Rabbit Offal Transformed Into Antioxidant Peptides by Enzymatic Hydrolysis

Every year, the global rabbit meat industry slaughters millions of animals and discards roughly a fifth of each carcass before a single cut of meat reaches the market. The viscera, a complete set of thoracic and abdominal organs including lungs, liver, kidneys, stomach, intestines, cecum, spleen and bladder, account for 20 to 25 percent of live weight, and most of this protein-rich material is rendered, dumped or otherwise wasted. A new exploratory study published in Food Science of Animal Resources suggests that this overlooked stream of slaughterhouse by-product could be converted into something far more valuable: bioactive peptides with measurable antioxidant capacity and the ability to reduce the viability of human colon cancer cells in laboratory cultures.

The research team, led by Nuly Z. Acosta-Acevedo of the Universidad Politécnica de Francisco I. Madero in Mexico, worked with viscera from commercial crossbred California × New Zealand rabbits raised at a farm in Hidalgo, Mexico. Six animals with an average live weight of about 2,047 grams were processed after routine commercial slaughter, and the researchers calculated a comprehensive viscera yield of 20.21 percent of live weight, a figure consistent with published ranges of 18 to 22 percent and notable because it included the full complement of organs rather than the selective subsets of edible offal that most studies report. Rabbit viscera are physiologically distinctive: the animal’s hindgut fermentation and cecotrophy shape a gastrointestinal tract that differs from those of fish, poultry and conventional livestock, potentially producing a unique protein matrix and amino acid profile when the tissue is broken down enzymatically.

Before any chemistry could begin, the raw material required careful preparation. The organs were cleaned, ground into a homogenate the authors call Fresh Rabbit Viscera, and then defatted using an adapted Bligh and Dyer method in which petroleum ether and methanol were stirred with the tissue in a 1:2 volume ratio before double centrifugation. Sterilization followed in an autoclave at 120 degrees Celsius for 15 minutes, and the material was then frozen at minus 45 degrees Celsius and freeze-dried for five days at minus 40 degrees Celsius and 0.210 millibar, yielding a stable powder designated Lyophilized Rabbit Viscera. Lyophilization drove moisture from 79.83 percent down to 2.40 percent and concentrated the protein content to 65.49 percent on a dry basis, placing the powder in the same league as porcine liver hydrolysates and chicken offal concentrates as a high-quality protein source.

Microbiological testing confirmed the pretreatment strategy worked. Fresh viscera carried aerobic mesophilic counts of 1.5 × 10⁴ colony-forming units per gram, comfortably below the 10⁶ threshold set by international quality standards, with no coliforms, molds or yeasts detected. After defatting, autoclaving and freeze-drying, the lyophilized powder showed no detectable microbial growth at all. The authors attribute this sterility to the combined disruption of microbial metabolism by organic solvents, spore inactivation by heat, and the collapse of cell membranes under low water activity, an outcome they emphasize is critical for ensuring that any subsequent proteolysis can be attributed solely to the enzymes the researchers added rather than to contaminating microbes.

The heart of the study was a head-to-head comparison of two industrial proteases with fundamentally different catalytic strategies. Alcalase, a serine endopeptidase from Bacillus licheniformis, cleaves internal peptide bonds and tends to generate medium-sized, hydrophobic peptides. Flavourzyme, a protease complex from Aspergillus oryzae, combines endopeptidase and exopeptidase activities, trimming peptides from their termini and pushing the reaction toward very short peptides and free amino acids. The team hydrolyzed the lyophilized viscera with each enzyme for 0, 2, 4, 6 and 8 hours at an enzyme-to-substrate ratio of 100:10, using pH 9 buffer and 55 degrees Celsius for Alcalase and pH 7 and 50 degrees Celsius for Flavourzyme, with three independent batches per condition to ensure biological replication.

Reverse-phase high-performance liquid chromatography revealed clearly divergent fragmentation patterns. Alcalase chromatograms showed a progressive shift toward shorter retention times as hydrolysis advanced, consistent with the endopeptidase first producing medium-sized peptides and then cutting them into smaller fragments; by six hours, more than 78 percent of the peptide signal corresponded to material below 1 kilodalton. Flavourzyme acted faster, generating strong signals for very small peptides and free amino acids almost immediately, with over 82 percent of the profile below 1 kilodalton by eight hours. The authors are careful to note that RP-HPLC estimates of molecular size are approximate, because retention depends on hydrophobicity and terminal composition as much as on mass, so the distributions should be read as relative hydrophobicity profiles, with mass spectrometry needed for definitive peptide identification.

Antioxidant testing produced the study’s most striking numbers. The DPPH radical-scavenging assay, a standard hydrogen-atom-transfer screen, was compromised by a visible precipitate that formed when the hydrolysates met the alcoholic DPPH reagent, a known artifact for hydrophobic, protein-rich samples, so those results were treated as tentative and likely underestimates. The team instead leaned on the oxygen radical absorbance capacity assay, run in both hydrophilic buffer and a lipophilic system containing 7 percent randomly methylated beta-cyclodextrin, which solubilizes hydrophobic peptides so they can neutralize peroxyl radicals. Alcalase hydrolysates climbed steadily from 31.86 micromoles of Trolox equivalents per gram of protein at time zero to a peak of 249.50 at six hours, a statistically significant increase, before declining at eight hours as over-hydrolysis shredded the active fragments. Flavourzyme peaked earlier and plateaued near 40 micromoles per gram, far below the Alcalase maximum.

The explanation, the authors argue, lies in peptide architecture. Short peptides rich in hydrophobic and aromatic residues donate hydrogen atoms efficiently to peroxyl radicals, and Alcalase preserves precisely this class of medium-sized hydrophobic fragments, whereas Flavourzyme’s exopeptidase activity grinds them down into free amino acids with little antiradical power. The pattern mirrors published results for porcine liver, pork by-products and freshwater carp hydrolysates, and the peak value for rabbit viscera compares favorably with reported figures for fish and poultry by-product hydrolysates. In both enzyme systems, prolonged hydrolysis diminished activity, underscoring that hydrolysis time is a tunable dial: stop too early and the bioactive fraction has not been released, stop too late and it has been destroyed.

The cell biology experiments added a provocative dimension. Using the MTT assay, which measures mitochondrial metabolic activity, the researchers exposed SW-480 human colon adenocarcinoma cells to hydrolysate concentrations of 25, 35, 40 and 45 percent for 24 hours. Both enzyme systems reduced viability in a concentration-dependent manner, but Alcalase was consistently stronger, with all five hydrolysis durations driving viability below 50 percent. Surprisingly, the greatest reduction came not from a fully digested sample but from the non-hydrolyzed, zero-hour Alcalase preparation, which cut viability to 24.20 percent at the highest concentration. The authors suggest that pasteurization and defatting alone may have exposed bioactive components in the native protein matrix, and they caution that the MTT assay cannot distinguish apoptosis from necrosis or nonspecific metabolic interference, nor can it speak to selectivity against healthy cells, which were not tested in this exploratory screen.

The researchers are candid about the limits of their work. No peptide sequences were identified, so no causal link between specific fractions and the observed effects can yet be drawn; sensory qualities, bitterness, solubility and stability in real food matrices remain unexamined; and the DPPH data are provisional. Still, the study makes a credible case that an underexplored slaughter by-product, one that rabbit production generates at a rate of roughly 860,000 tonnes of meat annually worldwide, can be upcycled into functional ingredients within a circular bioeconomy framework. The next steps the authors propose are peptide sequencing by liquid chromatography tandem mass spectrometry, testing against non-tumor cell lines to establish selectivity, in vivo validation, and ultimately industrial scale-up. If those efforts succeed, the humblest parts of the rabbit could find a second life not as waste, but as nutraceuticals on the shelf.

Subject of Research: Enzymatic hydrolysis of rabbit viscera to produce antioxidant bioactive peptides

Article Title: Valorization of rabbit viscera by enzymatic hydrolysis: bioactive peptides with antioxidant and cell viability properties

Article References: Acosta-Acevedo, N. Z., Lopez-Perea, P., Herrera-Hernández, M. G., Vergara-Castañeda, H. A., Pérez-Escalante, E., Martínez-Carrera, D., & Olloqui, E. J. (2026). Valorization of rabbit viscera by enzymatic hydrolysis: bioactive peptides with antioxidant and cell viability properties. Food Science of Animal Resources, 46(1), Article 72. https://doi.org/10.1007/s44463-026-00096-3

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00096-3

Keywords: rabbit viscera, enzymatic hydrolysis, bioactive peptides, antioxidant capacity, ORAC assay, DPPH assay, Alcalase, Flavourzyme, cell viability, SW-480 colon cancer cells, food by-product valorization, circular economy