酸奶通常含有5-7%的糖和3-5%的乳糖。由于β-半乳糖苷酶可以水解乳糖并改善甜味,它们有潜力生产无乳糖(LF)和无糖添加(NSA)酸奶。在这项研究中,通过位点饱和诱变对来自米曲霉的β-半乳糖苷酶AoBgal35A进行了工程改造。T955残基的19个变体的结果表明,T955R-AoBgal35A的乳糖水解率高达90.7%,远高于野生型的78.5%。此外,T955R-AoBgal35A的最佳pH从pH4.5移至pH5.5,最佳温度从60°C降至50°C。突变体T955R-AoBgal35A在巴斯德科马卡特氏菌中成功表达,细胞外产生4528U/mL的β-半乳糖苷酶活性。突变体T955R-AoBgal35A用于生产LF酸奶。LF酸奶的嗜热链球菌计数从7.9增加到9.5lgcfu/g,显着高于对照组(8.9lgcfu/g)。LF酸奶的残余乳糖含量为0.13%,符合“无乳糖”标签的要求(<0.5%,GB28050-2011,中国)。此外,用乳清粉代替酸奶中的糖生产LF-NSA酸奶。乳清粉的最佳添加量为7.5%。纹理,LF和LF-NSA酸奶的WHC和可滴定酸度在保质期内具有良好的稳定性。因此,这项研究为β-半乳糖苷酶在乳制品行业中的技术意义提供了见解。
Yogurt usually contains 5% to 7% sugar and 3% to 5% lactose. As β-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, the β-galactosidase AoBgal35A from Aspergillus oryzae was engineered by site-saturation mutagenesis. Results of 19 variants of T955 residue showed that the lactose hydrolysis rate of T955R-AoBgal35A was up to 90.7%, which is much higher than the 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 Komagataella pastoris, which produced extracellularly 4,528 U/mL of β-galactosidase activity. The mutant T955R-AoBgal35A was used to produce LF yogurt. The Streptococcus thermophilus count of LF yogurt increased from 7.9 to 9.5 log cfu/g, which is significantly higher than that of the control group (8.9 log cfu/g). The residual lactose content of LF yogurt was 0.13%, meeting the requirements of the national standard in
China for the \"lactose-free\" label (<0.5%). 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, water-holding capacity, and titratable acidity of LF and LF-NSA yogurt achieved good shelf life stability. Therefore, this study provides an insight for technological implications of β-galactosidases in the dairy industry.