Benhken design, results showed that the highest enzymatic activity occurred with a 24 hours fermentation, with a 10% whey medium and 85% of ethanol reaching 0.6 U/ mL. According to variance analysis, the model had an adequate fit with R2 of 0.96. The influence of ethanol was significant (p<0.01) and affected positively the enzymatic activity, the fermentation time influenced (p<0.05) negatively. Thus, higher the ethanol percentage and lower the fermentation time, higher the enzymatic activity. Whey concentration did not exert significant effect on Beta-galactosidase activity, all concentration can be used, but to optimize the production, a 10% concentration is recommended. Therefore, to optimize lactase production, is recommended novel experimental design reducing fermentation time among with a separate experimental design optimizing the permeation process.