The structural basis of cooking loss in beef: Variations with temperature and ageing.

Meat loses fluid during cooking, resulting in textural changes and loss in cook yield. To understand the structural basis of cooking losses, this work used 10 bovine semitendinosus muscles and two ageing periods (1 vs 14days) to examine micro- and macro-level dimensional changes in muscle during heating. Muscle blocks, muscle fibre fragments and myofibrils all showed similar maximum shrinkage in cross sectional area (20-24%) but maximum length shrinkage was less in myofibrils (15%) than muscle blocks and fibre fragments (25%). Dimensional changes were dominated by shrinkage in individual muscle fibres and myofibrils, indicating that connective tissue does not play a major role. Transverse shrinkage predominantly occurred over 50-65°C whereas the longitudinal shrinkage predominantly occurred over 70-75°C; we attribute these two separate shrinkage events to denaturation of myosin and actin respectively. Higher cook losses in samples aged for 14days versus 1day suggests that desmin, nebulin and titin denaturation are not major drivers of fluid expulsion as these proteins are degraded during ageing. We postulate that proteolysis during ageing produces protein fragments which are more easily lost from the structure during cooking, along with water.

A structural approach to understanding the interactions between colour, water-holding capacity and tenderness.

The colour, water-holding capacity (WHC) and tenderness of meat are primary determinants of visual and sensory appeal. Although there are many factors which influence these quality traits, the end-results of their influence is often through key changes to the structure of muscle proteins and their spatial arrangement. Water acts as a plasticiser of muscle proteins and water is lost from the myofibrillar lattice structure as a result of protein denaturation and consequent reductions in the muscle fibre volume with increasing cooking temperature. Changes in the myofilament lattice arrangement also impact the light scattering properties and the perceived paleness of the meat. Causes of variation in the quality traits of raw meat do not generally correspond to variations in cooked meat and the differences observed between the raw muscle and cooked or further processed meat are discussed. The review will also identify the gaps in our knowledge and where further investigation would beneficial.

Bimodal collagen fibril diameter distributions direct age-related variations in tendon resilience and resistance to rupture.

Scaling relationships have been formulated to investigate the influence of collagen fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (D(D1)) and larger (D(D2)) mean Ds, respectively. Both D(D1) and D(D2) increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) u(E) [from initial loading until the yield stress (σ(Y))], which contributes primarily to tendon resilience, and 2) u(F) [from σ(Y) through the maximum stress (σ(U)) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities u(E)/σ(Y) and u(F)/σ(U), both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in u(E)/σ(Y) (resilience energy) are associated with decreases in D(D1) and increases in D(D2), whereas u(F)/σ(U) (rupture energy) is associated with increases in D(D1) alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.

Meat Science and Muscle Biology Symposium: manipulating meat tenderness by increasing the turnover of intramuscular connective tissue.

Controlled reduction of the connective tissue contribution to cooked meat toughness is an objective that would have considerable financial impact in terms of added product value. The amount of intramuscular connective tissue in a muscle appears connected to its in vivo function, so reduction of the overall connective tissue content is not thought to be a viable target. However, manipulation of the state of maturity of the collagenous component is a biologically viable target; by increasing connective tissue turnover, less mature structures can be produced that are functional in vivo but more easily broken down on cooking at temperatures above 60°C, thus improving cooked meat tenderness. Recent work using cell culture models of fibroblasts derived from muscle and myoblasts has identified a range of factors that alter the activity of the principal enzymes responsible for connective tissue turnover, the matrix metalloproteinases (MMP). Fibroblasts cultured from 3 different skeletal muscles from the same animal show different cell proliferation and MMP activity, which may relate to the different connective tissue content and architecture in functionally different muscles. Expression of MMP by fibroblasts is increased by vitamins that can counter the negative effects of oxidative stress on new collagen synthesis. Preliminary work using in situ zymography of myotubes in culture also indicates increased MMP activity in the presence of epinephrine and reactive oxidative species. Comparison of the relative changes in MMP expression from muscle cells vs. fibroblasts shows that myoblasts are more responsive to a range of stimuli. Muscle cells are likely to produce more of the total MMP in muscle tissue as a whole, and the expression of latent forms of the enzymes (i.e., pro-MMP) may vary between oxidative and glycolytic muscle fibers within the same muscle. The implication is that the different muscle fiber composition of different muscles eaten as meat may influence the potential for manipulation of their connective tissue turnover.

Epinephrine-induced MMP expression is different between skeletal fibroblasts and myoblasts.

Skeletal fibroblasts and myoblasts are among the cell types currently being considered in cell therapy for ischaemic heart disease. To investigate whether the expression of the tissue-remodelling proteolytic enzymes matrix metalloproteinases (MMPs) and the cellular energy regulator AMP-activated protein kinase (AMPK) is comparable between the two cell lines in response to epinephrine treatment, mouse skeletal fibroblasts (NOR-10) and myoblasts (C2C12) were treated with or without a low (11 nmol·l(-1) ) or high (55 nmol·l(-1) ) dose of epinephrine for 2 or 6 h. Cellular MMP-3 expression was increased by the high-dose epinephrine at both treatment periods in both cell lines. Cellular MMP-2 and MMP-13 expressions were amplified by the 2- or 6-h epinephrine incubation in fibroblasts. However, in myoblasts, such an increase was only seen at the longer treatment time. An elevated AMPKα expression was observed after a 2-h presence of epinephrine in both cell lines, which matches temporally with the early increased cellular MMP-2 and MMP-13 expression in fibroblasts. Activity of secreted MMP-2 increased only after 6-h epinephrine treatment in both cell types. Our data suggest that skeletal fibroblasts respond earlier to epinephrine application in terms of endogenous synthesis of the proteolytic and the energy homeostasis enzymes, whereas such response occurs later and to a milder dose of the beta adrenergic agonist in myoblasts.

Phenotypic differences in matrix metalloproteinase 2 activity between fibroblasts from 3 bovine muscles.

Different muscles in a beef carcass are known to respond differently to the same stimulus or animal growth pattern or both. This may complicate the search by the meat industry for production methods to render meat tender. One of the major differences between muscles in the same carcass is in the expression of intramuscular connective tissue. Current study investigates the existence of a phenotypic difference among fibroblasts from 3 bovine skeletal muscles as exemplified by the expression of matrix metalloproteinases (MMP) the main enzymes responsible for connective tissue turnover. The sensitivity of phenotypic differences to cell culture conditions (passage number, presence of growth factors from fetal serum) was also examined. Fibroblasts, the main cells responsible for the production and turnover of collagen were isolated from LM, semitendinosus (STN), and sternomandibularis (SMD) muscles from a bull calf and grown in DMEM, 10% fetal bovine serum, and 5% CO(2). Cell doubling times, survival time, resting expression, and activity of MMP and the effect of serum withdrawal in the culture media on MMP expression and activity were determined for each cell line during 15 passages. Fibroblasts isolated from the 3 muscles had different growth potentials. The shortest (P < 0.0001) cell doubling times for almost every passage were found in cells from STN muscle. Cells from the LM had a shorter (P < 0.0001) survival time in comparison with STN and SMD. Cells derived from the STN had greater values (P > 0.05) of MMP-2 activity in comparison with LM and SMD cells until passage 4. At passage 15, no activity was detected for any cell line. Serum withdrawal generally reduced MMP-2 activation but did not eliminate differences in activity between fibroblasts from the 3 muscles. These results suggest that fibroblasts from different locations are phenotypically different and may respond differently to the same growth or nutritional stimulus in vitro. This may be related to in vivo differences in accumulation, maturity, and turnover of collagen, and ultimately meat tenderness. These findings may be important for selecting a management strategy for improving meat tenderness by manipulation of animal growth; a strategy applied to the whole animal may work for some muscles but not for others.

Disparity of dietary effects on collagen characteristics and toughness between two beef muscles.

Manipulation of growth rate and/or diet has been shown to affect protein turnover and may be used to improve beef quality. This trial was conducted to evaluate the effects of average daily gain (ADG) and diet on animal performance, collagen characteristics and beef quality of two different muscles; longissimus dorsi (LD) and semitendinosus (ST). Seventy six Hereford and Angus steers were assigned to three dietary management regimens for finishing: high grain diet based on corn (n=28), alfalfa pasture (n=22) and grass pasture (n=26). Average daily gains were greater (P<0.001) in Herefords vs. Angus and for corn- vs. pasture finished cattle. Overall, total collagen content was greater (P<0.001) and the percentage of total collagen that was heat soluble was lower (P<0.0001) for ST than for LD muscle. The lowest (P<0.05) values for both total and heat soluble collagens were found in animals finished on corn. WBSF values for LD were greater (P<0.01) in grass-fed vs. alfalfa- and grain-finished cattle while there was no difference in WBSF values for ST between grass- and corn-fed animals. No correlation between ADG and WBSF was observed for any muscle. ADG was not correlated with collagen solubility in ST, but was correlated (P<0.05) with collagen solubility in LD. A key finding is that growth rate affected heat soluble collagen in the two muscles to a different extent. In conclusion, this study shows that different feeding strategies may not influence the tenderness of all muscles in a similar way.

Matrix metalloproteinases are less essential for the in-situ gelatinolytic activity in heart muscle than in skeletal muscle.

In order to determine whether the contribution of matrix metalloproteinases (MMPs) to the tissue gelatinolytic activity is similar between myocardium and skeletal muscle tissue, in-situ zymography was applied to myoblasts originated from myocardium or skeletal muscle of rodents as well as tissue sections of heart and soleus muscles of rats. Gelatinolyic activity was observed in cytoplasm and nucleus of both heart and skeletal myoblasts. The chelating agent EDTA blocked much of the gelatinolytic activity and the organomercurial activator of MMPs increased the activity in cells of both muscle origins. However, the inhibition of gelatinolytic activity by a broad spectrum MMP inhibitor was less profound in heart myoblasts than that in skeletal myoblasts. Gelatinolytic activity was also expressed in the endomysium and perimysium of tissue sections of heart and soleus muscles. Similar with findings in the cell studies, the gelatinase activity was increased by the MMP activator, mostly blocked by EDTA and partially inhibited by the MMP inhibitor. In the presence of the MMP inhibitor, the remaining gelatinolytic activity in the tissue sections was again higher in myocardium than that in soleus muscle. This observation was further supported by the gelatinolytic activity examined in tissue homogenates. Our findings suggest that other proteinases, in addition to MMPs, are more responsive for the tissue gelatinolytic activity in heart muscle as compared with that in skeletal muscle.

Ageing changes in the tensile properties of tendons: influence of collagen fibril volume fraction.

Connective tissues are biological composites comprising of collagen fibrils embedded in (and reinforcing) the hydrated proteoglycan-rich (PG) gel within the extracellular matrices (ECMs). Age-related changes to the mechanical properties of tissues are often associated with changes to the structure of the ECM, namely, fibril diameter. However, quantitative attempts to correlate fibril diameter to mechanical properties have yielded inconclusive evidence. Here, we described a novel approach that was based on the rule of mixtures for fiber composites to evaluate the dependence of age-related changes in tendon tensile strength (sigma) and stiffness (E) on the collagen fibril cross-sectional area fraction (rho), which is related to the fibril volume fraction. Tail tendons from C57BL6 mice from age groups 1.6-35.3 months old were stretched to failure to determine sigma and E. Parallel measurements of rho as a function of age were made using transmission electron microscopy. Mathematical models (rule of mixtures) of fibrils reinforcing a PG gel in tendons were used to investigate the influence of rho on ageing changes in sigma and E. The magnitudes of sigma, E, and rho increased rapidly from 1.6 months to 4.0 months (P-values <0.05) before reaching a constant (age independent) from 4.0 months to 29.0 months (P-values >0.05); this trend continued for E and rho (P-values >0.05) from 29.0 months to 35.3 months, but not for sigma, which decreased gradually (P-values <0.05). Linear regression analysis revealed that age-related changes in sigma and E correlated positively to rho (P-values <0.05). Collagen fibril cross-sectional area fraction rho is a significant predictor of ageing changes in sigma and E in the tail tendons of C57BL6 mice.

Modelling quality variations in commercial Ontario pork production.

This study explores the interactions of sensory and nutritional environment with genotype occurring in current commercial pork production in Ontario, Canada, which may interact to result in poor quality meat. The study focussed on identifying factors and signalling mechanisms that contribute to poor meat quality, in order to develop strategies to reduce the incidence of unacceptable product quality. In the first phase of the work reported here, animal behaviour and muscle metabolism studies were related to meat colour, tenderness and water-holding capacity measurements from commercially-produced pigs killed in a commercial packing plant. A partial least squares analysis was used to determine the most important of the principal production variables, peri-mortem biochemical measures and post-mortem carcass condition variables studied, in terms of their influence on water-holding, toughness and colour (L*-value). Variations between producer and kill day at the slaughterhouse were very strong contributors to variability in these three meat quality parameters, followed by pH variations. A second phase of the study is currently underway to characterize patterns of gene expression related to extremes of end-product quality and to reduce quality variations by nutritional and behavioural management strategies.

Effect of muscle type on the rate of post-mortem proteolysis in pigs.

Post-mortem proteolysis was examined in muscle homogenates from porcine m. longissimus dorsi (LD), m. semitendinosus (ST), m. semimembranosus (SM), m. vastus intermedius (VI), and m. soleus (S). During post-mortem storage, desmin and troponin-T degraded faster in LD and SM than in ST, VI and S. ST exhibited the same rate of degradation as VI and S. These differences could not be explained solely by differences in fibre type distribution, indicating that other muscle-specific traits independent of fibre type determine myofibrillar degradation post-mortem. Thus, the rate of post-mortem proteolysis seems to depend more on muscle-to-muscle variations than on fibre type composition.

Effect of added µ-calpain and post-mortem storage on the mechanical properties of bovine single muscle fibres extended to fracture.

The effects of µ-calpain and post-mortem storage on the strength of single muscle fibres were investigated. During the 10 min of incubation at pH 7.5, µ-calpain became evenly distributed throughout the fibre. µ-Calpain-incubation resulted in thinner (P <0.001) Z-lines and reduced (P <0.001) the strength of the fibres compared to controls. These results demonstrate that µ-calpain is capable of mechanically weakening the muscle fibres. Post-mortem storage of meat for 10 days at 2 °C weakened (P <0.001) the muscle fibres compared to 24-h fibres. The presence or absence of Ca(2+) affected fibre stiffness. Fibres incubated at pH 7.5 in 100 µM Ca(2+) were less stiff than fibres incubated in 200 µM EGTA. Breaking stress and strain were not affected by Ca(2+). We hypothesise that Ca(2+) causes conformational changes in some of the load-bearing proteins, which alters their initial resistance to extension, but does not affect the breaking strength of the fibres.

Effect of proteolytic enzyme activity and heating on the mechanical properties of bovine single muscle fibres.

The effect of proteolysis and heating on the mechanical properties of single muscle fibres isolated from bovine M. semitendinosus was investigated. Short incubations with µ-calpain (pH 7.5) and cathepsin B (pH 5.6) at room temperature reduced (P<0.01) the raw fibre strength by 50%. After subsequent heating, µ-calpain-incubated fibres were 20% weaker and cathepsin B-incubated fibres 50% weaker than heated controls. Fibres were incubated for 8 days at 2 °C under post-mortem like pH conditions (pH 5.6) or pH conditions optimal for the calpains (pH 7.5). At pH 5.6 no significant weakening of the fibres was observed when incubations were performed in 0.1, 1 or 10 mM Ca(2+). In contrast, incubation in 1 or 10 mM Ca(2+) at pH 7.5 decreased the strength of the fibres. Addition of the protease inhibitors E-64 or PD150,606 prevented this weakening, suggesting that the weakening is caused by proteolyses and not directly by Ca(2+).

Dietary-induced changes of muscle growth rate in pigs: effects on in vivo and postmortem muscle proteolysis and meat quality.

The effects of various growth rates in pigs induced by four different feeding strategies on the activity of the calpain system and on postmortem (PM) muscle proteolysis and tenderness development were studied. An increased growth rate may be caused by an increased protein turnover, which results in up-regulated levels of proteolytic enzymes in vivo that, in turn, possibly will affect PM tenderness development. It can be hypothesized that increased proteolytic activity pre-slaughter will increase the PM tenderization rate. From postnatal d 28 to d 90 (phase 1) the pigs were divided into two groups, given either ad libitum (A) or restricted (R, 60% of ad libitum) access to feed. The two groups were then divided into two subgroups, given either restricted or ad libitum access to feed from d 91 to slaughter at d 165 (phase 2). Measurements of the activity of mu-calpain, m-calpain, and calpastatin; concentrations of total collagen and the percent of soluble collagen; and RNA, DNA, and elongation factor-2 where made at slaugther. Myofibrillar fragmentation index (MFI) was determined at slaughter and 24 h PM. Warner-Braztler shear force was determined 1 d and 4 d PM. Pigs fed restricted diets in phase 1 and fed ad libitum in phase 2 (RA pigs) had increased growth rates in the last phase compared to pigs fed ad libitum during both phase 1 and phase 2 (AA pigs). The increased growth rate (compensatory growth) was followed by an increased proteolytic potential (mu-calpain:calpastatin ratio), increased MFI values, and higher tenderization rates. There was a positive correlation between the activities of m-calpain and growth rates (r = 0.35, P = 0.03), and between RNA levels and growth rates (r = 0.43, P = 0.006). The proposed hypothesis is largely supported by the results. The activities of both mu- and m-calpain at slaughter were highest in fast-growing pigs. The calpain activity was highest in RA pigs, which in turn also had the fastest growth rates prior tslaughter among the four groups. This implies that the synthesis of these enzymes was up-regulated during the second feeding period to a larger extent in RA pigs. The proteolytic potential and the MFI values indicate that the up-regulated in vivo calpain activity had an effect on PM protein degradation, which also is supported by the higher tenderization rate in RA pigs.

Role of Ca(2+) for the mechanical properties of fibrillin.

Fibrillin-rich microfibrils are important structural elements widespread throughout connective tissues. Genetic defects identified in the Ca(2+) binding sites of fibrillin have severe effects and in addition Ca(2+) has a marked effect on the microfibrillar structure. We have studied the role of Ca(2+) on the mechanical behavior of fibrillin-rich microfibrils using the micro-needle technique. We find that Ca(2+)-depletion results in a 50% decrease in rest length and reduces the stiffness of fibrillin-rich microfibrils. At high strain, irreversible damage occurs. This behavior is consistent with Ca(2+) stabilization of interactions between consecutive EGF-like domains and breakdown in the quaternary structure upon over-extension.

Development of components of the extracellular matrix, basal lamina and sarcomere in chick quadriceps and pectoralis muscles.

1. The purpose of this study was to investigate differences in the development of components of the cell/matrix linkage in two functionally different muscle types: the pectoralis muscle, a major locomotory muscle in birds but not particularly functional in chickens, and the quadriceps muscle, a smaller and more functionally active muscle in the chicken. 2. The development of the extracellular matrix, basal lamina and sarcomere in the pectoralis and quadriceps muscles in chick embryos was examined biochemically to determine differences in the rate of development between these two muscles. Samples of these muscle types were dissected out from chick embryos from embryonic day 10 until 8 weeks post hatch. 3. Using SDS-PAGE electrophoresis and western blotting with antibodies against sarcomeric actin, laminin and collagens I, III and IV, it was apparent that muscle development begins earlier in the quadriceps muscle than in the pectoralis, and that late in the developmental process (d 18) both muscle types were well differentiated. The final concentration of collagens in the mature muscle remained higher in the quadriceps than in the pectoralis muscle. 4. The onset of development of the extracellular matrix, basal lamina and sarcomere was earlier in the quadriceps than the pectoralis, which could have functional implications for these muscles as a whole.

Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.

Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.

The effect of ageing on the water-holding capacity of pork: role of cytoskeletal proteins.

The water-holding capacity (WHC) of pork decreases post-mortem but has been shown to increase during subsequent ageing. In order to test a hypothesis that water-holding capacity increases during ageing due to degradation of the cytoskeleton, WHC was followed 10 days post-mortem and related to the extent of proteolysis of cytoskeletal proteins. A fast method for measuring WHC in small meat samples was developed by the use of centrifugation. The WHC of fresh pork decreases in the first part of post-mortem storage after which it increases to the level of 1 day PM. No changes in total water content of the meat were observed which could explain changes in WHC during ageing. Vinculin and desmin degrade gradually during ageing while talin degrades rapidly. These observations are consistent with the hypothesis that degradation of the cytoskeleton slowly removes the linkage between lateral shrinkage of myofibrils and shrinkage of entire muscle fibres, so removing the force that causes flow into the extracellular space. Inflow of previously expelled water is then possible, so increasing WHC as observed in later periods of storage.

Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific.

Myoblast cell lines are grown and differentiated readily in cell culture. Two cell lines typically used for investigating the growth and differentiation of muscle are the mouse cell line C2C12 and the rat cell line L6. The differentiation of these cells in vitro requires a switch from a serum-rich medium to a less rich medium after the cells have reached confluence. Since the components present in serum are not well characterized, the use of a better defined medium for these studies was investigated. C2C12 and L6 myoblasts were differentiated in both serum-containing and serum-free media. The differentiation state of these cultures was then tested both microscopically and biochemically. Cultures were checked for myotube formation, the activity of creatine phosphokinase and the presence of sarcomeric actin. In C2C12 cells, the extent of differentiation was greater in the serum-free than in the serum-containing system. In both media types, the C2C12 cells produced sarcomeric actin, showing the presence of sarcomere structure in the myotubes. In L6 cells, however, myotubes were readily formed in medium containing 2% horse serum, but not in the serum-free system. In addition, the ability of C2C12 cells to differentiate on substrates coated with extracellular matrix proteins was shown to be media-dependent. The presence of extracellular matrix proteins did not enable L6 cells to form myotubes when cultured in serum-free media. Primary cultures of chick myoblasts were able to differentiate in both media tested, with Dulbecco's modified Eagle medium containing horse serum being a more efficient medium for cell fusion. This study shows a divergence in muscle cell line responses in three cell lines, two of which are typically used as 'model systems' for understanding muscle growth and development.

Epinephrine upregulates calpain activity in cultured C2C12 muscle cells.

C2C12 cells were grown to confluence at 37 degrees C under a continuous 5% CO(2) stream and myotube formation was stimulated. The cultures were then incubated with or without 2 microg/mL epinephrine for 18 h prior to harvesting and calpain extraction. Epinephrine treatment resulted in a three-fold increase in extractable mu-calpain activity (P < 0.05), a three-fold increase in extractable m-calpain activity (P < 0.05), a 36% increase in calpastatin activity (P < 0.001), and a 16% decrease (P < 0.05) in the total protein content in the C2C12 cell homogenate. These results suggest that calpains may play a role in protein metabolism and that the hormone epinephrine may be directly involved in the regulation of their cellular expression.