This study sought to assess the ideal conditions under which hydrolysate can be produced from the split gill mushroom
proteins through the microbial
protease,
Alcalase. The research employed a central composite design and response surface methodology. Three specific parameters were varied for the purposes of the experimental process, while a fixed pH value of 8 was used in all cases. The variables were hydrolysis temperature (set as 45 °C, 50 °C, or 55 °C), hydrolysis time (set as 60 min, 120 min, or 180 min), and the ratio of
enzyme to substrate (set
as 2%, 4%, or 6% w/v). The variables under investigation exert a significant influence upon degree of hydrolysis (DH) in addition to 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (
ABTS) radical-scavenging activity (p < 0.05). Fractionation of the hydrolysate was accomplished using molecular weight (MW) cut-off membranes, while the greatest radical-scavenging capability was observed in the < 0.65 kDa fraction. The MW < 0.65 kDa fraction underwent separation through RP-HPLC in order to create five sub-fractions. Among these, the greatest
ABTS radical-scavenging capability was observed in the F5 sub-fraction, which was therefore chosen to undergo additional examination using quadrupole-time-of-flight-electron spin induction-mass spectrometry-based de novo
peptide sequencing. Via this process it was possible to determine five
antioxidant peptides. Furthermore, the MW < 0.65 kDa fraction was able to demonstrating cellular
antioxidant activity in the context of a human
intestinal cancer cell line (HT-29). The extent of this activity was shown to depend upon the concentration levels of the
peptide.