CHANGES IN TEXTURE AND MICROSTRUCTURE OF PRESSURE-TREATED FISH MUSCLE TISSUE DURING CHILLED STORAGE

Activities of four endogenous enzymes (cathepsin C, collagenase, chymotrypsin- and trypsin-like enzymes), as well as firmness/strength and elasticity of pressurized fish tissues were monitored over 3 weeks of storage (4–7C). Results indicate that pressurization of fish muscle at 1,000 atm increased firmness whereas pressurization at 2,000 atm or 3,000 atm caused an opposite effect. Changes in tissue elasticity also showed similar trends with correlation between firmness and elasticity. During storage, pressure-inactivated enzymes were reactivated to various extents depending on level of pressurization. Scanning electron microscopy of the tissues revealed some morphological changes with pressurization. At 1,000 atm, there were no significant changes in the myofibers while pressurization at 2,000 atm and 3,000 atm resulted in breakdown of myofibers and connective tissue networks. The results indicate that pressurization may be used to enhance and maintain fresh seafood texture during storage.     

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.     

Structural studies of rigor bovine myofibrils using fluorescence microscopy. II. Influence of sarcomere length on the binding of myosin subfragment-1, alpha-actinin and G-actin to rigor myofibrils

Sarcomere length influences the textural quality of meat, yet the molecular basis for the mechanism of post-mortem shortening and the toughness associated with shortened muscle remain obscure. Bovine and rabbit myofibrillar structure was investigated over a range of sarcomere lengths (SL), using the high affinity of the myosin head for actin and of alpha-actinin for the Z-line. Myofibrils were incubated with fluorescent conjugates of myosin subfragment-1 (S1), alpha-actinin and actin. When S1 and alpha-actinin were added together, S1 bound in the I-band and A-band but not at the Z-line, while alpha-actinin bound at the Z-line. The pattern of S1 binding was highly influenced by SL, and could be explained using a model with the ratio of myofibrillar actin to myosin heads in the overlap region of 2:1 and thin filament penetration into opposing half sarcomeres. Double staining with S1 and actin demonstrated that, once the tip of the thin filament reached the bare zone, few intrinsic myosin heads were available for fluorescent actin. The patterns observed for both S1 and actin staining suggest that myofibrillar rigor bonds form even at very short SL. These observations lead to the hypothesis that the toughness associated with short sarcomeres is due to thick-filament interactions and not to the density of rigor bonds in the myofibril. Regulation of S1 binding was investigated by incubating myofibrils with low levels of fluorescent S1 in the presence and absence of calcium; S1 binding was in the overlap region when calcium was absent, but in the I-band when it was present. These results suggest that the thin filament can be activated for muscle shortening by the binding of myosin heads, a mechanism that may contribute to post-mortem muscle shortening.     

Myofibrils of cooked meat are a continuum of gap filaments

When cooked meat is subjected to high degrees of stretch it becomes apparent in high magnification electron micrographs that A-filaments have ceased to exist. The A-band is filled with a coagulum of actomyosin. Fragmentation of this coagulum during stretch reveals an array of fine filaments (identified as gap filaments). This result is obtained irrespective of rigor temperature, state of contraction or degree of cooking. If the meat is first aged, the gap filaments surviving in the I-band are too weak to open up the A-band. The results show that myofibrils in cooked meat are entirely dependent on heat-stable gap filaments for structural continuity and tensile strength. Theories of meat tenderness must be revised accordingly.     

Structure of beef Longissimus dorsi muscle frozen at various temperatures: Part 2-ultrastructure of muscles frozen at -10, -22, -33, -78 and -115°C

The changes in electron micrographs of muscles frozen at -10, -22, -33, -78 and -115°C were analyzed. The ultrastructure of muscle successively changed with decreasing freezing temperature whereas light microscopy indicated anomalous behaviour at -22°C. It appeared that, in muscles frozen at -10°C, there was no freezing of water intracellularly; in those frozen at -22°C, water was frozen intracellularly (but only in the I-band region); whereas, in muscles frozen at -33°C, water was frozen inside the fibres, both in the I- and the A-bands. In muscles frozen at -78 and -115°C, water is frozen intracellularly. These findings can be explained on the basis that, in the I-band region, the major protein is actin, which has a relatively high proportion of non-polar residues and holds water weakly, whereas the predominant protein in the A-band is myosin, which contains many polar residues and has a high water-holding capacity.     

Pressure treatment of meat: Effects on thermal transitions and shear values

The effects of pressure treatment (150 MN m(-2) for 3 h at 0°C) on the pH, thermal transitions, ultrastructure and Warner-Bratzler shear values of post-rigor beef semimembranosus and longissimus dorsi muscles have been investigated. Pressure treatment resulted in a slight but significant increase in pH. Differential scanning calorimetry revealed large changes in the thermograms of muscle samples as a result of pressure treatment, in particular a transition attributed to F-actin was absent in the pressure-treated sample. Examination of the ultrastructure also revealed extensive change as a result of pressure treatment, particularly in the I-band and M-line region. Pressure treatment either did not change shear values or increased them, according to whether the muscle was in the stretched or contracted state, respectively. The results are thought to support a theory for contraction state toughness proposed by Voyle (1969) in which increasing toughness is caused by an increasing incidence of sarcomeres in which thick filaments have been compressed onto the Z-line, thus removing the I-band as a zone of weakness.     

ULTRASTRUCTURAL CHANGES IN BOVINE LONGISSIMUS MUSCLE CAUSED BY THE HYDRODYNE PROCESS

Transmission electron microscopy was used to examine the influence of the Hydrodyne process, a new technology for tenderizing meat in the raw state, on the ultrastructural characteristics of bovine longissimus muscle. Myofibrillar fiagmentation in the region adjacent to the Z-lines was clearly evident. Fragments of Z-lines were attached to the A-band on both sides of the fractures. These fractures resulted in increased intrumyofibrillar spaces with longitudinal gaps or splits in the myofibril lattice (lattice of filaments formed inside the myofibril). These observations offer evidence as to why there is a significant improvement in meat tenderness when meat is treated with the Hydrodyne process.     

Structural changes in meat during ageing

Summary. During the ageing of bovine muscle the most notable changes in the pattern of myofibrillar cross-striations are the complete disappearance of the Z lines and the lengthening of the A bands at the expense of the I zones. It is suggested that with the disintegration of the Z lines, the actin filaments of the I zones collapse onto the myosin rods, leading to apparent A-band lengthening. It is concluded that the weakening of the normally refractory Z lines of the myofibrillar structures is an event closely related to meat ageing.     

Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.     

Relationship between the translocation of paratropomyosin and the restoration of rigor-shortened sarcomeres during post-mortem ageing of meat-A molecular mechanism of meat tenderization

The weakening effect of paratropomyosin on rigor linkages formed between actin and myosin was determined by measuring the restoration of rigor-shortened sarcomeres of chicken, pork and beef. We observed the rate of the post-mortem translocation of paratropomyosin from the A-I junction region of sarcomeres onto the thin filaments in the A-band, where rigor linkages had been formed; this agreed well with the rate of increase in length of rigor-shortened sarcomeres. The sarcomere lengths were found to be maximum at 1, 7 and 10 days post-mortem in chicken, pork and beef, respectively. Thus, translocated paratropomyosin weakens rigor linkages and brings about the recovery in the length of rigor-shortened sarcomeres. Paratropomyosin stimulates the resolution of rigor mortis, and is a key factor in meat tenderization during post-rigor ageing. These results powerfully support the ‘Calcium theory of meat tenderization’ which we have proposed.     

Muscle tissue structural changes and texture development in sea bream, Sparus aurata L., during post-mortem storage

Sea bream, Sparus aurata L., specimens were studied in pre-rigor (3 h) and during the following post-mortem days: 1, 5, 10, 15 and 22. Muscle and textural parameters were evaluated on 6 specimens/stage. Structural results showed scarce fibre-to-fibre detachment on pre-rigor, which increased during the post-mortem degradation. Ultrastructural changes revealed rapid muscle degradation. In pre-rigor myofibrils were detached to both sarcolemma and endomysium. Intermyofibrillar spaces increased and some mitochondriae and sarcoplasmic reticulum were swollen. After 1 day, the sarcolemma appeared occasionally disrupted and the interfibrillar spaces increased. From 5 to 10 days, the I-band and Z line presented some alterations, although these were more severe at 15-22 days. Thus, in these two last stages, loss of I-band, Z line and actin filaments was observed, that coincides with the alteration of the hexagonal arrangement in these advanced stages. Also, the fragmentation of myofibrils increased from 5 to 10 days on. Sarcolemma and endomysium were gradually disrupted throughout the post-mortem stages with total loss at 22 days. Consequently, the interfibrillar spaces increased at last stages. Autophagic mechanisms increased from 5 days on, with an intense destruction of all the intracytoplasmic organelles. Textural parameters decreased from pre-rigor until 5-10 days, mainly associated to detachment of myofibers to sarcolemma-endomysium.     

Pressure-induced changes in the connectin/titin localization in the myofibrils revealed by immunoelectron microscopy

Changes in the connectin/titin localization in post-mortem and pressurized chicken muscles were investigated by immunoelectron microscopy. The anti-connectin monoclonal antibody, 1D11, strongly labeled the sides of thick filaments near the H-zone and weakly labeled the sides of Z-line in the sarcomere prepared immediately after death. With the development of the muscle contraction, the shortening of the sarcomere and the dispersion of the connectin epitope near the H-zone were observed. With the gradual increase of the sarcomere length during further storage, the apparent increase of the width of the epitope in the A-band region stained by the antibody was observed, but the distance from the epitope to M-line remained almost the same length. In the case of high pressure treatment, significant changes in the labeling pattern of the antibody were observed with the increase of the pressure applied. The increase of the distance from the epitope to M-line and dispersion of the epitope were observed in the fiber pressurized at 100 MPa. These phenomena were accelerated with the increase of the pressure applied. The discontinuous dense materials labeled by the antibody at the thick filament near the H-zone were observed in the fiber bundles pressurized at 200 MPa or more. This is probably due to the accumulation of connectin molecule from ordinary location in the sarcomere, because of the pressure-induced destruction of the thick and connectin filaments. In the fiber bundles pressurized at 300 MPa, a significant increase in the distance from the epitope to M-line accompanied with the increase of the sarcomere length was observed. From the results obtained, it was clear that the changes in the location of the connectin epitope induced by the brief exposure to high pressure were drastic in comparison with that in the sarcomere during post-mortem storage.     

EFFECTS OF HYDROSTATIC PRESSURE ON ALPHA-MACROGLOBULIN AND SELECTED PROTEASES

The enzymes, chymotrypsin, trypsin, collagenase and cathepsin C, as well as the protease inhibitor, α₂-macroglobulin, have been shown to be susceptible to hydrostatic pressure inactivation. a₂-Macroglobulin lost about 15% activity at a pressure of 2,000 atm and as much as 80% at 3,000 atm pressure applied for 1 h in both cases. Thermal stability studies also indicated the protein to be completely denatured at about 75C. The enzymes were also inactivated to various extents depending on the amount of pressure and the duration of application, The degree of susceptibility to pressures ranging between 2,000–4,000 atm applied for time periods ranging from 5–60 min was found to be as follows: chymotrypsin < cathepsin < trypsin < collagenase. The hydrostatic pressure effects were irreversible when pressure-treated enzymes were stored at room temperature (20–25C), but showed various levels of reactivation when stored at refrigerated temperatures (4–7C).     

SURVIVAL OF LISTERIA INNOCUA IN APPLE JUICE AS AFFECTED BY VANILLIN OR POTASSIUM SORBATE

Survival of stationary phase Listeria innocua ( as surrogate microorganism for L. monocytogenes) inoculated in apple juice (pH 3.3 or 3.8) supplemented with vanillin (1,500 ppm or 3,000 ppm) or potassium sorbate (500 ppm or 1,000 ppm) and stored at room temperature was studied. L. innocua survived in apple juice without the preservatives at pH 3.3 or 3.8, with minimal population reductions. In the juices with the incorporation of potassium sorbate or vanillin , L. innocua behavior depended on the pH value, the type of antimicrobial and its concentration. At pH 3.3, the presence of vanillin (3,000 ppm) or potassium sorbate (1,000 ppm or 500 ppm) decreased L. innocua counts, with population reductions ranging from 4 to 5 log cycles after a 4 h – 8 h exposure at 30C. However, at pH 3.8 , L. innocua showed sensitivity only to 3,000 ppm vanillin. Survival curves were successfully fitted using a Weibull type distribution of resistances.
The results suggest that the use of potassium sorbate or vanillin could prevent the survival of L. innocua in contaminated unpasteurized and pasteurized apple juice. Vanillin, a natural antimicrobial, would be particularly suitable as an antilisterial additive for less acidic apple juice.     

新鲜猪肉和经盐腌后在加热过程中超微细结构变化的研究

取新鲜的、未发生僵直的猪背最长肌并分为两组,其中一组样品分别在45、50、55、60、65、70、80和90℃水中加热;另一组样品先分别在1%、3%和5%的NaCl溶液中进行腌制,而后再加热到7275℃。所有样品在透射电镜及扫描电镜下对肌原纤维的结构进行观察及分析。加热对肌原纤维的结构具有明显的破坏作用。45～50℃会导致肌原纤维发生超收缩;在55、60和65℃时,部分肌原纤维超收缩加剧,而另一部分则不发生超收缩,另外由于肌丝和肌浆蛋白发生变性,肌原纤维的原始结构遭到一定程度破坏;若加热温度为70、80和90℃, 未发生超收缩肌节的数量随着温度升高而增多,肌丝发生热变性而凝固加剧,导致A带缩短、I带断裂,肌原纤维结构严重破坏,其崩解程度随温度升高而增大。用NaCl腌制对加热时肌原纤维的崩解起到促进作用,并可避免肌原纤维发生强烈收缩,其中3%～5%NaCl溶液的作用尤为明显。     

Effect of Hydrostatic Pressure on Aggregation and Viscoelastic Properties of Tilapia (Orechromis niloticus) Myosin

ABSTRACT: Hydrostatic pressures from 500 to 2000 atmospheres (atm) were applied at 0°C to determine the aggregation and viscoelastic properties of tilapia ( Orechromis niloticus ) myosin. Native myosins were present as long, linear, and single filaments. After a 500-atm treatment, these filaments unfolded and their volume decreased. Upon 1000-atm and 1500-atm treatments, myosins aggregated and formed inseparable network structures. Further, they transformed from viscous sol to elastic gels with a pressure of 500 to 1000 atm. At 2000 atm, the myosin formed irregular aggregates. This study reveals that at 500 atm, myosins unfolded; at 1000 atm, they aggregated, and beyond 1500 atm, they formed both a precipitate and gel.     

MOLECULAR PROPERTIES OF POSTMORTEM MUSCLE. 10. Effect of Internal Temperature and Carcass Maturity on Structure of Bovine Longissimus

SUMMARY– A study was carried out with light and electron microscopic techniques to discover the effect of heating and carcass maturity upon the connective tissue and myofibrillar proteins of longissimus from veal (5–6 months), A (12–20 months) and D (54–60 months) maturities. Longissimus was heated to internal temperatures of 1) 50°C; 2) 60°C; 3) 70°C; 4) 80°C and 5) 90°C. Light microscopy indicated that connective tissue fibers increased in size and degree of aggregation with carcass maturity. Also, the more aggregated fibers from D maturity were more heat resistant than the less aggregated fibers of veal and A maturity. Endomysial connective tissue shrinkage was initiated at 50°C and it was completed at approximately 70°C. On the other hand, perimysial connective tissue shrinkage required internal temperatures of 70°C and higher before any significant fiber changes were observed. However, if endomysial and perimysial connective tissue fibers showed similar degrees of aggregation, they appeared to react similarly to heat regardless of maturity group. Electron and phase contrast microscopy showed that myofibrillar proteins compressed and sarcomeres shortened at 50°C. Heating to 60°C caused loss of M-line structure, initiation of disintegration and coagulation of thin and thick filaments and further myofibrillar protein shrinkage. Heating to 70 and 80°C caused more disintegration of thin filaments and coagulation of thick filaments. At 90°C, an amorphous structure resulted, but regardless of the internal temperature the principal banding features of the sarcomere could be identified. These changes are discussed in relationship to changes in meat tenderness.     

The non-sliding filaments of the sarcomere

A model is proposed for the 'gap' or 'third' filaments of muscle, here renamed 'T-filaments'. These consist of single titin molecules, spanning the half sarcomere from M-line to Z-line. Of several possibilities for stoichiometry and arrangement, the one most favoured here consists of six T-filaments lying longitudinally on the surface of the A-filament, one against each peripheral subfilament of myosin. C-protein molecules over-lie the T-filaments in transverse and axial rows, with their long axes following a helix. Each C-protein molecule helps to bind a pair of T-filaments to one A-strand (but not to bind A-strands together), and hinders the immune response of titin in the C-zone. The gap filaments arise from the coalescence of T-filaments in the I-band, and their extreme extensibility from an unravelling of a beaded structure in the titin molecule. A layout is presented for the molecule in zones of configuration and function. Possible arrangements of the T-filaments in the M- and N(2)-zones are discussed.     

Pressure-heat treatment of meat: Changes in myofibrillar proteins and ultrastructure

The effects on muscle of a combined pressure-heat (P-H) treatment that overcomes myofibrillar toughness have been investigated using SDS gel electrophoresis and electron microscopy. Densitometer scans of polyacrylamide gels of muscle extracts revealed that P-H treatment caused greater degradation of connectin than did heat treatment alone. Breakdown of connection by P-H treatment was reduced in muscle that had been injected with the protease inhibitor pepstatin. However, pepstatin treatment did not reduce the effectiveness of P-H treatment for tenderizing meat, as would be expected if connectin was responsible for myofibrillar toughness. P-H treatment resulted in an increase in the intensity of a peak with M(r) ～ 150 000, but this peak was also produced by non-tenderizing pressure treatments. The ultrastructural studies revealed that P-H treatment disrupted the thick and the thin filaments, leaving voids at the M-line region. It is suggested that P-H treatment achieves most of its effect by an irreversible disaggregation of the myosin of thick filaments.     

浸渍冻结对凡纳滨对虾冻藏过程中肌肉组织的影响

本文以肌纤维轮廓、细胞间隙、肌丝、肌节以及肌纤维间隙等为指标,研究浸渍冻结和静止空气冻结对凡纳滨对虾冻藏过程中肌肉组织结构的影响。结果发现,浸渍冻结对虾的肌纤维排列更加致密,形成的冰晶间隙更加细小,分布均匀,肌原纤维直径变短的程度比静止空气冻结的小;而静止空气冻结的肌纤维排列疏松,形成的冰晶间隙宽大。随着贮藏时间的增加,浸渍冻结对虾的冰晶逐渐增大,间隙逐渐变大,细胞核也逐渐分解变少。冻藏90 d后,静止空气冻结对虾中很难发现细胞核的存在,肌丝结构大部分消失,明暗带难分辨,肌节长度变短程度严重,肌节消失,且没有完整结构的线粒体。但是浸渍冻结对虾中肌丝结构及明暗带依然存在,肌节长度变短程度比静止空气冻结的小,部分线粒体结构完整。结果表明,浸渍冻结更有利于对虾的肌肉结构的保持,能更好地保持对虾的品质。