RESUMO
This study examined the antioxidant capabilities of peptides derived from chicken feather meal (CFM) protein hydrolysates which were produced using 3 different microbial proteases (Neutrase, Alcalase, and flavourzyme) and tested at varying concentrations, namely 1, 2, and 5% by weight. The highest levels of 2,2-diphenyl-1-picrylhydrazl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activities were presented by CFM hydrolysate derived using 5 wt% Neutrase and digested for 4 h. Fractionation of this particular hydrolysate was then performed by applying 10, 5, 3, and 0.65 kDa molecular weight cutoff membranes. It was then determined that the molecular weight (MW) < 0.65 kDa fraction achieved the greatest level of free radical scavenging activity in the context of DPPH and ABTS. The MW < 0.65 kDa fraction then underwent additional fractionation using reverse-phase high-performance liquid chromatography to derive 3 main fractions designated as F1, F2, and F3. All of these fractions presented a high level of activity in DPPH radical scavenging, although no significant ABTS scavenging was observed. Quadrupole time-of-flight tandem mass spectrometry was used in determining the peptide contents of the fractions as Phe-Asp-Asp-Arg-Gly-Arg-X for F1 (FDDRGRX, 875 Da), Val-Thr-Leu-Ala-Val-Thr-Lys-His for F2 (VTLAVTKH, 868 Da), and Val-Ser-Glu-Ile-X-Ser-Ile-Pro-Ile-Ser for F3 (VSEIXSIPIS, 1,055 Da). Moreover, the F2 fraction was shown to be capable of preventing DNA damage induced by hydroxyl radicals, as indicated in tests using the plasmids pKS, pUC19, and pBR322 via the Fenton reaction. This outcome was demonstrated through in vitro antiproliferative activity in human cell lines based on SW620 colon cancer, using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The F2 fraction at 0.5 wt.% was also shown to be capable of inducing weak early apoptosis, which could be measured by using the Fluorescein isothiocyanate Annexin V Apoptosis Detection Kit with Propidium Iodide Solution. Furthermore, an increase in caspase-3 and caspase-8 activity was observed in SW620 cells following exposure for 24 h and 48 h.
