Preparation,Property And Structure-Activity Relationship Of Antioxidant Peptides From Paeonia Ostii Seed Meal

Antioxidant peptides are active peptide sequences composed of 2-20 amino acids.They are momentous for scavenging free radicals,maintaining redox homeostasis,preventing tissue damage,aging,and preventing heart disease,tumors,and other diseases.Paeonia ostii seed meal(Po SM)as a by-product after oil extraction is rich in crude protein and dietary fiber,and can be used as a natural source of vegetable protein and antioxidant peptides.In this paper,Po SM is used as a raw material to determine the general and nutrient components;the preparation and functional properties of oil peony seed meal protein(Po SMP)and peptides are studied,and five novel antioxidant peptide sequences are successfully identified.Finally,quantum chemical calculations were operated to research the structure-activity relationship of the identified novel peptides.The results provided a basis for the utilization of Paeonia ostii by-products and the development of new antioxidant peptides.The details are as follows:1. Study on the general and nutritional components of Paeonia ostii seed meal.The general components,amino acids,trace elements,and vitamins of Po SM were measured.The results show that the content of protein in Po SM is 20.2%,the content of aspartic acid(Asp)and glutamic acid(Glu)are 1.83 g/100g and 4.85 g/100g,respectively,and the content of essential amino acids is 33.7%.In addition,the contents of trace elements phosphorus P and potassium K were 5.11×10~3 mg/kg and4.28×10~3 mg/kg,respectively.Po SM is rich in protein,essential amino acids and trace elements.Po SM can be operated as an excellent source of protein and antioxidant peptides.2. Study on the Preparation and Functional Properties of Proteins and Enzymatic Products.In the experiment,Po SMP was extracted by alkali-soluble acid precipitation method.The effects of material-liquid ratio(g/m L),extraction time(min),p H,and extraction temperature(℃)on protein extraction rate were investigated.The results showed that the maximum extraction rate of Po SMP was 55.17%at p H=10,52℃,76.92 min,and 1:14.3 g/m L.Taking the degree of hydrolysis and in vitro oxidation resistance as indicators,among the five hydrolases,alkaline protease has the highest degree of hydrolysis and good ABTS and OH radical scavenging activity.Physicochemical properties show that Po SMP is mainly distributed near 23 k Da and34 k Da and the protein at 23 k Da sho ws strong digestibility;Po SMP and AHs have the lowest solubility at p H 4.5 and 3.7,respectively,under neutral conditions Both showed good solubility;the denaturation temperatures of Po SMP and AHs were67℃ and 83℃,respectively.Po SMP is resistant to high temperature,strong acid and weak alkali,while AHs is resistant to high temperature,strong acid and strong alkali,and neither of them has obvious cytotoxicity.3. Purification and identification of antioxidant peptides.Ultrafiltration,strong anion exchange chromatography,and re versed-phase high-performance liquid chromatography were sequentially used to purify and enrich AHs.Five ne w antioxidant peptides were identified by Nano LC-MS/MS technology.The peptide sequences were FSAP(Phe-Ser-Ala-Pro),PVETVR(Pro-Val-Glu-Thr-Val-Arg),QEPLLR(Gln-Glu-Pro-Leu-Leu-Arg),EAAY(Glu-Ala-Ala-Tyr)and VLRPPLS(Val-Leu-Arg-Pro-Pro-Leu-Ser).In vitro antioxidant experiments showed that the EAAY peptide sequence had the highest antioxidant activity,with ABTS and OH radical scavenging activities of 98.5±1.1%and 61.9±1.3%at 0.5 mg/m L,respectively.4. Study on Structure-activity Relationship of Antioxidant Peptides by Quantum Chemistry Calculations.The experiment is based on quantum chemical calculations.After the spatial distribution of five antioxidant peptides obtained from PEP-FOLD,the semi-empirical method PM6 and the density functional theory(DFT)B3LYP/6-31G*basis set are used for conformation optimization The orbital energy levels and active sites of the five antioxidant peptides were studied.The calculation results indicate that the difference in energy gap between HOMO(highest occupied molecular orbital)and LUMO(lowest molecular unoccupied orbital)has a certain degree of agreement with in vitro antioxidant free radical activity,and this value can be operated as one of the indicators for theoretical evaluation of antioxidant activity.In addition,the natural bond orbital(NBO)results indicate that the sites of hydrogen atom transfer related to free radical scavenging are mainly distributed on the terminal amino group,the guanidine group of arginine,and the phenolic hydroxyl structure of tyrosine.