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

Rhomboid proteases belong to a class of enzymes called intramembrane serine proteases, meaning they carry out protein cleavage deep inside the lipid bilayer of cell membranes rather than in the watery environment where most proteases operate. First identified in fruit flies through genes governing embryonic development, rhomboids have since been found across nearly all branches of life, from bacteria to humans, where they regulate processes as diverse as growth factor signaling, mitochondrial dynamics, and bacterial quorum sensing. The malaria parasite Plasmodium falciparum carries eight of these enzymes, and the new review systematically assembles what scientists have learned about their biology, structure, catalytic mechanics, and potential as therapeutic targets.

The catalytic ingenuity of rhomboid proteases lies in how they solve a fundamental chemical problem. Peptide bond cleavage requires water, yet the interior of a membrane is an aggressively hydrophobic environment. Structural studies of the bacterial rhomboid GlpG, whose crystal structure was solved in 2006, revealed how these enzymes thread a substrate protein’s transmembrane helix into an internal active site, use helix-breaking residues to unwind the substrate, and recruit water molecules through specialized internal sites to perform the cut. Substrate recognition depends on specific sequence motifs within the transmembrane domain rather than simple affinity, and kinetic studies have shown that proteolysis inside the membrane is a rate-governed reaction with a two-stage catalytic mechanism. These details matter enormously for drug design, because they define the chemical features an inhibitor must exploit.

In Plasmodium, the best-characterized members of the family are PfROM1 and PfROM4, and their roles map directly onto the parasite’s most vulnerable moments. During the invasion of red blood cells, the merozoite stage of the parasite deploys an arsenal of adhesive proteins on its surface to latch onto the host cell. Once invasion is complete, these adhesins must be removed, partly because lingering surface proteins would present targets to the host’s antibodies. PfROM4 serves as the principal protease responsible for shedding key invasion-related adhesin families, including the erythrocyte binding-like proteins, the reticulocyte binding-like homologs, and the thrombospondin-related anonymous protein family member MTRAP. This shedding reaction, first demonstrated in 2006, is a sequence-independent juxtamembrane cleavage that also helps the parasite evade invasion-inhibitory antibodies.

The importance of rhomboid-mediated shedding extends well beyond the blood stage. In the sporozoite stage, which is injected by mosquitoes and travels to the liver, the adhesive protein TRAP must be cleaved by a rhomboid protease for the parasite to achieve the gliding motility essential for infectivity. Meanwhile, ROM1 has been implicated in the proper formation of the parasitophorous vacuole, the compartment the parasite builds around itself inside host cells. PfROM1 has also been localized to a newly identified secretory organelle in merozoites called the mononeme, hinting at additional functions that remain poorly understood. Other family members, such as ROM6 and ROM8, have been linked to the apicoplast, the parasite’s remnant chloroplast-like organelle, and to processes whose details are still being worked out.

Genetic evidence has been crucial in establishing which of these enzymes the parasite cannot live without. Early systematic knockout experiments in the rodent malaria model Plasmodium berghei showed that the genes encoding PbROM4, PbROM6, and PbROM7 could not be deleted, strongly suggesting they are essential for parasite survival. More recently, inducible knockout technology applied directly to P. falciparum has independently confirmed that PfROM4, PfROM6, and PfROM8 are essential in the human malaria parasite itself. This distinction matters, because essentiality in a rodent model does not always translate to the human parasite, and direct confirmation in P. falciparum substantially strengthens the case for these proteins as drug targets. At the same time, loss-of-function analyses have revealed that some family members perform redundant or nonessential functions, meaning any drug development effort must carefully select which rhomboid to pursue.

The most exciting development highlighted in the review is the arrival of parasite-selective chemical inhibitors of PfROM4. Researchers designed two classes of compounds, a peptide boronate and an alpha-ketoamide, that covalently engage the catalytic machinery of the parasite protease while sparing its human counterparts. In laboratory experiments, these inhibitors blocked erythrocyte invasion by the parasite and cleared blood-stage parasitemia, providing the first proof of concept that rhomboid proteases can be pharmacologically targeted in malaria. The selectivity is a critical achievement, because humans possess their own rhomboid proteases with essential functions, including the mitochondrial enzyme PARL involved in mitophagy, and any therapeutic must avoid interfering with these. Related alpha-ketoamide chemistry has since been used to inhibit human PARL in other disease contexts, demonstrating that the inhibitor platform itself is versatile.

Further support for the druggability of the family comes from an independent line of research published in 2025, in which N-aryl acetamide compounds with antimalarial activity were shown to be susceptible to resistance mutations arising in ROM8 and the protein CSC1. That work suggests ROM8 may be the direct or indirect target of a novel chemical class, adding a second rhomboid-linked vulnerability to the parasite’s armor. Taken together, these findings indicate that the rhomboid family offers multiple points of chemical attack, each potentially independent of the mechanisms by which parasites evade existing drugs such as chloroquine, antifolates, atovaquone, and the artemisinin derivatives whose declining efficacy has alarmed the field since resistance emerged in Southeast Asia.

Yet the review is candid about the substantial gaps that remain. Most of the eight Plasmodium rhomboid proteases are only partially characterized or entirely uncharacterized, and remarkably, no experimental three-dimensional structure of any Plasmodium rhomboid has been solved to date. All structural and mechanistic knowledge has been extrapolated from bacterial and other model rhomboids, which may not fully capture the substrate preferences and regulatory quirks of the parasite enzymes. Moreover, the promising inhibitor studies were performed in vitro, and the authors emphasize that validation in animal models and careful assessment of cross-reactivity against human rhomboids are essential next steps before any therapeutic development can proceed.

Even with those caveats, the trajectory of this research field is unmistakable. Rhomboid proteases sit at the intersection of parasite motility, host cell invasion, immune evasion, and survival, all processes central to the malaria parasite’s life cycle and all processes that existing drugs do not touch. As artemisinin resistance spreads and combination therapies come under increasing pressure, the identification of genetically validated, chemically tractable targets becomes ever more urgent. The demonstration that parasite-selective rhomboid inhibitors can block invasion and clear parasitemia, however preliminary, transforms this enzyme family from a subject of basic biological curiosity into a credible platform for antimalarial drug discovery, one that the next decade of structural biology and medicinal chemistry will be watching closely.

Subject of Research: Rhomboid proteases in Plasmodium parasites as antimalarial drug targets

Article Title: Rhomboid Proteases in Plasmodium spp. : Biology, Functional Roles, and Therapeutic Potential

Article References: Basumatary, D., & Lhouvum, K. (2026). Rhomboid Proteases in Plasmodium spp. : Biology, Functional Roles, and Therapeutic Potential. Acta Parasitologica, 71(5), Article 219. https://doi.org/10.1007/s11686-026-01402-1

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01402-1

Keywords: rhomboid proteases, Plasmodium falciparum, malaria, intramembrane proteolysis, erythrocyte invasion, PfROM4, antimalarial drug targets, drug resistance, parasite-selective inhibitors, Acta Parasitologica, Rhomboid, Proteases