The Impact of Extrinsic Amino Acids and Solvent Fractionation on the in vitro Antioxidant Activity of Plastein Reaction-Stressed Casein Hydrolysates

Papain was used to prepare a casein hydrolysate with a degree of hydrolysis of 9.4 %. The hydrolysate had the in vitro antioxidant activity with a DPPH radical scavenging activity of 38.7 % and an EC50 of 1.63 mg/mL. Extrinsic phenylalanine or tyrosine was added to the hydrolysate for a papain-catalyzed plastein reaction. The temperature, substrate mass per volume fraction, and the levels of enzyme and amino acid addition during the reaction were optimized using response surface methodology with a fixed reaction time of 5 h, and were found to be 30 °C, 50 %, 3 kU per g of peptides and 0.74 mol per mol of the free amino groups of the hydrolysate, respectively. Some modified hydrolysates were prepared and their antioxidant activity was evaluated in terms of DPPH radical scavenging activity and reducing power. The results revealed that all prepared modified hydrolysates had significantly higher (p<0.05) scavenging activity and reducing power than the original hydrolysate, and among them one showed the lowest EC50 of 1.09 mg/mL against DPPH radical. When the modified hydrolysate with the highest activity was fractionated using ethanol/ water solvents in volume ratios of 3:7, 4:6, 5:5 and 6:4, the supernatant or precipitate fraction exhibited an enhanced or decreased activity or reducing power, especially with the solvent of lower polarity (e.g. 6:4 by volume). The obtained supernatant with the highest activity thus exhibited an EC50 of 0.69 mg/mL. The results show that extrinsic phenylalanine or tyrosine addition in the plastein reaction of casein hydrolysate and further solvent fractionation of the modified hydrolysate is applicable to improve the antioxidant properties of products.