Preparation Of Antioxidant Peptides From Fishscale By Coupling Of Enzymatic Hydrolysis And Membrane Separation

The objective of this research was to study the preparation of antioxidant peptides from Fishscale by coupling of enzymatic hydrolysis and membrane separation technology.To explore the optimum preparation methods of antioxidant peptides from Fishscale by coupling of enzymatic hydrolysis and membrane separation, the effects and kinetic parameters of three reaction modes, which included the batch reaction, continuous membrane reaction without feeding the material and continuous membrane reaction with feeding the material by the way of gradient dilution, were analyzed and compared.Seven different commercial proteases were employed to produce antioxidant peptides from hydrolyze scale collagen by the batch reaction mode. According to the DPPH free radical scavenging effect of hydrolysate, the Alcalase 2.4 L was chose for the further study. And then, the response surface experiments were conducted to optimise the condition of enzymatic hydrolysis.Based on the DPPH free radical scavenging effect of hydrolysate and conversion rate of protein, the optimised experiment of batch reaction was carried out. Hydrolysates were separated by flowing through the ultrafiltration membrane with molecular weights of 10,8,5 and 3 kDa successively. The groups with molecular weight less than 3 kDa were collected and analyzed to calculate the hydrolyzation properties and kinetic parameters. The optimum conditions of batch reaction were substrate concentration of 4%, enzyme concentration of 1%, pH 9.0, temperature of 50℃, and time of 30mins. Under the optimum conditions, the conversion rate of protein was 95.31%, the peptides yield was 71.47%, the peptides mass per gram of enzyme was 71.58 g·g-1, the EC50 value was 14.04 mg-mL-1, and the kinetic parameters of Michaelis constant (Km) and turnover number (kcat) were 73.64g·L-1 and 5.81min-1, respectively.According to the conversion rate of protein, the optimised experiment of continuous reaction without feeding material was carried out. Hydrolysates were separated by flowing through the ultrafiltration membrane with molecular weight of 3 kDa. The groups of permeation were collected and analyzed to calculate the hydrolyzation properties and kinetic parameters. The optimum conditions of continuous reaction without feeding material were substrate concentration of 4%, enzyme concentration of 1.5%, time of 30mins, temperature of 30℃, pump rotation speed of 120 r·min-1 and pH 9.5. Under the optimun conditions, the conversion rate of protein was 97.70%, the peptides yield was 50.56%, the peptides mass per gram of enzyme was 842.67 g·g-1, the EC50 value was 14.84 mg·mL-1, and the kinetic parameters of Michaelis constant (Km) and turnover number (kcat) were 35.47 g·L-1 and 47.15 min-1, respectively.Based on the results of continuous reaction mode without feeding material, and the conversion rate of protein, the optimised experiments of continuous reaction mode with feeding material by the way of gradient dilution were carried out. The optimimum conditions were substrate concentration of 4%, enzyme concentration of 1.5%, pump rotation speed of 120 r-min-1, temperature of 50℃, pH 9.0, and feeding material concentrations of 3.5%,3%,2.5%,2%,1.5%,1%,0.5% and 0%, respectively. The reaction was stopped when the pollution of membrane was severe very much. The results showed that the total consumed volume of material was 8000 mL, the reaction time was 190mins, the conversion rate of protein was 97.42%, the peptides yield was 73.83%,the peptides mass per gram of enzyme was 258.65 g·g-1, the EC50 value was 18.24 mg·mL"1, and the kinetic parameters of Michaelis constant (Km) and turnover number (kcat) were 35.41 g·L-1 and 1.27min-1, respectively.The coupling of enzymatic hydrolysis and membrane separation can avoid the inhibition effects of products, improve the reaction process, and enhance the conversion rate of protein. By the online separation of products from reaction solution, the over-degradation of peptides was avoided effectively, and the activity of products was increased. The peptides mass per gram of enzyme was increased by recycling the enzyme.