ACE-inhibitory Peptides Derived From Milk Protein By Gradient Dilution Continuous Coupling Of Enzymatic Hydrolysis And Membrane Separation: Modeling And Experimental Studies

In order to study and investigate the optimum process model of preparing ACE-inhibitory peptides from milk protein by enzymatic membrane reactor, three models of integration of enzymatic hydrolysis and membrane separation(IEM),including the IEM with feeding material by the way of gradient dilution, the IEM with continuous feeding and the IEM with adding water after continuously supplying raw material, were analyzed and compared. On the basis of previous research, the process stability and effects of three models were investigated in the course of reaction. Then,the reaction process and kinetics of the gradient dilution of IEM were studied. The detailed results are as follows:Five different proteases were applied to hydrolyze milk protein to produce ACE peptides respectively. According to the ACE-inhibitory activity and IC50, Neutrase was chosen for the further study. Based on the single factor experiment, orthogonal design was adopted and the optimal conditions of enzymatic reaction were obtained as follows: reaction time of 60 min, substrate concentration of 11%, enzymatic dosage of2000 U/g, temperature of 50℃ and pH 7.0.Using the membrane flux, permeates’ ACE-inhibitory activity and IC50 as factors,the molecular weight of ultra-filtration membrane was determined as 5 kDa. The effects of reaction time, substrate concentration, enzymatic dosage and cycle pump speed on conversion rate of protein were investigated respectively. The optimal conditions obtained from the single factor experiments were: substrate concentration7%, enzymatic dosage 2000 U/g, reaction time 100 min and cycle pump speed 100r/min. Under the above conditions, conversion rate of protein reached 53.16% with productivity of 39.59 g peptide·(g protease)-1 and the inhibitory rate of ACE as76.81%. Compared with the control group, the three contents were increased by16.99%, 16.61% and 18.55% respectively. The relative molecular weight of permeates was measured by high-performance size-exclusion chromatography(HPSEC). The results showed that the molecular weight could be effectively controlled by ultra-filtration and 86.60% of sample was less than 3 kDa.According to the reaction process curve of three kinds of feeding models, it is illustrated that the gradient dilution feeding system was more stable than the other two feeding system with high permeates’ concentration(12.748 mg/mL), the smallest change of membrane flux(120.403 mL·(m2·min)-1) and relatively stable protein concentration in reactor(22.637 mg/mL). In addition, the method of gradient dilution feeding had the highest conversion ratio of protein(67.58%) and peptide yield(67.07%). Compared with the method of adding water after continuous feeding, the two contents were increased by 12.35% and 9.93% respectively. In comparison to the method of continuous feeding, the two contents were increased by 62.30% and34.03% respectively. The gradient dilution continuous integration of enzymatic hydrolysis and membrane separation was an effective polypeptides preparation method due to its high enzymatic hydrolysis efficiency and stability.By making some suitable hypothesis during the process of IEM with gradient dilution feeding material, the equation of degree of hydrolysis(DH) with substrate concentration(S0) and hydrolysis time(t) was built. On basis of the equation, it could predict the variation tendency of hydrolysis degree at other substrate concentration with time. A kinetic model for the hydrolysis process was built. The kinetic constants,Km of 69.481 g/L and Vmax of 0.752 g·l-1min-1, were obtained. At different substrate concentrations, the enzymatic hydrolysis process of gradient dilution feeding model was simulated. It was confirmed that the experimental values were in a good accordance with those of simulation values. The optimal substrate concentration obtained by the kinetic constant was 70 g/L.Under the optimal substrate concentration, the gradient dilution feeding system can work continuously and stably for 720 min. The results showed that the conversion rate of protein was 74.51%, the peptide yield was 75.13%, the production of peptide was 142.27 g peptide·(g protease)-1, the IC50 value was 0.77 mg/mL, the ash and moisture of the product were 8.04% and 11.68%, and the peptide content was 37.40%.In conclusion, the model, gradient dilution continuous coupling of enzymatic hydrolysis and membrane separation, could not only overcome the low utilization rate of enzyme and substrate deficiency at late stage of reaction, but also absorb theadvantages of other feeding models to improve the production efficiency. It could provide a more effective method for the preparation of ACE-inhibitory peptides from milk protein.