Study On The Modification Of Glutenin And The Stability Of Hesperetin/Hesperidin Delivery System Constructed By Glutenin

Glutenin has the advantages of a wide range of sources,hypoallergenicity and good biological value,and can be used as a main protein in dairy simulants,functional drinks and other protein-rich products.However,glutenin has limited applicability in the food industry due to its poor water solubility and emulsifying properties.Therefore,new strategies are needed to improve the physicochemical and functional properties of glutenin.The complexation of modified glutenin with bioactive compounds（such as hesperetin and hesperidin）can be developed as effective and promising soluble nanocomplexes to improve the dispersibility and stability of hesperetin and hesperidin in aqueous environment.These lays the foundation for the application of hesperetin（Ht）,hesperidin（Hd）and glutenin（Glu）in health food or the optimization of glutenin-based products.In order to improve the function and physicochemical properties of glutenin and construct a delivery system for hesperetin and hesperidin,this study used glutenin as raw material to improve the water solubility and emulsification of glutenin through the strategy of p H-shifting combined with heat treatment and fibrillated modification,and characterized the binding mechanism between glutenin and hesperetin or hesperidin and functional properties of glutenin.Finally,the nanocomposites were applied in emulsion and the stability of the emulsion was studied.The main results are as follows:Both hesperetin and hesperidin quenched the fluorescence of glutenin through a static process and formed Ht-Glu and Hd-Glu complexes at one binding site.The binding constants between glutenin and hesperetin and hesperidin were 4.20×10⁴L mol^-1and 3.59×10⁴L mol^-1at 25 ^oC,respectively.The exposed hydrophobic groups in glutenin formed hydrogen bonds and hydrophobic interactions with hydroxyl groups or glycosides in hesperidin and hesperetin,the secondary and tertiary structures of glutenin were significantly altered.The content ofβ-sheet of glutenin increased,and the carbon chain of glutenin shrank.Moreover,the skeleton structure of glutenin polypeptide chain was more affected by hesperidin than hesperetin.Hesperetin entered into the active cavity of glutenin and interacted with amino acid residues Gln 739,Pro734,Gln 520,Gln 744 and Trp 518,while hesperidin interacted with key amino acid residues Leu 107,Pro 154,Pro 233,Gln 288,Ala 225,Pro 211,Gln 231 through different docking positions.Molecular dynamics results showed that the formation of Ht-Glu and Hd-Glu complexes both enhanced the stability of glutenin.Tyr was more involved in the binding process between hesperetin and glutenin,while Trp was more involved in the binding process between hesperidin and glutenin.After glutenin was combined with hesperetin and hesperidin,the solvent accessible surface area of the glutenin decreased to different degrees,and Ht-Glu<Hd-Glu.Hesperetin and hesperidin induced the exchange reaction between the sulfhydryl group and disulfide bond of glutenin,which increased the free sulfhydryl group content of glutenin,and the increase degree affected by hesperidin was lower than that of hesperetin.Hesperetin and hesperidin changed the conformation of glutenin,exposing hidden hydrophobic region of glutenin and increasing its hydrophobicity.When combined with hesperetin and hesperidin,respectively,the surface polarity of glutenin increased,which improved emulsifying activity of glutenin but decreased emulsifying stability.Hesperetin and hesperidin increased rigid clusters and decreased structural flexibility of glutenin,resulting in improved thermal stability of the protein.Theα-helix ratios of glutenin modified by p H-shifting combined with heat treatment showed an upward trend,and from 4.00±0.51%increased to 29.04±0.06%.In addition,high molecular weight glutenins depolymerized at high temperatures.The combined treatments of p H-shifting and heating disrupted disulfide bonds in protein aggregates and induced conformational changes in glutenin,thereby reducing the size of protein aggregates.Therefore,smaller square clusters can be observed.These changes increased the solubility of glutenin to 85.00±1.99%at p H 7.Compared with untreated glutenin,the combined treatments of p H-shifting with heating increased the solubility of glutenin by 21.3 times.In addition,more hydrophobic groups were exposed in the modified glutenin,and better emulsifying properties were obtained at the same time.The lag period of the fibrillated modification of glutenin was very short,and glutenin entered the self-assembly maturation stage when heated for 8 hours,forming a large number of glutenin fibrils.Moreover,the glutenin fibrils were compact and theα-helix ratio of the treated glutenin was finally increased to 59.90±0.01%.Smooth tubular fibrils of approximately 70-100 nm in diameter and approximately 5μm of glutenin in length were also observed.In addition,the solubility of fibrillated glutenin was also increased to 14.00±0.32%,the fibrillation process increased the solubility of glutenin by 3.5 times compared to untreated glutenin.In addition,the fibrillated glutenin exhibited excellent emulsion stability(EAI:50.84±0.51 m²g^-1)and thermal stability（peak temperature increased from 109.58 to 149.05°C）.Both hesperitin and hesperidin strongly quenched the fluorescence of modified glutenin in a static mode,and formed hydrogen bonds and hydrophobic interactions with the hydroxyl or glycoside of hesperitin and hesperidin.Hydrogen bonds force was enhanced between fibrillated glutenin and hesperitin/hesperidin.The complex binding constants between hesperetin/hesperidin and glutenin modified by combined treatments of p H-shifting with heating were 4.24×10⁴L mol^-1and 3.19×10⁴L mol^-1,respectively,while the binding constants between hesperetin/hesperidin and fibrillated glutenin were 3.73×10⁴L mol^-1and 3.49×10⁴L mol^-1at 25^oC,respectively.The modified glutenin was used to construct the delivery system,and the final process optimized by single factor and orthogonal experiments was as follows:hesperitin/hesperidin:orange peel pectin:fibrillated glutenin 5:2:40,and the p H was 6.In fibrillated glutenin-stabilized emulsion,the solubility of hesperitin and hesperidin was increased to 23.12±0.96μg m L^-1and 64.84±3.21μg m L^-1,respectively(400μg m L^-1added).By complexing with orange peel pectin,the solubility of hesperitin and hesperidin increased to 102.16±4.82μg m L^-1and 220.57±10.82μg m L^-1,respectively.In addition,the fibrillated modification increased the degree of aggregation of glutenin molecules,resulting in greater steric resistance,thereby improving the storage stability of the emulsion.