Abstract:
The optimum conditions for hydrolysate production from split gill mushroom protein using the microbial protease, Alcalase were investigated. The study was conducted using central composite design and response surface methodology. Three independent factors: hydrolysis temperature (45, 50, and 55 °C); hydrolysis time (60, 120, and 180 min); and enzyme to substrate ratio (2%, 4%, and 6% w/v) were varied, while the pH was fixed at pH 8. These three factors had a significant effect on the DH and ABTS radical-scavenging activity (p < 0.05). The optimum conditions obtained from experiments were: enzyme to substrate ratio 2%; hydrolysis time 161.4 min; and temperature 55 °C. The coefficient of determination (R2) of total protein and the IC50 value for ABTS radical-scavenging activity were 0.972 and 0.205 µg/mL, respectively, and the DH value was 50.882%. The hydrolysate was fractionated by molecular weight (MW) cut-off membranes (10, 5, 3, and 0.65 kDa). The < 0.65 kDa fraction had the highest radical scavenging ability (IC50 = 0.026 ± 0.004 µg/mL). The MW < 0.65 kDa fraction was separated by reversed-phase HPLC to yield four subfractions (F1–5). The F4 subfraction showed the highest maximum ABTS radical-scavenging activity and was selected for further analysis by quadrupole-time-of-flight-electron spin induction–mass spectrometry-based. Five antioxidant peptides were identified. In addition, the MW< 0.65 kDa fraction protected against oxidation-induced DNA damage in pBR322, pKS, and pUC19 plasmids. Furthermore, the MW < 0.65 kDa fraction, exhibited cellular antioxidant activity in a human intestinal cancer cell line (HT-29), which was dependent on the peptide concentration. These results suggested that split gill mushroom protein hydrolysate is a good source of bioactive peptides and has potential as a natural antioxidant in food production and cosmetic products.
