Enzymatic Synthesis Of High Polymerization Isomalto-oligosaccharides And Their Functional Evaluation

Isomaltooligosaccharides（IMO）are oligosaccharides mainly composed ofα-1,6glycosidic bonds linked glucose groups.As low-digestible oligosaccharides,IMO have a low glycemic index,which plays a critical role in maintaining blood glucose homeostasis.Owning to their physiological functions such as lowering cholesterol levels and alleviating intestinal chronic inflammation,IMO are widely used in the food and health care market.The current commercial IMO production has low yield and contains high isomaltose content with poor probiotic function.Therefore,this study proposed a new process based onα-glucosidase（AGL）and cyclodextrin glycosyltransferase（CGTase）for the synthesis of high polymerization IMO（IMO^H）,which improves the polymerization degree and yield of the product.The main research contents are as follows:（1）Establishment and optimization of the system for the synthesis of IMO^H.Firstly,the experimental enzymes and reaction conditions for the synthesis of IMO^H by AGL were optimized by single-factor experiments.When AGL from Aspergillus nidulans was used as the production enzyme and 30%(w·v^-1)maltodextrin with dextrose equivalent（DE）value of 15～20was used as the substrate,under the reaction conditions of 50℃,p H5.5,7 U·g^-1 substrate of enzyme addition and 12 h reaction time,the yield could reach 57.13%with up to 95.46%of the high polymerization fraction.By comparing the kinetic parameters of transglycosylation and hydrolysis between the two sources of AGL,it was analyzed that A.nidulans AGL had strong transglycosylation of substrate but weak hydrolysis ability to the substrate and product,which explained its higher product yield in single enzyme synthesis of IMO^H.（2）Optimization of the system for the synthesis of IMO^H through dual enzyme synergistic catalysis and exploration of the process.Based on the results of single-factor optimization,cyclodextrin glycosyltransferase（CGTase）was selected for compounding.Using Bacillus stearothermohilus NO2-derived CGTase（Bs CGTase）as compounding enzyme,when the dosage of enzyme was 10 U·g^-1 substrate and the reaction time was 12 h,the yield could reach80.13%with 97.82%of the high polymerization fraction.The kinetic analysis of the acceptor specificity of Bs CGTase,combined with the molecular docking results,indicated that Bs CGTase could utilize small molecular sugars withα-1,6 bond structures that are not easily hydrolyzed,such as isomaltose and isomaltotriose,as acceptors in disproportionation reaction to improve the polymerization and yield of IMO^H.（3）Structural characterization and physicochemical properties identification of IMO^H.HPSEC,¹H NMR and enzymatic analysis were used to characterize the structure of IMO^H.The results showed that the average molecular weight of IMO^H was 1107 Da,the average polymerization degree was 6.83,and theα-1,6 bond ratio was about 70.63%.The product contained high polymerization components with complex structure.The physicochemical properties of IMO^H were determined and the results showed that the IMO^H product was stable,and no obvious browning reaction occurred in the range of p H 4～6.Meanwhile,in the in vitro anti-digestive performance test,the proportion of anti-digestive component was 57.71%,showing a good anti-digestive performance.（4）In vitro probiotic function evaluation of IMO^H.The effects of IMO^H and commercial IMO50 on the growth status of probiotics,intestinal commensal bacteria and potentially pathogenic bacteria,and the in vitro fermentation performance of probiotics were investigated separately using in vitro pure culture.The results suggested that IMO^H has the potential to create an intestinal environment with richer organic acids and better microbial structure than IMO50.In the in vitro intestinal simulated fermentation research,based on 16S r RNA amplicon sequencing technology,the effects of IMO^H on human fecal microbiota was analyzed combined with various bioinformatics methods.The results suggested that IMO^H has intestinal probiotic function and has the potential to become highly polymerized functional oligosaccharides.