Study On Preparation Of Bioactive Peptides From Walnut(Juglans Regia L.) Meal Using Ultrasonic Enhancement And The Coupling Of Enzyme Hydrolysis And Membrane Separation

Walnuts?Juglans regia spp.?,one of the top four most consumed nuts have assumed a large-scale global production reaching over 4 million tons per year.China ranks first?1.78 million tons,42.5%?on the world production scale.A vast amount of walnut meal with 40–80%protein on a dry weight basis is being generated.Although there are many studies on the extraction of walnut protein and its conversion into an active polypeptide by enzymatic hydrolysis,the walnut meal resource has not been extensively explored.The reasons being the shortfalls of traditional methods of protein extraction and enzymatic hydrolysis,such as low extraction yield,low product activity,and large enzyme consumption.The production cost is high,and the product quality is poor,which limits the wide application of the technologies and the product.To this end,this paper uses ultrasonic enhanced protein extraction and enzymatic hydrolysis,using enzyme membrane coupling technology to prepare a walnut meal active peptide.The main research contents and results are as follows:?1?Ultrasound-assisted extraction?UAE?and traditional alkaline extraction?TAE?of walnut meal protein were assessed.The optimal conditions for the traditional alkali extraction?TAE?were;1:20 g/mL material to liquid ratio;50°C extraction temperature,60 min extraction time and62.34%protein yield.For the UAE;power density of 80 W/L,46.6°C of extraction temperature,45 min sonication time,1:20 g/mL material/liquid ratio and 91.23%protein yield were obtained and experimentally verified.Protein extraction yield,purity,and the chemical score increased by30.15%,16.27%,and 9.74%,respectively,with a 25%reduction in extraction time in the UAE relative to the TAE.The best frequency mode was 20/40 kHz/kHz.?2?The effect of the UAE on the functional properties and structure of walnut meal protein isolates?WMPIs?was studied.The results showed that UAE improved the functional properties of WMPIs relative to the control.Dual-frequency increased fat absorbability,emulsion/foam stabilities and bulk density by 13.2%,34.5%,39.8%,and 6.1%respectively,while water absorbability reduced by 25.4%compared to the control.Surface hydrophobicity?SH₀?also increased by 6.6%,4.94%and 3.67%via dual,mono and triple frequency treatments respectively.The secondary structural components;-sheet,-turns,and random coil increased and?-helix decreased significantly?p<0.05?by 95.76%,101.3%,105.1%and 85.7%correspondingly in the dual-frequency ultrasound treatment compared to the control.It means that the protein structure is expanded,which is more beneficial for subsequent enzymatic hydrolysis.?3?Enzymatic hydrolysis of walnut meal protein first stage:effect of ultrasonic-assisted enzymatic hydrolysis was studied.The walnut meal was pretreated with ultrasonic-assisted alkaline extraction;the protein conversion degree?PCD?was taken as an index to study the enzymatic hydrolysis conditions of walnut protein in the first stage.The results showed that the best protease was trypsin.The conversion rate of the substrate to product increased by 20.29%?40kHz?,30.07%?20/28/40 kHz/kHz/kHz?and 32.54%?20/40 kHz/kHz?via sonication treatment over the control.The optimum conditions were enzyme dosage of 2000 U/g,hydrolysis time of 90minutes,pH 7.5,30 g/L substrate concentration and hydrolysis temperature of 50°C.Under these conditions,the conversion rate reached 48.14%?20/40 kHz/kHz?,which is 32.54%higher than36.32%protein conversion rate for the control treatment without ultrasound.The dual-frequency combination?20/40 kHz/kHz?was more efficient compared with the single and the triple frequency modes.?4?The effects of ultrasonication on the kinetic and thermodynamic parameters of trypsin hydrolysis of the walnut meal were assessed.Results revealed that both the ultrasound and the control processes obeyed first-order kinetics within the study constraints.The Michaelis constant?K_M?in the ultrasonic process declined by 58.66,61.53 and 45.60%respectively for mono,dual and triple frequencies compared with the control.The reaction rate constant significantly increased at varying temperatures?293-323 K?by 84.75-37.79%,100-55.26%and 88.25–9.15%for mono,dual and triple frequency treatments.Generally,dual-frequency sonication reduced the bond energy?E_a?,enthalpy change??H?,entropy change??S?and Gibbs free energy??G?by36.61,28.05,18.22 and 5.21%magnitudes compared with the control respectively.?5?The effect of ultrasound-assisted enzymolysis on antioxidant and the ACE inhibitory activity of walnut meal peptide fractions was assessed.The results revealed an equitably balanced amino acid profile;Glu,Arg,and Asp were the dominant amino acids.Largely,peptides with a molecular weight cut off?MWCO?of<5 kDa exhibited higher antioxidant and ACE inhibitory capabilities.The peptide fractions with lower?<1,1-3,and 3-5 kDa?MWCO had the highest reducing power ranging from 1.05–1.31 and 1.15–1.41 compared with higher?5-10,10-30,and>30 kDa?MWCO with 0.84–0.49 and 0.92–0.65 for control and ultrasound peptide fractions respectively.The ABTS^*+and DPPH scavenging activities were higher in 1-3 kDa peptides;2.59TEAC mM and 56.98–75.56%as well as 2.78 TEAC mM and 61.59–84.19%for the control and ultrasound samples respectively.ACE inhibition was concentration-dependent with 1-3 kDa peptide fraction inhibiting 50%ACE(IC₅₀)at 0.2 mg/mL.Therefore,the ultrafiltration membrane with a molecular weight of 3 kDa was selected for the additional enzymatic membrane coupling reaction.?6?Enzymatic hydrolysis of walnut meal protein second stage:studies on enzyme membrane coupling reaction was undertaken.Using trypsin,the result of the batch process of the coupled enzyme hydrolysis and membrane separation?CEH-MS?was;hydrolysis time?60 min?,substrate concentration?5%?,enzyme dosage?2000 U/g?,hydrolysis temperature?55°C?,pH?7.5?and permeate flux?11 mL/min?.The protein conversion degree?PCD?was 61.78%compared with48.14%of the traditional enzymolysis.Using the CEH-MS batch process optimal conditions,the CEH-MS-gradient dilution feeding?CEH-MS-GDF?was efficient for a reaction time of 660 min achieving a maximum PCD of 89.95%in that period.Comparatively,the CEH-MS-GDF improved the PCD,peptide production yield,and production efficiency by 86.85%,82.90%,and 239.624%margins respectively relative to the traditional process.ACE inhibitory activity increased significantly by 17.19 and 73.82%for the CEH-MS batch and the CEH-MS-GDF processes respectively relative to the traditional enzymolysis.