Antioxidant Properties Of A Plastein Reaction-Stressed Casein Hydrolysate With Two Added Extrinsic Amino Acids

Casein, as one of mik proteins of high-quality, has desired functional properties such as solubility, emulsibility, foamability, gelation capacity and some biological properties including antibiosis, antioxidanty, angiotensin-Ⅰ-converting enzyme inhibitory activity and others. Plastein reaction is a protease-catalyzed reaction and can be used for food proteins to modify their important qualities, e.g., increasing of added value, improving functional properties and incorporating some essential amino acids for better nutritive value.In the present study, papain was used to prepare casein hydrolysate in vitro. Extrinsic phenylalanine or tyrosine was added into hydrolysate to carry out a papain-catalyzed plastein reaction. The modified hydrolysate with the highest activity was thus selected and subjected to solvent fractionation, with the original hydrolysate as control. Four ethanol-or methanol-water solvents in proportions of3:7,4:6,5:5or6:4(by volume), respectively, were used to fractionate the prepared casein hydrolysate and the modified hydrolysate. Some antioxidant properties of casein hydrolysate, modified hydrolysate and fractionated hydrolysate were evaluated in vitro. The results obtained in this study are as follows.1) Casein was hydrolyzed by papain to prepare casein hydrolysate. The degree of hydrolysis (DH), peptide recovery and scavenging activity on DPPH radical of the otained hydrolysate was assayed. A casein hydrolysate with hydrolysis time of2hours had a DH of9.4%and a scavenging activity on DPPH radical of38.7%, were selected as the substrate of the plastein reaction.2) Extrinsic phenylalanine or tyrosine was added into the prepared hydrolysate to carry out the papain-catalyzed plastein reaction. When reaction time was fixed at5h, response surface method was used to optimize the plastein reaction conditions, which were reaction temperature of30℃, substrate concentration of50%(by mass per volume), enzyme addition level of3kU/g peptides and amino acid addition level of0.74mol/mol, respectively.3) Under the condition of optimization, some modification was produced and evaluated for their antioxidation with the original hydrolysates, the mixtures of hydrolysates and phenylalanine or tyrosine as controls. The evaluation results showed that the carried out plastein reaction without phenylalanine or tyrosine addition could improve the three investigated antioxidant properties of the modified casein hydrolysates, including scavenging activity on DPPH radical, reducing powder and scavenging activity on hydroxyl radical. The modified casein hydrolysates with phenylalanine or tyrosine addition had better antioxidant properties as their scavenging activities on DPPH radical increased from38.7%to45.6-49.3%, reducing powder increased from0.497to0.608-0.662and scavenging activity on hydroxyl radical increased from17.2%to21.2-29.7%. Both addition of phenylalanine or tyrosine and reaction time applied in the plastein reaction did not give obvious impacts on antioxidant properties of the modified hydrolysates.4) The solvent fractionation was able to further enhance the antioxidant properties of the modified hydrolysates. Upon this solvent fractionation treatment, the obtained supernatant (or precipitate) fraction had enhanced (or decreased) antioxidant activity. With the concentration of ethanol or methanol in the fractionation solvent increased, antioxidant activity of the supernatant fraction increased, but the precipitate fraction was on the contrary. Analysis results showed that the IC50values of the supernatant and precipitate fractions of one modified casein hydrolysates4on DPPH radical were0.69mg/mL and2.09mg/mL, respectively, revealing a remarkable difference in activity. The solvent fractionation had potential application to separate antioxidant peptides with better activity.5) Analysis results for amino acid compositions showed that compared with the precipitate fraction, the obtained supernatant fraction contained more hydrophobic amino acids, i.e., the fractionated hydrolysates was in higher antioxidant activity and more hydrophobic amino acids. But detailed research is needed to clarify the relationship between amino acid compositions and antioxidant properties of antioxidant peptides.