Study On The Preparation,Antioxidant Activity Evaluation And Purification Of Fermented Wheat Bran Glycoprotein

Glycoprotein is a kind of biological macromolecule which is composed of carbohydrate and protein by covalent bond.It is widely distributed in animals,plants and some microorganisms.It has the functions of enhancing immune regulation,inhibiting tumor,reducing blood sugar and antioxidation.Nowadays,the bioactivity of glycoprotein extracted from many plants,such as soybean,sweet potato,yam,oats and so on,has been proved.Fermented bran is a kind of wheat bran prepared by microbial fermentation.The co fermentation of probiotics and enzymes can change the fiber structure of wheat bran,so as to increase the content of nutrients and enhance its antioxidant capacity.At present,the research on wheat bran is mainly focused on polysaccharides,polyphenols,ferulic acid and other active substances,but there is no research report on fermented bran glycoprotein at home and abroad.Therefore,it is of great theoretical and practical significance to extract glycoprotein from fermented wheat bran and further study its properties,functional activities and structural composition.In this paper,the optimal fermentation conditions of wheat bran were determined by single factor experiment through the cooperative fermentation of probiotics and enzyme preparation.The glycoprotein was extracted and separated,and its antioxidant activity in vitro and in vivo was studied.Finally,the glycoprotein was further purified by column chromatography,and the structure of the purified sample was characterized.The paper mainly includes the following four parts:1.Preparation of fermented wheat bran glycoproteinIn this experiment,the technology of breath bag fermentation was used.Probiotics and enzyme were used to ferment bran together.Single factor method was used to set up different amount of compound bacteria,cellulase,fermentation temperature,fermentation time and water content.Taking protein content and polysaccharide content as indicators,the optimal fermentation parameters were obtained as follows:0.1%of compound bacteria,2g of cellulase and 28℃ of fermentation temperature,fermentation time 3 days,water content 40%.The extract of fermented bran was obtained by hot water extraction.The best ethanol concentration was 100%by alcohol precipitation and screening.The free protein was removed by Sevage method,and dialysis the fermented bran glycoprotein(FWBG)was obtained.2.Detection of fermented wheat bran glycoproteinThrough a series of color reactions,the glycoprotein was detected to contain protein and acid polysaccharide,but not tannin and starch.SDS-PAGE showed that fwbg was a single band,and Coomassie brilliant blue staining and sugar staining were in the same position,molecular weight was about 40 kDa,which was confirmed to be glycoprotein.3.Evaluation of antioxidant activity of fermented wheat bran glycoproteinThe antioxidant activity of fwbg in vitro was evaluated by DPPH,the scavenging ability of hydroxyl radicals and their reducing power.The results showed that fwbg had strong antioxidant capacity,and with the increase of concentration,the scavenging capacity and reducing power of DPPH and hydroxyl radicals were stronger,showing a certain dose effect relationship.Using zebrafish embryo as the model,the antioxidant activity of FWBG in vivo was analyzed.The safe concentrations of FWBG were 0.05mg/mL,0.1mg/mL and 0.2mg/mL.The effect of FWBG on the survival rate of zebrafish embryos in the oxidative stress induced by AAPH was verified.The results showed that the survival rate of zebrafish embryos treated with different concentrations of FWBG(0.05mg/mL,0.1mg/mL,0.2mg/mL)was significantly higher than that in the AAPH group,and FWBG could protect zebrafish from the damage of AAPH.After being treated with DCFH-DA,DPPP and AO fluorescent dyes,the fluorescence microscope was used to take photos and analyze the fluorescence intensity.It was found that FWBG also has the effect of removing ROS,reducing lipid peroxidation and cell death rate in zebrafish,with better antioxidant effect and better correlation with its concentration.4.Isolation and structural characteristics of fermented wheat bran glycoproteinFWBG was purified by DEAE-52 cellulose column.The concentration(5mg/ml,10mg/ml),eluent(distilled water,0.1,0.2mol/L NaCl)and flow rate(0.5ml/min,1ml/min)were single factors.The absorption peak of protein and polysaccharide was the detection index.The final elution conditions were determined.Finally,it was determined that the concentration of the sample was 10mg/mL,the eluant was distilled water and O.lmol/L NaCl,and the elution speed was 0.5mL/min.After elution and purification by DEAE-52 cellulose column,a main component FWBG-1 was obtained.The infrared spectrum,amino acid composition,monosaccharide composition and SEM of FWBG-1 were analyzed.The results show that:at 4000-400 cm-1 FWBG showed several typical absorption peaks of polysaccharide and protein in the infrared absorption spectrum of cm-1,and it was concluded that the glycoprotein chain of FWBG-1 was β-glucoside bond structure of pyranose;the glycoprotein contained 17 different types of amino acids,among which the content of glutamic acid,proline,aspartic acid,glycine and argini was high;FWBG-1 was determined by HPLC It contains 10 kinds of monosaccharides,and the mole percentage of each monosaccharide is mannose,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose,arabinose,fucose=4.18:0.57:0.28:0.21:0.07:33.33:4.59:31.29:25.31:0.16,in which the content of glucose is the highest,reaching 33.33%;.The morphology of FWBG-1 was observed by scanning electron microscope,and it showed a relatively dispersed sheet structure with uneven size and smooth surface.