In situ galacto-oligosaccharide (GOS) synthesis in milk using β-galactosidases is an effective method for developing prebiotic dairy products. However, the low lactose concentration in milk (∼4.6%, w/w) reduces the GOS yield. In this study, a modified β-galactosidase from Bacillus circulans (mBgaD-D) with enhanced transglycosylation activity at low lactose concentration was developed through directed evolution and saturation mutagenesis. The GOS yield by mBgaD-D increased from 22.8% (wild type) to 30.8% in 50 g/L lactose (phosphate buffer). P_mgut was a strong sorbitol-inducible promoter from Bacillus subtilis. The expression of mBgaD-D in B. subtilis, coupled with the P_mgut promoter, resulted in a 6.4-fold increase (compared to the P₄₃ promoter) in extracellular enzyme activity. Additionally, adding whey powder to boost the initial lactose concentration further improved the GOS yield, which reached 43% under the optimized conditions. Combining mBgaD-D and whey powder enhanced milk sweetness, producing no sugar-added, GOS-enriched yogurt (GOSY). The GOS content in GOSY was 4.1/100 g, providing an appropriate level of sweetness and yielding a yogurt that is elastic as well as firm. GOSY also increased the population of Bifidobacterium spp. during a 24 h in vitro fecal fermentation. Thus, fortifying yogurt with mBgaD-D and whey powder can enhance its technological properties and health benefits.

中文翻译：

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改性 β-半乳糖苷酶原位合成乳清粉低聚半乳糖及其对酸奶技术功能特性的影响


使用 β-半乳糖苷酶在牛奶中原位合成低聚半乳糖 （GOS） 是开发益生元乳制品的有效方法。然而，牛奶中的低乳糖浓度（∼4.6%，w/w）会降低 GOS 的产量。在本研究中，通过定向进化和饱和诱变开发了一种来自环状芽孢杆菌 （mBgaD-D） 的修饰 β-半乳糖苷酶，在低乳糖浓度下具有增强的转糖基化活性。mBgaD-D 在 50 g/L 乳糖（磷酸盐缓冲液）中的 GOS 产量从 22.8%（野生型）增加到 30.8%。P_mgut 是来自枯草芽孢杆菌的强山梨糖醇诱导型启动子。枯草芽孢杆菌中 mBgaD-D 的表达与 P_mgut 启动子相结合，导致细胞外酶活性增加 6.4 倍（与 P₄₃ 启动子相比）。此外，添加乳清粉以提高初始乳糖浓度进一步提高了 GOS 产量，在优化条件下达到 43%。mBgaD-D 和乳清粉的结合增强了牛奶的甜味，生产出不添加糖的富含 GOS 的酸奶 （GOSY）。GOSY 中的 GOS 含量为 4.1/100 克，提供适当的甜味，并产生富有弹性和坚实的酸奶。GOSY 还在 24 小时的体外粪便发酵期间增加了双歧杆菌属的数量。因此，用 mBgaD-D 和乳清粉强化酸奶可以增强其技术特性和健康益处。