Enzymatic Synthesis Of Isomalt/malt Polysaccharide And Its Properties

The daily diet of Chinese residents is mostly rice,noodles and its products.Its main ingredient is starch,which is the main source for body’s energy intake,and it has a high glycemic index.Long-term consumption will increase the risk of some chronic diseases such as diabetes and high blood pressure.Dietary fiber can effectively interfere with the health of the body.Through enzymatic modification,starch and its hydrolysate can be used to synthesize dietary fiber.Glucosyltransferase（GTase）has the function of transglucosylation on starch and its hydrolysates.It can convertα-1,4 glycosidic bonds into other glycosidic bonds,thereby enhancing the anti-digestibility of starch.In this study.In this project,the enzymatic properties of GTase derived from Lactobacillus reuteri 121 will be studied.By analyzing the modification effect of GTase on different starches/maltodextrins,maltodextrin（DE12）（MD）was selected as the most suitable substrate.Then MD was used as the substrate to prepare the product（GPMD）.The preparation process was monitored and its physical and chemical properties were characterized.Finally,its intestinal probiotic function was evaluated by simulated fermentation in vitro.First of all,in this study,the GTase gene derived from L.reuteri 121 was heterologously expressed to obtain GTase.The enzymatic properties of the purified GTase were studied.The results showed that the relative molecular mass of GTase derived from L.reuteri 121 was 105k Da.The optimum temperature and p H were 40°C and 5.0,respectively.The enzyme is stable between p H 7.0 and 8.5.Further studies have found that Ca²⁺has a significant activation effect on the GTase enzyme activity derived from L.reuteri 121,and 1 mmol/L Ca²⁺can make enzyme activity increased about 2.1-fold;The K_m and V_max of GTase to amylose were 1.18 g/L and 2.6×10^-6 g/s,respectively.Secondly,maltodextrin（DE12）,potato starch（PS）,corn starch（MS）and waxy potato starch（WPS）were used as substrates and modified by GTase derived from L.reuteri 121.The structure of the four modified products was analyzed,and the results showed that GTase derived from L.reuteri 121 had hydrolysis and transglycosidation effects and was more likely to act on linear glucose chains.The modified product is an isomalt/malt-polysaccharide（GPMD）,in whichα-1,6 glycosidic bonds exist in the form ofα-1,6-glc-α-1,4 structure,linearα-1,6 glycosidic bond andα-1-4,6 branching points.By comparing the structures of the four modified products,MD was selected as the most suitable substrate for GTase.Subsequently,isomalt/malt-polysaccharide（GPMD）was prepared with MD and analyzed the structure of GPMD during the modification process by HPLC,HPSEC-MALL-RI,and enzyme fingerprinting.The results showed that the smallest unit of action of GTase is maltotriose;The relative molecular weight of GPMD was reduced from the initial 1.2×10⁵ g/mol to 6.8×10⁴g/mol.The content ofα-1,6 glycosidic bonds was increased from 3.4%to 41.8%;The content of digestible starch（RDS）was reduced from 83.95%to57.99%,and the content of Slow Digested Starch（SDS）and Resistant Starch（RS）increased from 1.16%and 14.89%to 10.13%and 32.53%,respectively.It was found that the digestibility of GPMD was 79.85%,which was about 15.62%lower than that of MD.Then the physical and chemical properties of GPMD were characterized,the results showed that GPMD is anα-polysaccharide with an average particle size of 92.09 nm,which is a nano-sized particles.The rheological properties of GPMD was analyzed,the results showed that the GPMD solution is shear thinning pseudoplastic fluid.Using differential scanning calorimeter and thermogravimetric analyzer to analyze the thermodynamic characteristics of GPMD,it was found that GPMD will undergo strong thermal degradation at 170℃,and the phase transition temperature is 129.05℃.Analysis of the temperature and p H stability of GPMD showed that GPMD remains stable in the environment of p H 4.0～9.0 and 4～70℃.Finally,the intestinal function of GPMD was evaluated by in vitro simulated fermentation experiment.The results showed that compared with fructooligosaccharides（FOS）and inulin,GPMD can promote the reproduction of intestinal flora and promote the production of short-chain fatty acids（SCFAs）,and create a low p H environment for the gastrointestinal tract.It showed that GPMD has the function of intestinal probiotics and can regulate the health of the body.It is a new type of water-soluble dietary fiber and is expected to be a prebiotic.