Preparation And Activity Of Antioxidant Peptides From Antioxidant Peptide Of Cucumaria Frondosa

Cucumaria frondosa viscera is the primary by-product of the manufacturing process of Cucumaria frondosa.Because of its rich protein and polysaccharide content,it is considered to have great prospects in food processing.This thesis taking the viscera of Cucumaria frondosa as the main experimental material,studied the extraction of visceral protein from Cucumaria frondosa,the preparation of antioxidant peptides of Cucumaria frondosa and the optimization of process conditions,the antioxidant peptides of Cucumaria frondosa were separated and purified by ultrafiltration,and the activities of antioxidant peptides of Cucumaria frondosa with various molecular weights were investigated,the protective effect of antioxidant peptides ultrafiltration componenton on oxidative damage induced by H₂O₂ in PC12 cells was confirmed.The main results are as follows:（1）The effects of different extraction methods on the extraction of visceral protein from Cucumaria frondosa were compared,and ultrasonic assisted extraction was determined as the extraction method of visceral protein from Cucumaria frondosa in this experiment,the protein extraction rate and purity were 70.05±0.95%and 74.95±1.25%,respectively;the single factor and response surface methodology was used to optimize the etraction process of Cucumaria frondosa visceral protein.Under the conditions of1:41 g/m L solid-liquid ratio,p H value of 11.2,temperature of 57.6°C,and power of 80W,ultrasonic extraction was performed for 38 min,and the protein extraction rate could reach 82.87±0.12%.Cucumaria frondosa visceral protein was mostly made up of proteins with molecular weights of 150 k Da,45 k Da,and 12 k Da,according to SDS-PAGE.Cucumaria frondosa visceral protein included 17 amino acids,which had the potential as a source of antioxidant peptides.（2）A variety of proteases were selected and applied to the enzymatic hydrolysis of visceral protein of Cucumaria frondosa,and the composite protease was determined as the best enzyme for hydrolysis,the single factor and the response surface methodology was utilized to optimization the manufacturing process of antioxidative peptides from Cucumaria frondosa.Under the conditions of substrate mass fraction of 4.4%,enzyme addition of 4887 U/g,p H value of 7.0,and temperature of 56°C hydrolysis was performed for 4.2 h.The DPPH radical scavenging rate of enzymatic hydrolysis products was as high as 89.18±0.11%.The analysis of molecular weight distribution and amino acid composition of enzymatic hydrolysis products showed that the fractions<1000 Da in antioxidant peptide of Cucumaria frondosa accounted for 85.01%,and the fractions<500Da accounted for 71.39%,among them,small molecular peptides are the main ones;acidic and hydrophobic amino acids accounted for 29.68%and 40.27%of the total amino acids,respectively.Therefore,the antioxidant peptides of Cucumaria frondosa showed strong antioxidant activity,which was inseparable from its lower molecular weight distribution and amino acid composition.（3）The antioxidant peptides of Cucumaria frondosa were separated and purified by ultrafiltration,and obtain five peptide components C1（>10 k Da）,C2（5～10 k Da）,C3（3～5k Da）,C4（1～3 k Da）,and C5（<1 k Da）.The best ultrafiltration separation conditions were discovered after adjusting the ultrafiltration conditions using a single factor test:p H value of 7.0,temperature 25°C,pressure 0.15 Mpa,and tangential flow rate 6 LMH.The results of antioxidant activity showed that the antioxidant capacity of antioxidant peptide C5 of Cucumaria frondosa was the strongest among all components,with obvious free radical scavenging activity,Fe²⁺chelating ability and reducing power.The IC50 values of DPPH,ABTS,hydroxyl radical scavenging rate and Fe²⁺chelating rate were 0.397 mg/m L,0.161mg/m L,0.209 mg/m L and 0.159 mg/m L,respectively;In addition,compared with other components,the Fe²⁺chelating capacity of C2 component is the most prominent,and the IC50 value is 0.189 mg/m L.（4）To create an in vitro H₂O₂-induced oxidative stress model of PC12 cells,to study the protective effect of Cucumaria frondosa antioxidant peptide ultrafiltration fraction C5on oxidative damage to PC12 cells.The results showed that the ultrafiltration fraction C5（10-1500μg/m L）of Cucumaria frondosa antioxidant peptide had no toxic effect on PC12cells;Determine H₂O₂ at 500μM at the concentration of PC12 cells were induced for 4hours to establish the oxidative damage model;The concentrations of ultrafiltration component C5 at low,medium and high doses were 25μg/m L,50μg/m L,and 100μg/m L,respectively,while the final concentration of VC in the positive control group was 200μg/m L;Ultrafiltration component C5 can obviously reduce the level of ROS and MDA content in oxidative damaged PC12 cells,reduce the release of LDH,maintained the structure and integrity of cell membrane,increased the activities of SOD,GSH-Px and CAT in cells,and enhance their ability to withstand oxidative damage,and the best effect was obtained at 100μg/m L.