Optimization Research On Hydrolysis Of Walnut Protein By Trypsin Protease

In recent years, lots of studies showed that diet of walnut have the function for delay aging and Health promotion. Although many results indicated that walnut oil, walnut protein, polyphenol, walnut polypeptide for antioxidant and other components might be active ingredients, However, the mechanism effecting health is not very clear. Storage proteins in walnut digested by pepsin, trypsin and chymotrypsin would produce polypeptides, which may play essential role in walnut efficiently. In this paper, optimum conditions for trypsin digesting walnut protein in vitro to get polypeptides will be conducted.Moreover, The experiment of utilization of trypsin showed that protection efficiently of fructose for trypsin in extreme conditions, i.e. extreme alkaline pH, metal ions, high temperature, was also conducted in this paper. This research will be valuable for trypsin digesting walnut crude protein. In this paper, the study mainly involved the following aspects:1 The L16(45) orthogonal experiment was used to evaluate the factors affecting trypsin protease hydrolyzing walnut crude protein. The optimum reaction conditions about hydrolysis of walnut protein by trypsin protease were:reaction time 5 hr, substrate concentration 50g/L(w/v), reaction temperature 35℃, pH8.5 and enzyme/substrate ratio 60 g/kg(w/w). The sequence of factors that worked on the hydrolysis of walnut protein were shown as below:temperature, substrate concentration, enzyme/substrate, reaction time, pH. On the other hand, our study indicated that the polypeptides of walnut on Bacterium pseudoanthracis and Staphylococcus aureus have the Bacteriostatic effect than others experiment bacterium.2 Our study found that the protective effect of trypsin by D-fructose hasn't been reported yet in alkaline conditions. Under alkaline conditions, the protective effect of fructose is best of fructose, the activity of trypsin remained 50% after the treatment of fructose, while the no treatment group was only 24%.3 Our study of temperature affect on the activity of trypsin showed that, trypsin activity gradually increase with the increasing of temperature, and get activity of protoease the maximum at 40℃, and then reduced gradually, the activity of trypsin was 72% of 20℃to 40℃, but only 13% of 70℃to 40℃. Protease activity of fructose-treated group are higher than the untreated group at the same time, and the greatest difference at 40℃.4 In alkaline conditions of pH12,10% concentrations of fructose have a better protective effect on trypsin, and fructose 5% treatment group was 1.55 times than the untreated group of activity of trypsin, when the fructose concentration was 10%, the activity of protease is 1.66 times than the control. However, when the concentration of fructose was 70%, the activity of protease was only 1.54 times than the control.5 In alkaline condition, the protect effect of fructose treatment on trypsin in water bath was no significantly changed with the increase of time, after 13h, the trypsin activity remained 99%, while the no treatment group was only 73%.6 Moreover, the fructose has the protective effect on the stabilization of trypsin against metal ions, detergent SDS, protease inhibitors, which lead to enzyme inactivation. After deal with 2% SDS for 30 minutes, the activity remained at 91% of treatment group. while untreated group was only 41%. The SDS-PAGE results have the dispersion phenomenon, So we Speculated that this may be a phenomenon of glycation on trypsin by fructose. Then we suggest that the protective mechanism of trypsin is glycosylation of trypsin by D-fructose.