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

Abstract

Za pripremu hidrolizata kazeina stupnja hidrolize od 9,4 % upotrijebljen je enzim papain. Dobiveni je hidrolizat imao in vitro antioksidativnu aktivnost tj. sposobnost uklanjanja DPPH radikala od 38,7 % i EC50 vrijednost od 1,63 mg/mL. Dodatkom fenilalanina ili tirozina potaknuta je plastein reakcija katalizirana pomoću papaina. Metodom odzivnih površina optimirani su sljedeći parametri pri vremenu reakcije od 5 h: temperatura od 30 °C, koncentracija supstrata od 50 % (m/V), udjel enzima od 3 kU/g peptida i udjel aminokiselina od 0,74 mol/mol slobodnih aminokiselinskih grupa hidrolizata. Pripremljeno je nekoliko modificiranih hidrolizata, te je ispitana njihova antioksidativna aktivnost, i to sposobnost uklanjanja DPPH radikala i reducirajuća snaga. Dobiveni su rezultati pokazali da su svi modificirani hidrolizati imali znatno veću sposobnost uklanjanja radikala (p<0,05) i reducirajuću snagu od izvornih hidrolizata, a među njima je bio i jedan s najnižom EC50 vrijednosti od 1,09 mg/mL. Frakcioniranjem modificiranog hidrolizata najveće antioksidativne aktivnosti pomoću etanola i vode u omjerima od 3:7, 4:6, 5:5 i 6:4 dobiveni su supernatanti ili precipitati veće ili manje antioksidativne aktivnosti i reducirajuće snage, naročito pomoću otapala male polarnosti (npr. omjera etanola i vode od 6:4). Supernatanti s najvećom aktivnošću imali su EC50 vrijednost od 0,69 mg/mL. Rezultati pokazuju da se dodatkom fenilalanina ili tirozina u plastein reakciji hidrolizata kazeina te daljnjim frakcioniranjem otapalom mogu dobiti modificirani hidrolizati veće antioksidativne aktivnosti.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