Studies On Preparation Of Wheat Germ Active Peptides With Multiplex Enzymes And Alkali Method

In this paper, a series of experiments were taken to prepare bioactive peptidesusing enzymatic preparation from Defatted wheat germ, and the main mechanism is thatthe protein was degraded to become soluble peptides and be separated by using protease.In the research, two proteases were chosen to hydrolysis defatted wheat germ for wheatgerm peptides, the influences of various experimental factors on the enzymatichydrolysis process were explored, the major factors of the parameters were analyzedand optimized, The optimum conditions were investigated by orthogonal test, andenzymatic hydrolysis water was extracted with alkaline method after enzymatichydrolysis; Molecular weight distribution of the mixed peptide solution was measured;According to the molecular weight distribution, the mixed polypeptide was ultrafiltrated consecutively by membrane and the optimal ultra filtration condition wasdetermined for the different molecular weight peptides.The main contents and results of this research are as followed.(1) The optimum enzymatic process of wheat germ proteins was studied in ourpaper, which was hydrolyzed by the mixture protease, through exploring the factors ofpH, substrate concentration, temperature, time and consumed enzyme. Enzymatichydrolysis water was extracted with alkaline method after enzymatic hydrolysis so as toimprove the rate of peptide extraction. The results showed that when ratio of papain toneutral protease was set as1∶1, the maximum rate of peptides extraction was up to89.87%under the condition of pH6, substrate concentration5%, temperature50℃,time90min and consumed enzyme400U/g. In addition, enzymatic and alkalineextraction methods were combined in this paper. The condition for extraction was pH8.5,45℃, alkaline extracting for60min. It was proved that the extracting ratio of wheatgerm peptides was increased from89.87%to93.76%.(2) The molecular weight distribution of the peptide mixture was determined by gelfiltration chromatography, Sephadex G-15, and the choice of reference material ismyosin (F3)(2500Da), vitamin B12(molecular weight1355Da), oxidation glutathione(600Da), reduced glutathione (300Da). The result of gelatin chromatographic analysisshowed: molecular weight of peptides in the mixed enzyme of papain and neutralhydrolysis accounted for58.91%was amount of2500-300Da; and accounted for27.48%was greater than2500Da; accounted for14.86%was amount of2500-1335Da; accounted for25.54%was amount of1335-600Da; accounted for18.51%was amountof600-300Da and accounted for13.57%was less than300Da.(3) According to the molecular weight distribution, the mixed polypeptide wasultra filtrated consecutively by membrane and the optimal ultra filtration condition wasdetermined through exploring the factors of pH, substrate concentration, temperature,time and consumed enzyme. The different molecular weight peptides were obtainedthrough spray drying and determined the proportion of each peptide fragment. Theresult showed: wheat germ protein hydrolysis that the degree of hydrolysis was19.03%were ultra filtrated consecutively by upgrade cellulose FP membrane with the molecularcut off of3000Da,1500Da and1000Da．The optimum conditions of the ultra filtrationwere material concentration1.5％-2％, temperature30℃，pressure0.15MPa and time80min．The recovery rate of wheat germ peptides after ultra filtration was up to90.12%.The proportion of four kinds of classification polypeptide respectively were peptide I(molecular weight>3000Da)25.36%, peptide II (3000-1500Da)20.79%, peptide III(1500-1000Da)22.81%, peptide IV (<1000Da)31.04%.