Study On Mechanism Of Electron Beam Denaturation To Improve Proteolysis Efficiency And Antioxidant Activity Of Rice Protein

Rice protein is a kind of high-quality cereal protein with reasonable amino acid composition,high biological titer and low sensitization.The development of antioxidant peptides by protease hydrolysis can improve the poor solubility of rice protein while retaining its nutritional properties.However,rice protein is hard to be hydrolyzed by enzyme due to it high hydrophobicity,resulting in low yield of bioactivity peptides.Electron beam irradiation?EBI?is a new cold modification technology with strong controllability and high degree of automation.In recent years,it has been found that EBI treatment can change the structural characteristics of proteins.Therefore,this paper intends to study the effect of EBI denaturation technology on the advanced structure of rice protein,enzymatic hydrolysis efficiency and antioxidant activity of rice protease hydrolysate?RPHs?,explore the molecular mechanism of EBI denaturation technology on antioxidant activity of rice protease hydrolysate,and lay a theoretical foundation for the development of high-activity protein peptides by EBI denaturation technology.The main conclusions of this study are as follows:EBI denaturation treatment of rice protein can effectively improve the degree of enzymatic hydrolysis of rice protein and the yield of polypeptide.SEM analysis showed that the intensity rice protein granules were destroyed by EBI and small granules appeared.The FTIR analysis indicated that the primary structure of rice protein was maintained but the secondary structure was changed;the tight ?-helix structure was transformed into the unfolded ?structure and disordered structure,which increases the molecular flexibility of the protein.The UV spectra,fluorescence spectrum and surface hydrophobicity analysis showed that conformational changes occurred because of EBI treatment,exposing more activity groups.The structural change of rice protein induced by EBI increased the surface area of rice protein and released more restriction site,leading to improvement of hydrolysis efficiency.The hydrolysis efficiency and peptide yield of rice protein was improved by EBI treatment and the degree of improvement was different between RPHs obtained with various proteases,in decreasing order of Alcalase>Protamex>Neutrase>trypsin.The DH and peptide yield of RPHs obtained with Alcalase were increased by19.02±0.37%and 13.5±0.29%,respectively.EBI denaturation treatment had a significant effect on the processing characteristics and antioxidant properties of rice protein hydrolysates.EBI pretreatment improved the emulsifying capacity and emulsifying stability of RPHs obtained with Neutrase,but not for RPHs obtained with Trpsin;the emulsifying capacity and emulsifying stability of RPHs obtained with Alcalase and Protamex increased with the increase of EBI treatment dose,and then decreased to the original level.EBI pretreatment could improve the foaming capacity of all RPHs?except alkaline protease hydrolysates?,but reduce the foam stability.On the other hand,with the increase of EBI treatment dose,the antioxidant activity of RPHs hydrolyzed by different proteases showed a trend of gradual enhancement and then flattening in different in vitro chemical systems.Among them,the product of EBI pretreatment coupled with alkaline protease hydrolysis has the highest antioxidant activity.However,excessive EBI treatment dose?40 kGy?destroys some amino acids and reduces the nutritional value of rice protein.Therefore,it is not possible to continuously increase the EBI treatment dose in pursuit of high activity.EBI denaturation treatment improved the antioxidant activity of modified rice protease hydrolysate?ERPHs?in cells.CAA test results showed that EBI denaturation treatment can significantly improve the antioxidant capacity of ERPHs.When the EBI treatment dose was 30kGy,the EC50 value of RPHs decreased from 1.18±0.05 mg/mL to 0.78±0.01 mg/mL,and the CAA value increased from 7.58±0.19?mol QE/g to 11.46±0.22?mol QE/g.A H₂O₂-induced oxidative damage model was constructed,the H₂O₂ concentration was 0.4?mol/L and processing time was 4 h.This model was used to investigate the inhibitory effect of ERPHs on cell oxidative damage.The results showed that ERPHs could effectively inhibit cell damage caused by oxidative stress.When EBI denaturation treatment dose was 30 kGy,the cell survival rate increased from 50.66 1.72% to 89.62 1.63%.The antioxidant mechanism of ERPHs was elucidated using the cell oxidative damage model:?1?ERPHs could remove excess ROS in cells,interrupt free radical chain reaction,maintain the balance of intracellular REDOX,and reduce the damage caused by oxidative damage to cells;?2?ERPHs could improve the activity of SOD,CAT,GSH-Px and GSH-Rx in cells,and protect cells by improving the antioxidant enzyme defense system of cells;?3?ERPHs could reduce the level of MDA in cells,inhibit the release of LDH,maintain the integrity of cell biofilm and improve cell survival rate by inhibiting lipid peroxidation.In addition,the effects of ERPHs on mitochondrial membrane potential?MMP?,caspase-3 activity and apoptosis rate were investigated.It was proved that ERPHs could effectively maintain MMP stability and reduce caspase-3 activity,thus inhibiting apoptosis caused by oxidative damage.When EBI degeneration treatment dose was 30 kGy,the apoptosis rate of cells decreased by 62.60%compared with the model group.The change of ERPHs structure can significantly affect the oxidation activity.EBI treatment changed the molecular structure of rice protein,exposed more enzyme cutting sites and decomposed rice protein into smaller peptides,leading to increase of the content of antioxidant amino acids in ERPHs.Furthermore,in the ERPHs treated by EBI,the?-helix structure was opened,and the disordered and loose structure was increased,which reduced the steric hindrance when interacting with free radicals and achieved the purpose of scavenging free radicals more effectively.The spatial structure analysis showed that more aromatic amino acid residues and hydrophobic groups in the ERPHs treated by EBI were exposed to the environment with a more hydrophilic surface and played an antioxidant role.The increase of Zeta potential value makes RPHs tend to disperse in the solution and the decrease of average particle size,thus increasing the effective concentration of active groups in the solution and increasing the antioxidant capacity of ERPHs.