Ultrastructural and biochemical changes in electrically stimulated dark cutting beef

Meat Science 12 (1985) 177-188

Ultrastructural and Biochemical Changes in Electrically Stimulated Dark Cutting Beef S. F a b i a n s s o n & A. Laser Reuterswfird Swedish Meat Research Institute, POB 564, S-244 00 Kfivlinge, Sweden

& R. Libelius University of Lund, Laboratory of Clinical Neurophysiology, University Hospital, S-221 85 Lund, Sweden (Received: 2 March, 1984)

SUMMARY In clark cutting bceJ; with a high ultimate pH, ultrastructural and biochemical changes were jollowed dur#zg the very early post-mortem period in non-stimulated and electrically stimulated muscles. The consumption of A TP was extremely rapid in the electricallystimulated dark cutting muscle. In these sample, s pronounced changes in the ultrastructure were seen in theJorm ojheat T contractions and complete disorganization oJ the tissue. The changes could have been caused by a combined eJfi'ct of super-contractions and proteolytic activity. The changes seen in ek'ctrically stimulated samples were not reflected in improvements of the instrumentally evaluated temterness.

INTRODUCTION The tenderness of meat depends mainly on the variations in myofibrillar proteins, connective tissue proteins, water content and state, as well as the

interaction between the components during cooking (Dransfield, 1981). 177

Meat Science 0309-1740;85/S03-30 ~ Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain

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Some of these parameters could be influenced by the speed of the postmortem process (e.g. electrical stimulation) or the ultimate pH reached (e.g. D F D - - t h e high pH meat with a dark, firm and dry appearance). Several researchers have shown a tenderizing effect with the use of electrical stimulation (reviewed by Cross, 1979 and Bendall, 1980), although the exact mechanisms of action are unknown. DFD beef, with a high ultimate pH, is more tender than normal beef with an ultimate pH of about 5-5, whether sensorily or instrumentally evaluated (Bouton et al., 1973). Fjelkner-Modig & Rud~rus (1983) found that DFD meat tested by sensory evaluation 1 day after slaughter, whether electrically stimulated or not, was significantly more tender than meat with an ultimate pH at or below 5.8. This difference disappeared for electrically stimulated meat after 5 days of storage and for non-stimulated meat after 14 days of storage. There was also a tendency for electrically stimulated DFD meat to be more tender than non-stimulated DFD meat as early as the day after slaughter, but the difference was non-significant with the sole exception of 14 days of ageing. No explanations for the findings were given. Dutson et al. (1982) found that electrical stimulation did not influence the tenderness of DFD meat when sensorily or instrumentally evaluated 48 h after slaughter. They suggested that the rapid pH decline normally associated with the electrical stimulation was necessary in order to produce an increase in tenderness. We have been able to follow the post-mortem process in a DFD carcass, half of which was electrically stimulated immediately after debleeding. Pronounced ultrastructural changes in the muscle fibres, not reported before, were found very early during the post-mortem process in the electrically stimulated part. However, tenderness was not improved by electrical stimulation.

MATERIALS AND METHODS During a study of the effects of electrical stimulation on ultrastructural and biochemical changes (Fabiansson & Laser Reutersw'ard, 1984; Fabiansson & Libelius, 1984), the ultimate pH in one carcass remained well above 6-2 and the carcass was thus designated as DFD. The present report mainly contains results from this carcass, but some data from the other nine carcasses in the study are included for comparison. The animal was stunned by the use of a captive bolt pistol,

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exsanguinated and electrically stimulated within 10min of stunning. Stimulation continued for 32 s with a peak voltage of 85 V (current flow approximately 0"65 A) and the use of square wave pulses of 5 ms duration repeated every 72ms. The current was applied through a clip in the nostrils, with the shackle and overhead rail as the negative electrode. M. longissimus dorsi was removed from one side of the carcass immediately before and from the other side immediately after stimulation and the two sides were transported to the laboratory. The muscles were wrapped in polyethylene bags and stored at 22 °C for the first 3 h, at 10°C for 21 h and at 4°C for the following 6 days. Samples from the muscles were taken at regular intervals during the first day for transmission electron microscopy, histochemistry, and determination of pH, adenosine triphosphate (ATP) and creatine phosphate (CP). Sarcomere length was determined 2 days, and peak shear force 7 days, after slaughter. Samples for transmission electron microscopy were prepared by excising bundles of muscle fibres which, while kept in isotonic saline at room temperature, were carefully cleaned, slightly stretched and pinned to Stylgaard plates under a dissecting microscope. The muscle specimens were then fixed in 4 °//oglutaraldehyde in 0-1,~l caccodylate buffer (pH 7.5) for at least 4 h at room temperature. Small pieces of muscle fibres were cut with a razor blade and rinsed in buffer. The samples were postfixed for _~h in 2 °//o osmium tetroxide containing ferrocyanide (15mgm1-1) in 0-1M caccodylate buffer. The specimens were then rinsed in buffer, dehydrated and embedded in Polar bed 812. Semi-thin sections (l-2~tm) were cut on glass knives and inspected using phase-contrast microscopy. Ultra-thin sections (grey to silver) were cut on a diamond knife (Juniper Ultra Micro), stained with uranyl acetate and lead citrate, and examined in a Jeol 100CX electron microscope. At each testing time about 5-13 specimens were examined using phasecontrast microscopy and about 4-7 different blocks were sectioned and examined in the electron microscope. Histochemical staining was performed on I 0 ¢tm thick cross-sections obtained from unfixed muscle pieces frozen in isopentane cooled by a mixture of dry ice and ethanol. Staining for acid phosphatase was performed according to Burstone (1958) as modified by Barka (1960) using naphthol AS-BI phosphate as the substrate. Staining for NADHdiaphorase, glycogen (PAS) and phosphorylase was performed according to laboratory routine.

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The pH was determined in homogenized samples according to Bendall (1978). Samples for the determination of A T P and CP were immediately frozen and later pulverized in liquid N z mixed with 5 parts by weight of frozen HC10,,, homogenized for 2 x 15s, using an Ultra Turrax, and centrifuged at 40 000 g. The supernatants were neutralized using K H C O 3 solution, the resulting precipitate was centrifuged down and the metabolites were analysed using an enzymatic/spectrophotometric method with the addition of glucose-6-phosphate dehydrogenase, hexokinase and creatine kinase according to Lamprecht et al. (1974). Sarcomere length was determined using phase-contrast microscopy (Cross et al., 1980-81) and shear force was measured using a Warner-Bratzler shearing device mounted in an Instron Universal Testing Instrument according to Nilsson et al. (1979).

RESULTS A N D DISCUSSION The ultimate pH of the D F D carcass was 6-65. Electrical stimulation hastened the pH drop slightly, but in relation to the non-stimulated counterpart the difference never exceeded 0-2pH units. Electrical stimulation did, however, influence the consumption of ATP and CP, as illustrated in Figs l and 2. 6-

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Time post mortem (h)

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Fig. 1. The influence ofelectrical stimulation on the post-mortem A T P turnover rate in M. longissimus dorsi of normal and D F D carcasses. O , PH~s < 5-8, not stimulated (NS); O , pH~s > 6.2, NS; A , pH~s < 5.8, electrically stimulated (ES); A , pH.~s > 6-2, ES.

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Fig. 2.

T h e influence of electrical s t i m u l a t i o n on the p o s t - m o r t e m C P turnover rate in

M. longissimus dorsi of n o r m a l and D F D carcasses. C), pH~s < 5'8, not stimulated (NS), Q , pHas _> 6.2, NS; pHas < 5.8, electrically stimulated (ES); at, pH4s > 6-2, ES.

Mothersill & McLoughlin (1977), when examining normal, unstimulated carcasses, reported an initial value of CP in M. longissimus dorsi of 7.5/.zmol =,,-', which is very, close to the value of 7-2/.~mol g-1 given here fbr the same type of carcasses. In contrast to these figures, the value of 15-1~mlot g-~ found immediately after slaughter in the unstimulated D F D muscle was unexpectedly high. An excessive antemortem breakdown of glycogen is known to occur in the development of D F D (Lister & Spencer, 1981). If muscle activity demands less energy than the amounts made awfilable through the rapid degradation of glycogen, then the extra energy could probably be stored in the form of CP. This would indicate that there is no real need for the high antemortem muscle energy turnover of animals later showing D F D characteristics. The a m o u n t of C P decreased very rapidly during or immediately after electrical stimulation. The decrease in A T P was also extremely rapid in the electrically stimulated D F D muscle. In less than 45 min half of the initial A T P content was consumed. When the A T P reserves are depleted the muscle cells are not able to maintain membrane potentials and an efflux of Ca 2 + from the sarcoplasmatic reticulum to the cytoplasm is seen (Nakamura, 1973). A Ca 2+ activated neutral protease (CANP) responsible for Z-disc degradation has been reported (Nagainis & Wolfe, 1982). Electron micrographs of muscle samples were produced at 2, 3, 4 and

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6 h after slaughter. The non-stimulated D F D samples looked fairly normal at 2, 3 and 4 h (Fig. 3), but some minor irregularities in the Z-discs could be seen. At 6 h the Z-discs in some muscle fibres were broadened, the I-bands were reduced in width and part of the muscle seemed to be in rigor (Fig. 4). In the electrically stimulated D F D muscle some fibres showed changes after only 2 h, with less densely occurring thin filaments and disorganized Z-disc material resembling Z-band streaming (Fig. 5). At 3 h the I-bands were reduced in width in some samples and the Z-discs were broadened, much the same as in the non-stimulated samples at 6 h (Fig. 6). Other samples showed a relatively normal picture and the variability was thus great. At 4 h many muscle fibres showed contractions of some sarcomeres and concomitant tearings of neighbouring sarcomeres with an altered appearance of the Z-discs (Fig. 7). At 6 h there were signs of heavy contractions which resulted in a very dense appearance with a new banding pattern, with condensed material in the Z-disc region and the complete disappearance of the I-band regions (Fig. 8). Large parts of complete disorganization were also seen, with no identifiable structural components (Fig. 9). This picture is not seen in normal carcasses even

Fig. 3. U nstimulated sample 4 h post mortem. Well preserved ultrastrt,cture with slight local broadening of the Z-band (arrows). m, mitochondrion" × 11 750; bar = 1pm.

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Fig, 4. U n s t i m u l a t e d sample 6 h post mortem. Note s h o r t e n i n g of sarcomeres, slightly b r o a d e n e d Z - b a n d s and reduced width of the I-band. m, m i t o c h o n d r i o n ; x 11 750: b a r = 1 g~m.

Fig. 5,

Electrically stimulated sample 2 h post mortem. Z - b a n d streaming is indicated by asterisks, x 11 750" bar = 1/~m.

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S. Fabiansson, A. Laser Reuterswiird, R. Libetius

Fig. 6. Electrically stimulated sample 3 h post mortem. Shortening ofsarcomere length, slight broadening of the Z-band (Z) and reduced width of the I-band (I). x 11 750; bar = t Fire.

after ageing for 9 days (Gann & Merkel, 1978) or in electrically stimulated normal carcasses aged for 24 h (Fabiansson & Libelius, 1984). The mechanism of the Z-disc degradation seen in the present investigation cannot be determined, but may have been caused by CAN P. The activity of C A N P on Z-discs has been studied on myofibrils from rabbits (Suzuki et al., 1982), pork (Dayton et al,, 1981) and beef (Slinde & Kryvi, 1984). Prolonged digestion with C A N P removes the entire Z-discs. The activity of CAN P is enhanced by elevated C a 2 + concentrations and a pH value of around neutrality, conditions possibly prevalent very early in the electrically stimulated D F D muscle. Using histochemical staining it was possible to demonstrate the presence of oxidative enzymes at all the times tested (15 min, 5 h, 12 h and 24h) with a feasible differentiation between type I and type II muscle fibres. No detectable staining reaction with PAS was found, indicating a low glycogen content. The phosphorylase activity was lost after 12 h in the electrically stimulated muscle and after 24 h in the non-stimulated muscle. There was no response in the staining for acid phosphatase at any time tested and thus no evidence was found for an increase in the lysosomal enzyme activity.

Fig. 7. Electrically stimulated sample 4 h post mortem. Pronounced sarcomere shortening and contractures with concomitant tearings of I-band regions (asterisks). Arrowhead indicates visible. Z-band. x 1 [ 750; bar = 1 itm,

Fig. 8. Electrically stimulated sample 6 h post mortem. Advanced shortening of sarcomeres with loss of normal striation banding pattern and dissolution of myofilaments. Remnants of Z-bands are indicated by arrowhead, x 11 750; bar = 11~m.

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S. Fabiansson, A. Laser Reutersw~rd, R. Libelius

Fig. 9. EIcctrically stimt, lated sample 6 h post mortem. Advanced shortening of sarcomeres ~vith myoiibrillar dissolution and loss of myofibrillar continuity with replacement by a fine granular material (asterisk). x 11 750: bar = t .urn.

The shear forces for the non-stimulated normal (n = 90) and D F D carcasses ( n = 10) were 3.32 ( S D = 0 . 6 8 ) k g and 2.45 ( S D = 0 - 5 9 ) k g respectively. For the electrically stimulated normal (n = 90) and D F D carcasses (n = 10) the shear forces were 3.06 (SD = 0 - 5 9 ) k g and 2.70 (SD = 0-77) kg respectively. The difference was highly significant for the non-stimulated carcasses ( P < 0 - 0 0 1 ) , but non-significant for the electrically stimulated carcasses (P > 0.05). The results of FjelknerM odig & Rud~rus (1983) for sensory evaluation of tenderness after 5 days of storage were in accordance with our results, but the results of their shear force measurements did not show any significant differences between normal and D F D meat at this time, whether electrically stimulated or not. However, Bouton et at. (1973) showed a significant influence of pH on tenderness evaluated by shear force measurements for non-stimttlated carcasses. Chrystall et al. (1982) found that meat from exercise-stressed sheep, with a high ultimate pH, was more tender than meat from non-stressed animals. Electrically stimulated loin muscles of the exercise-stressed animals showed a tendency to be tougher than the non-stimulated muscles, much the same aswas found in the present study.

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Measurements of sarcomere length at 48 h post-mortem using phase contrast microscopy, showed very large variations. Sarcomere lengths from 1-15/~m up to 2-60pm were recorded for both the electrically stimulated and the non-stimulated D F D samples. The mean sarcomere lengthwas 1-57/~m for the electrically stimulated sample and 1-68 Ftm for the non-stimulated sample. The distance between the outer border of two adjacent bands in the banding pattern in Fig. 5 was of the same length as t h e myosin filament measured before the contraction, indicating overlapping of myosin filaments. As described by Marsh et al. (1974) the overlap of myosin from one sarcomere with actin from the next would produce continuously linked actomyosin extending for considerable length. This part of the tissue becomes very tough. The extensive shortenings found in some parts of the tissue could possibly counteract the tenderizing effect that would be assumed to occur because of the disintegration of the Z-discs (Nagainis & Wolfe, 1982) and the complete disorganization of parts of the tissue. Because of the variability f o u n d among and within muscle fibres, very tough and very tender parts could be dispersed over the structure. Such a variability could explain the inferior correlation between shear force measurements and sensory evaluated tenderness for D F D meat found by Fjelkner-Modig & Rud4rus (1983). Dutson et al. (1982) suggested that in the absence of the rapid pH decline normally associated with electrical stimulation, neither the stimulation itself nor the contractions produced were sufficient to produce the desired effects on palatability. The findings reported here indicate that electrical stimulation has a profound influence on D F D meat without a marked pH drop, but it does not produce the increase in tenderness that has been found for non-stressed animals. ACKNOWLEDGEMENTS This study was partly financed by grants from the Swedish Council for Forestry and Agricultural Research and the Swedish Medical Research Council, Stockholm, Sweden (14X-5953). REFERENCES Barka, T. (1960). Nature, 187, 248. Bendall, J. R. (1978). Meat Sci., 2, 91.

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Bendall, J. R. (1980). In: Developments in meat science--I (Lawrie, R. (Ed.)). Applied Science Publishers, London, p. 37. Bouton, P. E., Carroll, F. D., Fisher, A. L., Harris, P. V. & Shorthose, W. R. (1973). J. Fd Sci., 38, 816. Burstone, M. S. (1958). J. Nat. Cancer Inst., 21, 523. Chrystall, B. B., Devine, C. E., Snodgrass, M. & Ellery, S. (1982). N.Z.J. Agric. Res., 25, 331. Cross, H. R. (1979). J. Food Sei., 44, 509. Cross, H. R., West, R. L. & Dutson, T. R. (1980-81). Meat Sci., 5, 261. Dayton, W. R., Schollmeyer, J. V., Lepley, R. A. & Cortes, L. R. (1981). Biochem. Biophys. Acta, 657, 48, Dransfield, E. (1981). In: The problem of dark-cutting in beef(Hood, D. E. & Tarrant, P. V. (Eds)). Martinus Nijhoff Publishers, The Hague, p. 344. Dutson, T. R., Savell, J. W. & Smith, G. C. (1982). Meat Sci., 6, 159. Fabiansson, S. & Laser Reutersw~ird, A. (1984). Meat Sci., in press. Fabiansson, S. & Libelius, R. (1984). J. Fd Sci., in press. Fjelkner-Modig, S. & Rud+rus, H. (1983). Meat Sci., 8, 203. Gann, G. L. & Merkel, R. A. (1978). Meat Sci., 2, 129. Lamprecht, W., Stein, P., Heinz, F. & Weisser, H. (1974). In: Methods oJ enzymatic analysis (Bergmeyer, H. U. (Ed.)). Verlag Chemie, Weinheim/ Bergstr., p. 1777. Lister, D. & Spencer, G. S. G. (1981). In: The problem of dark-cutting in beef (Hood, D. E. & Tarrant, P. V. (Eds)). Martinus Nijhoff Publishers, The Hague, p. 129. Marsh, B. B., Leet, N. G. & Dickson, M. R. (1974). J. Fd Technol., 9, 141. Mothersill, C. & McLoughlin, J. V. (1977). Biochem. Soc. Trans., 5, 1741. Nagainis, P. & Wolfe, F. H. (1982). J. Fd Sci., 47, 1358. Nakamura, R. (1973). J. FdSci., 38, 1113. Nilsson, H., Rud~rus, H. & Fabiansson, S. (1979). Proc. 25th Eur. Meeting Meat Res. Workers, Budapest, Vol. I, p. 2.2. Slinde, E. & Kryvi, H. (1984). Eur. J. Biochem., in press. Suzuki, A., Matsumoto, Y. & Nonami, Y. (1982). Meat Sci., 7, 269.