Antioxidant Peptide Production Using Keratin from Feather Waste: Effect of Extraction and Thiol Blocking Method

Keratin-made biomaterials, including feathers, are considered a protein-rich bioresource due to their intrinsic properties, including biocompatibility, biodegradability, mechanical resistance, and biological abundance. Beta-keratin exists as an insoluble stringy protein due to the high presence of disulfide cross-links, and as a result, it is mechanically stable and resistant to enzymatic digestion. Because of this, it is not easily decomposed, and this has made the application of feathers difficult. In this study, after dissolving feathers in NaOH, sodium sulfide, and 2-Mercaptoethanol (2-ME), the relative molecular mass of beta-keratin was calculated. Thin-layer chromatography was also used to display proteins with lower molecular weights. The antioxidant activities of the samples were evaluated by Fe-chelating and free radical scavenging tests with 2,2-diphenyl-1-picrylhydrazyl (DPPH). To investigate the effect of blocking thiol groups on the antioxidant activity of dissolved keratin, iodoacetamide and H₂O₂ were used. According to the three methods-(A) sodium hydroxide, (B) sodium sulfide, and (C) urea and 2-ME-used to extract and dissolve the feathers, method C caused the least change in the chemical structure of keratin molecules. Method A destroyed the primary structure of keratin and drastically reduced its molecular mass, but method B caused a drastic increase in the molecular mass from 9.6 kDa to higher masses, due to intermolecular bonds. For the keratin molecules dissolved by method C, the Fe-chelating activity was 93.18% and free radical scavenging was 77.45%. Blocking the thiol group with iodoacetamide initially reduced the free radical scavenging activity with DPPH by 42%, but blocking it with H₂O₂ did not affect this activity. Also, blocking of the thiol group did not initially affect Fe-chelating activity and free radical scavenging activity. After a kinetic study of the activities, an interesting observation was that both blocking agents had negative effects on radical scavenging activity, but had positive effects on Fe-chelating activity. This indicates the complexity of the role of disulfide bonds in keratin's antioxidant behavior types. According to the observed antioxidant activities, it can be expected that beta-keratin extracted from chicken feathers is a suitable candidate for application in industrial, pharmaceutical, and health applications.