Effect of glycolysis on water holding capacity during postmortem aging of Jersey cattle-yak meat.

BACKGROUND: Postmortem muscle moisture loss leads to a decrease in carcass weight and can adversely impact overall meat quality. Therefore, it is critical to investigate water holding capacity (WHC) to enhance meat quality. Current research has primarily focused on examining the correlation between signaling molecules and meat quality in relation to the glycolysis effect on muscle WHC. But there exists a significant knowledge gap regarding the mechanism of WHC in Jersey cattle-yak meat. RESULTS: Jersey cattle-yak meat pH decreased and then increased during postmortem aging. Lactate content, cooking loss, pressing loss, drip loss and centrifuging loss of Jersey cattle-yak meat increased and then decreased during postmortem aging. The glycogen content of Jersey cattle-yak meat was significantly higher than that of yak meat at 6-120 h, being 8.40% higher than that of yak meat at 120 h. The activity of key glycolytic enzymes hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) in Jersey cattle-yak meat was lower than that in yak meat. Correlation analysis showed that Jersey cattle-yak meat WHC was positively correlated with the activity of HK, PK, PFK and LDH. CONCLUSIONS: The WHC of Jersey cattle-yak meat was higher than that of Gannan yak meat, and it was significantly positively correlated with the activity of key enzymes of the glycolytic signaling pathway. Therefore, the glycolysis rate can be reduced by inhibiting enzyme activity to improve Jersey cattle-yak meat WHC and meat quality. © 2023 Society of Chemical Industry.

Effect of tremella polysaccharides on the quality of collagen jelly: insight into the improvement of the gel properties and the antioxidant activity of yak skin gelatin.

BACKGROUND: The present study aimed to investigate the effects of tremella polysaccharides on the gel properties and antioxidant activity of yak skin gelatin with a view to improving the quality of collagen jellies. The preparation of composite gels were performed by yak skin gelatin (66.7 mg mL-1) and tremella polysaccharides with different concentrations (0, 2, 4, 6, 8 mg mL-1), and finally the collagen jelly was prepared by composite gel (yak skin gelatin: 66.7 mg mL-1; tremella polysaccharides:6 mg mL-1) with the best performance. RESULTS: Tremella polysaccharides not only improved the hardness, springiness, gel strength, water holding capacity and melting temperature of yak skin gelatin, but also enhanced the composite gel's scavenging activity against ABTS radicals, DPPH radicals, O2 and OH radicals. The filling of tremella polysaccharides into the gelatin network increased the number of crosslinking sites inside the gel, which resulted in the gel network structure becoming dense and orderly. The gel particles became finer and more uniform, and the thermal stability was improved. Furthermore, the sensory score of commercially available gelatin jelly decreased more rapidly during storage compared to the composite gel jelly. CONCLUSION: The gel properties and antioxidant activity of yak skin gelatin were improved by adding tremella polysaccharides, and then the quality and storage properties of the jelly were improved, which also provided technical reference for the development of functional gel food. © 2024 Society of Chemical Industry.

Reactive oxygen species-mediated oxidative stress accelerates glycolysis via activation of the CaMKKß/AMPK pathway in the yak longissimus dorsi postmortem.

BACKGROUND: Adenosine monophosphate-activated protein kinase (AMPK) is instrumental in the initiation of early postmortem glycolysis and the advent of pale, soft, and exudative (PSE) meat when cellular energy is altered. However, conflicting studies show that AMPK activation without corresponding energy level changes in PSE meat challenges this long-held notion. Here, we examined the effects of reactive oxygen species (ROS)-mediated oxidative stress on AMPK activation in the context of glycolysis, protein solubility, and water-holding capacity (WHC) in the postmortem yak longissimus dorsi (LD) muscle. Further, we explored the mechanisms underlying these effects. RESULTS: Hydrogen peroxide (H2 O2 ) significantly augmented the degree of oxidative stress, increasing the production of ROS and malondialdehyde excessive production and reducing the activity of the anti-oxidants superoxide dismutase and glutathione peroxidase. In turn, oxidative stress dramatically promoted AMPK activation and glycolysis by increasing glycogen depletion and promoting hexokinase and phosphofructokinase activity. Subsequently, lactic acid accumulation increased, leading to a rapid decline in pH, which aggravated protein solubility degree and centrifugal loss in the early postmortem yak LD muscle. Importantly, these changes caused by oxidative stress were eliminated by the AMPK inhibitor. Mechanistically, oxidative stress elevated calcium ion (Ca2+ ) levels, which mobilized calcium/calmodulin-dependent protein kinase ß (CaMKKß) and AMPK. Rescue experiments confirmed that the increases were attenuated using Ca2+ and CaMKKß chelators, respectively. CONCLUSION: These results indicated that oxidative stress caused by ROS hastened early-stage postmortem glycolysis and reduced the WHC of yak meat. These effects were likely mediated by the alternative and energy-independent CaMKKß/AMPK signaling pathway. © 2022 Society of Chemical Industry.

Synergistic modification of collagen structure using ionic liquid and ultrasound to promote the production of DPP-IV inhibitory peptides.

BACKGROUND: Dual modification of collagen was performed using ionic liquid (IL) and ultrasound (US) to modulate the activity of collagen hydrolyzed peptides and reveal the production mechanism of cowhide-derived dipeptidyl peptidase (DPP-IV) inhibitory peptides. RESULTS: The results revealed that dual modification (IL + US) significantly improved the hydrolytic degree of collagen (P < 0.05). Meanwhile, IL and US tended to promote the break of hydrogen bonds, but inhibit the crosslinking between collagens. The double modification reduced the thermal stability and accelerated the exposure of tyrosine and phenylalanine of collagen, and improved the proportion of small molecular (< 1 kDa) peptides in collagen hydrolysates. Interestingly, the hydrophobic amino acid residues and DPP-IV inhibitory activity of collagen peptides with small molecular weight (< 1 kDa) was increased further under the combination of IL and US. CONCLUSION: Enhanced hypoglycemic activity of collagen peptides can be attained through the dual modification of IL and US. © 2023 Society of Chemical Industry.

Study on the apoptosis mediated by cytochrome c and factors that affect the activation of bovine longissimus muscle during postmortem aging.

This study investigates whether bovine longissimus muscle cell apoptosis occurs during postmortem aging and whether apoptosis is dependent on the mitochondria pathway. This study also determines the apoptosis process mediated by cytochrome c after its release from mitochondria and the factors that affect the activation processes. Results indicate that apoptotic nuclei were detected at 12 h postmortem. Cytochrome c release from the mitochondria to the cytoplasm activated the caspase-9 and caspase-3 at early postmortem aging and the activation of caspase-9 occurs before the activation of caspase-3. The pH level decreased during the first 48 h postmortem, whereas the mitochondria membrane permeability increased from 6 to 12 h. Results demonstrate that an apoptosis process of bovine muscle occurred during postmortem aging. Apoptosis was dependent on the mitochondria pathway and occurred at early postmortem aging. Increased mitochondria membrane permeability and low pH are necessary conditions for the release of cytochrome c during postmortem aging.

Preservation of chilled beef using active films based on bacterial cellulose and polyvinyl alcohol with the incorporation of Perilla essential oil Pickering emulsion.

In this study, Perilla essential oil (PEO) Pickering emulsions, prepared using soybean protein isolate-chitosan nanoparticles (SPI-CSNPs) as emulsifiers (SCEO), were used to improve the performance of bacterial cellulose/polyvinyl alcohol (BC/PVA) films for application in chilled beef preservation. The SCEO has a smaller particle size (185 nm), higher viscosity, a more uniform dispersion and was more stable at an oil phase volume fraction of 80 %. An increase in the films' surface roughness and in the hydrogen bonding between SCEO and the films' matrix was also observed, resulting in a lower tensile strength (TS, 94.75-62.02 MPa) and higher elongation at break (EAB, 26.78-55.62 %). Moreover, the thermal stability, water vapor permeability, antioxidant and antibacterial properties of the composite films improved as the SCEO content increased. Furthermore, the Pickering emulsion method was effective in preventing the loss of PEO during storage. Overall, one particular composite film, BP/SCEO3, could prolong the shelf life of chilled beef by up to 14 days, and hence was promising for food preservation.

Molecular Characterization of Resistance and Virulence Factors of Trueperella pyogenes Isolated from Clinical Bovine Mastitis Cases in China.

Purpose: The present study was designed to investigate the resistance determinants and virulence factors of 45 Trueperella pyogenes isolates from clinical bovine mastitis in Hexi Corridor of Gansu, China. Methods: Minimum inhibitory concentrations (MICs) was tested by E-test method. Gene of antimicrobial resistance, virulence integrase and integron gene cassettes were determined by PCR and DNA sequencing. Results: The T. pyogenes isolates exhibited high resistance to streptomycin (88.9%) and tetracycline (64.4%), followed by erythromycin (15.6%) and gentamicin (13.3%). Resistance to streptomycin was most commonly encoded by aadA9 (88.9%); and to tetracycline, by tetW (64.4%). Importantly, all streptomycin-resistant isolates carried aadA9 alone or in combination with aadA1, aadA11 and strA-strB. Similarly, all tetracycline-resistant isolates harbored tetW alone or in combination with tetA33. Meanwhile, ermX was detected in 13.3% isolates, only one erythromycin-resistant isolate was not identified for this gene. Moreover, all T. pyogenes isolates carried class 1 integrons, and 17.8% of them contained gene cassettes, including arrays aadA1-aadB (4.4%), aad A24-dfrA1-ORF1 (2.2%) and aadA1 (2.2%). Furthermore, all tested isolates harbored virulent genes plo and fimA, followed by fimC (88.9%), fimE (86.6%) nanP (75.6%), nanH (40.0%), cbpA (35.6%) and fimG (6.7%). Conclusion: To our knowledge, this is the first report of integron gene cassettes of T. pyogenes isolates from bovine mastitis cases in China. These findings are useful for developing the prevention and the virulence factors of T. pyogenes could be promising candidates for vaccine antigens for bovine mastitis caused by T. pyogenes in China.

Potato oxidized hydroxypropyl starch/pectin-based indicator film with Clitoria ternatea anthocyanin and silver nanoparticles for monitoring chilled beef freshness.

Potato oxidized hydroxypropyl starch (POHS)/pectin (P) functional and smart beef freshness indicator films were prepared using butterfly pea (Clitoria ternatea) anthocyanin (BA) and silver nanoparticles (AgNPs). BA exhibited significant pH-responsive color changes. BA and AgNPs were evenly distributed within a polymer matrix to create a compatible film with POHS/P. The films containing BA and AgNPs had good UV resistance and maintained strong mechanical strength, barrier properties, and color stability. The color of the indicator film changed from purple to green when exposed to ammonia, with the 1 % POHS/P/BA/AgNPs film showing the most sensitive response. The films also demonstrated strong antibacterial and antioxidant properties. The freshness of beef was monitored using 1 % POHS/P/BA/AgNPs films and was identified as sub-fresh and spoiled on days 4 and 7, respectively. The relationship between the color change of the indicator label and the freshness of chilled beef was established: purple for fresh meat, blue for less fresh meat, and green for spoiled meat. Thus, the new POHS/P/BA/AgNPs film can serve as a smart packaging material to indicate food freshness and extend shelf life. These results suggest that POHS/P/BA/AgNPs films have significant potential as an active and smart food packaging material.

Potential Effects of NO-Induced Hypoxia-Inducible Factor-1α on Yak Meat Tenderness during Post-Mortem Aging.

The objective of this study was to investigate the mechanism underlying nitric oxide (NO)-induced hypoxia-inducible factor-1α (HIF-1α) and its impact on yak muscle tenderness during post-mortem aging. The Longissimus thoracis et lumborum (LTL) muscle of yak were incubated at 4 °C for 0, 3, 6, 9, 12, 24, and 72 h after treatment with 0.9% saline, NO activator, or a combination of the NO activator and an HIF-1α inhibitor. Results indicated that elevated NO levels could increase HIF-1α transcription to achieve stable expression of HIF-1α protein (P < 0.05). Additionally, elevated NO triggered HIF-1α S-nitrosylation, which further upregulated the activity of key glycolytic enzymes, increased glycogen consumption, accelerated lactic acid accumulation, and decreased pH (P < 0.05). These processes eventually improved the tenderness of yak muscle during post-mortem aging (P < 0.05). The results demonstrated that NO-induced activation of HIF-1α S-nitrosylation enhanced glycolysis during post-mortem aging and provided a possible pathway for improving meat tenderness.

Characterization of sodium alginate-carrageenan films prepared by adding peanut shell flavonoids as an antioxidant: Application in chilled pork preservation.

This study prepared and characterized sodium alginate and carrageenan (SAC) composite films incorporated with peanut shell flavonoids (PSFs). PSFs compound identification research was implemented. The physicochemical features of PSFs-SAC composite films and their ability to preserve chilled pork in a 4 °C refrigerator were determined. PSFs consist of luteolin, eriodictyol, 5,7-dihydroxychromone, and 8 other components. They significantly improved the mechanical properties, barrier properties, thermal stability, and antioxidant properties of SAC composite films (P < 0.05). PSFs were also responsible for increasing the density of the film structure between the sodium alginate and carrageenan molecules. During storage, compared with the control group, the prepared PSFs-SAC composite films did not allow the total viable count (TVC), pH and total volatile base nitrogen (TVB-N) of the chilled pork to increase rapidly. Further, they were able to inhibit lipid oxidation more effectively (P < 0.05). For these reasons, the use of the PSFs-SAC composite films prolonged shelf life of chilled pork from 6 days to the 12 days. Therefore, PSFs-SAC composite films are expected to be used as bioactive substances in food preservation.

Cowhide gelatin peptide as a source of antioxidants for inhibiting the deterioration of pudding quality during storage.

To investigate the effect of gelatin peptide on the inhibition of quality deterioration in stored pudding, gelatin peptide with antioxidant properties was added to pudding products. For this purpose, a pudding recipe containing gelatin peptides was created. The gelatin peptides were characterized based on their antioxidant activity and protein structure. It was found that gelatin peptides had better antioxidant properties, lower thermal stability and crystallinity, higher hydrophobic amino acid content, and greater surface hydrogen bond exposure than commercially available peptides. Properties such as the pH, colony growth, and sensory characteristics of the pudding were characterized at 4 °C and 25 °C. The results showed that the addition of 0.5-1.0 % gelatin peptide to pudding was capable of significantly (P< 0.05) slowing down the decline in pH and sensory scores of the pudding and significantly inhibiting colony growth. It could prolong its storage life by five days at 4 °C and three days at 25 °C.

Development of amine-sensitive intelligent film with MIL-100(Fe) as function filler based on anthocyanins/pectin for monitoring chilled meat freshness.

To enhance the amine-sensitivity of intelligent films for accurate monitoring of chilled meat freshness, different additions (0, 1, 2, 3 wt%) of MIL-100(Fe) were incorporated into the matrix composed of anthocyanins (ANs) and pectin (P). Results indicated that the tensile strength, thermal stability, barrier performance and absorption capacity of the films with MIL-100(Fe) were improved significantly (p < 0.05). Especially, the film with 2 % MIL-100(Fe) exhibited the best performance due to its compact structure and the highest crystallinity. Additionally, adsorption isotherms of the films with MIL-100(Fe) were fitted on the Langmuir and the Freundlich isotherm, and adsorption kinetics were fitted on the pseudo-second-order model and Elovich model, respectively (R2 > 0.96), suggesting a combined mechanism of chemisorption and intraparticle diffusion. Besides, when the films were exposed in ammonia environment, they changed color from purple to blue-violet, finally to green. Ultimately, film with 2 % MIL-100(Fe) was used to monitor the chilled meat freshness, as expected, similar color variation was observed at three stages of meat freshness (fresh, sub-fresh, and spoiled), which enabled the accurate differentiation of meat freshness. Thus, films with MIL-100(Fe) demonstrated the potential to be amine-sensitive intelligent packaging for monitoring chilled meat freshness in real time.

Impact of Nitrite Supplementation on Bioactive Peptides during Sausage Processing.

The goal of this investigation was to examine the impact of nitrite supplementation on the concentration, antioxidant properties, and species of antioxidant peptides in fermented sausages. The polypeptide concentration in nitrite-supplemented sausages was markedly elevated during sausage processing compared to the blank control (p < 0.05). Moreover, nitrite supplementation in fermented sausages markedly enhanced the DPPH, as well as the ABTS, hydroxyl radical, and superoxide anion free radical scavenging abilities (FRSA) of polypeptides (p < 0.05). The ferrous ion chelating ability was also significantly enhanced (p < 0.05). Based on the liquid chromatograph-mass spectrometer (LC-MS) analysis of the sausage, LPGGGHGDL, TKYRVP, FLKMN, SAGNPN, GLAGA, LPGGGT, DLEE, GKFNV, GLAGA, AEEEYPDL, HCNKKYRSEM, TSNRYHSYPWG, and other polypeptides exhibited antioxidant properties. Moreover, the number of species of antioxidant polypeptides in the nitrite-supplemented sausage was greater in comparison to the controls. Based on this evidence, it may be concluded that nitrite supplementation positively modulated antioxidant polypeptide formation in fermented sausages, thereby providing strong evidence that nitrite supplementation significantly enhances sausage quality.

High oxygen-modified packaging (HiOx-MAP) mediates HIF-1α regulation of tenderness changes during postmortem aging of yak meat.

In the present study, we studied the effect of high oxygen-modified packaging (HiOx-MAP) on yak meat tenderness and the underlying mechanism. HiOx-MAP significantly increased the myofibril fragmentation index (MFI) of yak meat. In addition, western blotting showed that the expression of hypoxia-inducible factor (HIF-1α) and ryanodine receptors (RyR) in the HiOx-MAP group was reduced. HiOx-MAP increased the activity of sarcoplasmic reticulum calcium-ATPase (SERCA). The energy disperse spectroscopy (EDS) mapping showed gradually reduced calcium distribution in the treated endoplasmic reticulum. Furthermore, HiOx-MAP treatment increased the caspase-3 activity and the apoptosis rate. The activity of calmodulin protein (CaMKKß) and AMP-activated protein kinase (AMPK) was down-regulated leading to apoptosis. These results indicated that HiOx-MAP promoted apoptosis during postmortem aging to improve the tenderization of meat.

Effects of muscle-specific oxidative stress on protein phosphorylation and its relationship with mitochondrial dysfunction, muscle oxidation, and apoptosis.

This study aimed to investigate the effects of muscle-specific oxidative stress on phosphorylation and its relationship with mitochondrial dysfunction, muscle oxidation, and apoptosis of porcine PM (psoas major) and LL (longissimus lumborum) during the first 24 h postmortem. The global phosphorylation level decreased and the mitochondrial dysfunction, oxidation level, and apoptosis increased significantly at 12 h postmortem compared with 2 h postmortem, suggesting that lower phosphorylation level was related to more mitochondrial dysfunction and apoptosis during the early postmortem, regardless of muscle type. PM exhibited a higher global phosphorylation level but showed greater mitochondrial dysfunction, oxidation level, and apoptosis than LL, regardless of aging time. The increased mitochondrial dysfunction and oxidative stress accelerated apoptosis, but their relationship with phosphorylation was different in various muscle types at different aging times. These findings provide insight regarding the roles of coordinated regulation of phosphorylation and apoptosis in development of quality of different muscles.

The potential mediation of hypoxia-inducible factor-1α in heat shock protein 27 translocations, caspase-3 and calpain activities and yak meat tenderness during postmortem aging.

The present study aimed to characterize the influence of hypoxia-inducible factor-1α on heat shock protein 27 and cytochrome c translocation, yak meat microstructure destruction, and endogenous enzymes activities, refining the understanding of the tenderization process after slaughter. Postmortem yak longissimus thoracis et lumborum muscles were incubated with 0.9% saline or hypoxia-inducible factor-1α stabilizer dimethyloxaloylglycine at 4 °C for 6, 12, 24, 72, and 120 h. Results showed that hypoxia-inducible factor-1α activation promoted heat shock protein 27 migration and cytochrome c release, facilitating (P < 0.05) caspase-3 activity by mediating the heat shock protein 27/caspase-3 interaction but did not exert (P > 0.05) significant effects on the calpain-1 activity. Additionally, hypoxia-inducible factor-1α activation contributed to the mitochondrial apoptosis cascade, leading to a higher (P < 0.01) apoptosis rate. Therefore, these observations indicate that hypoxia-inducible factor-1α affects caspase-3 activity and tenderness of postmortem muscle through distinct regulatory mechanisms, possibly, in part, with heat shock protein 27 and cytochrome c mediation.

Hydroxypropyl methyl cellulose/soybean protein isolate nanoparticles incorporated broccoli leaf polyphenol to effectively improve the stability of Pickering emulsions.

This study prepared SPI-Pol-HPMC (SPH) nanoparticles from soybean protein isolate (SPI), hydroxypropyl methyl cellulose (HPMC), and broccoli leaf polyphenol (Pol) and used them as a stabilizer for the Pickering emulsion. The SPH (2:1) nanoparticles have the best ability to encapsulate broccoli leaf polyphenols, with uniform particle size distribution, and a more dense and stable structure. The chemical and hydrogen bonding forces between the SPH nanoparticle components were enhanced. Additionally, the 1.5 % SPH nanoparticle-stabilized emulsions exhibited good physical stability, manifesting as small particle droplets with good rheological properties and uniform dispersion. The volume fraction of the emulsified phase of the 1.5 % SPH nanoparticle-stabilized emulsions was the greatest after 21 days of storage. Interestingly, SPH nanoparticles also improved the oxidative stability of the emulsions, as evidenced through their lower peroxide values and thiobarbituric acid active substances. The aforementioned results suggest that SPH nanoparticles may be used as food-grade emulsifiers that stabilize emulsions and inhibit their lipid oxidation.

Chitosan coating with grape peel extract: A promising coating to enhance the freeze-thaw stability of beef.

This study investigated the effect of chitosan coating with grape peel extracts (CH + GPE) on the physiochemical properties, protein and lipid oxidation, microstructure, and bacterial community diversity of beef during freeze-thaw (F-T) cycles. The results indicated that the CH + GPE groups had lower pH values, total aerobic count, total volatile base nitrogen, and thiobarbituric acid reactive substance values and better protection against color, water holding capacity, and sensory quality after five F-T cycles. The CH + GPE coating effectively inhibited beef microstructure destruction during the F-T cycles. High-throughput sequencing analysis revealed that the CH + GPE coating contributed to a decline in the bacterial diversity of beef and inhibited the growth of pathogenic bacteria. Interestingly, the CH + GPE coating affected the correlation between quality parameters and bacteria in beef. Consequently, the CH + GPE coating can be used as a novel packaging for preventing the loss of frozen meat quality due to temperature fluctuations.

Development and characterization of tapioca starch/pectin composite films incorporated with broccoli leaf polyphenols and the improvement of quality during the chilled mutton storage.

This study aimed at the composition of active packaging film from tapioca starch/pectin (TSP) incorporated with broccoli leaf polyphenols (BLP) was prepared and applied to improve the qualities of the chilled mutton during storage. The results indicated the addition of BLP significantly improved the thickness, density, barrier ability, mechanical properties, water solubility and antioxidant activity of the composite films while inducing decreases in the brightness (p < 0.05), enhancing inter-molecular interactions of TSP + BLP composite films. The WVP, oxygen permeability and elongation at break of the composite film reached the minimum when BLP concentration was 3 % while exhibiting the highest tensile strength and the best performance. This composite film delayed microbial growth and minimized oxidative rancidity during chilled mutton storage, causing the improvement of its quality and extending its shelf life to 12 days. Therefore, TSP + BLP composite films possessed the promise to be applied as bioactive materials in food packaging sectors.

Green tea catechin prevents oxidative stress-regulated autophagy and apoptosis signaling, and inhibits tenderness in postmortem bovine longissimus thoracis et lumborum muscle.

Although green tea catechin has been reported to be an antioxidant and preservative in meat, the extent to which it affects the tenderization of bovine muscle remains largely unknown. This study seeks to evaluate the effect of catechin on the interplay between apoptosis and autophagy, and subsequently, the development of bovine muscle tenderness. The results indicate that catechin significantly alleviated oxidative stress. A concomitant reduction of autophagic markers LC3-II/LC3-I ratio, Beclin-1, and Atg7 levels were caused by catechin. Besides, aforementioned autophagy inhibition was further augmented by PI3K/Akt/mTOR activation. Additionally, catechin protected against mitochondrial dysfunction and inhibited mitochondria-dependent caspase apoptosis pathway. Furthermore, there was a reciprocal inhibition between autophagy and apoptosis. Ultimately, tenderness at 24 and 120 h, an increase in the gap between muscle fiber bundles, and disintegration of myofibrillar architectures were all inhibited by catechin. Therefore, despite alleviating oxidative stress, catechin may hamper tenderization pattern of postmortem bovine muscle.