Study On Enzymatic Production And Synthesis Of Cellooligosaccharides And Its Performance In Promoting The Growth Of Intestinal Probiotics

Cello-oligosaccharides are short chain glucose polymers formed by β-1,4-glucoside bonds with D-glucose as monomer,usually referred to as cellobiose to cellodecose.Cello-oligosaccharide is a new kind of prebiotics,which can promote the growth of a variety of intestinal bacteria,and has various physiological functions such as preventing dental caries,promoting bone growth and improving body immunity.It has application potential in the fields of dairy products and probiotics.At present,the main preparation methods of cello-oligosaccharides are: enzymatic degradation of lignocellulose to produce cello-oligosaccharides;Cello-oligosaccharides were synthesized by enzyme conversion.Among them,enzyme conversion is the main method for industrial production of oligosaccharides because of its simple and rapid reaction process,easy purification of end products,easy access to raw materials and low cost.Therefore,the production process of enzymatic conversion of synthetic cellooligosaccharides was established in this paper.A new type of whole cell catalyst was constructed by screening the heat-resistant cello-oligosaccharide synthetase.The multienzyme cascade reaction system was optimized,and the subsequent separation and purification of cello-oligosaccharides were conducted to explore their probiotic function.The main research contents and results are as follows:1.Three kinds of thermostable sucrose phosphorylase,cellobiose phosphorylase and cellodextrin phosphorylase from different sources were cloned in E.coli.By optimizing the enzyme activity,the optimum reaction temperature and the optimum p H of the three phosphorylases,the thermostable cello-oligosaccharides synthetase with excellent performance was obtained.2.The co-expression complex of double enzyme and triple enzyme was constructed by adjusting the sequence of three phosphorylase genes on the vector,and the production cost of cello-oligosaccharases was reduced.In order to improve the synthesis efficiency of oligosaccharides,conversion conditions such as the amount and ratio of enzymes added in the reaction,the ratio of two substrates,reaction temperature,p H and time were optimized.The optimal reaction conditions were as follows: The ratio of sucrose to glucose was 5:2,the reaction p H was 7.0,the reaction temperature was50℃,the ratio of COS 01 to Cc 01 was 10:5,the reaction time was 8 h,and 97 g/L soluble oligosaccharides were obtained.The purity of the purified product was about97%.3.The growth performance of intestinal probiotics promoted by cellooligosaccharides was investigated.Five kinds of intestinal probiotics were selected for growth determination with glucose,cellobiose,inulin,xylo-oligosaccharides(XOS)and fructo oligosaccharides(FOS)as control,except Bifidobacterium longum BL 01,When Lactobacillus casei WSH 004,Lactobacillus paracei WSH 005,and Lactobacillus acidophilus WSH 006 were grown using cello-oligosaccharides(DP 2-6)as carbon sources,the biomass of probiotics increased by about 2 times compared with other prebiotics(FOS,XOS,and inulin).When Bifidobacterium adolescentis BA 03 grew in the medium containing cello-oligosaccharides,the biomass was second only to inulin.In summary,this paper established the production process of enzymatic conversion of synthetic cello-oligosaccharides,which can use cheap substrates glucose and sucrose to efficiently produce high-purity oligosaccharides by multistage enzyme-linked method.This oligosaccharide can promote the growth of probiotics,maintain the balance of human intestinal microecology,and can be used as a potential new prebiotics to promote intestinal health.