Breeding Of Galactose Mutant Strains Of Lactobacillus Delbrueckii Subsp. Bulgaricus And Its Application In Fermented Milk Production

Lactobacillus delbrueckii subsp.bulgaricus is widely used in a variety of fermented dairy products as a starter,and its functions are important for the development of the dairy industry.In this study,L.delbrueckii subsp.bulgaricus IMAU 40078,which has good fermentation characteristics,was used as the object of study.The strains were mutagenized with nitrosoguanidine(NTG)as the mutagen.Screening the mutant strain for metabolizable galactose.On this basis,the mutant strains that can metabolize galactose were fermented,to study and analyse its characteristics and metabolites during the fermentation and storage.The results obtained were as follows.1.L.delbrueckii subsp.bulgaricus IMAU40078 isolated from fermented dairy products with good fermentation characteristics,was used as the original strain and mutagenised using different concentrations of NTG,the best mutagenic concentration was determined by lethality at 300 μg/m L.2.By comparing the activities of β-galactosidase(β-GAL),hexokinase(HK)and pyruvate kinase(PK)of the original strain and 20 mutant strains,selected the mutant strain IMAU40078 Y that can metabolize galactose.Compared with the original strain,the β-GAL activity of the mutant strain increased by 12.5%,HK and PK activities decreased by 31.21% and 38.98%.The results of gene sequencing showed that the Lac Z gene of the mutant strain IMAU40078 Y was inserted with "T","A" and "A" at the three sites of 385,1567 and 1576,respectively;the pyk gene at position 325,"A" was mutated to "T".The results of passage experiments showed that the mutant strain IMAU40078 Y had good genetic stability,and there was no significant difference in the activities ofβ-GAL,HK and PK among the strains of each generation(P>0.05).3.L.delbrueckii subsp.bulgaricus IMAU40078 and its mutant strain IMAU40078 Y were used as starters to analyze its fermentation characteristics during the fermentation and storage.The results showed that the acid production capacity,viscosity,water holding capacity and viable count of the mutant strain were higher than the original strain.It shows that the mutant strain has good fermentation characteristics and can be used as a starter in the production of dairy industry.4.High performance liquid chromatography(HPLC)was used to analyze the metabolites of L.delbrueckii subsp.bulgaricus IMAU40078 and its mutant strain during the fermentation and storage.The results showed that compared with the fermented milk of the original strain,the ability of the mutant strain to metabolize lactose was increased by 17.03%,the accumulation of galactose was reduced by 34.3%,and a small amount of glucose accumulated in the late fermentation period.This indicates that the mutant strain metabolizes lactose/galactose better than the original strain.The results of the heatmap clustering analysis of volatile flavour substances in fermented milk showed that there were significant differences between L.delbrueckii subsp.bulgaricus IMAU40078 and its mutant strain in fermented milk,indicating that NTG mutagenesis had an effect on the type and content of volatile flavour substances produced by the strains during fermentation and storage.