# Characterization of Bromelain‐Soluble Sheepskin (Ovis aries) Proteins and Effect of In Vitro Gastrointestinal Digestion on Angiotensin Converting Enzyme Inhibition Activity

## TL;DR

This study identifies bioactive peptides from sheepskin proteins that can inhibit a blood pressure-related enzyme after simulated digestion.

## Contribution

Novel ACE-inhibitory peptides from sheepskin collagen were identified using in silico analysis after in vitro digestion.

## Key findings

- Bromelain-soluble sheepskin proteins include keratin and collagen, with major amino acids like arginine and lysine.
- In vitro digestion increased ACE inhibition activity from 0% to 19.94% in ultrafiltrated peptide fractions.
- Two potent ACE-inhibitory peptides, GPAGPAGPR and QGPPGPAGPR, were identified from sheepskin collagen.

## Abstract

This work characterizes proteins extracted from sheepskin using bromelain and examines angiotensin converting enzyme (ACE) inhibition activity of the peptides generated by in vitro gastrointestinal digestion (GID). Gel electrophoresis revealed three major protein bands corresponding to keratin and collagen. In vitro gastrointestinal digestion facilitated protein breakdown into peptides, yielding below 3 kDa of molecular weight peptides. Arginine, tyrosine, lysine, and leucine were identified as the major residues of amino acids in the bromelain‐soluble protein hydrolysate (BSPH). The action of inhibiting ACE of 1 mg/mL of BSPH was 48.99%, whereas the activity of the < 3 kDa ultra‐filtrated fractions obtained before and after in vitro GID were 0% and 19.94%, respectively. To acquire the peptide profile and fractionate the ultra‐filtrated peptide fractions, reverse‐phase high‐performance liquid chromatography (RP‐HPLC) was utilized. Among all the RP‐HPLC fractions, 2, 3, 12, and 15 had the most active ACE inhibition. Subsequently, the peptide sequences from four active fractions were recognized using tandem mass spectrometry (MS/MS) and followed by in silico analysis to examine their bioactivity. Two potent ACE‐inhibitory peptide candidates from sheepskin collagen, GPAGPAGPR, and QGPPGPAGPR, were identified by in silico analysis.

Novel bioactive peptides resulted from in vitro gastrointestinal digestion of soluble sheep skin protein.