Postmortem ph, Myofibrillar Fragmentation, and Calpain Activity in Pectoralis from Electrically Stimulated and Muscle Tensioned Broiler Carcasses

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1 Postmortem ph, Myofibrillar Fragmentation, and Calpain Activity in Pectoralis from Electrically Stimulated and Muscle Tensioned Broiler Carcasses G. I. VEERAMUTHU and A. R. SAMS1 Department of Poultry Science, Texas A&M University, College Station, Texas ABSTRACT Broiler carcasses were conventionally processed or were electrically stimulated (ES) and had their Pectoralis muscles tensioned during rigor mortis development (ES/MT) to evaluate the relationship between the course of postmortem muscle ph decline and myofibrillar fragmentation. Samples from Pectoralis fillets harvested at 0, 0.5, 1, 2, and 24 h postmortem were analyzed for ph, myofibrillar fragmentation index, and calpain activity. The ES/MT treatment resulted in a more rapid ph decline and less myofibrillar fragmentation at all postmortem times sampled than conventional processing. Two distinct periods of fragmentation were noted for samples from both conventionally processed and ES/MT carcasses. These results agreed with the previous studies that this ES/MT treatment accelerated the normal postmortem ph decline, inhibited calpain activity, and thereby prevented postmortem proteolysis occurring during meat aging. (Key words: electrical stimulation, muscle tensioning, tenderness, fragmentation, calpain) 1999 Poultry Science 78: INTRODUCTION Postmortem electrical stimulation (ES) has been studied as a means to reduce the aging time required before deboning to prevent toughening the meat. Thompson et al. (1987) reported that low voltage ES improved the tenderness of hot-boned fillets and that high voltage ES was required to produce a tenderizing response in fillets deboned immediately after chilling. These authors also reported that tenderizing response was more highly related to myofibrillar disruption and increased sarcomere length than to ph or rate of adenosine triphosphate (ATP) disappearance. Sams et al. (1989) reported that ES did not have a significant effect on the rate of postmortem depletion of ATP as measured by R value. Froning and Uijttenboogaart (1988) reported that breast muscle ph was significantly lowered by ES. Electrical stimulation followed by deboning at 0 or 15 min actually toughened the breast muscle, but ES significantly tenderized muscles deboned at 120 and 240 min compared to controls. Restraining the wings during rigor mortis development, known as muscle tensioning (MT), was reported to greatly decrease shear values of early harvested prerigor breast fillets, but not to a level low enough to considered tender by consumers (Papa et al., 1989; Janky et al., 1992). However, when ES was combined with MT, their interaction resulted in significantly lower shear values that were acceptable (Birkhold and Sams, 1993). Birkhold et al. (1992) reported that shear values of fillets treated with ES/MT were positively correlated with the gravimetric fragmentation index (GFI). Further studies indicated that ES/MT treated carcasses exhibited less myofibrillar fragmentation than carcasses treated with MT only (Birkhold and Sams, 1993), but when comparing shear values between the two treatments, the ES/ MT shear values were significantly lower than those treated with MT only. Upon examination of electron micrographs of ES/MT treated fillets, Birkhold and Sams (1995), revealed that ES prevented z-line dissolution, suggesting the inhibition of calpain proteolytic activity, as this is thought to be the protease responsible for postmortem z-line dissolution. The few studies conducted on the effect of ES on the calpain proteolytic enzyme system in chicken have been inconclusive. Etherington et al. (1990) noted that m- calpain is inactivated by ES but m-calpain is not. Their studies verified the effects of ES immediately after application and in aged carcasses. Walker et al. (1995) indicated that m-calpain was inactivated in aged carcasses, whereas m-calpain had higher residual activity in ES treated carcasses than in controls. A study of beef Received for publication March 2, Accepted for publication October 6, To whom correspondence should be addressed: asams@poultry.tamu.edu Abbreviation Key: ATP = adenosine triphosphate; ES = electrical stimulation; ES/MT = electrical stimulation combined with muscle tensioning; GFI = gravimetric fragmentation index; MCE = 2- mercaptoethanol; MT = muscle tensioning; OFI = optical fragmentation index, TCA = trichloroacetic acid. 272

2 POSTMORTEM ph, MYOFIBRILLAR FRAGMENTATION, AND CALPAIN ACTIVITY 273 calpains indicated a relationship between declining ph and the decline of calpain activity in vitro postmortem conditions (Kendall et al., 1993). The objective of the present study was to elucidate the effect of ES/MT on the calpain proteolytic enzyme system over postmortem time and to relate this to the ph decline and myofibrillar fragmentation occurring during aging. Such information is necessary, as the accelerated ph decline with ES may influence the activity of the neutral protease, calpain, and the resulting fragmentation. MATERIALS AND METHODS In each of two 40-bird trials, male broilers were raised to 7 wk of age in wood shaving litter-covered floor pens while consuming a corn and soybean diet that was formulated to meet or exceed NRC requirements. Twelve hours prior to slaughter, feed and water were withdrawn. The birds were electrically stunned with 34 ma for 3 s and killed by allowing the bird to bleed through a neck cut that unilaterally severed the right carotid artery and jugular vein. Twenty birds received the ES/MT treatment and the other 20 birds served as controls. Electrical stimulation was applied by having a 12-gauge multistrand copper wire attached to the shackle and the other end clipped to the posterior end of the keel bone. Clipping was done in such a manner that the muscle and the skin were grasped by the clip without breaking the skin. The shackle rail system functioned as the ground. The carcasses were subjected to ES (440 V, ± 20 ma) by placing a charged kill knife2 across the anterior portion of the breast that had been moistened with tap water. The electricity was pulsed 2 s on and 1 s off for five pulses. After ES, all the carcasses were tagged to identify treatments, subscalded at 63 C for 45 s, defeathered using a rotary drum picker3 for 30 s and manually eviscerated. Muscle tensioning was applied to those carcasses that received ES by binding the wings behind the back at the elbows with plastic cable ties. All 40 carcasses were chilled together using a two-stage chilling regimen of 15 min prechill in tap water at 20 C, followed by chilling at 2 C in ice slush for 30 min. The wing restraints were removed after chilling. The 0 h breast fillets were removed before the carcasses entered the scald tank. The Pectoralis was removed by severing the humeral-scapular joint and pulling firmly on the wing in the caudal direction to strip the muscle from the carcass (Hamm, 1981). The Pectoralis was similarly removed from eight carcasses in each replicate (four ES/MT and four controls) at the end of the prechill period (0.5 h postmortem), after the chill period (1 h postmortem), after 1 h carcass aging in crushed ice at 4 C (2 h postmortem), and after 23 h aging in crushed ice at 4 C (24 h postmortem). Immediately after deboning, all fillets were cut parallel to the fiber 2Model VS200120, Midwest Processing, Minneapolis, MN Model SS-36, Brower, Houghton, IA direction into four equal-sized samples. The samples were sequentially numbered 1 through 4 for their respective posterior to anterior location, placed into plastic bags, and immediately frozen in liquid nitrogen. After freezing, the samples were held at 76 C until further analyses. Sample 1 was used for ph, Samples 2 and 3 for fragmentation index procedures (gravimetric and optical, respectively), and Sample 4 for the quantification of calpains. The ph of the samples was determined using the iodoacetate method of Sams and Janky (1986). The GFI was measured by the procedure of Davis et al. (1980) as modified by Sams et al. (1991). Optical fragmentation index (OFI) was determined by the modified procedure of Olson et al. (1976). Calpain extraction and assay conditions were similar to the procedures described by Wheeler and Koohmaraie (1991) with slight modification. The frozen samples were pooled (5 g per bird) by rigor treatment and boning time. Twenty grams of tissue from each pool were homogenized on ice in 2.5 vol of 50 mm Tris, 10 mm 2-mercaptoethanol (MCE), 1 mm EDTA, 0.2% triton X-100 at ph 8.5. Following centrifugation (34,000 g for 1 h), the supernatant was exhaustively dialyzed against 20 mm Tris, 5 mm EDTA, 10 mm 2-MCE at ph 7.5. Following a second centrifugation (34,000 g 1 h), the extract was loaded onto a cm diethylaminoethyl exchange column that was pre-equilibrated with 40 mm Tris, 0.5 mm EDTA, 10 mm 2-MCE at ph 7.5. The m- calpain was eluted with a linear gradient of 25 to 200 mm NaCl. The column was then washed with 400 mm NaCl to elute m-calpain. Absorbance (278 nm) of trichloroacetic acid (TCA) soluble material released following incubation of an aliquot of each fraction with 100 mm Tris, 1 mm of sodium azide, 5 mm calcium chloride, 5 mg/ml casein, 1 ml of 2-MCE/L (ph 7.5) for 1 h at 25 C was measured. One unit of calpain activity was defined as the amount of enzyme that produced a 1 unit absorbance increase. The fractions having activity of m-calpain or m-calpain were pooled by isoform and the activity in each pool measured as described for the individual elution fractions. Volume of the pooled fractions were recorded and protein concentration of the pools measured with the method of Bradford (1976). Activity was expressed as both activity per gram of muscle and activity per milligram of protein in the pool. The data were organized into a 2 5 factorial arrangement (two levels of stimulation and five postmortem times) and subjected to analysis of variance (SAS Institute, 1985). Results of the two trials were pooled because no interaction between stimulation, treatment, time, and trial was detected using the residual mean square error for the error term. Comparisons of means within time combinations for the effect of ES were made with the t test (SAS Institute, 1985). RESULTS AND DISCUSSION The ph of the Pectoralis in ES/MT treated carcasses was significantly lower than that of controls except at 24

3 274 h, when the ph of ES/MT and controls were similar. (Table 1). These results agree with the previous data published for poultry carcasses by Birkhold and Sams (1993) and Walker et al. (1994). This reduced ph indicates that the ES/MT treatment accelerated the process of rigor mortis development, possibly either by the transient increase in phosphorylase A as mentioned by Newbold and Small (1985) or by the metabolic channeling mechanism explained by Lawrie (1991). Lawrie (1991) reported that the glycolytic enzymes are bound to the myofibrillar protein actin in vivo and that the proportion of each glycolytic enzyme that is bound into the correct spatial arrangement may increase on stimulation of glycolysis (e.g., with ES) and decrease when such stimulation ceases. The OFI values are based on the amount of light refraction occurring in a suspension of myofibrils. As OFI values increase, there is a corresponding increase in the number of particles from increased fragmentation. In contrast, the GFI values are based on the amount of residue left on a 250-mm screen after a suspension of myofibrils has been vacuum-filtered through it, the smaller particles having passed through the screen leaving behind the larger particles. Consequently, the lower the GFI value the greater the degree of fragmentation. The OFI and GFI results of the present study (Tables 2 and 3, respectively) agree with the results obtained by Sams et al. (1991) and Birkhold et al. (1992). The results reveal an inhibition of fragmentation in ES/ MT muscles compared to that of controls in all hours of sampling except at 30 min. The sampling at 30 min corresponded to the approximate time of application of MT in these carcasses, which might have had some effect on the fragmentation. Sams et al. (1991) reported that ES and high temperature conditioning prevented postmortem proteolytic myofibrillar fragmentation and postulated that because these postmortem treatments accelerated the decline in intracellular ph, the activities of the calcium activated neutral proteases (calpains) were reduced. This reduction in myofibrillar fragmentation would seem to contradict the shear value reduction induced by ES/MT. However, the ES/MT process may induce a different type of fragmentation than that measured by OFI or GFI. These indices may indicate more uniform (i.e., at the Z-line) fiber disruption and high voltage ES of poultry may result in nonuniform fragmentation (tearing) throughout the myofibrils. Electron micrographs of VEERAMUTHU AND SAMS ES and ES/MT treated poultry carcasses have indicated that there was extensive, irregular fiber disruption and reduced z-line dissolution (Birkhold and Sams, 1995). A reduction in z-line disruption by ES/MT would suggest reduced m-calpain activity, and therefore contradicts the report of Etherington et al. (1990). However, the Etherington et al. (1990) study used a much lower stimulation voltage, in the range reported to not involve myofibrillar tearing in its mechanism of tenderization (Thompson et al., 1987). The activities of m-calpain and m-calpain are presented in Table 4 and Table 5, respectively. With ES, a large calcium release from the sarcoplasmic reticulum might induce a momentary increase in m-calpain activity and autolysis. This concept agrees with the results obtained by Etherington et al. (1990) in broiler carcasses and also with the results from beef carcasses by Ducastaing et al. (1985) and Uytterhaegen et al. (1992). Postmortem autolysis of m-calpain was also reported by Koohmaraie (1992). But it is also known that calcium ions are necessary for the protease to bind the inhibitors and that presence of calcium and inhibitor prevents autolysis of the enzyme (Kumamoto et al., 1991). The significant increase in m-calpain activity due to ES, when expressed as total activity per milligram protein, was noted at only 0 h and cannot be fully explained. Some activation mechanism by Ca +2 as suggested by Suzuki et al. (1981) may have occurred or the control-0 h mean may just be erroneously low. In the present study, m- calpain retained only about 25% of its original activity at 30 min after ES/MT treatment and the activity could not be detected at 2 h postmortem following ES. The control carcasses at the same time retained about 50 to 60% of their initial activity at 2 h postmortem. The m-calpain activity in ES treated and control muscles at 24 h was undetectable, indicating that m-calpain is an unstable enzyme and is inactivated by either autolysis or intermolecular rearrangement in postmortem storage as has been previously reported (Etherington et al., 1990). In contrast, m-calpain activity was only slightly affected by aging or ES/MT in both ES/MT and control carcasses. At 24 h postmortem, levels of m-calpain were 75 to 80% of the original activity in both control and ES/ MT treated carcasses. Similar results of m-calpain stability have also been reported for broiler and beef carcasses (Ducastaing et al., 1985; Etherington et al., 1990; Uytterhaegen et al., 1992). It is interesting to note that the slight decline in m-calpain activity during 24 h aging TABLE 1. Means 1 for ph of Pectoralis harvested at different postmortem times from broiler carcasses that received high voltage electrical stimulation (ES) Treatment 0 h 0.5 h 1 h 2 h 24 h Controls 6.67 ± 0.13 a 6.54 ± 0.09 a 6.55 ± 0.18 a 6.2 ± 0.26 a 5.65 ± 0.09 a ES/MT 6.40 ± 0.12 b 6.30 ± 0.06 b 6.21 ± 0.24 b 5.89 ± 0.17 b 5.69 ± 0.08 a

4 POSTMORTEM ph, MYOFIBRILLAR FRAGMENTATION, AND CALPAIN ACTIVITY 275 TABLE 2. Optical fragmentation index means 1 for Pectoralis harvested at different postmortem times from broiler carcasses that received high voltage electrical stimulation (ES) Controls ± a ± a ± 3.31 a ± 5.63 a ± a ES/MT ± b ± a ± 2.42 b ± b ± b Treatment 0 h 0.5 h 1 h 2 h 24 h TABLE 3. Gravimetric fragmentation index means 1 for Pectoralis harvested at different postmortem times from broiler carcasses that received high voltage electrical stimulation (ES) Controls ± b ± b ± 23.5 b ± b ± 9.49 b ES/MT ± 9.67 a ± a ± a ± 9.58 a ± a Treatment 0 h 5 h 1 h 2 h 24 h TABLE 4. Calpain activity per gram of muscle at different postmortem times of Pectoralis from electrically stimulated (ES) muscle tensioned (MT) and unstimulated broiler carcasses Enzyme Treatment 0 h 0.5 h 1 h 2 h 24 h m-calpain Control ± 0.34 a 1.46 ± 0.44 a 1.24 ± 0.12 a 0.63 ± ES/MT ± 0.16 a 0.36 ± 0.05 b 0.18 ± 0.03 b m-calpain Control ± 0.76 a 7.74 ± 0.04 a 6.15 ± 0.02 a 6.11 ± 0.18 a 5.55 ± 0.07 a ES/MT ± 0.41 a 6.72 ± 0.38 a 6.18 ± 0.01 a 5.57 ± 0.38 a 5.25 ± 0.15 a a,bmeans ± SD within an enzyme and column with no common superscript differ significantly (P < 0.05). 1No activity detected. 2n = 2 tissue pools for each mean. TABLE 5. Calpain activity per milligram of extracted protein at different postmortem times of Pectoralis from electrically stimulated (ES) muscle tensioned (MT) and unstimulated broiler carcasses Enzyme Treatment 0 h 0.5 h 1 h 2 h 24 h m-calpain Control ± 0.04 a 1.24 ± 0.04 a 1.22 ± a 0.76 ± ES/MT ± b 0.49 ± 0.05 b 0.29 ± 0.03 b m-calpain Control ± 0.21 a 4.90 ± 0.05 a 4.66 ± 0.05 a 4.89 ± 0.50 a 4.53 ± 0.51 a ES/MT ± 0.08 a 4.92 ± 0.03 a 4.72 ± 0.27 a 5.12 ± 0.04 a 4.44 ± 0.27 a a,bmeans ± SD within an enzyme and column with no common superscript differ significantly (P < 0.05). 1No activity detected. 2n = 2 tissue pools for each mean.

5 276 was not observed when the data were corrected for extractability by expressing the activity on a per milligram of protein basis (Table 5). This result suggests that the extractability of m-calpain (and possibly other proteins) declines during the first 24 h postmortem. A similar decline in extractability is known to occur as a result of the low ph and high temperature environment in pale, soft, exudative pork (Penny, 1969). The activity of m-calpain in postmortem muscles is influenced by several factors, including rate of ph and temperature decline, ultimate ph, initial calpastatin level and its postmortem decline, and the inactivation of m-calpain either through autolysis or denaturation. It is likely that the interaction of these factors determines the rate and extent of myofibrillar protein degradation (i.e., fragmentation) by m-calpain in postmortem muscle. It seems that with ES/MT, the faster decline in ph, along with the potential shifts in calcium concentrations and calpastatin degradation previously observed (Koohmaraie, 1992), cause a rapid decline in m-calpain activity. REFERENCES Birkhold, S. G., and A. R. Sams, Fragmentation, tenderness and post-mortem metabolism of earlyharvested broiler breast fillets from carcasses treated with electrical stimulation and muscle tensioning. Poultry Sci. 72: Birkhold, S. G., and A. R. Sams, Comparative ultrastructure of Pectoralis fibers electrically stimulated and muscletensioned broiler carcasses. Poultry Sci. 74: Birkhold, S. G., E. M. Hirschler, and A. R. Sams, Correlation between fragmentation index procedures and Allo-Kramer shear value of broiler breast fillets. Poultry Sci. 71(Suppl. 1):29. (Abstr.) Bradford, M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: Davis, G. W., T. R. 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