Study On Efficient Preparation Of Antioxidant Peptides From Silkworm Pupa Protein By Multifrequency Ultrasound Treatment

The protein content of silkworm pupae is about 60%,which is a good source of protein.There are few studies on the physicochemical properties and antioxidant activity of silkworm pupae protein after multi-frequency ultrasonic treatment.Therefore,in this thesis,the extraction rate and physicochemical properties of silkworm pupae protein were improved by comparing single-frequency,dual-frequency and triple-frequency ultrasonic treatment of silkworm pupae powder.The extraction kinetic model was used to describe the solubilization process of silkworm pupae protein treated with different frequency ultrasonic waves,and the mechanism and law of different frequency ultrasonic waves affecting the extraction of silkworm pupae protein were investigated in depth.The effect of tri-frequency ultrasonic treatment on the enzymatic properties of silkworm pupae protein was further investigated to improve the hydrolysis efficiency of silkworm pupae protein and the antioxidant activity of hydrolysis products.The main research contents and results are as follows:（1）Using silkworm pupa powder as raw material,different frequencies of ultrasound are used to treat silkworm pupa powder to achieve efficient extraction of silkworm pupa protein.Explore the feasibility of improving the extraction rate of silkworm pupa protein using ultrasound.In the ultrasound frequency combination of three frequencies of 22/28/40k Hz,the extraction rate of silkworm pupa protein was 39.98±0.13%,while the extraction rate of the control group was 21.02±0.64%,an increase of 90.20%.At the same time,establish extraction dynamics models based on logarithmic models,second-order rate models,Peleg models,and power-law models.The results showed that the linear correlation coefficients of the logarithmic model,power-law model,and Peleg model were 0.907,0.870,and 0.843,respectively.The linear correlation coefficient of the second-order rate model was 0.999,which is higher than the other three mathematical models.Therefore,the second-order rate model is more suitable for predicting the effect of ultrasonic extraction of silkworm pupa protein process.（2）Using triple-frequency ultrasonic treatment to extract silkworm pupae protein,the effects of different ultrasonic frequencies on silkworm pupae protein were evaluated from the perspectives of secondary structure,physicochemical properties and antioxidant activity of silkworm pupae protein.The results showed that triple-frequency ultrasound significantly reduced theα-helix content（p<0.05）.Meanwhile,ultrasound exposed the hydrophobic groups of silkworm pupae protein,which increased the surface roughness of silkworm pupae protein and increased the surface area of silkworm pupae protein,which facilitated the contact between enzyme and substrate and promoted the enzymatic reaction.Compared with the control,The DPPH clearance rate,Fe²⁺chelation rate,and reducing power of silkworm pupa protein treated with tri-frequency（22/28/40k Hz）ultrasound increased by 57.11%,52.99%,and 73.07%,respectively,and the solubility increased by115.27%.The emulsifying ability of dual frequency（28/40k Hz）ultrasound increased by44.23%,and the emulsifying stability increased by 45.02%.This indicates that the physicochemical properties and antioxidant activity of silkworm pupa protein have been significantly improved.Therefore,multi frequency ultrasound treatment is an excellent method for improving the physicochemical properties of silkworm pupa protein.（3）A triple-frequency ultrasonic treatment catalyzed enzymatic digestion process of silkworm pupae protein was established.The process conditions of single-frequency,dual-frequency and triple-frequency ultrasonic treatment for the enzymatic digestion of silkworm pupae protein were screened with the degree of hydrolysis as the index.The highest hydrolysis degree and the best enzymatic effect of triple-frequency ultrasonic treatment of silkworm pupae protein were obtained.The effects of different substrate concentrations,enzyme concentrations,temperature and p H on the hydrolyzed protein concentration of silkworm pupae protein were investigated.At the enzyme concentration of 0.2 g/L,substrate concentration of 2 g/L,enzymatic digestion temperature of 50℃,p H of 9,enzymatic digestion time of 60 min,and the combination of tri-frequency 22/28/40 k Hz,the hydrolyzed protein concentration was 0.27±0.02 g/L,and that of the control group was0.23±0.01 g/L,which increased by 20.89%.The results showed that the triple-frequency ultrasound significantly improved the enzymatic efficiency of silkworm pupae protein.Meanwhile,the effect of triple-frequency ultrasonic treatment on the kinetic parameters of enzymatic digestion of silkworm pupae was explored,and the correlation coefficients of the second-order rate models were all above 0.85,with most of them as high as 0.99.Therefore,the second-order models were suitable for the prediction of the results of the analysis of the enzymatic digestion process of silkworm pupae.Meanwhile,the effect of triple-frequency ultrasonic treatment on the thermodynamic parameters of silkworm pupae proteolysis was explored.Compared with the untreated group,the K_mvalue decreased by 26.1%and K_Aincreased by 7.3%after triple-frequency ultrasonic treatment.The improved affinity between the enzyme and the substrate can promote the occurrence of the enzymatic reaction of silkworm pupae protein,thus improving the enzymatic efficiency of silkworm pupae protein.The activation energy（E_a）,activation enthalpy（△H）,activation entropy（△S）and activation Gibbs free energy（△G）of silkworm pupae protein were reduced by 21.94%,24.71%,2.89%and 4.54%,respectively.Thus,ultrasound enabled the enzyme to hydrolyze the silkworm pupae protein with less energy.（4）The physicochemical properties and antioxidant activity of silkworm pupae protein hydrolysis products after triple-frequency ultrasound treatment were investigated.After the triple-frequency ultrasonic treatment,the enzymatic hydrolysis products of silkworm pupae protein became smaller molecular weight peptides and the surface hydrophobicity of silkworm pupae protein hydrolysis products was enhanced.The thermal stability was also enhanced,and the enthalpy of thermal change（ΔH）increased significantly from 2.68±0.22J/g to 10.54±0.09 J/g,which was 2.93 times higher.The results of atomic force microscopy scans illustrated that the surface roughness of the hydrolyzed products of silkworm pupa protein increased after the treatment.This shows that the triple-frequency ultrasonic treatment changed the spatial structure of the silkworm pupae proteolytic product,which in turn affected its physicochemical properties.Meanwhile,compared with the untreated group,the IC₅₀values of DPPH scavenging and Fe²⁺chelating power of the silkworm pupae proteolytic product decreased by 12.8%and 9.2%,respectively,and the reducing power of the silkworm pupae proteolytic product increased by 7%.Therefore,the physicochemical properties and antioxidant activity of silkworm pupae proteolytic products were enhanced after triple-frequency ultrasonic treatment.In conclusion,in this thesis,antioxidant peptides were prepared using silkworm pupae powder.The differences in the effects of single-frequency ultrasound and multi-frequency ultrasound on the extraction rate of silkworm pupae protein were investigated to improve the extraction rate of silkworm pupae protein,and the kinetic model of silkworm pupae protein extraction was successfully constructed.The effect of triple-frequency ultrasonic treatment on the kinetic parameters and thermodynamic parameters of enzymatic digestion of silkworm pupae protein was further investigated to provide a technical reference for the multiple utilization of silkworm pupae protein.