β-Galactosidase is a crucial glycoside hydrolase enzyme with potential applications in the dairy, food, and pharmaceutical industries. The enzyme is produced in the intracellular environment by bacteria and yeast. The present study reports yeast Kluyveromyces sp. PCH397 isolated from yak milk, which has displayed extracellular β-galactosidase activity in cell-free supernatant through the growth phase. To investigate further, cell counting and methylene blue staining of culture collected at different growth stages were performed and suggested for possible autolysis or cell lysis, thereby releasing enzymes into the extracellular medium. The maximum enzyme production (9.94 ± 2.53U/ml) was achieved at 37 °C in a modified deMan, Rogosa, and Sharpe (MRS) medium supplemented with lactose (1.5%) as a carbon source. The enzyme showed activity at a wide temperature range (4–50 °C), maximum at 50 °C in neutral pH (7.0). In addition to the hydrolysis of lactose (5.0%), crude β-galactosidase also synthesized vital prebiotics (i.e., lactulose and galacto-oligosaccharides (GOS)). Additionally, β-fructofuranosidase (FFase) activity in the culture supernatant ensued the synthesis of a significant prebiotic, fructo-oligosaccharides (FOS). Hence, the unique features such as extracellular enzymes production, efficient lactose hydrolysis, and broad temperature functionality by yeast isolate PCH397 are of industrial relevance. In conclusion, the present study unrevealed for the first time, extracellular production of β-galactosidase from a new yeast source and its applications in milk lactose hydrolysis and synthesis of valuable prebiotics of industrial importance.

β-半乳糖苷酶是一种重要的糖苷水解酶，在乳制品、食品和制药行业具有潜在应用。该酶由细菌和酵母在细胞内环境中产生。本研究报告酵母克鲁维酵母sp。PCH397 从牦牛奶中分离出来，其在整个生长期在无细胞上清液中表现出细胞外 β-半乳糖苷酶活性。为了进一步研究，对在不同生长阶段收集的培养物进行细胞计数和亚甲蓝染色，并建议可能的自溶或细胞裂解，从而将酶释放到细胞外培养基中。在补充有乳糖 (1.5%) 作为碳源的改良 deMan、Rogosa 和 Sharpe (MRS) 培养基中，在 37 °C 下实现了最大酶产量 (9.94 ± 2.53U/ml)。该酶在很宽的温度范围（4-50 °C）下表现出活性，在中性 pH 值（7.0）中的最大温度为 50 °C。除了乳糖（5.0%）的水解，粗β-半乳糖苷酶还合成了重要的益生元（即乳果糖和半乳糖寡糖（GOS））。此外，培养上清液中的 β-呋喃果糖苷酶 (FFase) 活性导致重要的益生元低聚果糖 (FOS) 的合成。因此，酵母分离物 PCH397 的胞外酶生产、高效乳糖水解和广泛的温度功能等独特的特性具有工业相关性。总之，本研究首次揭示了从新酵母来源胞外生产 β-半乳糖苷酶及其在牛奶乳糖水解和合成具有工业重要性的有价值的益生元中的应用。酵母分离物 PCH397 的广泛温度功能具有工业相关性。总之，本研究首次揭示了从新酵母来源胞外生产 β-半乳糖苷酶及其在牛奶乳糖水解和合成具有工业重要性的有价值的益生元中的应用。酵母分离物 PCH397 的广泛温度功能具有工业相关性。总之，本研究首次揭示了从新酵母来源胞外生产 β-半乳糖苷酶及其在牛奶乳糖水解和合成具有工业重要性的有价值的益生元中的应用。