Investigation Of The In Vitro Enzymatic Hydrolysis Mechanism Of Breadfruit Starch Self-assembled Ternary Complexes

During the thermal processing of tropical starch,starch self-assembled ternary complexes are formed,which can affect the physicochemical and digestive properties of the products.However,there is limited research on the digestion kinetics mechanism of highly branched tropical starch ternary complexes.Therefore,based on the relationship between the digestive properties of tropical starch and its multi-scale structure,this study selected breadfruit starch as the raw material to prepare self-assembled ternary complexes for further investigation.The extracted starch resources include locally grown the Spice and Beverage Research Institute species grafted on the jackfruit tree breadfruit,fluffy Spice and Beverage Research Institute species variant breadfruit,Shimei mountain species breadfruit,Comoros species breadfruit,Samoa species breadfruit,jackfruit seed,avocado seed,chempedak seed,Pouteria campechiana seed,sweetsop pulp,Musa ABB/MA,Musa AA Pisang Mas/MAM,Musa ABB Pisang Awak/MAA,Musa Basjoo Sieb/MBS,and Musa AAB-Prata/MAP all of which meet the standards of commercial starches that can be industrialized for edible purposes.The studied samples are all high-amylose starches and have good modification potential.The study found that all breadfruit starches and sweetsop pulp starches have a B-type crystal structure.MA,MAM,MBS,avocado seed,chempedak seed,Pouteria campechiana seed,jackfruit seed starches have an A-type crystal structure.MAA and MAP have a CA-type crystal structure.Based on the difference in glycemic index（EGI）,Xiangyinso breadfruit 13,banana,and avocado are classified as low EGI starches,while soft breadfruit,mountain apple breadfruit,and custard apple starches are medium EGI foods,and other starches are all high EGI starches.Further exploration found that low EGI starches benefit from their long branched cluster of long branch chains,which can self-assemble into dense microcrystals and relatively more long double helix structures.At the same time,the high content of straight-chain starch helps to form a V-shaped multi-crystal structure,thereby enhancing the electrostatic attraction between glycosidic bonds and the hydrogen bond force between molecules,making the electron cloud density between the semi-crystalline layers higher and the hydrogen bonding adsorption force between starch molecules stronger.Therefore,the morphology of low EGI starch particles is more compact,requiring more thermal energy and more digestive enzymes to degrade the molecules.This study provides a theoretical basis for the processing of non-traditional starch-based products.In addition,the preliminary research of this experiment found that five types of breadfruit starch belong to low,medium,and high EGI index starch,and the structural digestion characteristics differ greatly.They are representative for the same genotype but different types of fruit starch.Therefore,different types of fruit starch with the same genotype were selected as the control variable to study the comprehensive analysis of digestion characteristics and molecular structure when complexed with fatty acids and proteins.This aims to explore the mechanism of the differences in physicochemical properties of three-component complexes of different EGI index starches.Based on this,this study comprehensively analyzed the high-amylose（HA）seedless breadfruit starch from the Spice and Beverage Research Institute species grafted on the jackfruit tree（GJS）,fluffy Spice and Beverage Research Institute species variant（FSS）,Shimei mountain species（SMS）,Comoros species（CS）,and Samoa species（SS）,and endogenous protein-endogenous lipid complexes extracted by isoelectric deposition and supercritical headspace solid-phase microextraction.Based on the significant differences in resistant starch（RS）values,the natural starch samples were divided into high RS group（GJS,FSS,SMS）and low RS group（SS and CS）for convenience.The I and IIb type ternary complex peaks appeared in the FTIR and DSC spectra.The crystal type of all starches changed from type B to type V after complexation.In addition,compared with the low RS group,the high RS group showed significantly higherα-1,6-glycosidic bond,free side chain CH2OH cluster,double helix conformation,V-type multi-crystal,composite index（CI）,short-range order,relative crystallinity（Rc）,thermal decomposition temperature,weight average molecular weight,molecular density index value,gel hardness,final viscosity（p<0.01）,decreased semi-crystalline layer thickness,rotation radius（Rg）,"stopper structure"size（Rq）,porosity,swelling force,solubility,and gel brittleness,making their particle nanofractal structure uniformity higher and roughness lower.There are fewer nano-enzymatic cleavage sites near the particle surface,leading to a lower multi-level enzymatic rate constant（k₁ and k₂）and predicted enzymatic equilibrium concentration.Based on Circos,neural network,PCA,and other analyses,these results can be attributed to the natural high RS group starch having a more dense and ordered superhelix cross-linking network matrix of branched-straight chain starch compared to the low RS group.In addition,due to the lower spatial hindrance of the branched starch in the natural low RS group starch itself,the flexible interval is larger,so that the branched free long side chains and straight chain starch can complex with ligand residues.Moreover,some branched chains will produce superconjugated effects,which induce electrostatic interaction with short chains of straight chain starch and truncate them,resulting in an increase in branched starch B1 chain,C chain,Rc,and Rg,and a decrease in Rq and amorphous phase content after complexation.Therefore,compared with the original starch,the enzymatic cleavage sites of low RS group starch complex are unchanged,and the digestion kinetics are reduced.The natural high RS group starch in the complex process has a tightly ordered molecular nano-microstructure destroyed,particle morphology is destroyed,and the main chain of branched starch is sheared,resulting in a significant decrease in molecular weight,leading to the enzymatic cleavage site changing from near the surface to the center of the Maltese cross,thereby increasing the digestibility after complexation.This study provides theoretical guidance for the industrial production of multi-nutrient component foods.As the investigation of the above ternary complexes only analyzed the mechanism of digestion kinetics through macrostructural changes,there has been no research on the nanoscale basic structure of the refined structure.Therefore,this study explores the influence of the degree of polymerization（DP_w）of straight chain starch on the digestion kinetics of recombinant starch-lauric acid-β-lactoglobulin ternary complexes,aiming to identify the most important structural factors.This study discusses the impact of the nanoscale hierarchical polymerization index（DP_w）of straight chain starch on the logarithmic slope（LOS）model of the digestion kinetics mechanism of the recombinant starch-lauric acid-β-lactoglobulin ternary complex.In this study,the five types of high amylose（HA）breadfruit starch and GJS with the highest DP_w and RS were mixed in a ratio of 1:1 at high temperature to prepare recombinant starch ternary complex samples with different DP_w.The results showed that all the complex samples were similar to those in abstract（2）and showed a V-type crystal structure and a rod-shaped molecular configuration.The XRD,DSC,FITR,and GPC-RI spectra of the ternary complexes all exhibited significant ternary complex characteristic peaks,confirming that recombinant starch can also be complexed with ligands.In addition,as the DP_w of straight chain starch increased,the CI,Rc,short-range order,average molecular weight,molecular density index,gelatinization temperature,thermal decomposition temperature,RS,slowly digestible starch（SDS）,and rate constant of the second stage of enzyme digestion（k₂）all increased,while the semi-crystalline layer thickness,mass fractal structure parameter,characteristic crystal cell unit average length,Rg,Rq,nanoscale particle surface fractal structure difference,roughness,number of surface cavities of the particles,final viscosity,rate constant of the digested fragment from SDS to RS stage(k _SDS),equilibrium concentration,and EGI decreased.Based on chemometric analysis（Circos,neural networks,PCA,etc.）,there was a very significant correlation（r>0.99 or r<-0.99,p<0.01）between the first-order split digestion kinetics parameters,physical properties,and multiscale supramolecular structures.In summary,the experimental results confirmed that the DP_w of straight chain starch is an important structural factor affecting the digestion kinetics and mechanism of the ternary complex,and provided a new theoretical direction for the production of multi-nutrient component foods based on starch.