Study On The Reaction Mechanism Of Lactobacillus Reuteri 121 Glucanotransferase GtfBdN On Amylopectin And On The Properties Of Modified Starch Products

The product obtained from starch modified by Lactobacillus reuteri 121Glucanotransferase GtfB(GtfB)is a soluble dietary fiber and has prebiotic potential,so it has a good application prospect in the field of starch modification.However,the current research focuses on its conversion mechanism and product properties of amylose,and the mechanism of action on amylopectin,the other important group of starch,has not been confirmed.This also leads to a lack of in-depth understanding of the correspondence between the properties of the products obtained after the overall modification of starch and the mechanism of enzyme action on starch.Therefore,this study has explored the mechanism of its effect on amylopectin and determined the properties of its modified starch products,aiming to provide a basis for better modified starches and the application in food.The N-terminal truncated GtfB(GtfBdN),whose activity and product are the same as GtfB,but whose soluble expression level is greatly improved,was expressed in Escherichia coli BL21(DE3)*and purified by nickel affinity chromatography.The purity and molecular weight of the enzyme protein were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and it was found that the purified enzyme can reach electrophoresis purity with a molecular weight of about 100 k Da.The enzymatic activity of GtfBdN measured by amylose-iodine staining method was 4.12 U/mg.The basic enzymatic properties were characterized,and the optimal reaction conditions were measured to be 40?and p H 5.0.Six kinds of starches including sweet potato starch,tapioca starch,potato starch,pea starch,corn starch and waxy corn starch were modified by GtfBdN,and the structure of the modified products were determined.Nuclear magnetic resonance spectra testing results indicated that the(?1?6)linkages content in the product increased,and the content of(?1?6)linkages in the modified pea starch was up to 31.3%.High efficiency size exclusion chromatography measurement showed that the weight average of molecular weight(M_w)and radius of gyration of the product decreased,and the polydispersity index increased.The M_wof potato starch after modification was 7.2×10⁵g/mol,the decrease was the most obvious.High performance anion exchange chromatography measurement found that the relative content of the B₂chains and B₃chains of the product decreased,but the relative content of the A chains increased.Among them,the relative content of A chains of potato starch increased the most,increasing by 200.0%of the original.The above results indicated that the chain segment of amylopectin from the non-reducing end to the nearest branch point could be acted by GtfBdN.Then GtfBdN was used to act on?-limit dextrin,and it was found that?-limit dextrin could be used as a donor substrate of GtfBdN to produce reducing sugars,which proved that the part of the chain segment of amylopectin from the reducing end to the nearest branch point could also be acted by GtfBdN.Finally,the specific hydrolysis reactions of?-amylase and dextranase were used to explore whether amylopectin could be used as an acceptor substrate of GtfBdN,and it was found that amylopectin could not be used as an acceptor substrate of GtfBdN.That is,amylopectin is a donor substrate of GtfBdN instead of an acceptor substrate,but the chain segments of amylopectin from the non-reducing end to the nearest branch point and the reducing end to the nearest branch point can be used as a donor substrate of GtfBdN.Based on the study of the mechanism of GtfBdN on starch,the properties of the products obtained from six kinds of starches modified by GtfBdN were further explored,including the oligosaccharide production,gelatinization properties,viscoelastic properties,thermal properties and in vitro digestibility.High performance liquid chromatography results indicated that oligosaccharides with a degree of polymerization of 1-4 were formed in the product,of which glucose and maltose were the main ones.The rapid viscosity analyzer,rheometer,and differential scanning calorimetry determination results indicated that the peak viscosity,breakdown and setback of the product were all reduced,and each viscosity value was reduced below 30 cp;the product had transitional viscoelasticity within the test range,but it appeared as a fluid state for a long time;the short-term and long-term retrogradation of product slowed down.The In vitro digestion experiments showed that the content of slowly digestible starch content and resistant starch(RS)content of the product increased,and the RS content of the product derived from pea starch increased the most,which increased by 124.51%.The above results showed that the product derived from starch by GtfBdN modification could inhibit retrogradation and digestion,and was in a fluid state.It can be added to rice cereal,beverages and other foods with application potential.