Antioxidant and antihypertensive activities of bioactive peptides derived from fish collagen and milk lactoferrin

Abstract

Bioactive peptides (BPs) are short sequences of amino acids known for their physiological benefits, including antioxidant, antimicrobial, antihypertensive, and antidiabetic properties. BPs can be derived from various food proteins; from plant, meat, milk, and marine sources, through methods such as microbial fermentation or enzymatic hydrolysis. This study explored the potential of BPs derived from milk lactoferrin (LF) and fish collagen using in silico and in vitro analyses. The inhibitory potential of BPs derived from LF, a multifunctional milk glycoprotein found in bovine and human milk, against angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) enzyme were analysed. Initially, in silico digested bovine and human milk-derived-LF sequences were screened for potential ACE and DPP-IV inhibitory activity using AHTpin and StackDPPIV, respectively. Molecular docking results revealed that bovine LF peptides ‘EPYF’ (−10.1 kcal/mol) and ‘WQWR’ (−9.3 kcal/mol) had significantly lower binding energies with ACE and DPP-IV, respectively. In addition, in vitro analysis of bovine LF hydrolysates demonstrated IC50 values of 0.48 ± 0.01 mg/ml (ACE) and 0.93 ± 0.02 mg/ml (DPP-IV). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis identified the peptides ‘EPYF’ and ‘WQWR’ which are potential inhibitors of ACE and DPP-IV, respectively, present within the parent peptide sequences of the bovine LF hydrolysates. These findings suggested that bovine LF peptides, due to their bioactive potential and functional similarities to human LF, may serve as a viable alternative to human LF. Subsequently, the research evaluated the inhibitory potential of BPs derived from fish collagen. The first step involved extracting collagen from the pretreated snapper salmon skin using an acid-solubilisation technique, followed by characterisation through biophysical analyses, such as SDS-PAGE and CD spectroscopy. Thereafter, the extracted collagen was hydrolysed using a pre-digestive approach. Papain was used as the pre-digestive enzyme due to its broad specificity, which effectively breaks down collagen and releases a range of BPs. Pepsin and trypsin were also key enzymes utilised in the in vitro digestion process to mimic human gastrointestinal conditions, since they are present in the stomach and small intestine, respectively. In this regard, snapper salmon skin collagen was hydrolysed using two approaches: pre-digestion followed by pepsin-trypsin digestion (PDPTD) and pepsin-trypsin digestion alone (PTD). This yielded fish collagen hydrolysates (FCH), and the peptides were fractionated using various cut-off membranes (<3 kDa, 3–10 kDa, and >10 kDa). xv The FCH that did not undergo pre-digestion exhibited IC50 values of 0.54 ± 0.03 mg/ml (ACE inhibition), 2.25 ± 0.02 mg/ml (DPP-IV inhibition), and 0.04 ± 0.01 mg/ml (ABTS antioxidant activity). In contrast, pre-digested FCH showed lower IC50 values of 0.33 ± 0.06 mg/ml, 1.67 ± 0.08 mg/ml, 2.38 ± 0.31 mg/ml, and 0.03 ± 0.00 mg/ml for ACE inhibition, DPP-IV inhibition, DPPH antioxidant activity, and ABTS antioxidant activity, respectively. FCH (from both PDPTD and PTD methods) were subjected to peptide identification and the identified peptides underwent in silico analyses to assess their physicochemical properties and potential bioactivities. Molecular docking analysis of pre-digested FCH peptides identified the peptide, IGFPGFPG, with significant ACE and DPP-IV inhibitory activities, exhibiting low binding energies to ACE (−11.2 kcal/mol) and DPP-IV (−9.1 kcal/mol). The study concluded that pre digested collagen peptides demonstrated improved bioactive potential, as reflected by their lower IC50 values and docking score. Overall, the study concluded that BPs derived from bovine milk LF and fish collagen have potential as antioxidants and inhibitors of ACE and DPP-IV. These findings underscore their potential for use in developing dietary supplements and functional foods, which could offer a myriad of health-promoting properties.