Myofibrillar proteins are the workhorses of muscle tissue, accounting for roughly 55 to 65 percent of the total protein in meat. Composed primarily of myosin and actin, they form the structural framework of muscle fibers and govern the qualities that matter most to consumers and processors: solubility, gelation, water-holding capacity, and flavor development. When bromelain cleaves these proteins, the consequences ripple through every one of those attributes. Previous research had shown that the enzyme reduces shear force and hardness, degrades large protein molecules, and nudges ordered protein structures toward more disordered forms, but the dynamic molecular interactions between the enzyme and its protein targets had never been systematically characterized.

The research team, based at an institution supported by the Liaoning Provincial Basic Research Program, extracted myofibrillar proteins from beef longissimus dorsi muscle and treated them with bromelain at four enzyme-to-substrate ratios: 0.10, 0.25, 0.50, and 0.75 percent by weight, alongside an untreated control. Each sample was incubated for 30 minutes at 40 degrees Celsius before the reaction was halted with a protease inhibitor. This concentration range allowed the investigators to trace how escalating doses of the enzyme reshape protein behavior, from gentle depolymerization to outright fragmentation.

The results revealed a striking dose-dependent pattern with a clear optimum at 0.50 percent bromelain. At this concentration, protein solubility peaked at 81.53 percent, turbidity and average particle size fell to their lowest values, and the polydispersity index dropped to 0.338, down from 0.871 in the control, indicating a remarkably uniform dispersion of protein particles. The explanation lies in the balance between two competing processes. Moderate hydrolysis breaks apart myofibrillar bundles and exposes polar, charged groups that were previously buried, strengthening hydration interactions and boosting solubility. But when the enzyme dose climbed to 0.75 percent, excessive cleavage generated a flood of small peptides and exposed hydrophobic patches, which promptly reaggregated into insoluble clumps, dragging solubility back down.

Spectroscopic measurements painted a consistent picture of progressive protein unfolding. Ultraviolet absorption and intrinsic fluorescence both intensified as bromelain levels rose to 0.50 percent, signaling that aromatic amino acid residues such as tryptophan, tyrosine, and phenylalanine were being liberated from the protein interior into the surrounding polar environment. The fluorescence emission maximum also shifted slightly toward longer wavelengths, a hallmark of a more extended protein conformation. Surface hydrophobicity climbed in parallel, confirming that hydrophobic residues once hidden within the folded structure were now accessible at the molecular surface. At the highest enzyme dose, all of these signals reversed, as reaggregation re-buried the exposed groups.

Fourier transform infrared spectroscopy delivered perhaps the most dramatic evidence of structural transformation. The amide A band, sensitive to hydrogen bonding, shifted from 3304 to 3405 wavenumbers as enzyme concentration increased, indicating the progressive dismantling of the hydrogen-bond network that holds the protein together. Curve-fitting analysis of the amide I band showed that ordered secondary structures, alpha-helices and beta-sheets, declined from 70.97 percent of the total to just 50.37 percent, while disordered beta-turns and random coils expanded from 29.03 to 49.63 percent. In effect, bromelain converted a compact, well-ordered protein architecture into a loose, flexible tangle, a transition the authors describe as protease-driven structural disordering.

Scanning electron microscopy and gel electrophoresis corroborated the story at larger scales. Untreated protein displayed dense, block-like aggregates, while the 0.50 percent treatment produced the most dispersed and uniformly distributed microstructure of any group. Electrophoretic profiles showed the myosin heavy chain band, normally a prominent feature at approximately 220 kilodaltons, becoming progressively fainter with increasing enzyme dose, accompanied by diffuse staining at lower molecular weights that betrayed extensive fragmentation. Actin, the other major myofibrillar component, also showed changes at the higher enzyme levels, though the myosin heavy chain proved conspicuously vulnerable.

To probe the enzyme’s target at atomic resolution, the team built a three-dimensional model of the bovine myosin heavy chain by homology modeling, validated it with a Ramachandran plot showing 94 percent of residues in the most favored regions, and docked bromelain onto it. The best docking pose exhibited a binding energy of minus 12.4 kilocalories per mole, stabilized by a multipoint hydrogen-bonding network with bond distances ranging from 1.9 to 3.6 angstroms, involving residue pairs such as GLU211 of bromelain with THR424 of myosin and ASN78 with GLU577. Molecular dynamics simulations running 100 nanoseconds under physiological conditions then revealed what binding actually does to the protein: the complex showed higher root-mean-square deviation, greater radius of gyration, and larger solvent-accessible surface area than myosin alone, all signs of a looser, less compact, more flexible structure primed for further proteolytic attack.

The energetic accounting confirmed the partnership is spontaneous and thermodynamically favorable, with a total binding free energy of minus 59.97 kilojoules per mole calculated by the MM/PBSA method. Van der Waals and electrostatic interactions emerged as the primary driving forces, with electrostatics contributing most significantly, while residue decomposition identified MET549, GLU374, VAL420, LYS368, GLU598, THR547, LYS568, and LYS281 as key contributors to binding. The free energy landscape of the complex displayed multiple rugged low-energy basins, in contrast to the smoother, more concentrated landscape of free myosin, indicating that the enzyme destabilizes its target even as it grips it.

For the meat industry, the practical message is one of precision. The study demonstrates that bromelain’s benefits hinge on hitting a narrow optimal dose, because the same proteolytic power that disperses aggregates and unlocks solubility at moderate concentrations can trigger wasteful reaggregation and quality loss when overdone. By linking macroscopic properties like solubility and turbidity to specific molecular events, hydrogen-bond disruption, secondary-structure loss, and a computationally characterized enzyme-substrate interface, the work provides a rational framework for deploying bromelain in meat tenderization and quality regulation. It also offers a template for studying other enzyme-protein systems in food science, showing how experiments and simulations can converge to explain, at the level of individual residues, why a pineapple enzyme makes steak tender.

Subject of Research: Bromelain-induced structural and physicochemical changes in beef myofibrillar proteins and their molecular interaction mechanisms

Article Title: Bromelain-induced physicochemical and structural changes in beef myofibrillar proteins and their molecular interaction mechanisms

Article References: Huang, W., Fu, H., Geng, L., Qi, R., Jia, D., & Zhou, W. (2026). Bromelain-induced physicochemical and structural changes in beef myofibrillar proteins and their molecular interaction mechanisms. Food Chemistry: X, 39, Article 104560. https://doi.org/10.1016/j.fochx.2026.104560

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104560

Keywords: bromelain, myofibrillar proteins, meat tenderization, myosin heavy chain, molecular docking, molecular dynamics simulation, protein solubility, protein structure, food chemistry, enzymatic hydrolysis, beef quality, hydrogen bonding