The research team, led by Van-Ba Hoa and colleagues at the National Institute of Animal Science in Wanju, Korea, set out to develop cost-effective methods for converting livestock bones into value-added biomaterials. Their target molecules were collagen, the most abundant protein in the animal body and a mainstay of the nutraceutical and cosmetics industries, and chondroitin sulfate, a sulfated glycosaminoglycan recommended by the European League Against Rheumatism as a symptomatic slow-acting drug for osteoarthritis. Both compounds are traditionally extracted using commercial proteases such as pepsin and Alcalase, enzymes that cleave the chemical cross-links holding these molecules within the bone matrix. The problem, the authors note, is that these enzymes are expensive, driving up extraction costs and, ultimately, the price of the final products on the shelf.

The logic behind the pineapple approach rests on bromelain, a cysteine protease found throughout the pineapple plant but concentrated in the core, which makes up roughly sixteen percent of the fruit’s processing by-products. Pineapple is cultivated extensively across the globe, guaranteeing a stable and abundant supply of the raw material for enzyme production. To prepare their extract, the researchers ground fresh pineapple cores, filtered the pulp through cheesecloth, and centrifuged the liquid to obtain a crude preparation they call pineapple extract, or PE. Colorimetric assays revealed that the extract’s proteolytic activity rose from 1.18 units per milligram of protein at four degrees Celsius to 3.33 units per milligram at fifty-five degrees, confirming that the enzyme remains active across the temperature ranges relevant to industrial extraction.

For the collagen experiments, the team ground pork leg and backbone into a fine powder, stripped away non-collagenous proteins with sodium hydroxide, demineralized the bones with EDTA, and then soaked them in acetic acid before adding either pepsin at 0.5 percent or the pineapple extract at five percent by weight. After forty-eight hours of enzymatic digestion at four degrees Celsius, the released collagen was salted out with sodium chloride, centrifuged, dialyzed, and freeze-dried. The results were striking: the pineapple-extract-derived collagen, designated PESC, yielded 3.77 percent of the original bone weight, whereas pepsin-soluble collagen, or PSC, managed only 1.42 percent. That difference, statistically significant at p below 0.05, translates into more than two and a half times the collagen recovered from the same quantity of bone.

The researchers attribute this yield gap to the distinct cleavage specificities of the two enzymes. Pepsin preferentially cuts peptide bonds in the telopeptide regions at the ends of collagen molecules, a relatively restricted mode of attack. Bromelain, by contrast, hydrolyzes peptide bonds at multiple sites, including those adjacent to aromatic, basic, and hydrophobic residues, giving it a broader capacity to dismantle the intra- and intermolecular cross-links that lock collagen chains into the bone matrix. Previous studies have likewise shown that bromelain exhibits higher enzymatic activity than pepsin under identical hydrolysis conditions. For chondroitin sulfate extraction, performed at fifty-five degrees Celsius with either Alcalase or the pineapple extract, the two approaches proved essentially equivalent: PE yielded 11.98 milligrams of CS per gram of bone compared with 11.37 milligrams per gram for the commercial enzyme.

Quality control was central to the study, because a cheaper extract is worthless if it produces an inferior product. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that collagen from both extraction routes displayed the characteristic two-band pattern of type I collagen, with alpha-1 chains at roughly 130 to 120 kilodaltons and alpha-2 chains at 120 to 110 kilodaltons. The PESC sample also showed several lower-molecular-weight bands between about 75 and 20 kilodaltons, evidence that bromelain cuts not only within the telopeptide domain but also at specific sites inside the triple-helical region. Amino acid analysis confirmed that both products were dominated by glycine, at approximately 23.7 to 24.2 percent of total amino acids, followed by proline and glutamic acid, a profile consistent with authentic collagen and with no significant differences between the two extraction methods.

The chondroitin sulfate products passed equally rigorous scrutiny. Agarose gel electrophoresis revealed a single band for both PE- and Alcalase-derived CS, migrating in the same position as a commercial CS standard, and the bands disappeared entirely after digestion with chondroitinase ABC, indicating that the extracts consisted of pure glycosaminoglycans free of contaminants such as dermatan sulfate. Proton nuclear magnetic resonance spectroscopy at 600 megahertz then probed the sulfation patterns of the molecules. Both products showed the diagnostic signals for N-acetylgalactosamine methyl protons near 2.0 parts per million and, crucially, peaks at approximately 4.15 and 4.63 parts per million corresponding to the H4 and H6 protons of GalNAc-4-sulfate and GalNAc-6-sulfate. This confirmed that the pork bone CS contained both CS-A and CS-C structural units, the same forms found in commercially sourced products, which today come overwhelmingly from marine organisms processed in China.

Perhaps the most intriguing findings emerged when the researchers digested their two collagen products with the pineapple extract to generate peptide hydrolysates. Using ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry, they identified 28 peptides, seventeen of which appeared in both hydrolysates. Seven peptides, including Gly-Arg, Glu-Gly-Arg, Ala-Val-Gly, Leu-Ala, Leu-Val-Gly, Phe-Ala-Gly-Gly, and a longer proline-rich sequence, were found exclusively in the pepsin-derived material, while four others, including Leu-Pro, Leu-Thr-Gly, and two glycine-proline-rich sequences, appeared only in the pineapple-extract-derived hydrolysate. These differences trace directly back to enzyme specificity: pepsin favors bonds next to leucine and phenylalanine, whereas bromelain cuts beside aromatic, basic, and hydrophobic residues such as glycine. All identified peptides contained glycine, proline, or hydroxyproline, the structural backbone of collagen, and most fell within the two-to-twenty-residue range associated with strong biological activity.

Those structural differences translated into measurable functional advantages. In DPPH free radical scavenging assays, the PESC-derived hydrolysate achieved an EC50 of 0.76 milligrams per milliliter, outperforming the PSC-derived hydrolysate at 0.82 milligrams per milliliter. The same pattern held for ferrous ion chelation, where the PESC hydrolysate recorded an EC50 of 0.77 milligrams per milliliter against 0.83 for the pepsin product. Both fell short of the synthetic antioxidant butylated hydroxyanisole, which posted EC50 values between 0.65 and 0.69 milligrams per milliliter, but the researchers point out that the enhanced activity of the pineapple-derived peptides likely stems from their richer content of hydrophobic amino acids such as leucine, glycine, alanine, proline, and valine, which donate electrons or hydrogen atoms to neutralize radicals and bind metal ions that catalyze oxidative damage.

The implications extend well beyond the laboratory bench. Free radicals generated through normal metabolism and environmental exposure can overwhelm the body’s antioxidant defenses, contributing to oxidative stress and a range of chronic diseases, so natural antioxidant peptides from food sources are attracting growing commercial interest. By demonstrating that a crude, inexpensive extract from pineapple cores can outperform pepsin on collagen yield, match Alcalase on chondroitin sulfate recovery, and deliver collagen peptides with superior antioxidant activity, the Korean team has outlined a genuinely circular economy: fruit waste becomes an enzyme source, and bone waste becomes a biomaterial feedstock. The authors caution that further studies are needed to validate the biological activities of the identified peptides and CS and to assess their suitability for functional food and pharmaceutical applications, but the economic case is already compelling. For an industry that discards both of these raw materials by the millions of tons each year, the study suggests that two waste problems may, together, be the solution to one another.

Subject of Research: Extraction of collagen and chondroitin sulfate from pork bones using pineapple by-product bromelain extract as an alternative to commercial proteases

Article Title: Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases

Article References: Hoa, V.-B., Park, W.-S., Ham, J.-S., & Bae, I.-S. (2026). Extraction and characterization of biomaterials from pork bones: a potential alternative of pineapple by-product extract for commercial proteases. Food Science of Animal Resources, 46(1), Article 58. https://doi.org/10.1007/s44463-026-00066-9

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00066-9

Keywords: pork bones, collagen, chondroitin sulfate, bromelain, pineapple by-products, proteases, pepsin, Alcalase, antioxidant peptides, food waste valorization, biomaterials, functional foods