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Free Radical Scavenging, Redox Balance and Wound Healing Activity of Bioactive Peptides Derived from Proteinase K-Assisted Hydrolysis of Hypophthalmichthys molitrix Skin Collagen

Daniela Ilieorcid tiny, Andreea Iosageanuorcid tiny, Oana Craciunescu1*orcid tiny, Ana-Maria Seciu-Grama1orcid tiny, Catalina Sanda1orcid tiny and Florin Oancea2orcid tiny

1National Institute of R&D for Biological Sciences, 296, Splaiul Independentei, 060031 Bucharest, Romania

2National Institute for R&D in Chemistry and Petrochemistry – Icechim, 202, Splaiul Independentei, 060021 Bucharest, Romania

Article history:

Received: 21 December 2020

Accepted: 28 April 2022

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Key words:

fish peptides; protease; antioxidant activity; cell migration; oxidative stress


Research background. Various protocols of enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they target to minimize waste from industrial processing and environmental requirements. This study aimed to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulted bioactive peptides on free radicals scavenging, redox balance and regulation of fibroblast proliferation and migration processes.

Experimental approach. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K treatment, at specific sites, in controlled conditions. Ultrafiltration permeate was analyzed for molecular mass by gradient electrophoresis and gel filtration chromatography. The biologic activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions.

Results and conclusions. Enzymatic hydrolysis of extracted fish collagen was performed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblasts metabolism in a dose-dependent manner and up-regulated cell migration in a scratch-wound model. Pre-treatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of redox balance maintaining and skin wound healing processes improvement, which indicated further potential applications in the development of pharmaceutic and nutraceutic formulations.

Novelty and scientific contribution. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin aging phenomena.

*Corresponding author: +40212200882

§These authors have equally contributed to this study

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