Preparation Of ⁶⁰Co-γ-ray Irradiation-assisted Enzymatic Method Of Active Peptides Of Silkworm Pupae Origin And Its Application

Silkworm pupae（Bombyx mori）are the main by-product of the silk reeling industry and one of the novel resource foods approved by the Ministry of Health.Silkworm pupae protein（SPP）is a rich source of bioactive peptides,but these proteins’complex structure and insolubility limit the enzymatic preparation of active peptides.Enhancing the enzymatic catalytic process by physical field modification technology can increase the exposure of enzymatic cutting sites in proteins,which is beneficial to the enhancement of protein properties and the acquisition of small molecule peptides.However,the commonly used ultrasound and microwave techniques suffer from the problems of low sample volume and high energy consumption in a single treatment,which limits the industrial production of large quantities.In recent years,⁶⁰Co-γ-ray irradiation has become an emerging continuous protein modification technology that can be used to enhance the functional properties of proteins.Therefore,this thesis is the first to apply⁶⁰Co-γ-ray irradiation technology to SPP,coupled with an enzymatic method to prepare novel silkworm pupae active peptides,develop a novel process of microencapsulation and chelation processing of silkworm pupae active peptide,and investigate the bioavailability of novel active peptide from both in vitro simulated digestion and cellular absorption.The main results are as follows:（1）A novel process of ⁶⁰Co-γ-ray irradiation coupled with enzymatic hydrolysis of SPP to obtain small molecule active peptides was developed.The silkworm pupae were treated with ⁶⁰Co-γ-ray irradiation at a dose point of 10 k Gy,followed by alkaline protease-catalyzed hydrolysis to resolve the functional properties of the proteins and the bioactivity and molecular weight distribution of the hydrolysate.The results showed that the proportion ofα-helix in the secondary structure of SPP increased by 7.17%after irradiation,indicating that irradiation could improve in the structure of SPP.In addition,the solubility,hydrolysis,water-holding capacity,oil-holding capacity,and emulsification ability of the irradiated SPP were significantly higher than those of the control group（p<0.05）.The percentage of small molecular peptides（180～500 Da）prepared by irradiation-coupled enzymatic hydrolysis increased from 32.04±0.97%to 52.77±0.72%,and the percentage of small molecular peptides（0.5～1 k Da）increased from 19.65±0.52%to 24.75±0.79%,which was significantly higher than that of the control group（p<0.05）,indicating the feasibility of ⁶⁰Co-γ-ray irradiation coupled enzymatic hydrolysis process is feasible in improving the functional properties of proteins and increasing the percentage of small molecular weight peptides.（2）Bioactivity tracing isolated and identified a novel silkworm pupae-derived peptide product with antioxidant,hypoglycemic,and hypotensive activities.To detect the biological activity of the peptides,5 k Da molecular weight ultrafiltration and dextran gel G-15chromatography were used to isolate small molecule peptides from the hydrolysate of SPP prepared by ⁶⁰Co-γ-ray irradiation coupled with the enzymatic method,and the sequences of the isolated peptides were identified by nano-liquid chromatography-tandem mass spectrometry,and the peptides were simulated by Auto Duck Vina molecular docking on angiotensin-converting enzyme and the inhibition mechanism ofα-glucosidase was simulated by Auto Duck Vina molecular docking.The results showed that 14 novel peptides were identified from the control and irradiated groups,and the active peptides in the irradiated group were smaller in molecular weight.Among them,the novel peptides with amino acid sequences QASSGTPATLR,LLPR,FKVPN,and HHFP showed stronger DPPH radical scavenging activity(IC₅₀415.00±5.83μg/m L),α-glucosidase inhibitory activity(IC₅₀446.17±11.39μg/m L),Fe²⁺chelating activity(IC₅₀401.49±10.96μg/m L)and ACE inhibitory activity(IC₅₀119.61±12.40μg/m L).The molecular docking results showed that the number of hydrogen bonds bound to angiotensin-converting enzyme andα-glucosidase was increased in the irradiated group compared to the control group,and the binding affinity was enhanced,suggesting that⁶⁰Co-γ-ray irradiation-coupled enzymatic hydrolysis can be a key technique for the preparation of highly active small molecule peptides.（3）Caffeic acid grafted chitosan（CA-g-Ch）-sodium alginate encapsulated silkworm pupae active peptide microcapsules were prepared by the microfluidic complex coalescence method.The microfluidic preparation technique of a flow-focused chip was used to precisely prepare the peptides into micron-sized capsules of uniform size using CA-g-Ch and sodium alginate as wall materials and characterized by scanning electron microscopy,Fourier transforms infrared spectroscopy,X-ray diffraction,differential scanning calorimetry,and thermogravimetric analyzer to investigate their stability in INFOGEST standardized gastrointestinal digestion.The results showed that the encapsulation rate of microcapsules reached 97.20±1.66%under the conditions of selecting CA-g-Ch concentration of 0.3%,sodium alginate concentration of 3%,Ca Cl₂concentration of 0.6%and active peptide concentration of 40 mg/m L.Compared with the sodium alginate microcapsules,the thermogravimetric analysis curve showed that the weight loss of CA-g-Ch-sodium alginate microcapsules was 8.33%,which was significantly lower than that of the control（25.88%）,showing stronger thermal stability.The peptide release rate in CA-g-Ch-sodium alginate microcapsules was 9.76±0.23%,which was significantly lower than that of the control group（14.05±0.83%,p<0.05）,indicating that CA-g-Ch-sodium alginate microcapsules have anti-gastric disintegration properties.In addition,the bioactivity of CA-g-Ch-sodium alginate microcapsules loaded with active peptides was 310.21±11.99μg/m L,363.02±5.37μg/m L,and 331.64±9.13μg/m L.The results indicated that the microfluidic complex coalescence method for the preparation of CA-g-Ch-sodium alginate encapsulated silkworm pupae active peptide microcapsules is technologically advanced with anti-gastric disintegration properties and high thermal stability.（4）To develop key technology for the preparation of active peptides from silkworm pupae-calcium chelates to improve the bioavailability of active peptides.A Box-Behnken central composite design with the calcium chelation rate as the response value was used to fit the preparation parameters of the chelates which were characterized to investigate their digestive characteristics and Caco-2 cell absorption in the simulated gastrointestinal tract.The results showed that the Box-Behnken central composite design of chelates was prepared with the following parameters:peptide-calcium mass ratio of 3:1,chelation p H of 6.7,chelation temperature of 35.6℃,chelation time of 33 min,and the calcium chelation rate of84.67%.The absolute value ofζ-potential was significantly lower（p<0.05）compared to the active peptide,indicating that the stability of the active peptide was significantly improved after chelation.In addition,the chelates maintained higher antioxidant,hypotensive,and hypoglycemic activities than the active peptides.In the simulated intestinal phase,the calcium solubility of the chelates was 71.01±1.91%significantly higher than that of free calcium（59.34±1.24%）,indicating that the bioavailability of active peptide can be significantly enhanced by chelating active peptide with calcium.Therefore,the ⁶⁰Co-γ-ray irradiation coupled enzymatic hydrolysis process constructed in this thesis can improve the functional properties of SPP to efficiently prepare small molecule active peptides with antioxidant,hypotensive and hypoglycemic activities,and then develop microcapsules of silkworm pupae-derived active peptides with stomach acid resistance and high thermal stability and calcium chelates of active peptides with high bioavailability,which can broaden the application field of active peptides and provide theoretical and technical support to further extend the silkworm industry chain and break through the bottleneck of traditional agricultural products processing technology.