Preparation Of Antioxidant Peptides From Pine And Study On Its Antioxidant Activity

Pine protein is a high quality protein.It contains twenty kinds of amino acids,and most of them are essential amino acids.In recent years,the active peptides from plant protein have attracted people’s attention.so it is of great significance and broad application prospect to study the preparation and identification of natural active antioxidant peptides from pine nuts.This experiment mainly study on five aspects:the optimization of pine nuts protein extraction process,screening the best pine nutrient enzyme,preparation of pine nuts antioxidant peptides,the antioxidant activity in vitro and vivo of pine nit peptides,separation and purification of pine nut antioxidant peptides.The main results of the research are as follow:1 Optimization of pine nut protein extraction process:In this paper,Yunnan pine nuts,as raw materials,was grinded into pulp,and then the fatty was removed using Soxhlet extraction method to get the pine nut meal.Orthogonal test was used to predict optimum conditions for ultrasonic-assisted extraction technology of protein components from pine nut.The optimal extraction process was 800W of ultrasonic power,30min of ultrasonic time,9.5 of pH,1:30 of solid to liquid ratio.Under this condition,the extraction rate of pine nut protein was 67.4%.Alkali-soluble acid precipitation method was applied to extract pine nut protein.2 Screen the best enzyme:Two optimal enzymes were screened from Trypsin,Alkaline protease and Papain.The preparation of pine nut active peptide was determined by double enzymatic Sequential hydrolysis method.The degree of protein hydrolysis and DPPH free radical scavenging rate were used as evaluation indicators.After screening,using Alkaline protease and Trypsin step by step was the best combination.The hydrolysis degree of pine nut protein was 11.3%.Under the conditions of substrate concentration 50mg/mL,50℃ of temperature,3%of enzyme dosage,9.0 of Alkaline protease hydrolysis pH,Trypsin hydrolysis pH 8.0,Step-by-step hydrolysis time 1.5h.3 Isolation and purification of the pine nut active peptide:The mixture of pine nuts proteolytic peptides was separated by ultrafiltration to obtain three components,Fra.1(<5kDa),Fra.2(5k～10kDa)and Fra.3(>10kDa)Clearance rate as an indicator of different molecular weight scavenging capacity.The results showed that the ability of scavenging free radicals was:Fra.1>Fra.2>Fra.3.And the same component,with the concentration of active peptide increases,the ability to scavenge free radicals increased.The high activity components Fra.1 was selected for subsequent purification.First,the sample was passed through Q Sepharose XL anion exchange chromatography prefilled column and divided into four components,chronologically named Fra.1-1,Fra.1-2,Fra.1-3 and Fra.1-4 respectively.Four components were collected and lyophilized Activity study.The results showed that its ability to scavenge free radicals was:Fra.1-3>Fra.1-2>Fra.1-1>Fra.1-4.The Fra.1-3 enriched high activity fraction was purified by Sephadex G-25 gel chromatography and divided into two fractions Fra.1-3-1 and Fra.1-3-2.Base cleaning ability,the results showed that its ability to scavenge free radicals was Fra.1-3-2>Fra.1-31.Combined with the liquid chromatography tandem mass spectrometry(LC-MS/MS),fifteen peptides(1-15)with potential antioxidant activity were identified,and these amino acid sequences were:VEIIANQGNR(Val-Glu-Ile-Ilc-Ala-Asn-Gln-Gly-Asn-Arg);DLTDYLMK(Asp-Leu-Thr-Asp-Tyr-Leu-Met-Lys);MQIFVK(Met-Gln-Ile-Phe-Val-Lys);NQIKDK(Asn-Gln-Ile-Lys-Asp-Lys);EGIPPDQQR(Glu-Gly-Ile-Pro-Pro-Asp-Gln-Gln-Arg);ESTLHLVLR(Glu-Ser-Thr-Leu-His-Leu-Val-Leu-Arg);MASVPTK(Met-Ala-Ser-Val-Pro-Thr-Lys);EMVELPLR(Glu-Met-Val-Glu-Leu-Pro-Leu-Arg);VIPELNGK(Val-Ile-Pro-Glu-Leu-Asn-Gly-Lys);LTGMAFR(Leu-Thr-Gly-Met-Ala-Phe-Arg);VVLIGDSGVGK(Val-Val-Leu-Ile-Gly-Asp-Ser-Gly-Val-Glu-Lys);IQEAGTE VVK(Ile-Gln-Glu-Ala-Gly-Thr-Glu-Val-Val-Lys);KDELER(Lys-Asp-Glu-Leu-Glu-Arg);IKDAVEK(Ile-Lys-Asp-Ala-Val-Glu-Lys);YNQLLR(Tyr-Asn-Gln-Leu-Leu-Arg)。.4 The vitro test of pine nut antioxidant peptide:The total reducing power,DPPH free radical scavenging rate and ABTS free radical scavenging rate were used as indexes to determine the antioxidant activity of pine nut antioxidant peptide in vitro.The results showed that the pine nut antioxidant peptide has a strong reducing power and ability to scavenge free radicals,and displayed a certain dose-effect relationship.Within a certain range,as the concentration increases its antioxidant capacity gradually increased,and the gap between vitamin C gradually reduced.5 The vitro test of pine nut antioxidant peptide:Different concentrations of pine nuts antioxidant peptides were used on the D-galactose aging model,and with vitamin C as a control.The effects of pine nuts antioxidant peptide on the state of mice were observed.The changes of body weight,index of organ,apoptosis rate of brain cells,reactive oxygen species in cells of brain and liver,GSH-Px,SOD in serum,liver and heart Vitality,MDA content and other antioxidant indicators.The results showed that different concentrations of pine nuts antioxidant peptide can reduce the rate of apoptosis of brain cells,reduce brain and liver cells in the level of reactive oxygen species,brain cells from injury.At the same time,the activities of GSH-Px and SOD in the brain,serum and liver of mice were significantly increased,and the MDA production in brain tissue,serum and liver was also decreased.