Sarcomere Shortening of Prerigor Muscles and Its Influence on Drip Loss. K. O. Honikel, C. J. Kim*, R. Hamm. & P. Roncales

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1 Meat Science 16 (1968) Sarcomere Shortening of Prerigor Muscles and ts nfluence on Drip Loss K. O. Honikel, C. J. Kim*, R. Hamm nstitute for Chemistry and Physics, German Federal Centre for Meat Research, D-865 Kulmbacb, Federal Republic of Germany & P. Roncales Dept. Tecnologia y Bioquimica de los Alimentos, Facultad de Veterinaria, Miguel Servet 177, Zaragoza 13, Spain (Received: 25 February, 1985) SUMMARY Detailed studies of muscle shortening post mortem at incubation temperatures between - 2 C and + 38 C revealed that the sarcomeres in unrestrained, excised red bovine muscle (M. sternomandibularis) shortened less than 1% in the prerigor state between 6 C and 18 C. Below 6 C, sarcomeres contracted up to 7,'. Between 2 C and 38 C sarcomere shortening of 4 % was observed. n the red porcine M. cleidooccipitalis the minimum of shortening was measured at about 1 C, a higher degree of shortening--up to 5 lo--being obtained above and below this temperature. The drip loss of both muscle types increased linearly with increasing prerigor shortening. This latter relationship is discussed with regard to changes within the muscle post mortem. The influence of three events on water movement from the interfilamental space into the interfibrillar fluid and from there into the extraeellular space is critically evaluated. These events are." (1) the prerigor contraction of sarcomeres depending on the temperature of storage, (2) the changes due to the falling ph post mortem and (3) the onset of rigor mortis, with its irreversible association of aetin and myosin. * Present address: Seodaemoon-Gu, Mamgacha-Dong , Seoul, Korea. 267 Meat Science $3.5 Elsevier Applied Science Publishers Ltd, England, Printed in Great Britain

2 268 K. O. Honikel, C. J. Kim, R. Harem, P. Roncales NTRODUCTON During muscular contraction the contractile units of the myofibrils, the sarcomeres, shorten. Shortening is induced by the release of Ca 2 ions from the sarcoplasmic reticulum into the myofibrillar space. The movement of Ca a ions within the muscle cells post mortem depends on temperature, ph and ATP concentration present (Cornforth et al., 198; Whiting, 198; Honikel et al., 1983). Contraction of a muscle in a carcass shortly after slaughter, or of an excised prerigor muscle, can be, induced by electrical stimulation (Chrystall et al., 198) or by low (below 15 C) and high (above 2 C) temperatures (cold and rigor shortening, respectively) (Roncal~s et al., 1982; Locker & Hagyard, 1963). The degree of contraction of a muscle, especially of the muscle in a carcass, is furthermore dependent on the structure of the connective tissue network within the muscle, the cooperative action of adjacent muscles and the fixation of the muscle on the bones via tendons. n the last few years we have investigated the influence of temperature and subsequent prerigor shortening on biochemical changes and drip loss of bovine muscles (Honikel & Hamm, 1978; Honikel et al., 198a,b). Cold shortening and rigor shortening caused an increase in the drip loss after several days of storage, which Powell (1978) also reported. The minimum velocity of biochemical changes (Honikel & Hamm, 1978), minimum drip loss and minimum shortening of sarcomeres occurred at 5-15 C (Honikel et al., 198a,b). n this paper, the influence of temperature during the prerigor state on porcine muscles with regard to sarcomere shortening and drip loss is reported and compared with the results obtained with bovine muscles. MATERALS AND METHODS Bovine sternomandibularis muscle (weight about 25 g) and porcine M. cleidooccipitalis (weight about 1 g) were received within 3-45 min post mortem and trimmed of visible fat and connective tissue. These muscles were chosen as they contain similar high myoglobin concentrations (red muscles). The muscle was cut, in the direction of fibers, into two parts. One part was wrapped in alumina foil, put into a polyethylene pouch and

3 Sarcomere shortening and drip loss 269 incubated in a water bath at temperatures between - 2 C and 38 C. At intervals of approximately 45 to 6 min small pieces were cut off and used for immediate sarcomere length and ph measurements. The other parts of the samples were prepared for drip loss measurements and incubated in the same way. For each temperature, muscles of a different animal were taken. Measurement of sarcomere length At different times post mortem, 1-2 g of muscle samples were carefully cut with a knife and inserted in 2 ~ glutaraldehyde solution with 2 ~ glucose in a. lu phosphate buffer, ph 7., of similar temperatures as those at which the muscles were incubated, and sarcomere length was measured according to Voyle (1971) with a Helium-Neon laser. With this method a single fibre or a fibre bundle is placed into the laser beam. On determining the sarcomere length by laser diffraction we observed, under certain conditions at different positions of the fixed fibres, different diffraction lines within the first order of diffraction. n bovine muscles we measured the sarcomere length in duplicate; in the porcine muscles studied later we measured the sarcomeres in several samples up to six times in the same muscle. Measurement of ph About 5 g of muscle were cut into small pieces and homogenized with 1 ml of distilled water for 1 s in a Biihler homogenizer (Fa. Btihler, Tiibingen, Germany). The ph was measured immediately after homogenization. Measurement of drip loss A cube of muscle tissue about 3 cm long, was cut and weighed and hung up by a thread in a plastic pouch under atmospheric pressure. The pouch was sealed. For the first 24h such pouches with muscle cubes were incubated in the water bath at various temperatures; from day 2 on, all pouches were stored at C. After different times of storage the pouches were opened, the weight of the meat samples was measured and the samples were repacked for further storage. The measurements were taken in duplicate.

4 27 K. O. Honikel, C. J. Kim, R. Haram, P. Roncales RESULTS AND DSCUSSON nfluence of temperature and ph fall on sarcomere shortening Bovine M. sternomandibularis at ph 6.8 at about 1 h post mortem exhibited a resting sarcomere length of #m. On incubation at various temperatures for 24h the sarcomeres shortened. At C the sarcomeres contracted within 3min at ph by 1~o (Fig. 1), shortening further with falling ph. At ph 6.3 to 6. (6 to 1h post mortem) we repeatedly observed (one set of experiments is shown in Fig. 1) two different sarcomere lengths. These were obtained either on different samples taken at different positions in the muscle or at different positions of the same specimen taken for measurement. These results agree with the findings of Marsh et al., (1974) where a heterogeneity in sarcomere shortening was observed in cold shortened muscles. After 24 h at ph below 5.9, however, we again obtained only one spacing throughout the muscle which indicates a shortening of about 5 ~o. The sarcomere length was then about 1.~m. As we and others have shown earlier (Honikel et al., 1983; Powell, 1978) excised intact prerigor bovine muscles start to cold shorten at temperatures below 1 C-15 C at high ph shortly after reaching these temperatures. Cold shortening of intact muscles, particularly at low temperatures (5 C and below), sometimes occurred in a two-step process. The first immediate muscle shortening is limited to about 1-2 ~o (Honikel et al., 1983), followed by a lag phase, or sometimes e,ven a release at low load (Bendall, 1973). The second phase of shortening occurs with falling ph, depending on the load applied (Honikel et al., 1983). These observations have been made with muscles as a whole; however, what is true for muscles, should also apply for sarcomeres. According to Fig. 1, at C sarcomeres shortened immediately by 1~o, followed by a lag phase down to ph 6.2 when a further shortening of a part of the sarcomeres was observed together with a slower shortening of another part of the sarcomeres, both ending at an ultimate ph of 5.7 with about 5 ~o shortening. On incubation at 1 C the sarcomeres stayed nearly unchanged until the final ph was reached. At 15 C and 21 C, at ph values below 6. (8-1 h post mortem) a moderate shortening of about 1 ~o was observed. This shortening at low ph values increased considerably if the incubation temperatures were 3 C and higher. At these temperatures the shortening at the ultimate ph (24 h post mortem) was nearly as high as at C. n Fig. 2 the maximum shortening of sarcomeres after reaching the final ph at all

5 Sarcomere shortening and drip loss 271 Oo 5 4 h. c 1 l 1,,e ~ -or" 9---o i! f t i i 1 f 1 C o a~ 5 c if_ 4 "E o = 3 ~_ 2 c u 1 o o,.! t ls C i i!!, q]o i! 1 ~ ~ ~6 l,.i.. -l..-l_iiil--i -i d4 i i i l 3 C t! t o -1, p z "L_p_. -;---P-" - " C -, -- --~ 7--"1 38 C L _ ~_ - ~ ~ 1 6, , pH i e-- ~l,'l TpH Fig. 1. Shortening of sarcomeres of bovine M. sternomandibular& at various temperatures between + C and + 38 C as a function of the ph post mortem. The starting sarcomere length was tm. Each point ofsarcomere length is a mean of two measurements. n some muscle samples we found, at different positions, distinct sarcomere lengths. n this case two lines were drawn. t

6 272 K. O. Honikel, C. J. Kim, R. Harara, P. Roncales 7 --'1 } 6C ~, 5 "U o ~LO. 3 E 2O E l x O Fig L L +38 C temperature of incubation Maximum shortening of bovine sarcomeres at ultimate ph values, depending on the temperature of incubation. For details see Fig 1. temperatures of incubation between -2 C and + 38 C is shown. There exists a minimum of shortening between 6 C and 12 C, increasing slightly between 12 C and 22 C. Below 6 C and above 22 C there is an abrupt increase of sarcomere shortening. On incubation at low temperatures cold shortening started shortly after reaching the temperature at ph values above 6., whereas, at temperatures above 2 C, rigor contracture leading to a considerable shortening of the sarcomeres did not occur until shortly before, or at the onset of, rigor mortis at ph values below 6.. This is in agreement with our earlier findings (Honikel et al., 198b) where we reported minimal shortening of sarcomeres of bovine muscles at 8 o to 12 C. Figure 3 shows similar experiments to those in Fig. 1 but with the M. cleidooccipitalis of pork. On incubation at C this red porcine muscle, which had an initial ph of 6.4, also experienced, within 3min of incubation, considerable sarcomere shortening at ph values of 6.3 and below. At 11 C the degree of shortening was considerably lower with about 1~o shortening at ph values below 6.. Unlike bovine muscles (Fig. 1), however, at 15 C and 21 C porcine muscle sarcomeres shortened (after 2 to 3 h of incubation) below ph 6.2 considerably and extensively, like the muscles after 1.5 h at 3 C and 38 C (Fig. 3). Figure 4 shows that there was a minimal shortening of porcine muscle sarcomeres at about 1 C. Cold and rigor shortening have again taken place in these muscles. n porcine muscles, however, pronounced rigor

7 Sareomere shortening and drip loss 273 o o t~ c 5 E 4 '- 3 ffl '" 2 c- U a o 5O i _ O C l i ~) ~ 15 C _ ll C 1 21 C 3 C.. 35 C 8 ~-o~6 o -- ~.4 o o -- O.o'8 ~ oo O o16o 8 r! O O O (~ O TpH ph Fig. 3. Shortening of sarcomeres of porcine M. cleidooccipitalis (shoulder) at various temperatures from C to 35 C, depending on the ph post mortem. The starting sarcomere length was pm. Each point of sarcomere length is a mean of two measurements. n some muscle samples we found, at different positions, distinct sarcomere lengths. n these cases two lines were drawn.

8 274 K. O. Honikel, C. J. Kim, R. Harem, P. Roncales u % ~ 2 c li J ~ r r \ j \, \ \, \ \ \ \ \ Fig. 4. i, L, 2~8,, Z ' C temperature Maximum shortening of porcine sarcomeres at ultimate ph values, depending on the temperature of incubation. For details see Fig 3. shortening occurred at lower temperatures (above 12 C; Fig. 4) than in bovine muscles (above 22 C; Fig. 2). Also, with regard to ph values, a difference was observed; in porcine muscles rigor shortening started at ph , whereas, in bovine muscles, it occurred below 6.. Similar results with regard to the temperature range of minimum sarcomere shortening were obtained with the red porcine diaphragm muscle whereas, in the light longissimus dorsi muscle from pigs, the temperature of minimum shortening was found to be about + 4 C (Fischer et al., 198). SHORTENNG AND DRP LOSS Powell (1978) and Honikel et al., (198a,b) showed that prerigor bovine neck muscles exhibited minimum drip loss at storage between 8 and 15 C, in the same temperature range where minimum sarcomere shortening at the ultimate ph is observed (Fig. 2). The question arises as to whether the degree of sarcomere shortening is responsible for the extent of drip formation during the storage of muscle. n agreement with the earlier results (Honikel et al.,! 98b) we found, in the present work, that the drip loss of bovine muscles was lowest when the prerigor muscles were incubated at about 12 C (Fig. 5). This minimum corresponds well with

9 - x Sarcomere shortening and drip loss 275 i, i i L from day 2 O C 1 e ~ e o 6 \ ~. 7 \\ day! x A--&- - x x " x x1~ ~ Z, C muscle temperoture O-24hours Fig. 5. Drip loss of bovine muscles versus temperature of incubation at the first day post mortem. After 24 h the muscles were stored at C; the days of storage are indicated by the numbers 1, 4, 5, 6 and 7 on the right-hand side of the Figure. the minimum shortening of sarcomeres. The same is also true for porcine muscles, as shown in Figs. 4 and 6. As shown in Fig. 7, drip loss after 7 days of storage exhibits, in both muscles, a close and linear relationship with sarcomere shortening Drip loss is apparently due to the changes of the microstructure of the muscle cells as revealed by sarcomere shortening. Similar conclusions about the ~ol i,!! i!! from doy2 C 7 ~N _?4 D [] U 3 o Q oi o n o 13 ~ o ~ ~ ~ l,,,, ( C muscle temperqture Qt -24 hours Fig. 6. Drip loss of porcine muscles versus temperature of incubation at the first day post mortem. After 24 h the muscles were stored at C: the days of storage are indicated by the numbers 1, 3, 6 and 7.

10 276 K. O. Honikel, C. J. Kim, R. Harem, P. Roncales Fig '~~o ~ par ol L jura ) final sorcomere length Relationship between final sarcomere length and drip loss after 7 days of storage. changes in muscle cell structures were drawn by Marsh & Leet (1966) who observed, in prerigor frozen and rapidly thawed muscles (partially constrained on thawing), a relationship between drip formation and shortening. n these thaw-contracted muscles, however, a high drip was produced only if the shortening exceeded 4 ~o, i.e. in the unrestrained muscles. The reason why the less thaw-contracted muscles showed low drip remains unexplained. RELATONSHP BETWEEN POST-MORTEM CHANGES N MUSCLE AND DRP FORMATON t is well known that the fall of ph in muscles post mortem changes the water-holding capacity (WHC) of the muscle. The close, and almost linear, relationship between shortening of sarcomeres in the prerigor state and during the onset of rigor mortis and the extent of drip loss during the storage of meat post rigor (Fig. 7) is another factor which influences the WHC of meat. Water retention of meat is supposed to be caused primarily by an immobilisation of tissue water within the myofibrillar system (Hamm 196, 1972, 1975, 1984). This means, of course, that a remarkable part of the immobilised water must be located within the thick filaments and between the thick and thin filaments of the myofibril. This concept was recently confirmed by Offer & Trinick (1983) who studied the swelling and shrinkage of myofibrils in salt solutions by microscopical techniques. t can be expected that a shift of water from the intracellular into the

11 Sarcomere shortening and drip loss 277 extracellular space and, finally, on the meat surface (drip formation) is a result of structural alterations within the level of sarcomeres or of myofilament structure. The question arises how protein interactions within the thick filaments and between thick and thin filaments post mortem might cause shifts of cellular water. n the myofibrillar system three different processes take place post mortem: (1) a more or less pronounced shortening of the sarcomeres (contracture), depending on incubation temperature; (2) the development of rigor mortis, i.e. an irreversible association of thick and thin filaments after depletion of ATP (see Hamm, 1982); (3) the fall ofph which causes alterations of the structure in the myofibrillar proteins (mainly myosin and actin). The importance of the first process, namely, sarcomere shortening, for the extent of drip formation stimulates considerations concerning changes in the state of tissue water during contraction of the living muscle. X-ray diffraction studies on living muscles have shown that no water inflow or efflux occur so that the sarcomeres remain isovolumetric during both active contraction and passive stretch (Huxley, 1953; Elliot et al., 1963; Morel et al., 1976; Heffron & Hegarty, 1979) and that a constant volume relationship exists over a very wide range of sarcomere length (Millman, et al., 1981). Over a wide sarcomere length range, myofilamental lattice spacing of relaxed glycerinated muscle varied so that, as in living muscle, a constant lattice volume is maintained. (Rome, 1972). From these facts we conclude that, during contraction of the living muscle or ofa prerigor muscle post mortem the amount of water, not only in the cell but also within the filamental system of the fibre, does not change; thus contraction must cause an increase of the distance between myofilaments within the sarcomeres (stage B in Fig. 8). Accordingly, during relaxation the distance between filaments decreases (B--,A in Fig. 8). As X-ray studies with skinned frog muscle fibres showed, the sarcolemma is necessary to maintain constant lattice volume on the skeletal muscle fibre (Matsubara & Elliott, 1972). These authors believe that this effect of the sarcolemma is not a mechanical one but is due to osmotic phenomena. They write: 'f we assume that the total number of the negative poly-ions in the fibre (fixed charges of the myofilaments and of impermeable anions) does not change during the length change, then any increase in the fibre volume, if it were to occur, would reduce the concentration of these impermeable negative charges. Because of the Donnan equilibrium the internal concentration of the electrolytes would

12 278 K. O. Honikel, C. J. Kim, R. Harem, P. Roncales thin fitoment thick fitament Z-l.ine sorcomere A prerigor relaxation t ~ contraction B prerigor unshortened shorte ned (i5ovo[umetr,c) ~ rigor mort~5 oooo o C postrigor OO ooooo o ooooooooo ~oooooo Fig. 8. Scheme of changes in sarcomere structure during contraction and changes post rigor. Open circles indicate water molecules. The possible ph-dependent changes in the volume of thick filaments post mortem are not considered in this Figure. (A) Prerigor sarcomeres unshortened. (B) Prerigor sarcomeres; the right sarcomere is shown in the contracted state but isovolumetric to the left sarcomere which remains unshortened. (C) Postrigor sarcomeres at the same state of shortening as B. decrease, resulting in a decreased osmotic pressure in the fibre fluid. Consequently, water would move out of the fibre until the original volume was restored. Conversely, if the fibre volume were to decrease, an increased osmotic pressure would restore the original volume'. Elsewhere the authors claim: 'The constant volume phenomenon in whole muscle cannot be due to the unavailability of water from the interstitial spaces because it can be observed also with single muscle fibres' (Matsubara & Elliott, 1972). As to rigor contracture, it was found that the total muscle volume (fibres plus extracellular space) does not change during contraction (Heffron & Hegarty, 1974); this seems to be in agreement with our observation that post-mortem contracture and rigor development cause

13 Sarcomere shortening and drip loss 279 little or no immediate shrinkage of bovine muscle (Honikel et al., 198b; Hamm, 1982; Fig. 5) or porcine muscle (Fig. 6). Of course, constant volume of muscles during cold shortening or rigor contracture means that the muscle becomes thicker. This could be caused by an increase in the diameter either of the fibres or of the extracellular space, or of both. As was explained above, during contraction of the living muscle a constant sarcomere volume is maintained, primarily by an increase in the distance between the filaments, which results in a larger fibre diameter. s that also the case in rigor contraction? t has been demonstrated that, during rigor development, the muscle fibre diameters decrease (Hegarty, 197) and the extracellular space increases (Heffron & Hegarty, 1974; Currie & Wolfe, 198, 1983). Thus, during rigor development wa~ter must migrate from the intracellular into the extracellular space. n muscle with shortened sarcomeres this shift of water is apparently more pronounced because the extent of drip formation is greater after several days of storage (Fig. 7). The release of water post rigor from the filamental system of sarcomeres could be due to a decrease of the distance between thick and thin filaments because it was demonstrated that the interfilamentary spacing of skinned muscle fibres (frog) decreased from the relaxed to the rigor state. The magnitude of this change was roughly proportional to the overlap between thick and thin filaments (Goldman et al., 1979). Maughan & Godt (1981) observed, with the same type of fibres, that with rigor, fibre widths decreased with shrinkage, being greater at shorter sarcomere lengths. The question arises as to whether the release of water from the shortened sarcomeres takes place as a result of the sliding of filaments before the onset of rigor or as a result of cross-linking between thick and thin filaments during the development of rigor. There is apparently no information in the literature to answer this question. t should be mentioned that a shrinkage of the myofibrillar system with a decrease of water-holding capacity occurs post mortem before the onset of rigor mortis because of the effect of decreasing ph (Hamm, 1981, 1982). As mentioned above the constant volume phenomenon in the contraction of living muscle can be explained by osmotic effects due to the low permeability of the cell membrane for ions. The increase in permeability of the membrane for ions taking place post mortem will certainly eliminate this type of osmotic effect. t was suggested that, during rigor, the extracellular space becomes hypertonic and contributes to a movement of water into the extracellular space (Heffron & Hegarty,

14 28 K. O. Honikel, C. J. Kim, R. Harem, P. Roncales 1974). Such a hypertonia does not explain the effect of sarcomere shortening on the shift of water post mortem. Therefore, we suggest that the degree of overlapping of myofilaments, rather than osmotic effects, is decisive for drip formation. Also, Millman, et al., (1981) mentioned that the shrinkage of the filament lattice of frog muscle that takes place during the first hours of post mortem does not appear to be just a simple osmotic effect. These authors found it surprising that the shrinkage was greater at 8 C than at room temperature; they did not realize the possibility of a cold shortening effect. The release of water from the muscle cell post mortem might not be the result, but rather the cause, of osmotic effects. By the release of water from the myofibril into the space between the fibrils the sarcoplasm will be diluted and its osmotic pressure is reduced. Water would then be transferred across the cell membrane to appear in the extracellular space (Offer & Trinick, 1983). mmediately after rigor development even muscles with strongly shortened sarcomeres show no, or only little, drip formation; the effect of sarcomere shortening on drip formation becomes evident only during storage of muscle post rigor (Honikel et al., 198b; Hamm, 1984; Figs 5 and 6). Apparently, the rate of increase in drip formation post rigor is a function of the slow migration of extracellular water along the extracellular channels to the surface of muscle, not a function of shift of water from the interfibrillar spaces of muscle fibres into the extraceuular space, because Heffron & Hegarty (1974) showed that the decrease in fibre diameter in skeletal muscle reaches its maximum within few hours post mortem and does not significantly change between 4 and 24 h post mortem. Currie & Wolfe (1983) explained differences in the decrease of waterholding capacity (increase of juice expressible from incubated tissue) of semitendinosus muscle from different beef carcasses upon rigor mortis with differences in the permeability of the cell walls for water; the membrane would resist the movement of the water out of the intracellular space, and increasing disruption of the membrane would facilitate the release of water in the extracellular space. n these experiments, an increased permeability of the membrane was derived from an increased uptake of inulin. However, the molecule of inulin is very large; thus, an increase in the permeability for inulin does not necessarily also prove an increase in the permeability of the membrane for the small water molecules. The permeability of the cell wall for water may already be high in the living muscle, otherwise Donnan equilibria (or the constant volume

15 Sarcomere shortening and drip loss 281 phenomenon during contraction; see above) could hardly exist. A disruption of cell membranes in these normal bovine and porcine muscles is very unlikely as the drip is released from the meat after only a few days, whereas, in PSE porcine muscles where a leakage of the cell membranes is discussed (Gallant et al., 1979), the exudate appears within 2 to 4 h post mortem at the meat surface (own observations). Further research is necessary to clarify the relationship between membrane failure and drip formation or other water-holding phenomena. Taking all the evidence reported above into account, the release of drip from muscles seems to be dependent on the state of contraction (contracted sarcomeres, fibrils, or fibres) after the onset of rigor and is due to the shrinkage of filamental spacing (perhaps also to changes in the cell membrane) which results in the release of water into the extracellular space. ACKNOWLEDGEMENTS C. J. Kim wants to thank the German Federal Ministry of Agriculture, Forestry and Nutrition for a fellowship; P. Roncal6s thanks the Alexander von Humboldt Foundation for another fellowship sponsored by the Krupp Stiftung. We also appreciate the technical assistance of Mr R. Egginger. REFERENCES Bendall, J. R. (1973). 19th Europ. Meeting Meat Research Workers, Paris, Vol. 1,1. Chrystail, B. B., Devine, C. E. & Davey, C. L. (198). Meat Sci., 4, 69. Conforth, D. P., Pearson, A. M. & Merkel, R. A. (198). Meat Sci., 4, 13. Currie, R. W. & Wolfe, F. H. (198). Meat Sci., 4, 123. Currie, R. W. & Wolfe, F. H. (1983). Meat Sci., 8, 147. Elliot, G. F., Lowy, J. & Worthington, C. R. (1963). J. Mol. Biol., 6, 295. Fischer, C., Honikel, K. O. & Hamm, R. (198). Fleischwirtsch., 6, 263. Gallant, E., Godt, R. E. & Gronert, G. A. (1979). Muscle & Nerve, 2, 491. Goldman, Y. E., Matsubara,. & Simons, R. M. (1979). J. Physiol., 295, 8P. Hamm, R. (196). Advances Food Res., 1, 355. Hamm, R. (1972). Kolloidchemie des Fleisches. Verlag Paul Parey, Berlin, Hamburg. Hamm, R. (1975). n: Meat (Cole, D. J. A. & Lawrie, R. A. (Eds)). Butterworths, London, p. 321.

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