Isolation,Purification,Structure Identification And Antioxidant Activity Of Peptides From Porphyra Haitanensis

Porphyra haitanensis,mainly distributed along the coasts of Fujian,Zhejiang and Guangdong,is one of the unique large-scale economic algae in China.It has the characteristics of high protein,high fiber,low calorific value and low fat.Rich in amino acids?including 8 essential amino acids?,Porphyra haitanensis is an ideal protein source for bioactive peptides.Firstly,the nutritional components and quality of Porphyra haitanensis at different harvesting stages were analyzed and evaluated.Secondly,RSM were used to optimize the ultrasonic assisted extraction Porphyra haitanensis protein process,and the basic properties of protein were determined.Thirdly,RSM were used to optimize acid protease hydrolysis of Porphyra haitanensis protein,and the antioxidant activities of the hydrolysates were determined.Finally,GFC was used to separate and enrich the antioxidant peptides from Porphyra haitanensis with different molecular weights.Objective to investigate the repair effect of high antioxidant components on oxidative damage induced by H₂O₂ in HepG2cells.Meanwhile,the primary structure of high antioxidant peptides from Porphyra haitanensis was identified by LC/ESI/MS.The specific results are as follows:1.In order to discuss nutrition and quality of Porphyra haitanensis in Shantou area at different harvesting stages,the basic nutrient composition,amino acids,fatty acids,inorganic elements and the nutritional qualities of Porphyra haitanensis were analyzed and evaluated.The results showed that there were significant differences in protein content,total sugar content,crude fiber content,ash content and fat content with the harvest period?p<0.05?.17 kinds of amino acids were detected in Porphyra haitanensis,with the highest level of 34.62g·100g^-11 in the last harvest of Porphyra haitanensis and the lowest level of 30.87g·100g^-11 in the second harvest of Porphyra haitanensis.According to nutritional evaluation by AAS and CS,the first limiting amino acid was Met.16 kinds of fatty acids were detected in the first harvest of Porphyra haitanensis,and 14 kinds of fatty acids were detected in the fourth and last harvest of Porphyra haitanensis,as well as 13 kinds of amino acids were detected in the second and third harvest of Porphyra haitanensis.12 kinds of inorganic elements were detected in Porphyra haitanensis at different harvesting stages,including four major elements such as Na,Mg,K,and Ca,and eight trace elements such as Mn,Fe,Cu,Zn,As,Se,Cd,and Pb.2.The extracted method of protein from the Porphyra haitanensis and its basic characteristics were studyed in the paper.Based on the appropriate factor levels determined by single-factor experiments,the box-Behnken center combination experiment design and RSM were used to optimize the ultrasonic assisted extraction Porphyra haitanensis protein process.The results showed that the highest protein yield was 61.20%under ultrasonic power of 1278w,ultrasonic emission time of 6s and total ultrasonic time of 52min.The pI of the protein of Porphyra haitanensis was 4.2.The results of ultraviolet spectrum showed that the maximum absorption peak of Porphyra haitanensis proteins was at 260 nm and no obvious heterotopes.The results of SDS-PAGE showed that the main molecular mass of the protein of Porphyra haitanensis was 56.35kDa,20.45kDa and 16.52kDa.Porphyra haitanensis protein contained almost all kinds of amino acids with Glu,Asp,and Ala being the predominant amino acids.The content of essential amino acids was 40.29%,the mass fraction of non-essential amino acids was 59.71%,and the ratio of essential amino acids and non-essential amino acids was 0.675.3.Based on the appropriate factor levels determined by single-factor experiments,the box-Behnken center combination experiment design and RSM were used to optimize hydrolysis of Porphyra haitanensis Protein by Acidic Protease.The results showed that the highest DH was 11.40%under substrate concentration of 2 g/100 mL,enzyme dosage of 4240 U/mg,pH value of 3.7,temperature of 54?and hydrolysis time of 4 h.The relationship between DH and antioxidant activity of the hydrolysates was investigated.It was found that the DPPH radical scavenging activity of the hydrolysates was the strongest(IC₅₀=0.68 mg/mL)when the hydrolysis time was 4 h?DH=11.40%?.4.Different molecular weights of the antioxidant peptides from Porphyra haitanensis were separated and enriched by GFC column.After purification,the antioxidant peptides of Porphyra haitanensis showed strong antioxidant activity.The DPPH free radical scavenging rate was73.32%and the hydroxyl radical scavenging rate was 30.15%when the concentration of antioxidant peptide from Porphyra haitanensis was 1mg/mL.The primary structure of the high antioxidant activity peptide of Porphyra haitanensis was identified by LC/ESI/MS,and a 4 peptide with amino acid sequence of Asp-Lys-Ser-Thr and molecular weight of 449 Da was obtained.The oxidative stress model of HepG2 cells induced by H₂O₂was established.It was confirmed that the high antioxidant activity peptide of Porphyra haitanensis could repair the oxidative damage of HepG2 cells induced by H₂O₂.The high antioxidant activity peptide from Porphyra haitanensis can enhance the activity of SOD and reduce the content of MDA in a dose-dependent manner.