Gastrointestinal digestion behavior and bioavailability of greenly prepared highly loaded myofibrillar-luteolin vehicle.

In this study, the highly loaded myofibrillar protein (MP)-luteolin (Lut) complexes were noncovalently constructed by using green high-pressure homogenization technology (HPH) and high-pressure micro-fluidization technology (HPM), aiming to optimize the encapsulation efficiency of flavonoids in the protein-based vehicle without relying on the organic solvent (i.e. DMSO, ethanol, etc.). The loading efficiency of Lut into MPs could reach 100 % with a concentration of 120 µmol/g protein by using HPH (103 MPa, 2 passes) without ethanol adoption. The in vitro gastrointestinal digestion behavior and antioxidant activity of the complexes were then compared with those of ethanol-assisted groups. During gastrointestinal digestion, the MP digestibility of complexes, reaching more than 70.56 % after thermal treatment, was higher than that of sole protein. The release profile of Lut encapsulated in ethanol-containing and ethanol-free samples both well fitted with the Hixson-Crowell release kinetic model (R2 = 0.92 and 0.94, respectively), and the total phenol content decreased by ≥ 40.02 % and ≥ 62.62 %, respectively. The in vitro antioxidant activity (DPPH, ABTS, and Fe2+) of the digestive products was significantly improved by 23.89 %, 159.69 %, 351.12 % (ethanol groups) and 13.43 %, 125.48 %, 213.95 % (non-ethanol groups). The 3 mg/mL freeze-dried digesta significantly alleviated lipid accumulation and oxidative stress in HepG2 cells. The triglycerides and malondialdehyde contents decreased by at least 57.62 % and 67.74 % after digesta treatment. This study provides an easily approached and environment-friendly strategy to construct a highly loaded protein-flavonoid conjugate, which showed great potential in the formulation of healthier meat products.

Effect of pulsed electric field (PEF) on NaCl diffusion in beef and consequence on meat quality.

The impact of various field strength (2, 3, 4 kV/cm) and treatment time (60s and 90s) combinations on NaCl content and diffusion coefficient of beef were evaluated in the current study. Weight change, water content, water holding capacity, and texture of beef after brining were also explored. The results demonstrated pulsed electric field (PEF) pre-treatment significantly increased NaCl uptake when the brining time was 150 min (P < 0.05). The maximum NaCl content increased by 19.50% and the diffusion coefficient increased by 58.50%. Relatively mild PEF (60s) could improve beef qualities, but longer treatment time (90s) was detrimental to these qualities. Meanwhile, more complete myofibrillar structure and lower lipid oxidation extent were observed in the samples treated by PEF, contributing to the higher a* values. In conclusion, short processing time (60s) and high field strength (4 kV/cm) treatment is a potential strategy for meat brining acceleration and quality improvement in practical industrial production.

An unfolding/aggregation kinetic instructed rational design towards improving graft degree of glycation for myofibrillar protein.

Glycation is an effective strategy for the application of myofibrillar protein (MP) in beverage formulas by improving water solubility. In conventional glycation, the efficiency was limited as MP-saccharides conjugates mostly produced at low temperature due to thermosensitivity. This study was aimed to explore unfolding/aggregation kinetics of MP, including aggregate behavior, structural characteristics, and micromorphology, which guided the selection of temperature for glycation. It was shown that 40 °C/47.5 °C were critical temperature for MP unfolding/aggregation, respectively. Accordingly, an innovative technology of glycation (cyclic continuous glycation, CCG) was established by combining such temperatures. The results confirmed that cyclic continuous heating (CCH) inhibited excessive exposure of sulfhydryl and hydrophobic groups impeding protein aggregation. Importantly, it was revealed that rational designed CCG promoted covalent binding of MP to glucose by regulating unfolding-aggregation balance, exhibiting higher glycation degree. Overall, CCG-modified MP is expected to motivate the application of meat proteins in food formulations.

Effects of pre-formulation and post-cooking method on the rheological and gelation properties of 3D printed chicken products.

This study investigated the influence of oil type (olive, soybean, and peanut oil) and post-cooking methods (oven bake and microwave) on the quality of 3D printed chicken meat products. The Ostwald-de-Waele model was used to describe the flow behavior of chicken meat paste (R2 > 0.995). Oil-fortified groups present significantly lower consistency index (K) and flow behavior index (n), indicating better fluidity. A modified Cox-Merz rule was applied by multiplying angular frequency with shift factors (αSF). Surprisingly, the values of αSF are well-correlated with accuracy parameters of 3D printed cubes (|r| >0.8). For post-heating methods, baking results in higher fluid loss but contributes to a smoother surface. The microwaved gels showed better fluid retention ability and higher accuracy but lost the detail shape of the 3D printing model. Overall, the PO (peanut oil) meat emulsion group presented better textural properties and flat surfaces than other oil-added counterparts.

Revealing extracellular protein profile and excavating spoilage-related proteases of Aeromonas salmonicida based on multi-omics investigation.

Aeromonas is a ubiquitous aquatic bacteria, and it is a significant factor contributing to meat spoilage during processing and consumption. The abilities of Aeromonas salmonicida 29 and 57, which exhibit spoilage heterogeneity, to secrete protease, lipase, hemolysin, gelatinase, amylase, and lecithinase were confirmed by plate method. A total of 3948 proteins were identified by ITRAQ in extracellular secretions of A. salmonicida, and 16 proteases were found to be potentially related to spoilage ability. The complete genome sequence of A. salmonicida 57 consists of one circular chromosome and three plasmids, while A. salmonicida 29 consists of one circular chromosome, without a plasmid. Transcriptomic analysis revealed a significant number of DEGs were up-regulated in A. salmonicida 29, which were mainly enriched in metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism), indicating that A. salmonicida 29 had better potential to decompose and utilize nutrients in meat. Six protease genes (2 pepB, hap, pepA, ftsI, and pepD) were excavated by combined ITRAQ with transcriptome analysis, which potentially contribute to bacterial spoilage ability and exhibit universality among other dominant spoilage bacteria. This investigation provides new insights and evidence for elucidating metabolic and spoilage phenotypic differences and provides candidate genes and strategies for future prevention and control technology development.

Effects of different mixed starter cultures on microbial communities, taste and aroma compounds of traditional Chinese fermented sausages.

The aim of this study was to investigate and compare the effects of different mixed starter cultures (Lactiplantibacillus plantarum and Staphylococcus simulans) on the bacterial communities and flavor of fermented sausages. The results indicated that native starters grew well in fermented sausages and became dominant at the end of ripening. Among them, Lactobacillus spp. had the highest relative abundance, followed by Staphylococcus spp. In addition, the inoculation of the mixed starters promoted the formation of taste and aroma compounds that contribute to the overall flavor of the fermented sausages. Among them, the L. plantarum CQ01107 + S. simulans CD207 (CCA) treatment was found to have the highest umami amino acid, nucleotide, lactic acid, fatty acid and ketone contents (P < 0.05), as well as excellent sensory properties. In conclusion, the CCA starter may be a desirable starter culture to enhance the flavor of fermented sausages.

Effects of Wooden Breast Myopathy on Meat Quality Characteristics of Broiler Pectoralis Major Muscle and Its Changes with Intramuscular Connective Tissue.

This study aimed to investigate the effect of wooden breast (WB) myopathy on chemical composition, meat quality attributes and physiochemical characteristics of intramuscular connective tissue (IMCT) of broiler pectoralis major (PM) muscle. Thirty-six fillets were classified into varying degrees of WB condition, including normal, moderate and severe. Results show that WB myopathy altered the collagen profile in PM muscle by increasing total collagen content and decreasing collagen solubility. The composition of macromolecules in IMCT, including hydroxylysyl pyridoxine cross-linking, decorin and glycosaminoglycans, were increased with the severity of WB myopathy. Differential scanning calorimetry analysis indicated higher denaturation temperatures and lower denaturation enthalpy of IMCT for WB. Secondary structures of α-helix and ß-sheet in the IMCT of WB were changed to ß-turn and random coil. In addition, chemical composition and meat quality attributes showed a correlation with collagen profile and IMCT characteristics. Overall, this study emphasizes the effect of WB myopathy on IMCT and their contributions to meat quality variation.

Improved emulsifying properties of water-soluble myofibrillar proteins at acidic pH conditions: Emphasizing pH-regulated electrostatic interactions with chitosan.

Water-soluble muscle protein with enhanced functionalities has attracted great interest for low-salt food design. Electrostatic interactions of chitosan (CS) with myofibrillar proteins (MP) in water-aqueous solution at acidic pHs (4.0-6.5) were investigated, and how pH regulated complex formation, microstructures, conformation changes, and emulsifying capacity was systematically explored. At pH 4.0-4.5, MP and CS were positively charged and displayed a co-soluble system, exhibiting small particles and high solubility. When the pH increased to near the isoelectric point (pI) of MP (pH 5.0-6.0), electrostatic interactions largely inhibited the aggregation of MP by forming smaller particle complexes. The flexible structures and improved amphiphilic properties promoted protein absorption at the oil-water interface, further improving the emulsion stability. When the pH increased to 6.5, large aggregates were formed causing poor functionalities. This study could provide great insights to further exploit meat-protein-based low-salt functional foods in novel food design.

The evaluation of mixed-layer emulsions stabilized by myofibrillar protein-chitosan complex for delivering astaxanthin: Fabrication, characterization, stability and in vitro digestibility.

Muscle protein based functional foods have been attracted great interests in novel food designing. Herein, myofibrillar protein (MP)-chitosan (CH) electrostatic complexes were employed to fabricate mixed-layer emulsions to protect and deliver astaxanthin. The MP/CH complex fabricated mixed-layer emulsions displayed higher stability against pH and temperature changes, exhibiting smaller droplet and homogenous distributions. After UV-light irradiation for 8 h, the mixed-layer emulsions had higher astaxanthin retention (69.11 %, 1:1 group). During storage, a lower degree of lipid oxidation, protein oxidation and higher astaxanthin retention were obtained, indicating desirable protections of mixed-layer emulsions. The vitro digestion reveled the mixed-layer emulsions could decrease the release of free fatty acids. Meanwhile, the bioaccessibility of astaxanthin was higher (30.43 %, 2:1 group) than monolayer emulsion. In all, the MP/CH prepared mixed-layer emulsions could protect and deliver fat-soluble bioactive compounds, and contributed to develop muscle protein based functional foods to meet the needs of slow and controlled release.

Analysis of the suitable thawing endpoint of the frozen chicken breast using video recording analysis, shear force, and bioelectrical impedance measurement.

This study focuses on analyzing the texture properties and bioelectrical impedance characteristics of frozen chicken breasts during low-temperature thawing, meanwhile, we also compared the differences in physiochemical properties. Frozen chicken breasts were thawed at 4 ± 2°C for 2, 4, 6, 8, and 10 h separately, then the physiochemical properties (color, pH, water-holding capacity, water distribution), the texture properties (easy-to-cut level), and the bioelectrical impedance were determined and analyzed. The easy-to-cut level of the samples was evaluated by the sensory panel and two indexes, one is Warner-Bratzler shear force measured by texture analysis machine, and the other is cutting speed value calculated by the consumer-oriented cutting behavior analysis using frame-by-frame video recording analysis method. These two methods were used to characterize the easy-to-cut level of the frozen samples during thawing from the industrial processing and home cooking standpoint. Strong correlations were observed between the easy-to-cut level and the bioelectrical impedance of the frozen chicken breasts during thawing. The impedance magnitude at 100 kHz showed a high correlation coefficient (R2 = .9417) with Warner-Bratzler shear force, and the impedance magnitude at 50 Hz showed a high correlation coefficient (R2 = .8658) with cutting speed. Our results indicated the acceptability of using bioelectrical impedance to evaluate the easy-to-cut thawing endpoint for both industry processing and home cooking.

Aggregate Size Modulates the Oil/Water Interfacial Behavior of Myofibrillar Proteins: Toward the Thicker Interface Film and Disulfide Bond.

Myofibrillar protein (MP) aggregate models have been established to elucidate the correlation between their aggregate sizes and interfacial properties. The interfacial layer thickness was measured by the polystyrene latex method and quartz crystal microbalance with dissipation measurement. Interfacial conformations were then characterized in situ (front-surface fluorescence spectroscopy) and ex situ (reactive sulfhydryl group and secondary structure measurement following MP displacement). The viscoelasticity of the interfacial film and its resistance to surfactant-induced competitive displacement were reflected by the dilatational rheology and dynamic interfacial tension with the bulk phase exchange. Finally, we compared the findings of competitive displacement before/after adding a sulfhydryl-blocking agent, N-ethylmaleimide, to highlight the role of S-S linkage on interfacial film formation and stability. We substantiated that the aggregate size of the MP governed their interfacial properties. Small-sized aggregates exhibited more ordered secondary structures on the oil-water interface, which was conducive to the adsorption ratio of the protein and the adsorption dynamics. Although larger aggregates lowered the diffusion rate during interfacial film formation, they allowed the thicker and more viscoelastic interfacial film to be constructed afterward through more disulfide bond formation, resulting in greater resistance to surfactant-induced competitive displacement.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat.

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1-3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1-3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.

Silver Nanoparticle Sensor Array-Based Meat Freshness Inspection System.

The series of biochemical reactions, metabolic pathways, and regulatory interactions that occur during the storage of meat are the main causes of meat loss and waste. The volatile compounds produced by these reactions, such as hydrogen sulfide, acids, and amines, can directly indicate changes in the freshness of meat during storage and sales. In this study, a one-pot hydrothermal method based on a surface control strategy was used to develop nanoparticles of silver with different reactivities, which were further immobilized in agar powder to develop a colorimetric sensor array. Due to the different chemical interactions with various volatile compounds, the colorimetric sensor array exhibited distinct color changes. The study demonstrates significant differences between 12 different volatile compounds and provides a quantitative and visual method to reveal rich detection indicators. The colorimetric sensor array is an economical and practical multi-analyte identification method. It has many potential applications such as food packaging, anti-counterfeiting, health monitoring, environmental monitoring, and optical filters.

Unexpected variations in the effects of ultrasound-assisted myofibrillar protein processing under varying viscosity conditions.

The efficient synthesis of myofibrillar protein(MRN)-gallic acid (GAD) complex in ultrasound (UID)-assisted processing is a challenging problem in food manufacturing. In this investigation, the effect of viscosity characteristics on the efficiency of UID processing in MRN-based beverages was analyzed. Both viscosity and surface tension can increase sono-physico-chemical effects on the degradation of terephthalic acid and crystal violet, with surface tension having a more significant effect (negative correlation, R2 = 0.99) than viscosity (positive correlation, R2 = 0.79). The structural indicators and microstructure demonstrated that the reaggregation and refolding of the MRN structure during the modification procedure occurred with relatively small three-dimensional dimensions. Compared to the MRN/GAD4 group, the water contact angle of the MRN/GAD7 system enhanced by 129.44%, leading to greater system stability. The ABTS-scavenging capacity of the system increased by approximately 19.45% due to the increase in viscosity of these two categories.

Different protein-anthocyanin complexes engineered by ultrasound and alkali treatment: Structural characterization and color stability.

Through alkali treatment (AT) and ultrasound (UT)-assisted processing producing covalent protein-anthocyanin complexes, we investigated the impact of treatment methods and protein types on conjugation efficiency, protein structure, and color stability. Our findings revealed the effective grafting of anthocyanins (ACNs) onto proteins, with myofibrillar protein (MP) exhibiting the highest conjugation efficiency of 88.33% after UT (p <.05). UT accelerated the structure unfolding of distinct protein samples, leading to the exposure of sulfhydryl, and hydrophobic groups in proteins, and enhanced the oxidation stability of ACNs. Notably, the modified ACNs maintained a favorable pH-color relationship, while U-MP showed a significantly higher absorbance (0.4998) than the other groups (p <.05) at pH 9.0, demonstrating an outstanding color improvement. UT-assisted processing also accelerated the NH3 reaction. Thus, the combination of UT and MP holds the potential for pH-color-responsive intelligent packaging and increases the efficiency of UT processing.

Comparisons between Myofibrillar Protein-Luteolin Conjugates Fabricated through Covalent and Noncovalent Modification.

Protein-flavonoid conjugation is considered to effectively enhance the functionality of proteins, although how different binding modes affect the conformation and antioxidative properties of these conjugates has yet to be revealed. Herein, myofibrillar protein (MP)-luteolin (Lut) conjugates were noncovalently and covalently constructed using equivalent amounts of Lut (10.00, 20.11, and 69.60 µmol/g protein). Fluorescence quenching confirmed that hydrophobic interactions were the main forces in noncovalent MP-Lut conjugates and that the binding was entropy-driven. Liquid chromatography-tandem mass spectrometry results confirmed that Lut could be covalently grafted with MP after alkaline treatment. Proteomics analysis identified that most graft sites were located on the myosin subunits. Intriguingly, in vitro results showed that the antioxidant activity was barely affected by the MP-Lut binding modes. This work provides a theoretical basis for the application of MP-Lut noncovalent/covalent complexes as functional components.

Effects of ultrasound treatment on the morphological characteristics, structures and emulsifying properties of genipin cross-linked myofibrillar protein.

Genipin is a natural crosslinker that improves the functional properties of proteins by modifying its structures. This study aimed to investigate the effects of sonication on the emulsifying properties of different genipin concentration-induced myofibrillar protein (MP) cross-linking. The structural characteristics, solubility, emulsifying properties, and rheological properties of genipin-induced MP crosslinking without sonication (Native), sonication before crosslinking (UMP), and sonication after crosslinking (MPU) treatments were determined, and the interaction between genipin and MP were estimated by molecular docking. The results demonstrated that hydrogen bond might be the main forces for genipin binding to the MP, and 0.5 µM/mg genipin was a desirable concentration for protein cross-linking to improve MP emulsion stability. Ultrasound treatment before and after crosslinking were better than Native treatment to improve the emulsifying stability index (ESI) of MP. Among the three treatment groups at the 0.5 µM/mg genipin treatment, the MPU treatment group showed the smallest size, most uniform protein particle distribution, and the highest ESI (59.89%). Additionally, the highest α-helix (41.96%) in the MPU + G5 group may be conducive to the formation of a stable and multilayer oil-water interface. Furthermore, the free groups, solubility, and protein exposure extent of the MPU groups were higher than those of UMP and Native groups. Therefore, this work suggests that the treatment of cross-linking followed by ultrasound (MPU) could be a desirable approach for improving the emulsifying stability of MP.

Insights into Intramuscular Connective Tissue Associated with Wooden Breast Myopathy in Fast-Growing Broiler Chickens.

Wooden breast myopathy (WBM) is a meat abnormality affecting pectoralis majors (PMs) of fast-growing broiler chickens. WBM-affected PMs exhibited varied meat qualities with increasing WBM severity. Normal PMs (NOR), mild WBM-affected PMs (MIL), moderate WBM-affected PMs (MOD), and severe WBM-affected PMs (SEV) were selected as raw materials. The structure and organization of connective tissue and fibrillar collagen were investigated through immersing with sodium hydroxide solution, Masson trichrome staining, and using an electron microscope. The mechanical strength of intramuscular connective tissue was analyzed via the shear force of samples treated with sodium hydroxide solution. The thermal property and secondary structure of connective tissue were analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The obtained connective tissue was dissolved in a sodium hydroxide solution for the evaluation of the physicochemical properties of proteins, including particle size, molecular weight, surface hydrophobicity, and intrinsic fluorescence. In particular, the particle size was measured using a zeta potential instrument. The molecular weight was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The surface hydrophobicity and intrinsic fluorescence were measured by spectroscopy technology. Histologically, macrophage infiltration, myodegeneration and necrosis, regeneration, fibrous connective tissue, and thickened perimysial connective tissue were observed in WBM-affected PMs, especially SEV with fibrosis, including blood vessels. Compared with NOR, WBM led to increased average diameter of the collagen fibrils in perimysial (36.61 nm of NOR to 69.73 nm of SEV) and endomysial (34.19 nm of NOR to 56.93 nm of SEV) layers. A significant increase (p < 0.05) was observed in the mechanical strength (2.05 N to 5.55 N) of fresh PMs and the thermal transition temperature (onset temperature (TO), 61.53 °C to 67.50 °C; maximum transition temperature (TM), 66.46 °C to 70.18 °C; termination temperature (TE), 77.20 °C to 80.88 °C) of connective tissue from NOR to SEV. Cooking decreased the mechanical strength, and MOD samples showed the highest mechanical strength (1.24 N, p < 0.05), followed by SEV (0.96 N), MIL (0.93 N), and NOR (0.72 N). For proteins in connective tissue, random coil (19.64% to 29.61%, p < 0.0001), particle size (p < 0.05), and surface hydrophobicity (p < 0.05) increased with the decrease in the α-helix (14.61% to 11.54%, p < 0.0001), ß-sheet (45.71% to 32.80%, p < 0.0001), and intrinsic fluorescence of proteins from NOR to SEV. The molecular weights of intramuscular connective tissue proteins were in the ranges of >270 kDa, 180-270 kDa, 110-180 kDa, 95-100 kDa, and <15 kDa. Taken together, WBM resulted in thickened organization, tightly packed collagen fibrils, increased mechanical strength and thermal temperature, and increased particle size, surface hydrophobicity, and intrinsic fluorescence of proteins in connective tissue, as the WBM severity increased.

Cytostatic Activity of Sanguinarine and a Cyanide Derivative in Human Erythroleukemia Cells Is Mediated by Suppression of c-MET/MAPK Signaling.

Sanguinarine (1) is a natural product with significant pharmacological effects. However, the application of sanguinarine has been limited due to its toxic side effects and a lack of clarity regarding its molecular mechanisms. To reduce the toxic side effects of sanguinarine, its cyanide derivative (1a) was first designed and synthesized in our previous research. In this study, we confirmed that 1a presents lower toxicity than sanguinarine but shows comparable anti-leukemia activity. Further biological studies using RNA-seq, lentiviral transfection, Western blotting, and flow cytometry analysis first revealed that both compounds 1 and 1a inhibited the proliferation and induced the apoptosis of leukemic cells by regulating the transcription of c-MET and then suppressing downstream pathways, including the MAPK, PI3K/AKT and JAK/STAT pathways. Collectively, the data indicate that 1a, as a potential anti-leukemia lead compound regulating c-MET transcription, exhibits better safety than 1 while maintaining cytostatic activity through the same mechanism as 1.

The interaction of an effector protein Hap secreted by Aeromonas salmonicida and myofibrillar protein of meat: Possible mechanisms from structural changes to sites of molecular docking.

Microbial spoilage of meat products is a significant problem in the food industry. Aeromonas salmonicida is a significant microorganism responsible for spoilage in chilled meat. Its effector protein, hemagglutinin protease (Hap), has been identified as an effective substance for degrading meat proteins. The ability of Hap to hydrolyze myofibrillar proteins (MPs) in vitro demonstrated that Hap has obvious proteolytic activity, which could alter MPs' tertiary structure, secondary structure, and sulfhydryl groups. Moreover, Hap could significantly degrade MPs, focusing primarily on myosin heavy chain (MHC) and actin. Active site analysis and molecular docking revealed that the active center of Hap was bound to MPs via hydrophobic interaction and hydrogen bonding. It may preferentially cleave peptide bonds between Gly44-Val45 in actin, and Ala825-Phe826 in MHC. These findings suggest that Hap may be involved in the spoilage mechanism of microorganisms and provide crucial insights into the mechanisms of meat spoilage induced by bacteria.