Two Enzymes Are Better Than One: Egg White Protein Unlocked for Antioxidant Peptides

Egg white is one of the most abundant and affordable high-quality proteins on the planet, yet its molecules guard their secrets well. Native egg white protein is folded into compact, highly organized structures that resist the digestive enzymes meant to break them apart, which limits how efficiently the body—and food manufacturers—can release the small bioactive peptides increasingly linked to antioxidant, antihypertensive, and anti-inflammatory effects. A new study published in Food Chemistry: X offers a deceptively simple solution: instead of attacking the protein with a single enzyme, deploy two in sequence. Researchers Jialei Shi, Xianzhen Li, Xunyu Song, and Siqi Wang show that a combined alcalase-papain treatment, abbreviated AlPa, dismantles egg white protein more thoroughly than either enzyme alone, produces a striking surplus of low-molecular-weight peptides, and—crucially—yields hydrolysates whose antioxidant power survives prolonged simulated digestion.

The logic behind the two-enzyme strategy rests on complementary specificity. Alcalase is a serine endoprotease with a relatively narrow range of peptide bonds it will cut, while papain, a cysteine protease, cleaves more promiscuously. Used alone, each enzyme is constrained by which parts of the folded protein it can physically reach. Used in tandem, the first enzyme acts as a molecular crowbar: alcalase attacks the accessible regions of the protein, loosening the native structure and exposing fresh peptide bonds that papain can then target. The result is a broader collective cleavage repertoire than either enzyme could achieve individually, a principle previously demonstrated in chicken breast protein but untested for egg white until now.

The experimental design was straightforward. Fresh egg whites were homogenized, diluted to 10 milligrams of protein per milliliter, and preheated at 55 degrees Celsius for 30 minutes before hydrolysis at 50 degrees Celsius. In the single-enzyme treatments, alcalase or papain was added at 2 percent by weight and allowed to work for six hours. In the composite treatment, 1 percent alcalase ran for three hours, followed by 1 percent papain for another three. Enzymes were then inactivated by heating, and the soluble hydrolysates were recovered by centrifugation. The team tracked everything from peptide yield and molecular-weight distribution to secondary structure, surface morphology, and behavior during a modified INFOGEST 2.0 simulated digestion extended to mimic elderly or patient gastrointestinal conditions—two hours of salivary digestion, six hours of gastric, and six more of intestinal.

The electrophoresis results told the first part of the story. Untreated egg white protein displayed prominent bands between 29.0 and 64.4 kilodaltons, corresponding mainly to ovalbumin, plus a faint lysozyme band below 14.3 kilodaltons. All hydrolyzed samples showed cleavage at the 44.3-kilodalton band and new fragments below 29 kilodaltons, but the high-resolution Tricine-SDS-PAGE revealed the real difference: the composite treatment generated markedly smaller products, dominated by peptides under 10 kilodaltons and including a substantial fraction below 1.7 kilodaltons. Alcalase alone outperformed papain alone, but their sequential use pushed hydrolysis further than either could go, confirming that the initial alcalase pass genuinely widened the door for papain.

Quantitative measures backed the gels. The hydrolysate yield peaked at 72.85 percent for the composite treatment, compared with 67.43 percent for papain and 63.77 percent for alcalase. More tellingly, the proportion of hydrolysate below 10 kilodaltons reached 63.71 percent for AlPa, against 50.22 percent for alcalase and just 39.11 percent for papain. This matters because small peptides tend to show stronger antioxidant potential: their shorter chains expose redox-active residues more effectively and interact more readily with radical species. By maximizing both recovery and the low-molecular-weight fraction, the composite strategy delivers precisely the peptide population that functional-food formulators want.

The structural analysis revealed something more surprising than simple fragmentation. Particle size actually increased after hydrolysis, most pronouncedly for AlPa, because proteolysis exposes hydrophobic residues that were previously buried inside the folded protein. These newly exposed nonpolar surfaces promote peptide-peptide aggregation, a tendency reinforced when electrostatic repulsion weakens—and indeed, the absolute zeta potential dropped for all hydrolysates, most sharply for the composite sample. Surface hydrophobicity rose significantly across treatments and was highest for AlPa, while intrinsic fluorescence told a matching story: the native protein’s emission maximum at 334 nanometers red-shifted to 361 nanometers for the composite hydrolysate, with the strongest quenching, indicating that aromatic residues such as tryptophan had been thrust into a far more polar, solvent-exposed environment.

Fourier-transform infrared spectroscopy added a second-structure dimension. Native egg white protein was fitted as 21.05 percent alpha-helix, 51.60 percent beta-sheet, 12.18 percent beta-turn, and 15.17 percent random coil. After composite hydrolysis, the beta-turn component dominated the fitted spectrum at 86.44 percent, while alpha-helical and random-coil contributions shrank to near-undetectable levels—possibly reflecting hydrophobic interactions, particularly involving phenylalanine and proline, that drive partial refolding. Scanning electron microscopy completed the picture: where single-enzyme treatments produced thin sheets and rough particulate aggregates, the composite hydrolysate showed a collapsed, globular morphology with smooth edges, surface protrusions, and conspicuous cavities never seen in the single-enzyme products—visual confirmation of the most extensive proteolytic degradation of all.

The digestion experiments addressed the question that most determines whether a functional ingredient is worth developing: does the bioactivity survive the journey? Before digestion even began, the composite sample already showed 78.64 percent free amino groups, the highest initial cleavage level, and all samples eventually converged on comparable apparent digestibility, confirming complete breakdown. But the antioxidant assays revealed functional persistence rather than simple loss. In the DPPH radical-scavenging assay, the composite hydrolysate consistently ranked highest across every digestion stage, ahead of papain, alcalase, and untreated protein. In the ABTS assay, alcalase and composite samples remained among the strongest scavengers throughout, an effect attributed to greater exposure of electron-donating groups, including sulfur-containing residues and tyrosine-associated phenolic groups, plus better radical access to hydrophobic peptide regions.

Amino acid analysis of the final digesta added a compositional explanation. The composite treatment produced the highest mean concentrations of branched-chain amino acids at 79.41 nanograms per milliliter and hydrophobic amino acids at 183.61 nanograms per milliliter—increases of 13.92 percent and 4.90 percent, respectively, over untreated egg white digest—alongside relatively high cysteine and methionine levels that favor radical-scavenging chemistry. Essential and total amino acid levels did not differ significantly among treatments, suggesting the composite strategy reshapes which residues remain in the peptide pool rather than the overall nutritional payload. The authors are appropriately cautious, noting that antioxidant performance also depends on peptide size, sequence, and residue accessibility, so individual amino acid groups cannot be assigned credit from composition alone.

The broader significance lies in the structure-composition-function chain the study establishes. The benefits of composite hydrolysis do not flow merely from a higher degree of hydrolysis; they arise from coordinated changes in peptide size, molecular conformation, and chemical composition that together produce antioxidant functionality resilient to further digestive proteolysis. For elderly people and patients whose prolonged digestive timelines can erode the activity of conventionally prepared hydrolysates, that stability is the key selling point. The researchers, supported by the Foundation of Liaoning Province Education Administration of China, point to a clear next step: identifying the specific bioactive peptide sequences responsible and verifying their bioavailability and physiological efficacy in vivo. If those trials succeed, the humble egg white—already a kitchen staple—may find a second career as the raw material for digestive-stable antioxidant ingredients, unlocked by nothing more exotic than two well-chosen enzymes working in relay.

Subject of Research: Synergistic dual-enzyme hydrolysis of egg white protein to enhance peptide release, structural unfolding, and digestive-stable antioxidant activity

Article Title: Synergistic Alcalase-papain hydrolysis of egg white protein: enhancing structural unfolding, antioxidant activity, and gastrointestinal stability

Article References: Shi, J., Li, X., Song, X., & Wang, S. (2026). Synergistic Alcalase-papain hydrolysis of egg white protein: enhancing structural unfolding, antioxidant activity, and gastrointestinal stability. Food Chemistry: X, 39, Article 104581. https://doi.org/10.1016/j.fochx.2026.104581

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104581

Keywords: egg white protein, bioactive peptides, alcalase, papain, enzymatic hydrolysis, antioxidant activity, gastrointestinal digestion, protein structure, Food Chemistry: X, functional foods, molecular weight distribution, FTIR spectroscopy