Steven M. Lonergan

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.

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.

Influence of early postmortem protein oxidation on beef quality

The objective of this study was to examine the effect of early postmortem protein oxidation on the color and tenderness of beef steaks. To obtain a range of oxidation levels, the longissimus lumborum muscles (LM) from both strip loins of 20 steers fed either a finishing diet with vitamin E (1,000 IU per steer daily, minimum of 126 d [VITE]; n = 10 steers) or fed the same finishing diet without vitamin E (CON; n = 10 steers) were used. Within 24 h after slaughter, the LM muscle from each carcass was cut into 2.54-cm-thick steaks and individually vacuum packaged. Steaks from each steer were assigned to a nonirradiated group or an irradiated group. Steaks were irradiated within 26 h postmortem, and were aged at 4 degrees C for 0, 1, 3, 7, and 14 d after irradiation. Steaks from each diet/irradiation/aging time treatment were used to determine color, shear force, and degree of protein oxidation (carbonyl content). Steaks from steers fed the VITE diet had higher (P < 0.01) alpha-tocopherol contents than steaks from steers fed the CON diet. Immediately following irradiation, steaks that had been irradiated had lower (P < 0.05) L* values regardless of diet. Irradiated steaks, regardless of diet, had lower a* (P < 0.05) and b* (P < 0.01) values than nonirradiated steaks at all aging times. Carbonyl concentration was higher (P < 0.05) in proteins from irradiated steaks compared to nonirradiated steaks at 0, 1, 3, and 7 d postirradiation. Immunoblot analysis showed that vitamin E supplementation decreased the number and extent of oxidized sarcoplasmic proteins. Protein carbonyl content was positively correlated with Warner-Bratzler shear force values. These results indicate that increased oxidation of muscle proteins early postmortem could have negative effects on fresh meat color and tenderness.

Progress in reducing the pale, soft and exudative (PSE) problem in pork and poultry meat.

Research in the area of the pale, soft and exudative (PSE) pork and poultry meat is reviewed in this article with an emphasis on genetic, biochemical and metabolic factors contributing to the problem. Over the past five decades, there has been much more work in the pork meat area where a few genetic markers have been identified, and are currently used to remove susceptible animals from the herd. Some of the markers are linked to aberrant calcium regulation in the early postmortem muscle. The poultry industry is still not at the point of using genetic marker(s); however, some recent work has revealed several potential markers. The review also discusses environmental factors such as antemortem stress and early postmortem processing practices (e.g. chilling rate) that can influence the development and severity of the PSE phenomenon. Some of these factors are known to cause protein denaturation at the early stage of postmortem and directly contribute to poor water-holding capacity and inferior texture in fresh meat and later in processed products. A newer hypothesis suggesting that variation in protein oxidation, in response to antemortem stress and early postmortem tissue environment, can contribute to development of PSE pork is also discussed. Finally, a few recommendations for future work are proposed.

High-oxygen modified atmosphere packaging system induces lipid and myoglobin oxidation and protein polymerization.

Beef steaks from longissimus lumborum, semimembranosus, and adductor muscles (n=10; respectively) were cut at 24h postmortem, randomly assigned to either high-oxygen modified atmosphere packaging (HiOx-MAP; 80% O(2), 20% CO(2)) or vacuum (VAC), and displayed for 9days at 1 degrees C. HiOx-MAP packaged beef steaks had a rapid increase in lipid oxidation and a decrease in color stability during display. The steaks in HiOx-MAP had significantly lower tenderness and juiciness scores, and higher off-flavor scores compared to steaks in VAC. HiOx-MAP condition did not affect the postmortem degradation of troponin-T or desmin. Furthermore, autolysis of micro-calpain was not influenced by packaging. SDS-PAGE, immunoblotting, and diagonal-PAGE revealed oxidative cross-linking of myosin heavy chain in meat packaged in HiOx-MAP. These results suggest that the HiOx-MAP system may negatively affect meat quality characteristics by inducing lipid and myoglobin oxidation and cross-linking/aggregation of myosin by protein oxidation.

Contribution of postmortem changes of integrin, desmin and μ-calpain to variation in water holding capacity of pork

The purpose of this study was to examine the relationship between integrin, desmin, μ-calpain and water holding capacity in fresh pork. High levels of intact integrin at one day postmortem were negatively correlated with day 1 (P<0.05) and days 1-5 (cumulative) drip loss (P<0.05). High levels of intact integrin at five days postmortem were negatively correlated with days 1-7 (cumulative) purge loss (P<0.05). Intensity of intact desmin at one day postmortem was positively correlated with days 1-7 purge loss (P<0.01). There were positive correlations between intensity of intact desmin at day 7 and day 1 (P<0.01), days 1-5 drip loss (P<0.01) and days 1-7 purge loss (P<0.05). Autolysis of μ-calpain was associated with the degradation of desmin and drip or purge loss postmortem. Our results indicate that low levels of degradation of integrin and high levels of desmin degradation were associated with low drip loss values in fresh pork.

Rate and extent of pH decline affect proteolysis of cytoskeletal proteins and water-holding capacity in pork

The objective of this study was to determine the extent to which early postmortem (PM) pH decline influences proteolysis of the intermediate filament protein desmin, the costameric proteins vinculin and talin and autolysis of μ-calpain in the longissimus muscle (LM) of pigs from two genetic lines. Based on the LM 3h pH (H=3h pH of LM>6.0; L=3h pH of LM pH<5.7) PM, 10 carcasses per line and pH group were selected. The average 3h pH within pH group was 6.23 (H) and 5.44 (L). The LM samples were collected 24, 48, 72, and 120h PM and percent drip loss was measured after 1, 2, and 4d of storage. Samples collected at 24, 48, 72, and 120h PM were used to monitor desmin, vinculin, and talin degradation and samples collected at 24h PM were used to determine the extent of μ-calpain autolysis by immunoblotting. Higher (P<0.01) pH values at 45min, 6h, and 24h PM and lower (P<0.01) drip losses after 1, 2, and 4d of storage were recorded in the H-compared to the L-group. Abundance of the 76kDa μ-calpain autolysis product was greater (P<0.01), proteolysis of talin at all measured time points and proteolysis of desmin after 24 and 48h PM was greater (P⩽0.03) in the H-group than in the L-group. The current findings indicate activation rate of μ-calpain may be associated with proteolysis of desmin and talin and could play a role in the development of drip loss. The rate of early PM pH decline can partly explain the variation of desmin and talin degradation by affecting the activation of μ-calpain.

Growth performance, carcass characteristics, meat quality, and tissue histology of growing pigs fed crude glycerin-supplemented diets.

The effects of dietary crude glycerin on growth performance, carcass characteristics, meat quality indices, and tissue histology in growing pigs were determined in a 138-d feeding trial. Crude glycerin utilized in the trial contained 84.51% glycerin, 11.95% water, 2.91% sodium chloride, and 0.32% methanol. Eight days postweaning, 96 pigs (48 barrows and 48 gilts, average BW of 7.9 +/- 0.4 kg) were allotted to 24 pens (4 pigs/pen), with sex and BW balanced at the start of the experiment. Dietary treatments were 0, 5, and 10% crude glycerin inclusion in corn-soybean meal-based diets and were randomly assigned to pens. Diets were offered ad libitum in meal form and formulated to be equal in ME, sodium, chloride, and Lys, with other AA balanced on an ideal AA basis. Pigs and feeders were weighed every other week to determine ADG, ADFI, and G:F. At the end of the trial, all pigs were scanned using real-time ultrasound and subsequently slaughtered at a commercial abattoir. Blood samples were collected pretransport and at the time of slaughter for plasma metabolite analysis. In addition, kidney, liver, and eye tissues were collected for subsequent examination for lesions characteristic of methanol toxicity. After an overnight chilling of the carcass, loins were removed for meat quality, sensory evaluation, and fatty acid profile analysis. Pig growth, feed intake, and G:F were not affected by dietary treatment. Dietary treatment did not affect 10th-rib backfat, LM area, percent fat free lean, meat quality, or sensory evaluation. Loin ultimate pH was increased (P = 0.06) in pigs fed the 5 and 10% crude glycerin compared with pigs fed no crude glycerin (5.65 and 5.65 versus 5.57, respectively). Fatty acid profile of the LM was slightly changed by diet with the LM from pigs fed 10% crude glycerin having less linoleic acid (P < 0.01) and more eicosapentaenoic acid (P = 0.02) than pigs fed the 0 or 5% crude glycerin diets. Dietary treatment did not affect blood metabolites or frequency of lesions in the examined tissues. This experiment demonstrated that pigs can be fed up to 10% crude glycerin with no effect on pig performance, carcass composition, meat quality, or lesion scores.

Identification of genetic markers associated with residual feed intake and meat quality traits in the pig

Residual feed intake (RFI) has become increasingly important and is being considered as a more reasonable approach to evaluate feed efficiency in livestock. However, the cost and technical difficulties in measuring this trait restrict the extensive adoption of RFI selection, and this makes marker assisted selection (MAS) a feasible tool. In addition, the effects on meat quality caused by low RFI selection have yet to be clarified. In this study, 11 SNPs from eight candidate genes were evaluated in a Yorkshire pig experimental population (n=169) consisting of a low RFI selection line and a randomly selected control line. Associations of these SNPs with RFI, growth rate, carcass composition, and meat quality measures including water holding capacity, pH at 2d postmortem, meat color and sensory traits were analyzed. The SNPs FTO p.Ala198Ala and TCF7L2 c.646+514A>G showed significant (P<0.05) and suggestively significant (P<0.1) associations with RFI, respectively. The MC4R SNP p.Asp298Asn was associated with backfat but it was not with ADG and meat quality attributes. Both SNPs within HNF1A were associated with intramuscular lipid content and sensory juiciness. The SNPs ACC1 c(*)384C>T and TCF7L2 c.646+514A>G were significantly (P<0.05) associated with ADG. The SNPs CTSZ p.Arg64Lys and TCF7L2 c.646+514A>G were associated with both visual scoring of meat color and the objective L-value measure of meat color. This study has identified potential genetic markers suitable for MAS in improving RFI, ADG, and meat color traits, but these associations need to be validated in other larger populations.

Disulfide bond within µ-calpain active site inhibits activity and autolysis

Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.