Researchers have demonstrated that pre-treating marine collagen extracted from fish skin with the plant-derived protease papain prior to simulated gastrointestinal digestion substantially accelerates protein breakdown and releases potent bioactive peptides. Utilizing skin by-products from the snapper salmon, Otolithes ruber, the scientific team established a multi-stage enzymatic digestion strategy designed to overcome the dense triple-helical structure characteristic of native collagen. By combining papain pre-treatment with sequential pepsin and trypsin hydrolysis, the resulting hydrolysates exhibited robust in vitro inhibitory activity against both angiotensin-converting enzyme and dipeptidyl peptidase-IV, alongside strong free radical scavenging capabilities. Detailed mass spectrometry and computational docking revealed that this dual-stage digestion protocol liberated twice as many unique peptide sequences as conventional digestion alone. Among these identified fragments, a specific octapeptide demonstrated exceptionally favorable binding affinities within the catalytic pockets of both target enzymes, highlighting an efficient strategy for converting discarded marine fisheries waste into functional biomolecules.

The extraction and valorization of marine structural proteins addresses substantial environmental and economic challenges associated with industrial fish processing by-products. Globally, massive quantities of fish skins, bones, and scales are discarded into landfills, generating severe organic pollution, unpleasant odors, and municipal waste management burdens. To recover valuable structural proteins from this biomass, investigators sourced fresh snapper salmon skins from a seafood market in Durban, South Africa, and subjected the tissue to chemical pretreatments. The raw skin was treated with zero-point-one molar sodium hydroxide for forty-eight hours to eliminate non-collagenous proteins, followed by immersion in ten percent butyl alcohol for twenty-four hours to remove lipid fractions. Following washing, the defatted tissue was solubilized in zero-point-five molar acetic acid for seventy-two hours at four degrees Celsius, filtered, centrifuged, and precipitated with sodium chloride. After dialysis and freeze-drying, acid-soluble collagen was obtained with an extraction yield of seventy-eight point seventy-three percent on a dry weight basis, markedly outperforming published yields for yellowfin tuna, seer fish, and Asian sea bass.

Biophysical and biochemical characterization verified the high purity and structural integrity of the acid-soluble collagen extracted from the snapper salmon skins. Ultraviolet-visible absorption spectroscopy revealed a prominent absorption peak at two hundred and thirty-two nanometers, corresponding directly to peptide carbonyl, carboxyl, and amide bonds within the triple-helical polypeptide chains, alongside a minor shoulder near two hundred and eighty nanometers reflecting low aromatic residue concentrations. Fourier-transform infrared spectroscopy confirmed native structural preservation, displaying characteristic amide B stretching bands at twenty-nine twenty-eight reciprocal centimeters, carbonyl stretching vibrations within the amide I region, and triple-helical hydrogen bonding near fifteen hundred and forty reciprocal centimeters. Differential scanning calorimetry recorded an initial endothermic denaturation peak at one hundred and four point ninety-one degrees Celsius and a secondary breakdown peak at two hundred and three point fifty-five degrees Celsius. Circular dichroism confirmed the intact triple helix through a negative band at two hundred nanometers, while electrophoresis displayed characteristic alpha-one, alpha-two, and beta chains alongside an amino acid profile rich in glycine, alanine, and proline.

To evaluate proteolytic efficiency and peptide release, the purified collagen was subjected to two distinct pathways representing simulated digestion with and without exogenous pre-treatment. In the experimental protocol, designated as papain pre-digestion followed by pepsin-trypsin digestion, a five percent collagen solution was incubated with one percent Carica papaya papain at pH six point zero and sixty degrees Celsius for five hours, followed by thermal enzyme inactivation and centrifugation. The supernatant was introduced into simulated gastric fluid containing porcine pepsin at pH three point zero for four hours at thirty-seven degrees Celsius, before being adjusted to pH six point eight and treated with trypsin for six hours. A control group subjected untreated collagen directly to identical pepsin-trypsin digestion. Membrane ultrafiltration demonstrated a profound shift in molecular weight distribution: while direct pepsin-trypsin digestion left over fifty-one percent of hydrolysates exceeding ten kilodaltons and yielded only eleven point eighty-three percent below three kilodaltons, papain pre-digestion produced thirty-three point thirty percent low-molecular-weight peptides under three kilodaltons.

Evaluation of the enzymatic hydrolysates revealed outstanding in vitro antihypertensive potential through the targeted inhibition of angiotensin-converting enzyme. Within physiological systems, this zinc metallopeptidase converts inactive angiotensin-I into the vasoconstrictor angiotensin-II while inactivating the vasodilator bradykinin, serving as a primary therapeutic target for blood pressure management. In fluorogenic microplate inhibition assays, hydrolysates generated through papain pre-digestion demonstrated a potent half-maximal inhibitory concentration of zero-point-zero eighty-eight milligrams per milliliter, demonstrating significantly higher inhibitory potency than hydrolysates produced by direct pepsin-trypsin digestion. When fractionated by molecular size, the low-molecular-weight fraction under three kilodaltons exhibited an almost identical half-maximal inhibitory concentration of zero-point-zero eighty-nine milligrams per milliliter, confirming that smaller peptide fragments drive enzymatic suppression in a clear dose-dependent manner. This measured inhibitory potency substantially exceeded benchmarks established in published literature for other marine species, including Shortfin scad hydrolysate fractions that recorded an inhibitory concentration of two point twenty milligrams per milliliter and Nile tilapia collagen hydrolysates that exhibited one point two milligrams per milliliter.

Parallel biochemical testing demonstrated that the pre-digested fish collagen hydrolysates also exhibited notable antidiabetic capabilities through the suppression of dipeptidyl peptidase-IV. This serine exopeptidase is responsible for the rapid physiological degradation of incretin hormones, specifically glucagon-like peptide-one and glucose-dependent insulinotropic polypeptide, which are essential for stimulating glucose-dependent insulin secretion and maintaining glycemic control. Suppressing dipeptidyl peptidase-IV activity prolongs the circulating half-life of these incretins, thereby enhancing postprandial endocrine regulation. In standardized in vitro microplate screening assays, the hydrolysate derived from papain pre-digestion achieved a half-maximal inhibitory concentration of one point sixty-seven milligrams per milliliter, which proved significantly superior to the two point twenty-two milligrams per milliliter observed for hydrolysates prepared through conventional pepsin-trypsin digestion alone. Mirroring trends observed in blood pressure regulation assays, the isolated low-molecular-weight fraction under three kilodaltons yielded an inhibitory value of one point sixty-nine milligrams per milliliter, comparing favorably against previously documented values for eel protein hydrolysates and simulated gastrointestinal digests of Alaska pollock skin collagen.

In addition to metabolic enzyme inhibition, the investigated marine peptides exhibited pronounced radical scavenging capabilities across multiple in vitro antioxidant testing models. When challenged with the stable nitrogen-centered organic radical DPPH, all peptide fractions and whole hydrolysates derived from the papain pre-digested series displayed significantly lower half-maximal inhibitory concentrations compared to the control group. The complete hydrolysate from the papain-treated protocol achieved a half-maximal inhibitory concentration of two point thirty-eight milligrams per milliliter, whereas hydrolysates produced solely through pepsin-trypsin digestion failed to attain meaningful clearance within the baseline experimental concentration boundaries and only showed scavenging at elevated concentrations. Strikingly, intact, unhydrolyzed snapper salmon skin collagen displayed zero detectable DPPH scavenging activity, illustrating that cryptic antioxidant peptide sequences remain dormant within native triple helices until released via enzymatic cleavage. The measured scavenging concentration of the papain-treated hydrolysate demonstrated superior potency when compared to published values for Blue shark skin gelatine hydrolysates and compared favorably to commercial salmon protein hydrolysates produced via alcalase, neutrase, or trypsin.

Complementary assessment using the ABTS decolorization assay confirmed that pre-digested collagen peptides act as effective chain-breaking antioxidants through electron-transfer and hydrogen-atom-donation mechanisms. In this spectrophotometric system, potassium persulfate oxidizes ABTS into a stable blue-green radical cation whose characteristic absorbance at seven hundred and thirty-four nanometers is diminished upon reduction by antioxidant molecules. Hydrolysates produced via papain pre-digestion exhibited an extraordinarily low half-maximal inhibitory concentration of zero point zero twenty-six milligrams per milliliter, substantially outperforming the zero point zero forty-six milligrams per milliliter recorded for standard pepsin-trypsin hydrolysates. Moreover, size-fractionated testing revealed that intermediate peptides within the three to ten kilodalton range exhibited the highest overall ABTS radical scavenging capacity, achieving an inhibitory concentration of zero point zero seventeen milligrams per milliliter. The authors attributed this heightened radical-quenching efficiency to the abundant presence of electron-donating aromatic and hydrophobic residues, particularly tyrosine and phenylalanine, alongside glutamine, asparagine, aspartate, and glycine, which supply excess electrons to neutralize reactive radicals, stabilize reactive intermediates, and terminate lipid peroxidation cascades.

High-resolution liquid chromatography-tandem mass spectrometry coupled with an array of in silico computational tools provided deep structural insight into the peptide profiles generated by each enzymatic approach. Mass spectrometric analysis using an Orbitrap mass spectrometer identified fifty-four unique peptide sequences among one hundred and sixteen detected fragments in the papain pre-digested hydrolysate, whereas direct pepsin-trypsin digestion generated only twenty-five unique sequences from forty-seven total fragments. Computational screening through PeptideRanker revealed that both digestion pathways produced bioactive candidates scoring above the zero-point-five threshold, with top-ranked papain-derived peptides reaching scores between zero point ninety-three and zero point ninety-six. Antioxidant prediction software AnOxPePred scored the papain-derived peptide GSAGPPGPPGPPGPPG at zero point sixty-eight, higher than the top candidate from pepsin-trypsin digestion. Furthermore, targeted screening using AHTpin and StackDPPIV identified forty-eight potential antihypertensive peptides and twenty dipeptidyl peptidase-IV inhibitory peptides from the pre-digested pool, compared to twenty-four and twenty-one from the control digest, while theoretical physicochemical profiling revealed molecular weights spanning zero point seventy-six to two point twenty-six kilodaltons.

Rigid molecular docking simulations conducted against crystal structures of the human angiotensin-converting enzyme receptor and dipeptidyl peptidase-IV illuminated the atomic interactions underlying these observed bioactivities while framing essential study boundaries. When docked against angiotensin-converting enzyme, top peptides from the pre-digested hydrolysate, including GSAGPPGPPGPPGPP and IGFPGFPG, exhibited binding energies of minus eleven point three and minus eleven point two kilocalories per mole, outperforming the clinical standard inhibitor captopril by establishing robust hydrogen bonds and critical catalytic zinc-ion coordination. In parallel, docking against dipeptidyl peptidase-IV demonstrated favorable affinities reaching minus nine point four kilocalories per mole, with the octapeptide IGFPGFPG demonstrating minus nine point one kilocalories per mole across active site subsites. Crucially, IGFPGFPG emerged as the sole sequence exhibiting dual-target binding stability, driven by hydrophobic interactions involving its isoleucine, proline, and phenylalanine residues. The authors emphasized that these promising observations are strictly derived from in vitro enzyme kinetics, cell-free chemical assays, and computational docking models, meaning that practical functional food development requires synthesized peptide validation and human clinical trials.

Subject of Research: Bioactive peptide release from fish skin collagen using sequential enzymatic hydrolysis

Article Title: ACE inhibitory and DPP-IV inhibitory activity of collagen peptides derived from the snapper salmon (Otolithes ruber) skin collagen via papain and pepsin-trypsin hydrolysis

Article References: Elisha, C., Bhagwat, P., Amobonye, A., & Pillai, S. (2026). ACE inhibitory and DPP-IV inhibitory activity of collagen peptides derived from the snapper salmon (Otolithes ruber) skin collagen via papain and pepsin-trypsin hydrolysis. Collagen and Leather, 8(1), Article 16. https://doi.org/10.1186/s42825-026-00248-7

Image Credits: AI Generated

DOI: 10.1186/s42825-026-00248-7

Keywords: Fish Collagen, Bioactive Peptides, Papain, ACE Inhibition, DPP-IV Inhibition, Antioxidant Activity, Otolithes ruber, Enzymatic Hydrolysis, inhibitory, DPP-IV, activity, collagen