Properties Of Wheat Malt Endopeptidase And Its Degradation Rule In Soybean Oil Meal

Wheat malt is rich in enzymes,among which endopeptidase mainly react on the peptide bonds inside the protein polypeptide chain to break down the long protein chain into short peptides.In many food raw materials and food processing processes,the activity of endopeptidase directly affects the content and composition of substrate and product protein,and which is also an important monitoring index for many fermented foods.Soybean oil meal has high protein content,which has large molecular weight,compact structure and low digestibility.When degraded by peptidase,large proteins of soybean oil meal would be broke down into small proteins,peptides and amino acids,and as a result the utilization rate of proteins is improved.As a natural compound enzyme,wheat malt should solve the problems of high molecular weight and low digestibility of protein molecules in soybean oil meal.In this study,we improved the method for the determination of activity of peptidase in wheat malt,explored the enzymatic properties and kinetic constants of peptidase in wheat malt,and determined the optimum conditions for the determination of activity.The effects of enzymatic hydrolysis time on the protein content,amino nitrogen content and protein molecular weight distribution of soybean oil meal were studied.The antioxidant peptides in soybean oil meal degradation products were investigated.The main research results are as follows:（1）In this experiment,on the basis of observing the protein bands distribution of reaction products,TCA was used to precipitate macromolecular proteins and to acquire peptide fragments,and the true activity of endopeptidase was determined by determine the peptide fragments of the action system.The experimental results,showed that adding TCA could better precipitate macromolecule proteins,and the experimental precision was good.The optimal conditions for the determination of endopeptidase activity in wheat malt were:reaction time was 5h,enzyme addition amount was 20u.（2）The optimal pH value of endopeptidase in wheat malt was 4,and the enzyme activity could keep stable for 60 minutes when pH=4,while the enzyme activity could not keep stable when pH=4.5,5,5.5.With the extension of culture time,the enzyme activity decreased to different degrees.The optimal reaction temperature of the enzyme is 50℃,and 40℃,45℃and 50℃is more suitable for the endopeptidase to remain stable.At 60℃,the endopeptidase activity decreases with the extension of culture time.Meanwhile,the measured value of linear fitting Km of Lineweaver Burk plot is 6.247mM,indicating that the affinity between the enzyme and the substrate was strong.The addition of 5mmoL/L Ca²⁺、Pb²⁺、Zn²⁺、Cu²⁺and EDTA significantly inhibited the activity of endopeptidase.（3）The effects of different degradation time on enzymatic hydrolysis of soybean oil meal were studied,and the optimal enzymatic hydrolysis time was 8h.The results showed that the amino acid nitrogen content of soybean oil meal reached to 5.19mg/g after enzymatic hydrolysis.The content of water-soluble protein（WSP）was significantly increased to23.85%.The content of salt-soluble protein reached 9.60%.The content of alcohol-soluble protein decreased to 6.27%.The alkali-soluble protein content was also significantly reduced to 6.03%.The results of SDS-PAGE showed that the the water-soluble protein at 8h and 10h after enzymatic hydrolysis had maximum number of bands and the deepest bands color.The alkali-soluble protein and alcohol-soluble protein bands were reduction to different degrees.Raman spectroscopy showed that the vibration mode of disulfide bond changed to t-g-t mode,which meaned that the secondary structure area of soybean oil meal increased significantly after enzymatic hydrolysis.The results of SEM showed that the surface of soybean oil meal protein was broken with degradation time.（4）In this experiment,two different methods of acid extraction and ethanol precipitation were used to precipitate macromolecular proteins to obtain product polypeptides.The results showed that there was no significant difference between the two methods in the precipitation of macromolecular proteins.Product polypeptides of soybean oil meal with molecular weight<3kDa had the highest activities,such as hydroxyl radical scavenging rate was the highest,iron reduction ability was the strongest,ABTS antioxidant capacity was the strongest.