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* Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506 and
and
Roman L. Hruska U.S. Meat Animal Research Center, USDA, ARS, Clay Center, NE 68933
2 Correspondence:
249 Weber Hall (phone: 785/532-1225; fax: 785/532-7059; E-mail:
mdikeman{at}oznet.ksu.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Beef • Chilling • Cooking • Proteolysis • Tenderness • Rate
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Differences in tenderness exist among muscles differing in location and function in the live animal. These differences are generally attributed to differences in collagen content and/or differences in contraction or stretching during rigor mortis.
In research, the effects of cooking on tenderness are generally considered to be constant; however, studies have shown that cooking methods differing in the rate and medium of heat transfer affect muscle tenderness differently (Lawrence et al., 2001). This suggests that the cooking process can create at least some variation in meat tenderness.
This study was conducted to evaluate the relationships among factors implicated in determining myofibrillar tenderness and how different muscles and cooking rates affect these relationships. Muscles and treatments used were intended to create variation in tenderness, sarcomere length, connective tissue content, and heating rate to study the interrelationships among these factors and proteolysis in determining beef myofibrillar tenderness. Specifically, our objectives were to quantify the effects of prerigor excision and rapid chilling vs. conventional chilling of two muscles on proteolysis during postmortem storage and the potential impact on tenderness, and to examine the effects of very fast and slow rates of heating on the myofibrillar characteristics and tenderness of muscles that had been either excised and rapidly chilled or conventionally chilled.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Approximately 45 min postmortem, the longissimus thoracis (LT) and the triceps brachii, long head (TB) were removed from the left side of each carcass. External fat was removed, and then the muscles were placed in plastic bags and immersed in an ice bath to induce cold shortening (excised, rapidly chilled). The right side was chilled intact in a 0°C cooler equipped with a spray chill (conventional chill). The spray chill cycle emitted water for 1 min, air for 1 min, and was off for 15 min. This cycle was repeated for a total of 8 h. Air chilling was then used for the remainder of the chilling time.
At 24 h postmortem, the right side was ribbed between the12th and 13th ribs. Trained University personnel determined yield and quality grade factors. Following grade data collection, the LT and TB were removed from the right side of each carcass. All muscles were cut into 2.54-cm steaks. Animal and location identity was maintained on all steaks, which were individually vacuum packaged and assigned randomly to a postmortem time (1 or 14 d) and to raw and cooking treatments. Steaks assigned to the 1-d treatment were frozen immediately at -40°C. Steaks assigned to the 14-d aging treatment were aged at 4°C for 14 d, and then frozen at -40°C until further analysis.
pH and Temperature Decline
The decline in muscle temperature and pH was monitored at 1, 2, 4, 8, 12, 16, 20, and 24 h postmortem using an Accumet AP 61 pH meter/thermometer (Fisher Scientific, Pittsburgh, PA). The pH decline of the first replication of six carcasses was monitored with a gel combination spear-type probe (catalog No. 476476, Corning Inc., Corning, NY). Due to equipment failure, pH readings were not taken at 16 and 20 h for the first six carcasses, and the 24-h pH reading was taken on those animals with an IQ 240 pH/mV/thermometer equipped with a nonglass ISEFT sensor probe (IQ Scientific, San Diego, CA). The pH decline on the second replication of six animals was measured using a flat-surface probe (Accumet Flat Surface Polymer Body Electrode, catalog No. 13-620-289, Fisher Scientific) attached to the Accumet AP 61 pH meter. Even though the pH component of the Accumet AP 61 pH meter stopped functioning for carcasses in the first replication, it was used for recording temperature throughout the study.
Cooking Treatments
Cooking was accomplished with a gas, forced-air convection oven (Blodgett, model DFG-102 CH#, G. S. Blodgett Co., Burlington, VT). To achieve a rapid cooking rate, the oven temperature was set at 260°C (FAST), whereas to achieve a slow cooking rate, the oven temperature was set at 93°C (SLOW). Steaks were thawed for 24 to 36 h prior to cooking, and then placed directly on the oven rack during cooking. Due to even heat and air circulation, uniform cooking was achieved without turning steaks. Internal temperature was monitored with copper constantan thermocouples inserted into the geometric center of the steaks and attached to a Doric model 205 temperature recorder (Vas Engineering, San Francisco, CA). When the internal temperature reached 70°C, the steaks were removed from the oven. Steaks were chilled at 4°C for 24 h before being cored for Warner-Bratzler shear force (WBSF) determination. Steaks were weighed before and after cooking to determine cooking loss, and cooking time was recorded.
Warner-Bratzler Shear Force Determination
Six 1.27-cm diameter cores were removed from each cooked, chilled steak parallel to the muscle fiber orientation. Each core was sheared once through the center with an Instron Universal Testing Machine (model 4201, Instron Corp., Canton, MA) equipped with a Warner-Bratzler V-notch blade, a 50-kg compression load cell, and a crosshead speed of 250 mm/min.
Sarcomere Length Measurement
For cooked steaks, a 5-mm portion was removed from the sheared edge of one-half of each of the six cores used for WBSF. For raw steaks, three 1.5 x 1.5 x 2.0-cm samples (one each from the lateral, center, and medial portions) were removed parallel to the muscle fiber orientation from each steak. Sarcomere length was measured using the protocol of Koolmees et al. (1986). Briefly, samples were fixed in a 5% solution of glutaraldehyde in 0.1 M NaHPO4 buffer at pH 7.2 and 4°C. After 4 h, the glutaraldehyde solution was replaced with a 0.2 M sucrose solution in 0.1 M NaHPO4 buffer at pH 7.2. Samples were held overnight at 4°C. Individual fibers (n = 6 for cooked samples; n = 10 for raw samples) were teased from each sample, placed on a glass microscope slide, and immersed in a drop of sucrose solution. Sarcomere length was measured by passing the beam of a He-Ne laser (model 102-3, Spectra-Physics Inc., Eugene, OR; 
= 0.6328) through the fiber. The sarcomere length was calculated from the distance between the first order diffraction bands, according to Cross et al. (1981).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blotting Analysis
Samples of raw steaks consisted of approximately 15 g of small muscle pieces, whereas for cooked steaks, SDS-PAGE samples consisted of the remainder of the cores used for WBSF with the outside, cooked edges removed. Muscle protein extraction, SDS-PAGE, Western blotting, and desmin detection were carried out as described by Wheeler and Koohmaraie (1999) and Wheeler et al. (2002). Muscle-specific standards were collected within 30 min of exsanguination from six animals and pooled. All samples from the same muscle and animal were run on the same gel. The appropriate standards were run in the center and outside lanes of each gel. The percentage of desmin remaining in each sample was determined by comparing the intensity of the band produced by that sample with the mean intensity of the bands produced by the three standards run on the same gel. Detection of protein was accomplished using a Pierce SuperSignal West Dura Extended Duration Substrate Kit (Pierce Endogen, Rockford, IL). Membranes were exposed for 3 min and then an image was captured using ChemiImager 4000 digital imaging system (Alpha Innotech, San Leandro, CA). The extent of desmin degradation was expressed as the difference in the density of the protein band from each sample relative to the at-death reference standard.
Some of the LT samples produced density values that were greater than the at-death standards (i.e., a value for desmin remaining greater than 100 was obtained). When this occurred, the sample with the largest density value was set at 100%. The remaining samples on that gel were adjusted accordingly.
Statistical Analysis
The pH and temperature decline data were analyzed as a split-plot design with repeated measures. The whole plot experimental unit was animal, with muscle as the treatment. The subplot experimental unit was muscle, and the treatment was chilling. These data were analyzed with the Proc MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The fixed effects included muscle and chilling treatment. Random effects included animal and chilling treatment within animal. Because two different pH probes were used, pH probe was included as a random effect for the pH data. Because the treatment x time interaction for these two traits was deemed to be important, least squares means for that interaction were generated and separated using the DIFF option with a Scheffe adjustment.
The sarcomere length, WBSF, desmin degradation, and cooking traits data were analyzed as a split-split plot design with a 2 x 2 factorial subplot. The whole plot experimental unit was animal and the treatment was muscle. The subplot experimental unit was muscle and the treatment was chilling. The sub-subplot experimental unit was steak and the treatments were aging and cooking rate. The data were analyzed with the Proc MIXED procedure of SAS. Fixed effects were muscle, chilling treatment, aging treatment, and cooking treatment. Random effects were animal and chilling treatment within animal. Least squares means were generated for significant interactions, and main effects not involved in higher order interactions were separated with the PDIFF option. Additionally, the changes in WBSF and percentage of desmin degradation during aging were calculated by subtracting the 14-d values from 1-d values.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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. These heifers produced carcasses that are typical of Charolais-crossbred cattle that would be found in a commercial slaughter facility.
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). By 2 h postmortem, the rapidly chilled muscles had reached a temperature of 16.8°C, whereas the conventionally chilled muscles did not approach that temperature until 8 h postmortem. The rapidly chilled muscles had a temperature of 3.7°C at 4 h postmortem, whereas the conventionally chilled muscles had a temperature of 3.6°C at 24 h postmortem.
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show that muscle pH was not statistically different (P > 0.05) between the chilling regimens at any specific time postmortem, but the rapidly chilled muscles had numerically higher pH values between 2 and 16 h postmortem. After 20 h, the pH was essentially identical between treatments. The excised, rapidly chilled and the conventionally chilled muscles had ultimate pH values of 5.56 and 5.54, respectively.
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) in the excised, rapid-chilling treatment. Additionally, an interaction between muscle and chilling treatment was observed for sarcomere length. The conventionally chilled TB had longer (P < 0.05) sarcomeres than did the conventionally chilled LT; conversely, the TB cold shortened to a greater extent (P < 0.05) than the LT (34 vs. 29%, respectively). Drastic shortening associated with rapid chilling of unrestrained muscle is consistent with the findings of Locker and Hagyard (1963), Marsh and Leet (1966), and Honikel et al. (1983). Geesink et al. (1995) reported a larger difference between the sarcomere lengths of these two muscles than we found with conventional chilling. Based on sarcomere length data, it is clear that the objective of creating a severely contracted state with which to study the effects of sarcomere length on tenderness and tenderization was met.
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). Based on the magnitude of shortening, it is surmised that the toughening observed in our study approached the maximum and was similar to that reported for the longissimus by Wheeler and Koohmaraie (1999).
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). This is in contrast to Koohmaraie et al. (1984) and Wheeler and Koohmaraie (1999), who reported no difference in proteolysis between cold-shortened and normal muscle.
The interaction means (Table 4) show a small increase (P < 0.05) in the sarcomere length of the TB during aging. However, no increase (P > 0.05) in sarcomere length of the LT was found during aging. Increased sarcomere length during aging has been reported by Stromer and Goll (1967a,b) and Wheeler and Koohmaraie (1994). Wheeler and Koohmaraie (1999) and Geesink et al. (2001) found no increase in sarcomere length with aging, and Geesink et al. (2001) suggested that increased sarcomere lengths reported with aging in some studies could be due to sampling error.
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). However, at 1 d postmortem, the TB tended (P = 0.06) to have a greater proportion of desmin degraded, but after 14 d, the LT tended (P = 0.12) to have more of its desmin degraded, thus the statistically significant (P < 0.05) interaction. Additionally, the mean difference in the extent of proteolysis between d 1 and d 14 was greater (P < 0.05) in LT steaks than in TB steaks (Table 4).
Even though proteolysis was reduced in the excised, rapidly chilled muscles, it is important to note that the mean change in the percentage of desmin that had been degraded between 1 and 14 d was not different (P > 0.05) between chilling treatments (Table 5). This indicates that the difference in proteolysis between treatments occurred during the first 24 h, after which, the rate of proteolysis did not differ.
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). The rapidly chilled muscles also declined to a lower ultimate temperature than the control samples. Decreasing temperature has been shown to lower the proteolytic activity of µ-calpain (Koohmaraie et al., 1986) and to slow the rate of tenderization in muscle (Davey and Gilbert, 1976; Whipple et al., 1990; Simmons et al., 1996). Wheeler and Koohmaraie (1999), who found no difference in proteolysis between normal and shortened muscle, induced shortening in a manner that did not affect the temperature decline of the muscles studied. Those findings, in conjunction with our results, support the theory that the reduction in proteolysis was due to reduced temperature rather than sarcomere shortening itself.
Aging improved (P < 0.01) WBSF values of both muscles, regardless of shortening treatment (Table 5). The improvement in tenderness with postmortem storage is common (Bouton et al., 1975; Locker and Wild, 1982; Campo et al., 2000). However, reports on the effect of aging on cold-shortened meat are varied. Goll et al. (1964), Herring et al. (1967), and Bouton et al. (1973) found that aging cold-shortened beef improved tenderness, whereas other studies found no improvement in tenderness as a result of aging (Davey et al., 1967; 1976; Locker, 1982).
Locker (1982) suggested that the gap filaments (titin and nebulin) were the primary determinants of meat tenderness. Other authors have also presented evidence that these two proteins are important to tenderness (Taylor et al., 1995; Huff-Lonergan et al., 1996; Koohmaraie, 1996). According to Locker (1982), cold-shortened meat does not improve in tenderness because the overlapping of the thick and thin filaments does not allow the calpain enzymes access to these proteins. If this were true, postmortem tenderization would not occur in cold-shortened muscle. However, improvements in the tenderness of cold-shortened meat with aging have been reported. Perhaps this disagreement can be partially explained by the findings of Davey and Gilbert (1977) and Locker and Wild (1982), who found that shear force and yield point (the point at which integrity is lost with longitudinal stretching) were highly correlated in normal muscle, and both shear force and yield point decreased with postmortem storage. In cold-shortened muscle, however, yield point decreased with aging but shear force did not (Locker and Wild, 1984). It has been suggested that yield point is an indicator of weakening of the cytoskeletal proteins in the I-band (Locker and Wild, 1982); thus, yield point would be an indicator of the proteolysis of titin and nebulin. It is suggested, then, that the reason some workers have not observed tenderization with aging in cold-shortened muscle is that the tenderization in response to proteolysis is masked by the overlapping thick and thin filaments.
In our study, postmortem storage improved WBSF of muscles, regardless of chilling treatment (Table 5). The change in WBSF during aging was greatest (P < 0.05) in the excised, rapidly chilled TB steaks and least in the conventionally chilled TB steaks. No difference (P > 0.05) in the rate of change in WBSF during aging was observed between conventionally chilled and excised, rapidly chilled LT steaks. Even though tenderization occurred to a greater extent in the excised, rapidly chilled vs. conventionally chilled TB muscle, the toughening effect of cold shortening was not eliminated by aging (Table 5). Geesink et al. (1995) reported small improvements in the tenderness of the TB compared with that of the longissimus. Perhaps the longer sacromere length in the conventionally chilled TB muscles reduces the impact of proteolysis on tenderness. When shorter sarcomeres are present, the impact of proteolysis is more pronounced.
Cooking Rate Effects
As expected, SLOW cooking required much longer (P < 0.05) cooking times than FAST cooking (Table 6). There was no difference (P > 0.05) in cooking time due to muscle, chilling treatment, or aging treatment when steaks were cooked FAST. However, when SLOW cooking was used, the TB steaks generally required longer (P < 0.05) cooking times than the LT steaks.
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). Cooking at both temperatures caused a reduction (P < 0.05) in sarcomere length. In conventionally chilled muscle, raw steaks had the longest sarcomeres. FAST cooking caused the greatest reduction in sarcomere length, with sarcomeres for SLOW cooking being intermediate to those of the raw and FAST cooked steaks. Reduced sarcomere length associated with cooking has been consistently reported (Lewis et al., 1977; Cheng and Parrish, 1979). Cooking also shortened the sarcomeres of the excised, rapidly chilled muscles but no difference (P > 0.05) was found between the two cooking treatments for conventionally chilled muscles (Table 7). This is in agreement with Hegarty and Allen (1975), Locker and Daines (1975), and Wheeler and Koohmaraie (1999), who reported that muscle shortening that resulted from cooking was dependent upon the sarcomere length in the raw state.
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). The raw LT steaks and SLOW-cooked TB steaks underwent more degradation of desmin than FAST-cooked LT steaks. Koohmaraie et al. (1986) found that the activity of µ-calpain at 5°C (pH 7.5) was about 60% of the maximal activity (25°C, pH 7.5). At a pH of 5.5, µ-calpain activity was lower at both temperatures and the difference in activity due to temperature was smaller. However, Inomata et al. (1984) reported that µ-calpain retained as much as 85% of its activity with heating up to 60°C (pH 7.5). Consistent with those results, Wheeler et al. (2002) reported greater desmin degradation in d-14 beef after belt-grill cooking than in raw samples, but did not detect that effect in pork, likely because 78% of desmin was already degraded in the raw pork samples. Furthermore, Davey and Gilbert (1976) found that the rate of tenderization of meat was maximized at 60°C, but approached zero at 70°C. Therefore, we hypothesized that extremely slow heating rates would increase the time that the meat was at elevated temperatures capable of producing rapid proteolysis. The SLOW-cooked LT and TB steaks combined had numerically greater amounts of desmin degradation (Table 8), but these differences were not statistically significant. The SLOW cooked TB steaks did have greater (P < 0.05) proteolysis of desmin than the FAST-cooked LT. We cannot explain the observation that, at 1 d postmortem, the RAW LT steaks had greater desmin degradation than the FAST-cooked LT steaks (Table 8). This could have been due to sampling variation.
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). Furthermore, LT steaks were not different (P > 0.05) from TB steaks when cooked FAST. However, SLOW cooking caused a reduction (P < 0.05) in WBSF for TB steaks. The small difference in proteolysis between cooking methods may have contributed to the increase in tenderness of the TB due to cooking rate. Greater sarcomere shortening due to FAST cooking was observed in both muscles as discussed earlier. Because no difference in tenderness was found between cooking rates in LT steaks, it is not likely that changes in sarcomere length resulting from cooking caused the differences in tenderness of the TB. Slower cooking rates have been shown to improve tenderness (Bayne et al., 1969; Cross et al., 1976; Lawrence et al., 2001). The myofibrillar contribution to shear force was influenced by the heating rate up to 80°C (Møller, 1981). However, the tenderizing effect of slow cooking is commonly attributed to the solubilization of collagen (Bayne et al., 1969; Penfield and Meyer, 1975). Møller (1981) found the connective tissue contribution to shear force was primarily influenced by a heating rate between 60°C and 80°C. Hence, the greater collagen content of the TB muscles (McKeith et al., 1985) in our study could be responsible for the differing response to cooking rate. The time at which steaks were in this temperature range was extended by our SLOW cooking treatment.
FAST cooking caused greater (P < 0.05) cooking losses than SLOW cooking in both muscles (Table 8). This is consistent with the results of Cross et al. (1976), who reported that faster heating rates caused greater evaporative and total cooking losses. Lawrence et al. (2001) found that cooking steaks from five beef muscles on an electric-belt grill at 163°C resulted in higher (P < 0.05) cooking losses than cooking on the belt grill at 93°C, even though cooking was relatively rapid at either temperature because of the efficient heat transfer of the belt grill.
The difference in cooking losses between cooking rates was much greater (P < 0.05) in LT steaks compared to TB steaks (7.27 and 4.06 percentage points for FAST and SLOW, respectively). However, the TB steaks had higher cooking losses than the LT steaks when cooked at the same rate. This could be due to the higher connective tissue content of the TB (McKeith et al., 1985). Davey and Gilbert (1974) found that the temperature at which cooking loss increased in meat corresponded to the temperature at which isolated collagen shrunk. It seems reasonable that the differences in cooking losses between cooking rates observed in our study could be due to a difference in the force generated on the myofibrils by collagen shrinkage. The collagen shrinkage might not have been as severe and, therefore, unable to generate as much force before being solubilized in the SLOW-cooked steaks.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication March 13, 2002. Accepted for publication February 5, 2003.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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