Screening Of Lactic Acid Bacteria With High Yield Beta-galactosidase Producing And Cloning And Expression Of The Enzyme

With the improvement of people’s living quality, milk and dairy products become one important part of people’s diet. They provide abundant protein, vitamins and trace elements which play an important role in human health. However, some people may have abdominal distension, abdominal pain, bowel exhaust, diarrhea and other symptoms during or after drinking milk or dairy products, which was called lactose intolerance in clinical. People suffering from lactose intolerance cannot eat directly or can eat only a small amount of milk and dairy products which has brought great trouble to human life.There are three types of lactose intolerance:congenital lactose intolerance, primary lactose intolerance （also known as adult beta-galactosidase deficiency）, secondary lactose intolerance. According to different causes, the solution is different. Generally, people with lactose intolerance are treated with two main therapeutic method. First, people can improve food habits who can absorb a certain amount of lactose every day. After a period, they can gradually increase lactose intake, together with other lactose-free diets. This method can alleviate symptoms of lactose intolerance. Data showed that, the first method may gradually raise the awareness of lactose tolerance value to a maximum intake of 6g. Second, to product dairy products with low lactose. Enzyme method, biological method and mechanical ultrafiltration are usually used. The first method is simple and easy, but it cannot solve the problem fundamentally; and the second method can avoid large intake of lactose which can effectively solve the lactose intolerance.In this study, the second method was selected to look strains breaking down lactose. Generally, strains with beta-galactosidase production ability were also able to reduce the content of lactose in dairy products. Beta-galactosidase production lactic acid bacteria were screened by microbial culture method combined with X-gal experiment. Then, beta-galactosidase gene was cloned and expression by gene engineering technique to investigate enzymatic properties and enzyme production conditions.Fermented soybean samples were collected in Gejiu, Jianshui, Lvchun, Honghe, Yuanyang and Kaiyuan in Yunnan province. A total of 23 strains with high production of beta-galactosidase were screened. Total DNA was extracted using a modified CTAB method and16S rRNA gene was amplified. The 16S rRNA gene sequences were aligned with 16S rRNA gene sequences with annotation information in GenBank database. The 23 strains could be assigned to six species:Lb brevis、P. acidilactici、Lb. plantarum、E.faecium、P. pentosaceus、 W.confusa.The beta-galactosidase activity of all 23 strains were determined and strains GJ₁₃ had the highest beta-galactosidase activity of 5.28 U/mL. GJ₁₃ was identified as Lb brevis by 16S rRNA gene sequence. Beta-galactosidase gene in GJ₁₃ was heterogeneous expression in E. coli BL21 and enzyme activity reached to 50U/mL which was 7 times higher than in the original strain.