Study On Structural Modification And Physicochemical Properties Of High-temperature Peanut Protein By Cold Plasma

High-temperature peanut meal is a by-product obtained from peanut oil production using high temperature pressing.Due to the comprehensive effect of high temperature and pressure during the processing,40%-55%of peanut protein contained in peanut meal is highly denaturated,which leads to its reduced physicochemical property,especially in poor solubility,and difficult to be recycled.Therefore,how to improve the hydration characteristics of highly-temperature peanut protein isolate（HPPI）is of great significance to realize resource recycling.This work is based on the question of poor hydration characteristics in HPPI,using cold plasma technology（CP）,combining high-speed shear,introducing reductive disaccharides and polysaccharides,respectively,to modify the structure of protein.The modification mechanism was discussed by characterizing the changes of structure,internal group,chemical bond,free amino acid,crystal structure and action site for the modified protein.Finally,the modified HPPI was applied to prepare functional oil-in-water emulsion,so as to realize the high-value utilization.The high denaturation of HPPI led to cross-linking and aggregation among its molecules.Therefore,the high-speed shear technology was first used to disperse and homogenize the HPPI molecules.The results showed that particle size of HPPI was decreased from 502.67±5.69 nm to 313.67±2.52 nm after shearing at 12000 rpm for 15min,and increasing the protein surface area.Meanwhile,during the shearing process,the tertiary structure and apparent structure of HPPI was gradually depolymerized and extended accompanied by exposing more hydrophobic amino acids to protein surface,and the thermodynamic stability was decreased.The secondary structure of HPPI was also changed from compact to loose after shearing,which was related to the improved molecular flexibility.In addition,the increase content of C-H,-OH and-NH₂ bonds and groups on the surface of protein improved the polarity of the solvent environment.Thus,the soluble protein concentration in high-temperature peanut meal,the water binding ability of HPPI,the stability of protein emulsion and viscoelasticity of protein solution were enhanced by shearing.Based on the expansion of HPPI molecular structure by shearing pretreatment,the surface structure of HPPI was further modified by CP to improve its interface hydration characteristics.The results showed that the soluble protein concentration in high-temperature peanut meal was increased from 0.22±0.01 mg/m L to 1.02±0.03mg/m L at 70 W for 2 min.Meanwhile,the number of hydration molecules on HPPI surface was decreased,which signified the improved hydration characteristics.The improvement reasons of hydration characteristics in HPPI were mainly divided into the following aspects:（1）The tertiary structure of HPPI after CP treatment showed loose conformation,and the protein molecular flexibility was improved by introducing NH₄⁺ions to the surface of protein;（2）The high-energy particles produced by CP etched the surface of HPPI,thus improving the roughness and expanding the area of interaction with water molecules;（3）CP treatment increased the content of hydrophilic amino acids and groups（including-COOH,-CO,-OH）on the surface of HPPI;（4）The decrease of sulfhydryl groups and the increase of dityrosine and oxygen in HPPI indicated that the protein structure was affected by oxidation during CP treatment;（5）CP treatment increased the surface free energy of HPPI,and accelerated the formation of hydrogen bond and electrostatic interaction between HPPI and water molecules.Additionally,CP treatment promoted the formation of crystal structure,which was closely related to the formation of its ordered secondary structure.Additionally,increasing the number of hydrophilic groups to the surface of protein was an important means to improve its hydration characteristics.Therefore,it was intended to use CP treatment to induce the covalent binding between hydrophilic reducing sugar-lactose and HPPI molecules,and the mechanism of action was discussed.The results showed that the structure of HPPI presented a dynamic process of depolymerization,binding,re-depolymerization and re-binding when it was combined with lactose under CP treatment.The degree of grafting between lactose and HPPI reached 18.89±0.31%at 80 W for 3 min,while the degree of browning in glycosylated products was decreased.Otherwise,the soluble protein concentration in glycosylated products increased from 0.61±0.02mg/m L to 1.34±0.04 mg/m L（90 W,3 min）,which meant that the protein hydration characteristics were improved.Due to the covalent binding of lactose molecules to the surface of HPPI,the particle size of the glycosylated products was increased,the surface hydrophobicity was decreased,and forming extended tertiary structure and stable ordered secondary structures.It was found that the amino acids,including Glu,Gly and Ala,etc,were involved in the maillard reaction induced by CP.Also,5 peptides including 7 graft sites（Lys and Arg）involved in the reaction were detected in the glycosylated products,which laid a foundation for the directed modification for HPPI.In order to further explore the effect of reducing sugar types on covalent binding,sesbania gum（SG）,a reducing polysaccharide,was selected to participate in the CP-induced reaction with HPPI molecules.The results showed that the degree of grafting was reached to 15.57±0.38%at 80 W for 3 min,and forming the final products of Maillard reaction.The soluble protein concentration in glycosylated products increased from 0.61±0.02 mg/m L to 1.58±0.01 mg/m L（70 W,3 min）.Compared with lactose,the covalent binding of SG to the surface of HPPI required lower sugar addition and energy consumption.Due to the strong steric hindrance between SG molecules,the order of secondary structure and tertiary structure of the glycosylated products were decreased,and therefore providing favorable conditions for the interaction of water molecules with the loose peptide chain structure.In addition,hydrophilic amino acids and aromatic amino acids in HPPI were the main reaction sites involved in CP-induced Maillard reaction,which also indicated that the type of reducing sugar has different effects on the occurrence of the reaction.The antioxidant capacity of the glycosylated products was improved.Finally,in view of the good hydration characteristics of CP-induced HPPI and SG glycosylated products（glycoproteins）,the expression of functional properties of HPPI and glycoprotein emulsions under different protein addition and their application in embeddingβ-carotene delivery were further investigated.The results showed that the glycoprotein emulsion had better stability,smaller droplet structure and stronger resistance to environmental changes than HPPI emulsion by forming a double-layer interface structure at the oil-water interface.The rheological behavior of the emulsion showed that the glycoprotein emulsion has a semi-solid gel state and thus has a more stable conformation.Whenβ-carotene was encapsulated by emulsion with 5%protein addition,the encapsulation rates of HPPI emulsion and glycoprotein emulsion were both exceed 97%.The release rate ofβ-carotene in glycoprotein emulsion was reduced from 67.32%to47.19%during simulated gastrointestinal digestion,realizing the sustained release effect ofβ-carotene in intestinal tract.At the same time,glycoprotein emulsions improved the bioaccessibility and the stability to transformation ofβ-carotene in the intestinal tract,which laid a foundation for its inclusion and delivery as a carrier for functional components.