Studies of the effect of hydrostatic pressure pretreatment on thermal gelation of chicken myofibrils and pork meat patty

The structures and characteristics of pressure–heat-induced gels of chicken myofibrils and pork patty were investigated. The M-line and Z-line in the chicken myofibril in 0.2 M NaCl were disrupted, and both of the thin and thick filaments were dissociated by pressure treatment. The microstructure of pressure–heat-induced chicken myofibrillar gel was composed of three-dimensional fine strands. Pressurization, at 200 MPa, prior to heating, increased the apparent elasticities of chicken myofibrillar gel and pork patty; however, pressure treatment above 200 MPa decreased it. The apparent elasticity of the pressure-treated (200 MPa) thermal myofibrillar gel was three times higher, and that of pork patty was twice higher than those of the unpressurized ones. The rheological properties of the low salt (1% NaCl) pork sausage can be improved by pressure treatment at 200 MPa prior to heating.     

Solubilization of Myofibrils and Inhibition of Autolysis of Squid Mantle Muscle by Sodium Citrate

ABSTRACT:  The effects of organic salts (sodium (Na)-citrate, Na-gluconate, and Na-succinate) on the solubilization of myofibrillar proteins and the inhibition of autolysis of squid mantle muscle were investigated. All of the organic compounds dissolved myofibrils as NaCl did. Na-citrate showed the strongest ability with half dissolving at 0.06 M. Half dissolving by Na-gluconate, Na-succinate, and NaCl were 0.27, 0.21, and 0.27 M, respectively. Dissolving myofibrils by Na-citrate was not accompanied by the acceleration of autolysis. Na-citrate above 0.25 M almost completely inhibited the autolysis. The inhibition of autolysis by Na-gluconate and Na-succinate was much less than that of Na-citrate. Consequently, it was concluded that Na-citrate was the organic salt to dissolve squid myofibrils without inducing autolysis.     

Is there a protease that preferentially cleaves the M-line in partially dehydrated muscle?

When a partially dehydrated muscle fibre bundle (PDM, 65% H(2)O, pH 5.5, at 4 °C) was treated with a supernatant fraction (M-line-cleaving fraction: MCF) of muscle homogenate for 5 hr, the M-lines disappeared. MCF was extracted from rabbit skeletal muscles by homogenization with 15 mM HCl containing 0.5 M NaCl (pH 3.7), fractionated with 25-65% (NH(4))(2)SO(4) and clarified by Sephadex G-75. Rabbit psoas PDM was obtained with an osmotic dehydration sheet and glycerinated. One end of the bisected fibre bundle was incubated with 10 mM Na-acetate (pH 5.5), 1 mM EDTA, 5 mM β-mercaptoethanol (β-MCE), 150 mM KCl, 10 mM NaN(3) with MCF at 25 °C for 5 hr, the muscle being stretched and relaxed several times. The other end was incubated in the same solution, except that MCF was omitted (control). Electron microscopy showed the myofibrils broken down at the M-line in the presence of MCF. The myofilaments were closely packed near the Z-line and flared out at both ends near the centre of the sarcomere (bow-tie shape). Thus, the Z-line is not the only target of proteases and structural decomposition can also occur at the M-line under specific conditions. An M-line cleaving protease may exist in the MCF muscle extract.     

Contribution of major structural changes in myofibrils to rabbit meat tenderisation during ageing

The contribution of major structural (myofibrillar fragmentation upon mechanical treatment) and ultra-structural (Z-line degradation, loss of electron density of M-line, transversal disruption of sarcomeres at N(2)-line level, longitudinal fissure of myofibrils, and loss of transversal alignments of Z- and M-lines) changes in myofibrils to rabbit (Oryctolagus cuniculus L.) meat tenderisation, during the ageing period (9 days at +4?°C), was studied for different types of muscle (type I, semimembranosus proprius; type IIB, semimembranosus accessorius; and type IID, psoas major). The results strongly suggest that myofibrillar structure weakening at N(2)-line level (evaluated by myofibrillar fragmentation upon mechanical homogenisation and observed by transversal disruption of sarcomeres), which is very likely mediated by cysteine endopeptidases, might be the major structural change responsible for rabbit meat tenderisation during ageing. Both myofibrillar fragmentation and transversal disruption of sarcomeres are good ageing indices for rabbit meat. The other major ultra-structural changes in myofibrils appear to have no major role in rabbit meat tenderisation at refrigeration temperatures. Finally, it is proposed that meat tenderisation during ageing depends mainly on the specific cleavage of titin molecules/filaments and nebulin molecules, at their susceptible sites located at or very close to the N(2)-line region (extensible segment and near C-terminus, respectively), mediated by cysteine endopeptidases (possibly calpains).     

Elucidation of a basic protein, glyceraldehyde-3-phosphate dehydrogenase, as a contributing factor to raise Mg-ATPase activity of myofibrils during meat conditioning

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCl concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0–0.15 M KCl increased time-dependently. This change was most remarkable in the range of pH 5.6–7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3–9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0°C for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCl concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Da), since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils.     

Protein extractability and thermal gel formability of myofibrils isolated from skeletal and cardiac muscles at different post-mortem periods

The effects of the post-mortem ageing period on the extractability of myofibrillar proteins from pork cardiac and rabbit skeletal muscles under various conditions of pH and ionic strength were studied with particular reference to the changes in the solubility of individual myofibrillar proteins and their denaturation characteristics. The ultimate influence of these changes on the heat-induced gel forming ability of myofibrils isolated from cardiac and skeletal muscles at different post-mortem stages was also investigated. Results showed that pork cardiac myofibrils always exhibited lower solubility than those from rabbit skeletal muscles under identical conditions of pH, ionic strength and temperature. SDS-PAGE profiles indicated several quantitative differences in the relative proportion of individual protein species present in cardiac and skeletal myofibrils. The solubility of various proteins present in myofibrils was also affected differently on heating in 0-1 and 0-6 _M NaCl solution at various pH values. Thermal denaturation of cardiac myofibrils occurred at about 10°C higher than that of skeletal myofibrils as revealed by differential scanning calorimetry. Cardiac myofibrils formed much weaker heat-induced gels than those produced by skeletal myofibrils under identical conditions of temperature, pH, ionic strength and protein content.     

Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure

Postmortem tenderization is caused by enzymatic degradation of key structural proteins in myofibrils as well as in extracellular matrix, and of proteins involved in intermyofibrillar linkages and linkages between myofibrils and the sarcolemma. The function of these proteins is to maintain the structural integrity of myofibrils. Current data indicate that calpains and cathepsins may be responsible for degradation of these proteins. Other phenomena occurring in cells postmortem (pH drop, sarcoplasmic Ca2+ increase, osmotic pressure rise, oxidative processes) may act in synergy with proteases. Our understanding of the underlying mechanisms of muscle degradation should be improved for an accurate evaluation of the postmortem muscle changes and consequently of the fish quality.     

The effect of conditioning on the myofibrillar proteins of pork muscle

Storage of pork muscle caused changes in the troponin complex of myofibrillar proteins. The changes were temperature dependent and progressive as conditioning proceeded. An alteration in actin also occurred but this became apparent only when myofibrils had been extracted with 5 mM Tris pH 8.2. About 60% of the proteins in an extract of myofibrils in 5 mM Tris pH 8.2 were bound to the precipitate formed with added F-actin. After conditioning of the pork muscle, the amount of proteins in a Tris extract which were bound to added F-actin was considerably reduced. Some of these changes were also observed in myofibrils which had been treated with a Ca²⁺ activated proteinase.     

Effects of pressure treatment on the ultrastructure of striated muscle

Pre- and post-rigor sheep semimembranosus muscles were subjected to a hydrostatic pressure of 100 meganewtons/m² at 25°C. The ultrastructure of the muscle fibres was compared with that of non pressure-treated samples. A conspicuous feature of pressure-treated post-rigor samples was the absence of the M-band in the central region of the A-band. Therefore it appeared that some, if not all, of the proteins in the M-line were very susceptible to disaggregation under high pressure. Another feature of the post-rigor pressure-treated sample was the loss of integrity and aggregation of I-band filaments which presumably involved an F-G transformation of actin. Pressure treatment of pre-rigor muscle resulted in extensive structural disruption with contraction band formation. It is suggested that a weakening of thin filaments and M-line bridges, when combined with a pressure-induced contraction, facilitates disruption of pre-rigor pressure-treated muscle.     

ABSENCE OF PROTEOLYSIS IN PURIFIED MYOFIBRILS OF POSTSPAWNED HAKE (MERLUCCIUS HUBBSI MARINI) DURING IN VITRO STORAGE AT 37C

The biochemical behavior of myofibrils from postspawned hake during in vitro storage at 37C was investigated. SDS‐PAGE, densitometric analysis of the band areas corresponding to the major myofibrillar proteins, and TCA soluble peptides determination showed no evidence of proteolysis in myofibrils after 44 h of incubation either in presence or in absence of a cocktail of protease inhibitors (1 mMPMSF+1mMiodoacetic acid + 1mM of EDTA). The absence of proteolysis in stored postspawned hake myofibrils contrasts with the autolysis in those from prespawned fish previously reported, indicating an influence of the reproductive cycle on the proteolytic activity closely associated to myofibrils.     

Sarcoplasmic proteins influence water-holding capacity of pork myofibrils

Water-holding capacity (WHC) is one of the main pork quality characteristics. The objective of this study was to determine the influence of (denaturation of) sarcoplasmic proteins on WHC. Myofibrils extracted from red, firm, non-exudative (‘normal’) and PSE (pale, soft, exudative) pork longissimus muscle were combined with sarcoplasmic extracts (with or without proteins) from PSE and normal pork longissimus samples. Weight increase of myofibrils (mg increase mg⁻¹ myofibrillar protein) was used as a measure of WHC. When combined with protein-containing sarcoplasmic extract from normal pork, WHC of myofibrils from PSE (2.6 mg mg⁻¹) and normal (2.8 mg mg⁻¹) pork was higher (P < 0.05) than when combined with sarcoplasmic extract from PSE meat (1.3 mg mg⁻¹ for PSE and 1.9 mg mg⁻¹ for normal myofibrils). Protein-free sarcoplasmic extracts were prepared by heating the extracts for 30 min at 80 °C. WHC of myofibrils combined with protein-free sarcoplasmic extract from PSE and normal pork was not significantly different. WHC of myofibrils combined with protein-free extract was lower than WHC of myofibrils combined with protein-containing extract. Ionic strength or pH could not explain the observed differences. It was concluded that sarcoplasmic proteins do influence WHC. The mechanism of this influence still needs to be determined. © 1999 Society of Chemical Industry     

Thermally Induced Interactions and Gelation of Combined Myofibrillar Protein from White and Red Broiler Muscles

The gelling properties of broiler myofibrillar protein were studied by determining protein-protein interactions during heating. Breast and leg salt-soluble protein (SSP) showed 1–3 transitions in protein-protein interactions within pH 5.5–6.5. The maximum transition temperatures of leg SSP decreased when leg SSP was mixed with breast SSP. The combined breast/leg myofibrils formed stronger gels than leg myofibrils alone at pH ≥ 6.0, and stronger gels than breast myofibrils alone at pH < 6.0. The results suggest that interactions existed between breast and leg myofibrillar proteins, and the transitions in these interactions were useful for predicting gel strength of the combined breast/ leg myofibrils.     

QUANTITATIVE CHANGES IN WHOLE MYOFIBRILS AND MYOFIBRILLAR PROTEINS DURING FROZEN STORAGE OF TRUE COD

The extractability of whole myofibrils from true cod decreased more rapidly during frozen storage at −40°C than did the extractability of the component myofibrillar proteins. There was a 95% decrease in extractable myofibrils after 6 months in storage, but only a 23% decrease in extractable myofibrillar proteins.     

Biochemical and Functional Properties of Myofibrils from Preand Post-Spawned Hake (Merluccius hubbsi Marini) Stored on Ice

Enzymatic activities assayed at beginning of storage in myofibrils from post-spawned hake were 3 X those in myofibrils from pre-spawned hake. Ca²⁺ sensitivity of myofibrils from pre-spawned hake was 40% less than that of myofibrils from post-spawned hake. The profiles of SDS-PAGE gels of pre-spawned myofibrils at beginning of storage showed a partially denatured myosin heavy chain, and polypeptide bands under myosin heavy chain. They probably represent proteolytic fragments produced by degradation of MHC in vivo. No proteolysis was detected in myofibrils during storage. These results help predict functional properties of fish proteins and changes during storage.     

The effect of proteasome on myofibrillar structures in bovine skeletal muscle

When bovine myofibrils are incubated with the 20S proteasome their structure is rapidly damaged with loss of material, particularly from the Z discs and I bands. After 24 hr of incubation the myofibrils rupture and debris appears. Certain myofibrillar proteins, including nebulin, myosin, actin and tropomyosin, are hydrolysed during the incubation; others are solubilised (α-actinin). The 20S proteasome completely and rapidly hydrolyses purified myofibrillar proteins in an energy-independent manner. This shows that the 20S proteasome probably plays a role in the postmortem transformation of muscle and more generally in the hydrolysis of cellular proteins.(1).     

Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes

Unacceptable water-holding capacity costs the meat industry millions of dollars annually. However, limited progress has been made toward understanding the mechanisms that underlie the development of drip or purge. It is clear that early postmortem events including rate and extent of pH decline, proteolysis and even protein oxidation are key in influencing the ability of meat to retain moisture. Much of the water in the muscle is entrapped in structures of the cell, including the intra- and extramyofibrillar spaces; therefore, key changes in the intracellular architecture of the cell influence the ability of muscle cells to retain water. As rigor progresses, the space for water to be held in the myofibrils is reduced and fluid can be forced into the extramyofibrillar spaces where it is more easily lost as drip. Lateral shrinkage of the myofibrils occurring during rigor can be transmitted to the entire cell if proteins that link myofibrils together and myofibrils to the cell membrane (such as desmin) are not degraded. Limited degradation of cytoskeletal proteins may result in increased shrinking of the overall muscle cell, which is ultimately translated into drip loss. Recent evidence suggests that degradation of key cytoskeletal proteins by calpain proteinases has a role to play in determining water-holding capacity. This review will focus on key events in muscle that influence structural changes that are associated with water-holding capacity.     

Hydroxyl radical oxidation destabilizes subfragment-1 but not the rod of myosin in chicken myofibrils

The thermal denaturation process of myosin in oxidized chicken myofibrils was investigated. Exposures of myofibrils to hydroxyl radical-generation systems (HRGS) resulted in an enhanced susceptibility of myosin to thermal inactivation of Ca-ATPase and a loss of salt solubility. The chymotryptic production of subfragment-1 (S-1) from myosin in oxidized myofibrils decreased more rapidly than that in un-oxidized myofibrils upon heating, which paralleled the Ca-ATPase decay. However, the heat-induced decrease in chymotryptic production of rod from myosin was not affected by the HRGS treatment. The results suggested that free radical oxidation promoted thermal destabilization of myosin in the S-1 portion instead of the rod portion. The altered myosin denaturation pattern due to hydroxyl radical oxidation was likely a cause for functionality changes in oxidatively stressed myofibrillar proteins in meat processing.     

Effect of high pressure on moisture and NaCl diffusion into turkey breast

High-pressure processing (HPP) affects food component diffusion. This study evaluated pressure (0.1-300 MPa) effect on the diffusion of NaCl and water into turkey breast at 25 °C. NaCl and water diffusion were found to be a function of pressure and holding time. During the pressure come-up-time, diffusion of water and NaCl into the sample was enhanced as compared to control. The diffusion coefficient of moisture infusing out of the sample was found to be a minimum at 150 MPa. The diffusion coefficient of NaCl infusing into the sample was maximum at 150 MPa. Within the range of experimental conditions studied, 150 MPa treatment (up to 15 min holding time) yielded meat samples with minimum hardness, gumminess, and chewiness. Ultrastructure images revealed that 150 MPa treatments caused swelling of myofibrils, disappearance of the M-line, reduced difference in the density of the A-band and I-band and breaking of segments of Z-line. HPP could be a useful technique for the salting of turkey meat.     

In vitro proteolysis of myofibrillar and sarcoplasmic proteins of European sea bass (Dicentrarchus Labrax L) by an endogenous m‐calpain

The effects of m‐calpain isolated from the skeletal muscle of sea bass on sarcoplasmic and myofibrillar proteins isolated from the same tissue were examined in vitro. Incubation of sarcoplasmic proteins with m‐calpain resulted in only a slight decrease (0.7 kDa) in the molecular weight (MW) of a 26.5 kDa protein. Degradation of myofibrils, monitored by quantification of TCA‐soluble peptides generated, resulted in the maximum amount of peptides being generated after 1 h of incubation at 25 °C. Noticeable modifications in the SDS‐PAGE profile of digested myofibrils were observed, including partial denaturation of myosin heavy chain and the release of tropomyosin, ～69 and ～27 kDa doublet bands and a few polypeptides of MW lower than 20 kDa in the soluble fraction. Examination of the degradation patterns of myofibrillar proteins using Western blotting showed that α‐actinin was partially degraded, with release of native α‐actinin and its fragments from myofibrils, whereas desmin was highly degraded after 2 h of digestion. © 2002 Society of Chemical Industry     

AUTOLYSIS OF PROTEINS FROM PRESPAWNED HAKE (MERLUCCIUS HUBBSI MARINI) DURING IN VITRO STORAGE OF MYOFIBRILS AT 20 AND 37C

The in vitro autolysis of hake myofibrillar proteins was investigated. No evidence of proteolysis was observed in myofibrils after 44 h of incubation either in presence or in absence of protease inhibitors at 20C. A 180‐kDa component present at zero time in the SDS‐PAGE gels of myofibrils from prespawning hake was degraded after 44 h of incubation at 37C. Degradation of the 180‐kDa component was accompanied by an increase in low molecular weight and TCA soluble peptides. These changes were not observed in myofibrils incubated in the presence of protease inhibitors (1 mM PMSF+ 1 mM iodoacetic acid + 1 mM of EDTA). Both PMSF and EDTA used alone partially inhibited the degradation of myofibrillar proteins. This inhibition was lower than that produced in the presence of both inhibitors, Azocaseinolytic activity was almost exclusively associated with the 180 kDa‐component eluted from SDS‐PAGE gels. These results suggest that both metallo and serine proteinases could be involved in the autolysis of myofibrils at 37C.