β-Galactosidase-Mediated Lactose Hydrolysis in Milk by Bacillus aryabhattai A Marine Bacterium

Abstract

β-Galactosidase hydrolyses lactose into glucose and galactose and has potential applications in dairy processing. In this study, 24 marine bacterial isolates were screened for β-galactosidase production. Isolate MBC1 showed the highest activity in quantitative screening and was identified as Bacillus aryabhattai using cultural, morphological, biochemical, VITEK 2, and 16S rRNA gene analyses; its sequence was deposited in GenBank (MG831954). Enzyme production was evaluated under different nutritional and physicochemical conditions. The enzyme was purified by ammonium sulfate precipitation, dialysis, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration. The purified preparation showed an activity of 1326 U/mL, a specific activity of 491.10 U/mg, 6.20-fold purification, and 60% recovery; its apparent molecular mass was approximately 45 kDa. Maximum catalytic activity was observed at pH 7.0 and 35 °C. During buffalo-milk treatment, reducing sugars increased from 20.87 ± 1.47 µg/mL at 1 h to 307.09 ± 4.01 µg/mL at 6 h, corresponding to 4.17 ± 0.29% and 61.42 ± 0.80% apparent hydrolysis, respectively. In a separate 5% lactose-solution assay, the recalculated 6-h reducing-sugar concentration was 407.39 ± 4.80 µg/mL in the 100-fold-diluted assay sample, corresponding to 81.48 ± 0.96% apparent hydrolysis when compared with the equivalent diluted substrate concentration. Sensory scores were generally higher for enzyme-treated milk, although the small panel and zero standard deviations reported for some attributes require careful interpretation. Overall, MBC1 is a potential source of β-galactosidase for further study. Since DNS estimates reducing sugars rather than residual lactose directly, the hydrolysis estimates should be confirmed using a validated lactose specific method.