Enzymatic Tailoring Structures Of Amylopectin And Formation Mechanism Of Low Digestibility Of Strcuture-Modified Amylopectin

As a new functional food,starch with low digestibility has many advantages,such as low digestion rate,low glycemic index,prevention and controling of diabetes,obesity and cardiovascular.Therefore,the preparation and application of low digestible starch has been widely concerned.On the basis of biological enzymes,the precise regulation of physicochemical properties and digestibility of starch can be achieved by the redesigin of molecular structure,and thus it is a way with great development prospect to manufacture novel modified starches in the field of carbohydrate polymers.In this study,amylosucrase and pulullanase were applied to modify the molecular structure of the starch,including the elongation of branch chains and the hydrolysis of branching points.The molecular structure,physicochemical properties and digestibility of the modified starches were evaluated by various technices and the formation mechanism of low digestibile starches are clarified.The formation mechanism of starch-lipid complexes was explored and the in vitro digestion of the complexes was thoroughly studied.The main research results of this work are as follows:The catalytic mechnisam of NpAS in the presence of starch.Waxy corn starxh was treated by dilute hydrochloric acid and the final viscosity of the starch decreased from 2112 cP to 17 cP.The native and acid-thinned starches were used as acceptors for NpAS in order to study the effect of substrate's mobility on the catalytic efficiency of the enzyme.The reaction kinetics suggest that the enzyme exhibited similar catalytic efficiency for both forms of starch,resulting from the inherent catalytic reaction mode of NpAS.The three-dimensional structure of NpAS reveal a non-catalytic binding site within the enzyme,which could enable the formation of the chain-enzyme complex as soon as NpAS was added to the reaction mixture.Afterward,the enzyme continuously elongated the combined chain via a zipper reaction mode,by which the binding interaction avoids the dissociation of NpAS from the complexed chains.Mechanistic insights into the structural modifications of the starch by PL A and NpAS.The amylopectin was modified by NpAS to produce modified starches MS-1,2,and 4.the number-average chain length((?))of the starch was increased from 17.8 of native starch to 19.4 of MS-1,22.3 of MS-2,and 26.9 of MS-4.Then,the NpAS-modified starches were debranched by pullulanase,yielding dual-enzyme treated starches(MSD-1,2,and 4),and((?))of the starches was further increased by 0.5-1.6.Scanning electron microscope(SEM)analysis suggested that the NpAS-modified starches displayed densely packed particles with diameter larger than 50 ?m while dual-enzyme treated starches showed the assembly of nanoparticles with diameter ranging from 200 to 400 nm.The A-type crystalline structure of native starch was changed to B type after NpAS treatment,and the relative crystallinity(Rc)of MS-1 was 21.1%while MS-4 showed a higher Rc(26.8)because of a longer branch chain length.In addition,the subsequent debranching treatment did not change the B-type crystalline structure of the starches,but further increased the Rc of the starch.The rapidly slowly digestible(RDS)and slowly digestible(SDS)contents in native amylopectin were 37.4%and 53.5%,respectively.The NpAS modification improved the RS content in MS-4 at a level of 54.2%whereas the RDS and SDS contents were lowered to 28.0%and 17.8%,respectively.Moreover,PLA treatment further lowered the digestibility of the starches and the highest conent of RS was obtained for MSD-4(67.4%).Digeston kinetics for the digestion of native amylopectin,MSD-1,MSD-2,and MSD-4,and the structural basis for the low digestibility of starches.The digestion process of native amylopectin,MSD-1,MSD-2 and MSD-4 was analyzed by logarithm of slope(LOS).The results suggest that the digestion of native amylopectin showed a single-phase digestion behavior with a rate constant of 0.0259 min-1.For all modified starches(MSD-1,MSD-2 and MSD-4),the LOS plots against digestion time were nonlinear,consisting of two linear phases,each characterized by a rate constant.The first digestion phase displayed high digestion rate constants ranging from 8.48×10-2 to 11.24×10-2 min-1 while the second digestion phase show low digestion rate constants ranging from 1.29×10-2 to 1.85×10-2 min-1.The digesta of MSD-1,2,and 4 was isolated after incubation with digestive enzymes for 20 and 120 min,and the molecular structures and physicochemical properties of the digesta were analyzed by HPSEC,SEM,and SEM.HPSEC results reveal that the long brancn chains were hydrolyzed by digesive enyzmes and anti-digestion fractions show relatively narrowed chain length distribution(DP 21-26).The Rc of the digesta was increased by 18.6-32.9%after 20-min digestion while the digesta show similar value of Rc after further 100-min digestion.In addition,the thermal stablility of the digeat was enhanced with peak(Tp)and conclusion(Tc)temperature increased by 5.3-9.5? and 3.4-6.0?,respectively.Meanwhile,the further digestion slightly lowered the Tp and Tc of the digesta at a magnitude of 2.5-4.5? and 0.7-5.5?,respectively.Overall,it was speculated that the retrogradtion of the starches formed bilayer structure with a outer shell consisting of amorphous materilas and a interior core containing interior crystallites,and that the digestibility of modified starches was mainly affected by the structure of the double helices.The lamellar structure of the MS-1,MS-4,and MSD-4 as affected by NpAS and PLA treatments.Native starch displayed relatively perfect lamellar structure,and its periodic distance(Bragg length)is about 9.8 nm.With the increase of chain length of the modified starches,the Bragg distance increased from 11.4 nm of MS-1 to 13.2 nm of MS-4.Under the same branch chain length distribution,the debranching by pullulanase further increased the Bragg distance of MS-4(15.0 nm for MSD-4).Besides,the SAXS curves of the modified starches were fitted by a two-phase nonparticulate system,and the crystallites thickness of MS-1,MS-4,and MSD-4 was eastimated to be 6.01 nm,7.22 nm,and 8.30 nm,respectively.The crtstallintes of native starch and the modified starches were isolated after mild hydrolysis,and the lamellar structure of native starch could be resolved by transmission electron microscope with a periodic distance of 9.0 nm.Platelet nanocrystals from native starch were about 40-50 nm in size and nanocrystals with sharp 60°acute angles were also observed.The crystallites from the modified starches showed polygonal in shape with a size of 20 nm,and the acute angles ranged from 40 to 60°.The thickness of the crystallites was further analyzed by HPSEC and the results indicate that the thickness of the crystallites from MS-1,MS-4,and MSD-4 was eastimated to be 5.25 nm,7.0 nm,and 8.05 nm,respectively,which was in aggrement with the results of SAXS model fitting.The thickness of crystallites was positively correlated with the peak gelatinization temperature of the starches but negatively correlated with the digestion rate constant of native starch and slow digestion fraction in the modified starches,verifying that the low digestibility of the starches was manily controlled by the structure stability of double helices.Fracbrication and charactertion of stach-lipid complexes:the effect of starch chain length on the physicochemical properties of the complexes.The branch chains of amylopectin were elongated to different extents by amylosucrase(mAP10,mAP20,and mAP60),followed by complexation with sodium palmitate(SP)to produce starch-SP complexes,which could inhibit the retrogradation of the starch at room temperature.The gel strength of starch-SP complexes was dependent on the chain length of mAP and SP content.A longer branch chain length led to a higher gel strength of starch-SP complexes while a higher content of SP could result in a lower gel strength.Besides,the fractal dimension of complex gels was analyzed by Wu-Morbidelli's scaling mode,and the fractal dimension of native starch was deceased after the addition of SP(5.0%).The complexes after dilution showed characteristics of poly electrolytes(-16.3 mv?-36.1 mv)and exhibited nanosized particles with an average diameter ranging from 70 to 1000 nm.Structural analysis implied that the side chains of the starches with efficient length could form left-handed single helices and the hydrophobic SP was entrapped in the helix cavity,leading to the formation of V-type crystalline structure.The V-type crystallity the complexes was increased from 4.7%to 20.1%with starch chain length increasing from 20.9 to 32.6.Moreover,the digestion degree of the complexes was lowered by 6.0%-17.5%due to the formation of V-type crystalline structure.Therefore,the preparation of low digestible starch can be obtained by the addtion of sodium palmitate.Overall,these results may provide theoretical basis and practical evidence to produce modified starches with low digestibility enabling novel applications.