Aging-induced changes in microstructure and water distribution in fresh and cooked pork in relation to water-holding capacity and cooking loss - A combined confocal laser scanning microscopy (CLSM) and low-field nuclear magnetic resonance relaxation study

Confocal laser scanning microscopy (CLSM) and low-field nuclear magnetic resonance (LF-NMR) relaxometry were combined to characterize microstructural changes and water distribution in fresh and cooked pork during an aging period of 14 days. At day 1 (24 h postmortem) a few muscle fibres, which appear swollen, were observed in both fresh and cooked meat. An identical microstructure was still apparent after 14 days, however, the number of muscle fibres showing distinguished characteristics was found to increase throughout the aging period. Hence, it was apparent that during aging the individual fibres swell and disintegrate at different rates. Development in water-holding capacity (WHC) was followed during the aging period using gravimetric methods, and an increase in the WHC in the fresh meat was observed, which resembled the amount of extramyofibrillar water measured by NMR relaxometry (T₂₂ population). This was consistent with the CLSM images, as a substantial increase in the number of myofibrils that appeared swollen, capable of holding more water, was observed during aging. In the cooked meat the width of the T_21c population, reflecting the myofibrillar water in the cooked meat, was seen to decrease during the entire storage period, which corresponds to the development of a more homogeneous structure. In the CLSM data a continuous degradation during the storage period was observed, which could resemble a shift to a more homogeneous structure. Comparison of CLSM of transverse sections of fresh and cooked pork revealed a pronounced shrinkage of muscle fibres upon cooking. This resulted in large gaps between the cooked muscle fibres, which also was visible as shrinkage at the level of the individual myofibrils. This pattern was also reflected in the NMR relaxation data. The cooking-induced shrinkage of the myofibrils occurred concomitantly with a decrease in the amount of intermyofibrillar water within the individual fibre and an increase in the larger extramyofibrillar spaces between fibres, i.e. water is expelled from the myofibrillar matrix upon cooking. Accordingly, the present study demonstrated that the use of CLSM together with NMR relaxometry can provide further information on the relationship between structural characteristics of meat and resultant water distribution.     

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.     

骨架蛋白肌联蛋白、伴肌动蛋白、纽蛋白的降解对冷却猪肉持水性的影响

肉的持水性可用许多指标衡量,而汁液流失率(drip loss)是其中一个重要指标.持水性受很多因素影响,但目前对形成汁液流失的机理的认识还很有限.本文综述了近几年来国内外学者对持水性的研究,主要分析了宰后骨架蛋白(肌联蛋白、伴肌动蛋白、纽蛋白)的降解及钙激活蛋白酶系统降解骨架蛋白对持水性的影响,以期为宰后猪肉持水性提高和肉品质改善提供参考.     

Changes of myofibrillar and centrifugal drip proteins and shear force of psoas major and minor and semitendinosus muscles from calves, heifers and cows during post-mortem ageing

Changes in myofibrillar protein content and centrifugal drip proteins of psoas major and minor (PM) and semitendinosus (ST) muscles of calves, heifers and cows taken from carcasses on the 1st, 6th and 12th day of post-mortem cold storage were estimated. Washed myofibrils and centrifugal drip from muscles were analysed using SDS-PAGE 10 and 12% polyacrylamide gels. No significant changes were observed in content of contractile proteins, α-actinin and regulatory proteins (except for TN-T). There were no significant differences between muscles from investigated groups and between muscles aged in chilled conditions. The levels of titin T1 during ageing varied slightly. The 30 kDa-fraction appearance was fastest in calf, slower in heifer and slowest in cow muscles. More pronounced differences in the level of protein degradation with regards to muscle type, age of animals and time of storage were found in the centrifugal drip of meat. In the drip, the level of high molecular weight proteins was higher in muscles from young animals and in the muscles stored longer. The opposite was observed in case of 26-28 kDa proteins. Their amount in muscle drip decreased with increased storage time. The rate of proteolysis and release of cytoskeletal proteins during cold storage of muscles were related to change in shear values of roasted meat. The highest rate of protein degradation was observed in PM calf muscle and the lowest rate in ST cow muscle. The fastest tenderization process was registered in calves muscles and the slowest tenderization in cows.     

滩羊肉宰后成熟期间ATPase活力变化对微观结构及保水性的影响

为研究宰后成熟期间ATPase活力变化对滩羊肉微观结构以及保水性的影响,以6 月龄滩羊背最长肌（Longissimus dorsi）为研究对象,分析其4 ℃成熟0、1、2、4、8 d时Na＋-K＋-ATPase、Ca2＋-ATPase、Caspase-3活力以及肌肉微观结构、pH值与滴水损失率的变化情况。结果表明：随成熟时间延长,Na＋-K＋-ATPase与Ca2＋-ATPase活力先升高后降低,成熟1 d时达到最大值;Caspase-3活力先升高后降低,成熟2 d时达到最大值;滴水损失率先升高后降低,pH值先降低后有所回升;总蛋白、低盐溶性蛋白及高盐溶性蛋白质量浓度均逐渐减少,水溶性蛋白质量浓度成熟2 d后显著降低（P＜0.05）;成熟至8 d时,肌原纤维断裂,肌纤维之间、肌束之间、肌纤维及肌膜之间形成间隙,Z线断裂,H带消失;相关性分析结果表明Na＋-K＋-ATPase活力与各指标均呈极显著相关性（P＜0.01）,Ca2＋-ATPase活力与pH值、Na＋-K＋-ATPase及Caspase-3活力均呈极显著相关性（P＜0.01）。结论：滩羊肉宰后成熟过程中Na＋-K＋-ATPase与Ca2＋-ATPase活力变化可能促使下游Caspase-3激活,Caspase-3水解结构蛋白可能导致肌肉组织在不同部位形成间隙,在重力作用下肌肉中的水分流入间隙中,引起滩羊肉滴水损失升高,保水性变差。     

Meat color is determined not only by chromatic heme pigments but also by the physical structure and achromatic light scattering properties of the muscle

Meat color is important for consumer acceptability, with excessively dark meat often associated with consumer rejection. It is determined chromatically by pigment content (measured by hue and chroma) and achromatically by scattering of light by the microstructure (measured by lightness), the latter of which has received minimal research focus. This review discusses the individual components of the meat microstructure that cause differences in achromatic contributions to color. Differences in achromatic light scattering between light and dark extremes of meat color are most likely explained by structural attributes within the muscle cell. These differences are proposed to arise from variations in (a) transverse shrinkage of the structural lattice of the myofilaments, myofibrils, and muscles fibers, (b) longitudinal shrinkage of the sarcomere, and (c) different protein composition of the surrounding medium (sarcoplasm and extracellular space). These are discussed at a mechanistic level, in relation to six parameters of the muscle cell: (a) protein surface charge altering the myofilament spacing, (b) protein solubility, (c) sarcoplasmic protein binding to myofilaments and myofibrils, (d) integrity of the cytoskeleton and cell adhesion proteins, (e) sarcomere integrity and myofibrillar proteins, and (f) myosin denaturation and rigor bond modification. New data are presented to support the proposed role of structural elements in muscle causing achromatic light scattering and their contribution to the surface color of meat. In addition, the relationships between lightness and water holding capacity and pH are explored and the economic impact of dark meat for the meat industry is discussed.     

An assessment of extracellular space measurements in post-mortem muscle

Extracellular space measurements of early post-mortem muscle were made by incubating muscle strips in inulin [(14)C] carboxylic acid. The extracellular space measurements provide a means to assess the functionality of the muscle membrane. This may lead to an understanding of variations in the water-holding capacity of meat and the effects of stress on the muscle membranes of stress susceptible animals. Secondly, a level of intrafibre water affinity between carcasses may be determined which may relate to the interfilamental spacings between myofibrils in the fibre and thus meat quality.     

Biochemistry of postmortem muscle — Lessons on mechanisms of meat tenderization

It is certain that meat tenderness is a highly valued consumer trait and thus definition of the multiple processes that influence meat tenderness will provide clues toward improving meat quality and value. The natural process by which meat becomes tender is complex. Tenderness development is dependent on the architecture and the integrity of the skeletal muscle cell and on events that modify those proteins and their interaction. Specifically protein degradation and protein oxidation have been identified as processes that modify proteins as well as the tenderness of meat. The intracellular environment is a major factor that controls these events. Ultimately, the interplay between these events determines the rate and extent of tenderization. Given the intricacy of the structure of the muscle cell, coupled with the complexity of the regulation of protein modification and the ever-changing intracellular environment it is not surprising that this area of research is a very dynamic field. Just as the overall integrity and function of muscle cells does not depend on a single protein, but rather on the coordinated interaction of several proteins, the structural weakening of muscle cells during postmortem aging also must not depend on the degradation of a single myofibrillar or other cytoskeletal protein. The proteins mentioned in this review are located in different regions of the muscle cell, and most have been implicated in some manner as being important in maintaining the structure and function of the muscle cell. Oxidation of myosin heavy chain, a predominant protein in the myofibril, is known to promote aggregation and toughening of meat. Degradation of proteins such as desmin, filamin, dystrophin, and talin (all located at the periphery of the Z-line) may disrupt the lateral register and integrity of the myofibril themselves as well as the attachments of the peripheral layer of myofibril to the sarcolemma. Degradation of the proteins within the myofibril that are associated with the thick and thin filaments may allow lateral movement or breaks to occur within the sarcomeres of postmortem aged samples. Titin, nebulin, and troponin-T, by their ability to directly interact with, or modulate the interaction between, major proteins of the thick and thin filaments and (or) the Z-line, play key roles in muscle cell integrity. Disruption of these proteins, especially titin and nebulin, could initiate further physicochemical and structural changes that result in myofibril fragmentation and loss of muscle cell integrity, and ultimately in tenderization of the muscle. In order to make real progress in this area, the scientific community must have a global appreciation of how both the structural proteins and the key proteases are influenced by the vast changes that occur during the conversion of muscle to meat.     

High pre rigor temperature limits the ageing potential of beef that is not completely overcome by electrical stimulation and muscle restraining

Two simultaneous trials were conducted to determine the effects of electrical input [electrical stunning and stimulation (ES)], wrapping, pre rigor temperature (15 °C and 38 °C) and different post rigor chilling rates on beef quality using M. longissimus lumborum (n=100). The high pre rigor temperature induced a faster pH decline than ES. The loins at 38 °C had significantly greater protein denaturation, more purge and drip loss, higher shear force values and less desmin degradation compared with the loins at 15 °C. No difference in sarcomere length was determined between the pre rigor temperatures regardless of ES and wrapping. Different post rigor chilling rates did not play a substantial role in water-holding capacity, proteolysis, or shear force values during ageing. These results suggest that high pre rigor temperature induces temperature-related toughness of muscle due to protein denaturation with subsequent limitation of proteolysis by μ-calpain, regardless of ES and wrapping treatments.     

Theoretical aspects of water-holding in meat

As myofibrils consist of a three-dimensional network of long, solid protein particles with the shortest dimension of less than 20 nm, the theoretical foundations of water-holding in meat should be studied from a colloid or surface chemistry point of view. The classical hypotheses for water-holding in meat are based on electrostatic forces or osmotic forces, which cause the swelling of the myofibrils. The more recent research adds to those the structure of water, whether it is low density water induced by kosmotropic effects dominating in the system, or high density water induced by chaotropes, respectively. The phenomena in the one to three molecules thick water layers on protein surfaces do not, however, explain the bulk water-holding. The interactions of ions and non-polar kosmotropes with water and proteins have a relevant effect on water-holding. The chaotropic/kosmotropic effects of different ions will be of importance especially when reducing sodium contents in meat-based foods.     

宰后猪肉pH值、骨架蛋白表达水平和持水性之间的关系

以20 头猪的背最长肌为材料,分别于宰后45 min和3、9、12、24 h测定猪背最长肌的pH值和整联蛋白、黏着斑蛋白、踝蛋白表达水平,并测定汁液流失率,探讨它们之间的关系。结果显示：高pH组（pH45 min≥6.00,n＝6）的汁液流失率和踝蛋白表达水平低于低pH组（pH45 min≤5.78,n＝14）,整联蛋白和黏着斑蛋白表达水平高于低pH组。pH值和整联蛋白、黏着斑蛋白表达水平与汁液流失率呈负相关,踝蛋白与之相反。pH值和整联蛋白、黏着斑蛋白、踝蛋白表达水平分别解释了汁液流失率变异的77%、41%、44%和34%。上述结果表明：pH值和骨架蛋白变化都会对猪肉持水性产生影响,但pH值的降低对猪肉持水性的影响比骨架蛋白降解影响更大。pH值会通过影响细胞骨架完整性,进而影响最终产品的持水性。宰后45 min的pH值可以用于预测宰后猪肉的汁液流失率。     

Effects of Salt and Pyrophosphate on the Physical and Chemical Properties of Beef Muscle

The effects of sodium chloride (NaCl) and pyrophosphate (PP) were examined by treating beef sternomandibularis muscle tissue and isolated beef myofibrils with various concentrations of NaCl, with and without 10 mM PP. Gel electrophoresis showed that higher NaCl concentrations (1.0M > 0.7M > 0.4M) increased the extraction of titin, myosin, and other myofibrillar proteins from beef tissue and that the inclusion of 10 mM PP to NaCl solutions enhanced the extraction of those proteins. Beef tissue water-holding capacity (WHC) was increased by higher NaCl concentrations and the presence of 10 mM PP. Increased myofibrillar/cytoskeletal protein extraction, especially titin, was associated with increased beef myofibril swelling and increased beef muscle WHC.     

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.     

The effect of simulated transport of fresh meats on their water-holding capacity as assessed by various methods

This study was designed to establish the effects of transport vibrations on the water-holding capacity of fresh non-stimulated veal and pork and electrically- and non-stimulated beef. Furthermore, drip production was followed during storage by various methods which were compared for their suitability in industrial practice and scientific research. During storage, the amount of drip increased, whilst the rate of drip formation decreased. Filter paper wetness measured at 1 day post mortem was positively correlated with drip lost over a period of 2 weeks of refrigerated storage in poly-ethylene bags (method 1) and poly-propylene containers (method 2). This suggests that the filter paper wetness test may be useful to predict drip loss of stored meat. Simulated transport did not result in higher drip losses, although a significant increase in filter paper wetness from the fresh cut surface of veal and pork was found, suggesting that transport affects the initial rate of drip production, but not the total amount of drip. In beef, transport simulation resulted in an increase in filter paper wetness from the surface of electrically-stimulated meat, stored for 1 or 2 weeks, indicating that fluid loss was elevated due to transport vibrations. However, this effect seemed to be related to the intrinsic water-holding capacity of the sample: meat with a low intrinsic water-holding capacity seemed to be more sensitive to transport vibrations than meat with a high intrinsic water-holding capacity.     

骨架蛋白对肉持水性的影响及机制研究进展

持水性是肉的重要品质指标, 直接影响肉的颜色、多汁性和嫩度。汁液流失常被用来衡量肉的持水性,宰后过多的汁液流失会给肉类行业造成一定程度的经济损失,因此, 研究肉的持水性是食品行业的一项重要内容。骨架蛋白作为肌肉细胞的主要成分,对维持细胞结构和功能起着重要作用,其在宰后肌肉成熟过程中发生的变化对肉的持水性有一定影响。本文综述了主要骨架蛋白的生理生化性质以及骨架蛋白对肉品持水性的影响, 探讨了影响骨架蛋白降解的重要因素和作用机制,以期为肉品持水性控制提供理论参考。     

The effect of various types of poultry pre- and post-rigor meats on emulsification capacity, water-holding capacity and cooking loss

In this study, the relationship between various kinds of poultry meat (quail, partridge, chicken and turkey) on pH, emulsification capacity, water-holding capacity and cooking loss was investigated. The effect of rigor state on pH, emulsification capacity, water-holding capacity and cooking loss was also determined. To investigate these parameters, immediately after slaughter and deboning, meat parts were submitted to both pre- and post-rigor analyses. The results indicated that the emulsification capacity of quail and chicken meat was higher than the values for partridge and turkey meat. Quail meat showed the highest water-holding capacity value in the post-rigor stage. The lowest cooking loss value was found in partridge meat, in both pre- and post-rigor stages. The state of rigor had a significant (P<0.05; P0.01) effect on pH and cooking loss values, respectively.     

Biochemical and technological characteristics of hot chicken meat

In the hot breast and leg muscles of broiler chicken the level of ATP, the 'R' value, the lactic acid content, the pH value, the length of sarcomers, the water and fat retention capacity, the fat emulsion stability, thermal drip, and the extractability of protein fraction were investigated. It was found that in the breast muscles the onset of rigor mortis commenced within 30–60 min, and in the leg muscles as early as 15–30 min after killing of the birds. The deepest rigor mortis occurred between the first and fourth hour, and then gradually declined, sooner in the leg than in the breast muscles. The addition of sodium chloride (2.0–2.5%) to the minced pre-rigor meat not later than 40 min after slaughter, or better, an injection of NaCl brine into intact muscles 15 min after slaughter of birds, preserved their good technological properties.
The tenderness and the thermal drip of hot salted and chilled salted muscles showed no significant differences, but water retention and fat emulsifying capacity were better in the hot salted meat samples. The hot salted and cooked muscles were preferred by the sensory panel to corresponding samples of chilled muscles.
From the hot salted chicken meat more sarcoplasmic and myofibrillar proteins were extracted than from meat salted after chilling. However, after frozen storage the extractability of myofibrillar proteins were higher in the salted chilled meat.     

生鲜肉持水性机理研究进展

持水性是生鲜肉的重要品质指标之一,其不仅与肉品的嫩度、多汁性、色泽等食用品质紧密相关,而且持水性直接影响肉及肉制品的出售质量,因此持水性是肉品行业关注的重点。本文对生鲜肉持水性基础理论及近年来的发展情况进行综述。首先,简要介绍肌肉的微观结构及肌肉中水分分布特点;其次,回顾肉制品经典的持水性作用力理论,即静电作用力、渗透压、毛细作用力;然后,介绍持水性的表征方法和测定方法;最后,围绕净电荷和微观结构两大因素重点介绍肉品持水性机理相关的最新研究进展,以期为肉品领域的研究与生产提供理论参考。