Engineering the ß-galactosidase from Aspergillus oryzae for making lactose-free and no-sugar-added yogurt.

ABSTRACT

Yogurt usually contains 5-7% sugar and 3-5% lactose. As ß-galactosidases can hydrolyze lactose and improve sweetness, they have the potential to produce lactose-free (LF) and no-sugar-added (NSA) yogurt. In this study, ß-galactosidase AoBgal35A from Aspergillus oryzae was engineered by site-saturation mutagenesis. Results of 19 variants of T955 residue showed that lactose hydrolysis rate of T955R-AoBgal35A was up to 90.7%, much higher than 78.5% of the wild type. Moreover, the optimal pH of T955R-AoBgal35A was shifted from pH 4.5 to pH 5.5 and the optimal temperature decreased from 60°C to 50°C. The mutant T955R-AoBgal35A was successfully expressed in Komagatella pastoris, which produced extracellularly 4528 U/mL of ß-galactosidase activity. The mutant T955R-AoBgal35A was used to produce LF yogurt. Streptococcus thermophilus counts of LF yogurt increased from 7.9 to 9.5 lg cfu/g, significantly higher than that of the control group (8.9 lg cfu/g). Residual lactose content of LF yogurt was 0.13%, meeting the requirement of "lactose-free" label (<0.5%, GB 28050-2011, China). Furthermore, sugar in yogurt was replaced by whey powder to produce LF-NSA yogurt. The optimal addition content of whey powder was 7.5%. The texture, WHC and titratable acidity of LF and LF-NSA yogurt achieved good stability during the shelf life. Therefore, this study provides an insight for technological implications of ß-galactosidases in the dairy industry.