可代谢半乳糖嗜热链球菌的诱变选育及其在发酵乳生产中的应用

【目的】嗜热链球菌(Streptococcus thermophilus)作为酸奶、奶酪等发酵乳制品的常用发酵剂，在乳品工业中应用广泛。大多数嗜热链球菌为半乳糖阴性(Gal^-)菌株，不能代谢半乳糖而将其排出到胞外，导致发酵乳中半乳糖含量增加。因此可通过化学诱变的方法处理嗜热链球菌，使其可代谢半乳糖，开发一种低半乳糖含量的发酵乳。【方法】利用亚硝基胍(nitrosoguanidine, NTG)对嗜热链球菌IMAU80846进行诱变处理，测定野生株嗜热链球菌IMAU80846与突变株β-半乳糖苷酶(β-galactosidase,β-Gal)、半乳糖激酶(galactokinase, GalK)、丙酮酸激酶(pyruvate kinase, PK)和葡萄糖激酶(glucokinase, GK)活性，分析编码这些酶的氨基酸序列，得到一株可代谢半乳糖的嗜热链球菌IMAU80846Y，同时对突变株进行全基因组测序，并检测野生株和突变株发酵乳中乳酸、乳糖、半乳糖和葡萄糖的含量，最后将野生株和突变株分别与德氏乳杆菌保加利亚亚种IMAU20450进行复配发酵，测定两组发酵乳在发酵与贮藏期间的发酵特性，制备一款低半乳糖含量的发酵乳。【结果】突变株嗜热链球菌IMAU80846Y的β-Gal、GalK活性高于野生株，PK和GK活性低于野生株，氨基酸序列与全基因组测序结果表明突变株与糖代谢有关基因发生突变，HPLC检测结果表明突变株发酵乳中乳糖、半乳糖的含量均低于野生株，而乳酸、葡萄糖的含量高于野生株。发酵特性的分析结果表明，与野生株复配组相比，突变株复配组发酵乳具有良好的滴定酸度、活菌数、黏度以及持水力。【结论】嗜热链球菌IMAU80846经NTG诱变后，菌株代谢半乳糖的能力发生了变化，利用该突变株可生产一款低半乳糖含量的发酵乳。

Streptococcus thermophilus capable of metabolizing galactose: mutagenesis and application in production of fermented milk

[Objective] Streptococcus thermophilus is widely used in the dairy industry as a common starter for fermented dairy products such as yogurt and cheese. Most strains of S. thermophilus are galactose-negative (Gal^-) and unable to metabolize galactose and excrete it extracellularly, which results in an increase in the galactose content in fermented milk. S. thermophilus can be treated by chemical mutagenesis to metabolize galactose and then used to develop the fermented milk products with low galactose content. [Methods] We used nitrosoguanidine (NTG) to induce the mutation of S. thermophilus IMAU80846. Furthermore, we measured the activities of β-galactosidase (β-Gal), galactokinase (GalK), pyruvate kinase (PK), and glucokinase (GK) in the wild-type and mutant strains of S. thermophilus IMAU80846 and analyzed the amino acid sequences encoding these enzymes. S. thermophilus IMAU80846Y that could metabolize galactose was obtained. We performed whole genome sequencing of the mutant strain and measured the content of lactic acid, lactose, galactose, and glucose in the fermented milk products produced with the wild-type strain and the mutant strain. We then compounded the wild-type strain and the mutant strain with Lactobacillus delbrueckii subsp. bulgaricus IMAU20450, respectively, and characterized the two groups of fermented milk products during fermentation and storage. Finally, we prepared a fermented milk product with low galactose content. [Results] S. thermophiles IMAU80846Y had higher activities of β-Gal and GalK and lower activities of PK and GK than the wild-type strain. The amino acid sequences and whole genome sequences showed that the mutant strain had mutations in the genes involved in carbohydrate metabolism. The HPLC results showed that the fermented milk produced with the mutant strain had lower content of lactose and galactose and higher content of lactic acid and glucose than that produced with the wild-type strain. Compared with the compound group with the wild-type strain, the compound group with the mutant strain improved the titration acidity, viable cell count, viscosity, and water holding capacity of fermented milk. [Conclusion] The mutagenesis with NTG changed the ability of S. thermophilus IMAU80846 to metabolize galactose, and the mutant strain could be used to produce the fermented milk with low galactose content.