Study On Enzymatic Modification Of Starch Structure And Properties

Starch is the typical component of the staple food for chinese residents?rice or noodle?and the main source of energy intake.The quality of starch directly affects the health of the body.In order to improve the quality and anti-digestibility of starch,enzymatic methods are used to modify starch.Glucanotransferase?GTase?is responsible for catalyzing transglycosylation reaction to break?-1,4-glucoside bond and form a new glucoside bond.This study focused on the enzymatic properties of GTase from Azotobacter chroococcum ATCC4412,and explored the mechanism of GTase conversion starch chain,in order to prepare indigestible dextrin maltodextrin?MD?was selected as substrate.The physical and chemical properties of indigestible dextrin and the effects of Caco-2 cells on glucose absorption and intestinal probiotic function were evaluated.First,the gene of GTase derived from A.chroococcum ATCC 4412 was cloned and expressed using molecular biological methods and the enzymatic properties were studied,and the results show that the optimum pH of GTase was 6.5-7.0,and the optimum temperature was55°C,the melting temperature was 62°C and 1 mM Ca²⁺activated the activities by approximately 2.4 times.The enzyme activity of GTase followed the kinetic behavior of Michaelis-Menten,and the catalytic efficiency(k_cat/K_m)is 141.97 L/?g·s?.Second,sucrose,maltose,maltodextrin?MD?,maize starch?MS?,waxy maize starch?WMS?and high-amylose maize starch?HMS?were modified by GTase.TLC analysis showed that GTase could not act on sucrose and maltose.The structure of different products was compared,indicating that GTase has the activity of hydrolysis/transglycoside,and the GTase is more likely to act on linear glucose chains.The structure of the GTase-treated starch was deduced as relatively highly branched glucan,consisting of maltooligosacharide element including maltose,maltotriose,maltotetraose and maltopentaose etc.linked by?-1,6-glc-?-1,4struce and?-1-4,6 branching points by enzyme fingerprint spectroscopy.The mechanism of GTase conversion starch chain was clarified,and the MD was selected as the most suitable substrate for the GTase.Third,preparation of indigestible dextrin with MD as substrate.During the preparation process,the content of?-1,6 glycosidic bonds in GMD gradually increased to 33.33%,the content of slowly digestible starch?SDS?and resistant starch?RS?also increased from 9.01%to 36.94%.Subsequently,a series of physical and chemical properties of indigestible dextrin were measured.It was showed that the average particle size of indigestible dextrin was 76.10nm,which suggested the indigestible dextrin is a typical nanoparticle.Indigestible dextrin of different concentrations shear thinned within a certain shear range,suggesting that the indigestible dextrin solution is pseudoplastic fluid.Thermogravimetric analyzer?TGA?and differential scanning calorimeter?DSC?were used to analyze the thermodynamic properties of indigestible dextrin,indicating that indigestible dextrin has good thermal stability.Indigestible dextrin was stored in different pH buffer and temperatures analyzed its stability,it was found that indigestible dextrin is easily acidolyzed in buffer of pH 3.0,and stored under other conditions,indigestible dextrin has good stability.Fourth,the effect of indigestible dextrin on the uptaken and glucose transport of Caco-2cell was studied.It was verified that 5 mg/mL indigestible dextrin can reduce the expression of two glucose transporters:SGLT1 and GLUT2 in Caco-2 cells.At 24 h of glucose uptaken experiment,the absorption of glucose by Caco-2 cells decreased 25.28%;at 5 h of glucose transfer experiment,the transfer rate of Caco-2 cells to glucose decreased 17.29%.This indicates that indigestible dextrin has the potential to maintain the post-prandial blood glucose homeostasis.In vitro fermentation experiment,the results showed that the bacterial concentration of the fermentation broth in the indigestible dextrin group was 1.411 times,1.274times,and 1.162 times compare with fructooligosaccharide?FOS?,galactose oligosaccharide?GOS?,and inulin groups,respectively;the short chain fatty acid?SCFA?content of indigestible dextrin group was 2.821 times,2.780 times and 2.617 times compare with GOS,FOS and inulin groups,respectively.The results showed that indigestible dextrin can promote the reproduction of enteric microorganism and the specific production of SCFA to play its probiotic function.