Studies On Preparation And Biological Activity Of Wheat Bran Peptide

Wheat is one of the three major grain crops in the world.The yield of wheat bran, byproduct of wheat processing,is very huge.At present,the effective utilization of wheat bran is very low,which is not fully,reasonably and efficiently utilized.Wheat bran peptide not only can broaden the range of wheat bran application and resolve the problem of bulk byproduct in the deep processing of wheat,but also enhance product added value.Wheat bran contains an abundant amount of protein,which is about 12～18%of the total weight,so it is a potential resource of plant protein.The study of wheat bran peptide made from wheat bran protein has not been reported yet.A series of experiments were taken to prepare bioactive peptides using enzymatic preparation from wheat bran protein,and wheat bran peptides were obtained successfully.The wheat bran peptide has good antioxidation,immunity enhancement and antifatigue effect.These researches can broad application area,develop new ways for further develop of wheat bran protein,provide theoretical basis and process parameters to industrial production of peptide and provide theoretical foundation and experimental basis for the more widely used of wheat bran peptide in health products and food.In this paper,preparation,purification and biological activity of wheat bran peptide was systematic studied.The wheat bran protein extraction was used by alkali method and salt method,the optimum extraction conditions were determined.The scavenging activity of hydroxyl radical and superoxide anion radical taken as index,the best conditions of enzymatic preparation of wheat bran peptide determined by orthogonal test method.The antioxidant capacity of wheat bran peptide was comprehensive evaluated by in vivo and vitro test.The function of immunoenhancement of wheat bran peptide were evaluated by lymphocyte transformation test,determination of half hemolysis value,phagocytic index,immune organ index.The antifatigue effect of wheat bran peptide was estimated by loaned swimming test and determination of liver glycogen and blood urea nitrogen.The toxicological and safety evaluation of wheat bran peptide were tested by acute toxicity test,Ames test,mouse bone marrow micronucleus test,the chromosome aberration test of mouse primary spermatocyte, comet assay.Wheat bran peptide was isolated and purified by ultrafiltration,SeaphadexG25, DEAE-32 and reversed-phase high performance liquid chromatography.The molecular weight is measured by mass spectrometry(MS).The stability of wheat bran peptide was studied. Microcapsule and liposome of wheat bran peptide were prepared by ultrasonic method and rotary film-ultrasonic method,the optimum preparation conditions were determined.The main results are as followed.1.The optimum extraction conditions of wheat bran protein by alkali method are as follows:solid-liquid ratio of 1:15,pH 11,temperature 50℃,time 2.5h,protein extraction rate is 57.86%.The optimum extraction conditions of wheat bran protein by salt method are as follows:concentration of 2%,temperature 40℃,time of 2h,solid-liquid ratio 1:15,protein extraction rate is 27.68%.Optimized by the two methods,alkali method is a better way.2.The best conditions of enzymatic preparation of wheat bran peptide prepared by double-enzyme fractional hydrolysis with Alkaline protease and Flavourzyme were as follows: pH8,enzyme activity 6000u/g material,time 3 h,temperature 50℃with Alkaline protease, pH6,enzyme activity 6000u/g material,time 3 h,temperature 55℃with Flavourzyme,under these conditions for hydrolysis of wheat bran protein,and clearance rate of O₂-^·and·OH were 81.60%and 86.16%.Orthogonal test results show that the influences of the effect of free radical scavenging capacity are:pH＞Time＞addition of enzyme activity＞temperature.3.Ultrafiltration were carried out to separate wheat bran peptide,less than 10KDa molecular weight is the strongest antioxidant activity,SephadexG-25 column chromatography was used to separate low molecular weight of wheat bran peptide,three separate peaks were got,of which the third peak with the strongest antioxidant activity.DEAE-32 cellulose column was used to separate the active component purified by the SephadexG-25,four separate peaks were got,of which the fourth peak with the strongest antioxidant activity. RT-HPLC was used to separate the active component purified by the DEAE-32 cellulose column,conditions are as follows:Column:SUPELLOSIL C₁₈ column(25cm×4.6mm), detection wavelength:215 nm,column temperature:35℃,velocity:0.5 ml/min,sample loaded volume:10μL,elution conditions are as follows:(80%methanol,20%water),two peaks were isolated,of which the first has higher antioxidant activity.The active component was collected for cellulose acetate membrane electrophoresis,a single band was obtained.The molecular weight is 1326.2Da analysised by mass spectrometry(MS).Wheat bran peptide has high purity.Preparation of Wheat bran peptide in the range of pH6.0～8.0 is more stable,pH＜4.0 and pH＞8.0,the stability of wheat bran peptide is decreased.4.The wheat bran peptide extracted by Sephadex G-25 has a higher scavenging activity of hydroxyl radical and superoxide anion radical,can inhibit hemolysis significantly,prevent the integrity of red blood cells effectively,inhibit mitochondria swelling induced by·OH, wheat bran peptide has good antioxidation in vitro.Wheat bran peptide has antioxidation of DNA in vivo,can promote mice serum and liver SOD,SOD,GSH-Px values and reduce the lipid peroxide MDA value,with better improvement of the body endogenous antioxidant enzyme activity,scavenging free radicals,inhibit lipid peroxidation injury.5.Immunological tests show that wheat bran peptides have enhanced the effectiveness of the immune system,through the promotion of the level of cell-mediated immunity mainly, and enhance of macrophage phagocytic function,increase of spleen index to achieve its role of the immune.Wheat bran peptide can extend mice swimming time,increase liver glycogen storage quantity in mice,reduce blood urea nitrogen significantly,enhance exercise tolerance and alleviate physical fatigue of animals.6.Toxicity test show that wheat bran peptide in mice acute toxicity test,LD₅₀＞20g/kg,is non-toxic class.Results of Ames test,mouse bone marrow micronucleus test,the chromosome aberration test of mouse primary spermatocyte and comet assay were negative. Wheat bran peptide is a non-toxic material.The present studies thus provide preliminary proof of safety for wheat bran peptide.7.Wheat bran peptide microcapsule was prepared with gelatin embedded as wall material,the influence order of entrapment efficiency is ultrasonic power＞core material:wall material＞ultrasonic temperature＞ultrasonic time.The best conditions of wheat bran peptide embedded with gelatin are as follows:core material:wall material for the 1:8,ultrasonic power of 60W,ultrasonic time of 70min,ultrasonic temperature 50℃,in these conditions,the rate of entrapment efficiency can be achieved 59.68%.Wheat bran peptide liposome was prepared with satured phospholipids and cholesterol, the influence order of entrapment efficiency is ultrasonic time＞amount of substance ratio of lecithin and cholesterol＞hydration temperature＞amount of wheat bran peptide,The best conditions of wheat bran peptide embedded with satured phospholipids and cholesterol are as follows:molar ratio of lecithin:cholesterol for the 1:1,the hydration temperature of 55℃, ultrasonic processing time for 3min,wheat bran polypeptide of 2mL,in these conditions,the rate of entrapment efficiency can be achieved 88.16%.