Mutagenesis Screening Of Metabolizable Galactose Streptococcus Thermophilus IMAU80285 And Its Application Research

Streptococcus thermophilus,as one of the basic ferments for fermented milk,is widely used in various fermented dairy products.In this study,S.thermophilus IMAU80285 with good fermentation characteristics was taken as the research object,and nitrosoguanidin(NTG)was used for mutagenesis treatment.By measuring the activities of β-galactosidase(β-GAL),hexokinase(HK),and pyruvate kinase(PK),mutant strains capable of metabolizing lactose were selected.On this basis,the mutant strains were co-cultured with 8 strains of Lactobacillus delbrueckii subsp.bulgaricus with good fermentation characteristics to select the best co-culture combination,and the fermentation process was optimized through single-factor and Box-Behnken experiments to determine the optimal culture conditions.The main research results are as follows:(1)By treating S.thermophilus IMAU80285 with varying concentrations of NTG,the optimal mutagenesis concentration was identified as 300 μg/mL.the mutant strain S.thermophilus IMAU80285 Y capable of metabolizing lactose was screened based on the activities of β-GAL,HK and PK.Compared with the original strain,the activity of β-GAL in the mutant strain increased by 16.8%,while the activities of HK and PK decreased by 28.6% and 12.6%,respectively.(2)Polymerase chain reactioon(PCR)method was used to amplify the gene sequences encoding β-GAL,HK and PK.The cloning and sequencing analysis results showed that in the mutant strain,the “C” at position 173 of the β-GAL gene(LacZ)mutated to “T”,High-performance liquid chromatography(HPLC)analysis results showed that the ability of the mutant strain to metabolize lactose/galactose was significantly stronger than that of the original strain.(3)After co-culturing S.thermophilus IMAU80285 Y with L.delbrueckii subsp.bulgaricus,the best co-culture combination was selected according to pH,titratable acidity,viscosity,water holding capacity,and sensory scores.On this basis,through single-factor experiments and Box-Behnken design-response surface method,the optimal process conditions were determined as co-culture ratio of 23 ∶ 1,sucrose addition of 5.3%,fermentation temperature of 42℃,and homogenization pressure of25 MPa.Under these optimal conditions,the fermentation characteristics of this coculture combination during cow milk fermentation and storage were analyzed to lay a foundation for the application of this starter culture in industrial production.