ELSEVIER

Meat Science 40 (1995) 217-234 ~'.) 1995 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0309- i 740/95/$9.50

0 3 0 9 - 1 7 4 0 ( 9 4 ) 0 0 0 5 4 - 9

Structural and Mechanical Changes in Raw and Cooked Single Porcine Muscle Fibres Extended

to Fracture

Gabriel Mutungi, a Peter Purslow'* & Chris Warkup b

~Muscle and Collagen Research Group, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol, BSI8 7DY, UK

hMeat and Livestock Commission, P.O. Box 44, Winterhill House, Snowdon Drive, Milton Keynes, MK6 lAX, UK

(Received 16 March 1994; revised version received 2 August 1994; accepted 7 September 1994)

ABSTRACT

Tensile tests on shlgh, muscle fibres .from raw and cooked porcine Iong- issimus thoracis muscle were per/brmed to explore the structural mech- anisms responsible for their d~brmation and fracture properties. Measurements of load and &f/brmation were made simultaneously with light microscopy observations of the structural changes which occur on extension. On extending the fibres to fracture, an r-shaped stress-strain curve was observed and the structural changes which occurred during this process could be divMed into three phases. Phase one, was characterised by a rapid increase in stress with little change in strain and ended at the yield point. Sarcomere length was uniform along the fibre in thL¥ initial phase. Raw fibres yielded at strains of between 2 and 5% of their resting lengths and cooked fibres at strains of between l0 and 20%. in phase two, there was rapid increase in strain with minimal changes in stress. In most fibres this phase was characterised by multiple cracks on the fibre surface and unequal sarcomere stretching. Sarcomeres in the regions where the surface had ruptured extended faster than those in areas still covered by the surface membrane, where sarcomere length remained relatively unchanged. In some cooked fibres, there was little or no surjbtce cracking and all the sarcomeres in these fibres extended almost uniformly. Phase three was characterised by a rise in stress as strain increased and then a final fall in stre.~s at the

*Author to whom correspondence should be addressed at: Anatomy Unit, Urfiversity of W'~les, P.O. Box 911, Cardiff, CF! 3US, UK.

2i7

218 G. Mutungi, P. Purslow. C. Warkup

breaking pohzt. This was accompanied by myofibrillar failure and finally breakage of the whole fibre. The myofibrils did not always fail as one unit; a progressive snapping o f small bundles ofmyofibrils was seen in some raw fibres. Muscle fibres could be stretched to 10"9 + 1.45% of their resting length before breaking when raw, but to 130 + 42% of their rest lengths after they were cooked for 1 h at 80°C.

Where multiple surface cracking was observed in phase two, sarcomeres in some cracked areas lengthened faster than others and the cracked areas which extended fastest were usually the focus o f the eventual failure o f the .fibre. hi raw fibres, sarcomeres in the areas where the fibre surface had ruptured could be stretched up to 107.7% before failure, while those in areas o f the fibre with an intact surface remained relatively unchanged. In cracked areas of cooked fibres the sarcomeres were more extensible and could be stretched to 169.7% before breaking. The order-of-magnitude ipcrease in overall extension to faih~re of fibres resulting from cooking is only partially due to this increase in sarcomere extensibility in cracked areas. Mechanically demembranating raw fibres depressed the stress at wl:ich yielding occurred and doubled their breaking strain. However, this process had no effect on the stress at which the fibres fractured. The results s;ww that de[brmation is not uniform along individual fibres, especially in the ra~, case and that the endomysium has an important contribution to this non-unijbrm deformation.

INTRODUCTION

Meat is usually cooked before consumption, a process which radically alters its structure and greatly influences its toughness and iuiciness. Toughness is prob- ably the most important determinant of meat eating quality. The extent to which meat eating quality is affected by the cooking process will depend on the con- nective tissue content of the muscles, post mortem chilling, storage regime, cook- ing methods, temperatures and times employed. Despite the obvious importance of cooking as the end-process in the production and preparation chain for meat, the structural changes which occur during this process and their contributions to the resultant alterations in the eating q~;ality of meat are not altogether under- stood.

Many of the current ideas on the effects of cooking meat on its eating quality, especially the myofibrillar contribution to meat toughness, are based on inference and not direct observation. For example, when meat is cooked and tested using the MIRINZ tenderometer, its toughness increases with cooking temperature in two phases. The first phase occurs at temperatures of between 40 and 60°C and the second between 65 and 80°C, with little changes in toughness at temperatures of 6065°C (Davey & Gilbert, 1974). The first increase in toughness has been attributed to heat denaturation of myofibrillar proteins, especially myosin. The solubility of myosin decrea;es at temperatures above 40°C and the onset of its denatmation as measured, by differential scanning calorimetry has been |ound to occur at temperatures b:tween 50 and 60°C depending on the pH of the meat (Wright et al., 1977; $tabursvik & Martens, 1980). The second rise in

Structural changes in single fibres 219

meat toughness as it is cooked to temperatures above 65°C has been ascribed to the toughening and shrinkage of intramuscular collagen whose denatura- tion has been found to occur at temperatures of 65-70°C (Martens & Void, 1976).

However, these often quoted results and inferences do not seem to agree with the results of earlier work using the Warner-Bratzler shear press, where tough- ness appeared to increase with cooking temperature to 50°C, but to decrease at temperatures between 50 and 60°C before rising again at temperatures above 60°C (Bouton & Harris, 1972). The decrease between 50 and 60°C is less marked in older animals, a result which led these authors to infer that connective tissue may be involved in determining meat toughness at temperatures below 60°C. Similarly, the initial yield value from Warner-Bratzler tests, which is thought to reflect the strength of the myofibrillar component of the muscle, showed the same increases with cooking temperature as the meat toughness results reported by Davey & Gilbert (1974). This led Harris & Shorthose (1988) to infer that the MIRINZ tenderometer, which Davey & Gilbert used, may measure only the myofibrillar contribution to meat toughness. If this is indeed the case, then the second rise in toughness reported by Bouton & Harris (1972) with cooking tem- peratures of between 65 and 80°C must be a reflection of changes in the myo- fibriilar structure and not ~olely in the con~:~cctive tissue as thought previously. These alternative inferences al~d explanations by Davey & Gilbert (1974) and Harris & Shorthose (1988) highlight the fact that simple empirical tests on whole meat do not provide direct and definitive evidence of the structural basis of toughness development during cooking.

To provide a fundamental explanation of the changes in meat texture as caused by structural alternations brought on by cooking, there is a requirement for basic studies of the structural and mechanical changes in the individual components of the muscle tissue (i.e. muscle fibres and connective tissue), as well as studies on the whole material to see how the properties of the two major components inter- act. D~rect observations of the structural and mechanical changes on cooking in the major collagenous component of meat (perimysium) have been provided by Lewis & Purslow (1989). They carried out experiments on perimysium excised from meat cooked to different temperatures, arguing that the mechanical prop- erties of the perimysium as defined by the amount of restraint when cooking in situ within the meat was most relevant to its contribution to cooked meat texture. Their results showed that the strength of perimysium increased with cooking temperatures until 50°C due to the crimped fibres straightening out, and decreased at higher temperatures. The thermally induced shrinkage of collagen observed at temperatures above 60°C may contribute to the increasing meat toughness at these temperatures by producing shrinkage in the myofibrillar material rather than by any increase in its intrinsic strength.

Whilst a good deal is therefore known about how the structural changes in the major collagenous component of meat result in changes in its mechanical prop- erties on cooking, similar information is not largely available for the myofibrillar components. Ultrastructural investigations have demonstrated some of the changes in the appearance of the sarcomere with cooking (Schmidt & Parrish, 1971; Locker et al., 1977; Locker & Wild, 1982), and how this appearance changes when cooked muscle is stretched to medium extensions (Locker & Wild, 1982; Locker et al., 1977), but these investigations have not been allied to mechanical

220 G. Mutungi, P. Purslow, C. Warkup

measurements. Although the extensibility of single muscle fibres has been reported to increase on heating (Wang et al., 1956; Hostetler & Cover, 1961) there are no reported studies to date of the detailed load-extension behaviour of cooked single muscle fibres.

The purpose of this study was to investigate the relationships between the structural and mechanical changes which occur on cooking single muscle fibres and to relate the changes in the myofibrillar component of muscle on cooking to changes in the whole tissue as a composite in which muscle fibres and connective tissue interact.

MATERIALS AND METHODS

Twelve pork loin samples, each 12 cm long from anterior to the last rib, were used in these experiments. All animals were reared at the MLC Stotfold pig development unit and were typical of pigs slaughtered in Great Britain, being in the weight range of 60-70 kg carcass weight. After evisceration and splitting, carcases were suspended from their achilles tendons and conventionally chilled overnight in a commercial abattoir (air temperature ~I°C). After chilling the samples of boneless loin were prepared, vacuum packaged and blast frozen to =30°C at approximately 24 h after slaughter. Subsequent storage was at-20°C in the laboratory until they were used. Eight of the samples used in the structural and mechanical studies on intact single fibres were frozen 1 day after slaughter. The samples were all obtained on t~le same slaughter date. A further four samples were stored in vacuum packs at I°C until 72 h post-slaughter and then blast frozen. These were used in a study of the effects of demembranation of single fibres. Plastic-wrapped loin samples selected for experiments were slowly thawed first under ice cold water and then under cold tap water. The way the muscle fibres run in the hmgissimus thoracis muscle was established and small muscle blo,'ks 1 cm wide and 5 cm long were carefully dissected out with all their fibres running parallel to each other. A quarter of the blocks were kept raw at 2°C. The rest were packaged in plastic bags and cooked in a water bath held at 80 + 0.5°C for ! h. The remaining muscle was then cut into small pieces and cooked at 80 :t: 0.5°C for 2 h and all the exudate collected, centrifuged and the supernatant collected for ~se as bathing medium for the mechanical experiments involving cooked samples.

A small strip of muscle was carefully dissected from each block and placed in a perspex dissecting dish half-filled with cured silicon elastomer (SYLGARD). Dissections were carried out under a solution containing either 400 mM mannitol with 50 mM potassium acetate for raw meat (Winger & Pope, 1981) or the supernatant obtained after centrifuging the exudate produced on cooking meat, for cooked muscles. Single muscle fibres were isolated under a binocular dissect- ing microscope at room temperature. The muscle fibres were then rapidly trans- ferred to thin aluminium plates, 4-5 mm wide and 25-30 mm long, so that they extended across a rectangular gap 2900 ± 50 #m cut on one side of the plate. Care was taken not to stretch the fibres during the transfer process. The fibre ends were then glued onto the plate using cyanoacrylate glue and transferred back into the original bathing solution. The whole attachment process took less than 1 min.

Structural changes in single fibres 221

In some preparations the membranes covering the surface of the fibres were mechanically removed.

The plate was then attached using cyanoacrylate glue onto two hooks, one attached to a motor and the other to the force transducer of a small mechanical testing device (Lewis & Purslow, 1989) mounted an the stage of a Leitz laborlux S microscope fitted with long working-distance objectives. The assembly was immersed in a chamber containing the same solution as that used during the iso- lation process. The piece of aluminium plate bridging the gap onto which the fibres had been mounted was then carefully cut leaving the fibre as the only structure joining the two plate ends. The initial length and diameter of the fibre was recorded via the microscope optics and the fibre slowly extended at a constant rate of 3.9 #m s -~ until it ruptured co w?!ete!y. The mechanical and structural changes which occurred as the fibre was extended until it fractured were monitored simultaneously using a chart recorder to register loads and extensions and a photographic camera and a video camera mounted on the microscope.

RESULTS

Figure 1 shows (A) the stress-strain curve of a single raw muscle fibre isolated from pig longissimus thoraeis muscle frozen at 24 h post-slaughter, and (B) the structural changes which occur in this fibre as it is stretched to breaking point. The breaking strain of fibres varied from animal to animal but little variation was noticed between samples from the same animal. Therefore, to minimise the

(A) 3

~-" 2

l r, cj

0

4 .5

3&

0 4 8 12

Strain (%)

Fig. 1. (A) A stress-strain curve for a typical single raw muscle fibre extended at a con- stant rate until fracture. Numbered arrows correspond to the point at which photographs in (B, overleaf) were taken. (B) The corresponding structural changes occurring at these

points in the stress-strain curve. Magnification x 530.

222 G. Mutungi, P. Purslow, C. Warkup

B

Fig. I. (B).

Structural changes in single fibres 223

5

. . . . . . , = o i ~ • ~ o '

Fig. 1. (B)--Continued.

224 G. Mutungi, P. Purslow, C. Warkup

variation iv. our observations, tour muscle samples from the same animal per treatment were used for each set of experiments comparing the effects of cooking, or of demembranation.

Raw single muscle fibres isolated from pig longissimus thoracis muscle frozen at 24 h post-slaughter could be stretched to 10"9 + 1"45% of their resting length before they broke. The stress-strain curve was r-shaped and the structural changes which occurred during this process could be divided into three phases. During phase one, no changes were observed in the appearance of the fibre; this phase was characterised by a rapid increase in stress with little change in strain and ended at the yield point. Raw fibres yielded at strains of between 2 and 5% of their resting lengths. The stiffness (Young's modulus) in the initial region of Fig. I(A) is 5.04 MPa. Sarcomere length is uniform along the length of the fibre in phase one, as shown by frame 2 of Fig. I(B).

Fig. 2. A single raw muscle fibre (a) at rest length, showing uniform sarcomere length, and (b) the same fibre after extension to beyond the yield point, showing heterogeneous sarcomere lengths; highly stretched sarcomeres occur in an area of the fibre where the surface membrane has cracked (arrow), whereas less extended sarcomeres are seen in

another area where the surf,ce membrane is intact (U). Magnification x 530.

Structural changes in single fibres 225

Fig. 3. A single raw muscle fibre (a) at rest length, (b) extended to the onset of fracture, and (c) after fracture has progressed by a small amount. Note in (b) & (c) the failure of

separated bundles or domains of myofibrils. Magnification x 530.

226 G. Mutungi. P. Purslow. C. Warkup

In phase two, strain increased rapidly with minimal changes in stress. This phase was characterised by rupture of the connective tissue and sarcolemmal membranes on the fibre surface at multiple sites along the length of the raw fibre. Surface cracking was followed by maequal sarcomere stretching, with those sarcomeres in the cracked regions extending faster than those in areas still covered by the surface membrane which remained relatively unchanged. This is demonstrated in frames 3-4 of Fig. 1 (b) and again in Fig. 2. In some raw fibres, the cracks were seen to occur at regular intervals of 92-5 + 2.8 #m. Phase two ended with the beginning of myofibrillar failure (Fig. 1 b, frame 5).

Phase three x~as characterised by the onset of myofibrillar failure and finally breakage of the whole fibre (Fig. I b, frames 5-6). The myofibrils in raw fibres often did not fail as one unit but in small bundles until the fibres broke com- pletely, as demonstrated in Fig. 3. As the fibre neared its fracture point, the banding pattern due to the registered arrangement of myofibrils disappeared in the extremely extended region. After the myofibrils ruptured they showed a slow but marked recoil and reduction in sarcomere length (Fig. 1, frame 6).

It was noted that if stretching was stopped before the yield point and the extension process reversed, there was total recoil of the fibre to its original length and the original ~tress-strain curve could be reproduced on extending the fibre a second time. However, if the extension was reversed after the yield point, the fibre never returned to its original length and the gradient of the stress-strain curve produced on extending the fibre a second time was lower than the first one, suggesting that total and irreversible damage had occurred in the myofibrillar structure above the yield point.

The surface cracking observed in phase two was accompanied by a propor- tionate but sometimes unequal lengthening of sarcomeres within the cracked areas, with sarcomeres in some cracks lengthening more than others. It was

120

too

,~ s0

0

~'~ 4 0

N 2o

0 ~ I I ~" / ~ I

0 2 4 6 8

&

&

10 12

Change in Fibre length (%)

Fig. 4. Sarcomerc length changes in the extensible cracked zones of a raw fibre vs length change of the fibre overall. Values shown are means (n = 4) with bars representing + one

standard error.

Structural changes in single fibres 227

usually the most compliant cracked zone that eventually failed. In these raw fibres, sarcomeres in the areas where the fibre surface had ruptured could be stretched up to 107.7 + 18.9% of their rest length before rupture, whilst those in areas of the fibre with an intact surface membranes remained relatively unchanged after the yield point. Thus, the compliance of the sarcomeres in these cracked areas was approximately an order of magnitude greater than that of the fibre as a whole, as shown in Fig. 4.

Fibres cooked at 80°C and pulled to their fracture point behaved in a similar manner to raw fibres in that they produced an r-shaped stress-strain curve (Fig. 5a). This was accompanied by similar structural changes as seen in raw fibres except that some of the cooked fibres did not show any surface cracking. In the fibres which showed no surface membrane cracking, there was almost uni- form sarcomere lengthening up to failure. However, cooked fibres were far more extensible than raw fibres; they could be stretched to 130 + 42% of their rest length before breaking and yielded at the higher strains of between 10 and 20%. The initial stiffness of the cooked fibres was also lower; the young's modulus of the initial portion of Fig. 5(a) is 1.21 MPa. Sarcomeres in cracked zones of cooked fibres were slightly more extensible than in cracked zones of raw fibres. Measurements on one fibre showed that Sarcomeres of 2.0 #m at rest length could be stretched to 5.3 #m before failure, representing a 169.7% change in sarcomere length. In contrast to the raw case, the extensibility of sarcomeres in the .:racked zones of cooked fibres is of a similar magnitude to that of the whole fibre', as shown in Fig. 6.

1'o investigate the contributions of the connective tissue and sarcolemmal membrane covering tb.e surface of the fibres to the structural and mechanical

(A) 4.5-

~ 3.0"

m 1.5

0.0

I

I i ! ' ~

0 50 100 150 Strain (%)

5

6 b,

Fig. 5. (A) A stress-strain curve for a typical single muscle fibre from meat cooked to 80 ° C extended at a constant rate until fracture. Numbered arrows correspond to the point at which photographs in (B, overleaf) were taken. (B) The corresponding structural

changes occurring at these points in the stress-strain curve. Magnification x 530.

228 G. Mutungi, P. Purslow, C. Warkup

B

Fig. 5. (B).

S t r u c t u r a l c h a n g e s in s ing le f i b r e s 229

~ . . . . ~ ~ i !iii i?i ̧~ ~ ~i! i ! !

5

e

Fig. 5. ( B ) - - C o n t i n u e d .

230 G. Mutungi, P. Purslow, C. Warkup

e

o

rl2

200

150

100

50

B

0

A

! ! ! !

50 !00 150 200

Change in Fii'.:e length (%)

Fig. 6. Sarcomere length changes in the extensible zones of a fibre cooked at 80°C for 1 h vs length change of the fibre overall. Values shown are means (n = 4) with bars repre-

senting + one standard error.

eq

r~

5 I0 15 20 25

Strain (%)

Fig. 7. Stress-strain curves of intact (filled symbols) and mechanically skinned (open symbols) single fibres isolated from raw longissimus thoracis muscle. Values are mean (n = 4)

stress at each strain level analysed. Bars = + one standard error.

Structural changes hi single fibres 231

events observed in raw fibres, load-extension measurements were repeated using some fibres from the muscle sample intact and some fibres whose surface mem- brane had been mechanically removed. Demembranating the fibres depressed the stress at which they yielded and doubled their breaking strain. For example, demembranated fibres yielded at stresses of 0.79 + 0"04 kg cm -2 and fractured at strains of 28-5:1: 3%. The values for fibres with intact surface membranes from the same muscle specimen were 1.05 + 0.09 kg cm -2 and 12.3 + 1.4%, respec- tively. However, removing the membrane had no effect on the stress at which the fibres fractured with fracture occurring at 1.41 + 0.08 kg cm -2 and 1.48 + 0.06 kg era-2 for the intact and demembranated fibres, respectively. The stress-strain curves for demembranated fibres is compared to the curve from corresponding intact fibres in Fig. 7. Demembranated raw fibres did not show any surface cracking and all its sarcomeres appeared to lengthen quite uniformly.

DISCUSSION

When muscle fibres are pulled slowly, they initially resist being stretched until a yield point, which occurs at strains of between 2 and 5% in raw fibres, and 10 and 20% in cooked fibres, is reached. After the yield point, the fibres are more compliant, i.e. require less force to be extended, thereby producing an r-shaped stress-strain curve (Figs la and 5a). To our knowledge this is the first time the stress-strain curves of single cooked muscle fibres have been reported. The steep initial gradient in the stress-strain curve suggests the presence of a structure or structures which resist extension until the yield point and probably become damaged after this. This is further supported by our findings that reversing the direction of the motor before the yield point leads to total recovery of the fibre and on re-extension, the original stress-strain curve can be reproduced. However, if this is done after the yield point, the fibre only recovers partially and the original stress-strain curve cannot be reproduced.

One structure which could be involved in this early steep rise in stress is the sarcolemmal-endomysial complex. Removal of this complex led to a 25% drop in the stress at which the fibres yielded and a decline in the initial gradient of the stress-strain curve (Fig. 7). Other structures which may play a role are the cross- bridges established during rigor mortis. The presence of undetatched cross- bridges would hold the thin and thick myofibrils together thereby preventing them from sliding past each other. In this case, one would expect the myofibrillar arrangement and the striated appearance of the fibres to persist until the yield point and become disrupted thereafter. However, the fibre striations and myo- fibriUar arrangement remains intact long past the yield point and in cooked fibres even when the sarcomere length has more than doubled (Locker & Leet, 1975; Currie & Wolfe, 1980; Locker & Wild, 1982; Present study). This appears to rule out cross-bridges as the sole cause of this steep rise in stress and irregular slippage of cross-bridges to occur after the yield point, so suggesting the presence of other structural connections between myofibrils which can resist the pulling process and are capable of holding the myofibrils together. Locker et al. (1977) and Locker & Wild (1982) have attributed this function to gap filaments. These authors found gap filaments to provide myofibrillar continuity and to be resistant to rupture after heating and stretching. The role and exact contribution of titin and other

232 G. Mutungi, P. Purslow. C. Warkup

myofibrillar proteins such as nebulin in this process is not known as yet and needs to be investigated in future. It was also noted that the intact raw fibres used as controls for testing the effect of demembranation on muscle samples frozen 3 days post mortem showed a lower breaking stress than intact raw fibres from muscle samples frozen one day post mortem. This indicates that there may be a significant effect of post-mortem conditioning on the properties of single fibres, as may be expected; again this is an area which merits further investigation.

It is revealing to compare the results from this study on the tensile properties of single muscle fibres to previous work on the tensile properties of larger muscle strips. Strips of raw rigor muscle loaded along the fibre direction yield at stresses of 1.2-1.4 kg/cm 2 and strains of 14-18%, but on cooking for 10-40 min at 70°C or above the yield point disappears and the stress strain curve is almost linear (Locker & Carse, 1976). The single fibres in the present study showed a different pattern of behavk~,u~; raw fibres yielded at much lower strains (2-5%) and the stress-strain curve of single fibres cooked to 80°C showed distinct yielding (Fig. 5). This difference in results may be due to the different specimens and muscles used in the two studies. Locker & Carse (1976) used strips of bovine sterno- mandibularis muscle, whereas single porcine longissimus muscle fibres were used in the present study. The bovine sternomandibularis has been shown to consist of short muscle fibres which terminate intrafascicularly (Purslow & Trotter, 1994). The fibres are linked together by the endomysial connective tissue which separates them (Trotter, 1990, 1993; Trotter & Purslow, 1992). It is possible that the series arrangement of short muscles linked by connective tissue has very different characteristics to the tensile properties of the indi- vidual fibres themselves. Yielding in larger strips from such a muscle could involve slippage between adjacent fibres rather than yielding of the fibres themselves. This would occur by irreversible shearing of the endomysial con- nective tissue between tibrcs. Tile loss of yield point on cooking observed in muscle strips by Locker & Carse (1976) may represent a heat-induced alter- ation in the connections between the muscle fibres. Schmitt & Parrish (1971) observed some shrinkage in endomysium at temperatures as low as 50°C. Clearly, this is an area which r.:quires further investigation. The endomysial connective tissue which links adjacent fibres is partially or wholly present on the surface of our isolated ttbres. However, this extracellular membrane can only contribute to the measured properties of raw and cooked single fibres in tension, not an shear. The results shown in the present study show that the extracellular membrane ruptures in tension early in the extension and its removal does not seem to affect the breaking stress of the fibres. Therefore the strength of the fibres must be provided by myofibrillar structures whose denaturation at temperatures up to 80°C and shrinkage in the cross-section due to fluid loss on heating results in a higher breaking stress (breaking force/cross-sectional area). The progressive denaturation of different myofibrillar proteins in rabbit muscle shown by the DSC studies of Wright et al. (1977), with a peak of myosin denaturation at 60°C and of actin denaturation at 80°C is also exhibited in DSC '~ermograms obtained for the porcine Iongiss#nus thoracis specimens used in the present study (Miles & Mutungi, unpublished results). Schmidt & Parrish (1971)showed that in bovine hmgissimus dorsi, cooking at 50°C produces myofibrillar shortening and some Z-line degradation, but otherwise leaves the thick and thin filament structural arrangement in the sarcomere essentially similar to the uncooked state. However,

Structural changes in single fibres 233

at 60°C, they report a coagulation of thick filaments and disrt~ption of the M-line and at 70°C there is disintegration of thin filaments and further coagulation of thick filaments. The fusing of the actin and myosin, during the coagulation of the thick and thin filaments observed at temperatures above 60°C, to form a gel-like mass may provide a strong structure in the overlap regions accounting for the increased force required to fracture the fibres.

As mentioned in the introduction, when meat is cooked and tested using the MIRINZ tenderometer, its toughness increases with cooking temperature in two phases. The first phase occurs at temperatures of between 40 and 60°C and the second between 65 and 80°C, with little changes in toughness at temperatures of 60-65°C (Davey & Gilbert, 1974). This is matched to an increased tensile fracture stress of individual fibres of porcine iongissimus thoracis on cooking; in the present study raw muscle fibres required stresses of 2.2 + 0.13 kg cm -2 to break and 3.4 4. 0.26 kg cm -2 after being cooked for 1 h at 80°C. However, unlike cooked muscle strips which become less extensible with cooking (Locker & Leet, 1975), single muscle fibres become more stretchable (Wang et al., 1956; Hostetler & Cover, 1961; Ccver et al., 1962; current study). For example, cooked muscle fibres isolated from pig longissimus thoracis could be stretched to 130 + 42% of their rest lengths but to only 10.9 + 1.45% when raw. Thus there seems to be a clear correlation between cooking temperature, fibre extensibility and meat toughness. This increase in meat toughness and its reduced extensibility as it is cooked to temperatures above 60°C, has been attributed to the toughening and shrinkage of intramuscular connective tissue (Bouton & Harris, 1972; Davey & Gilbert, 1974). Further investigations of the properties of single fibres at inter- mediate cooking temperatures will clarify this picture.

An important set of myofibrillar structures to which the toughness on cooking to various temperatures has been attributed are gap filaments. These filaments are continuous between adjacent sarcomeres, appear to be heat and stretch resistant and remain intact even when meat is cooked to temperatures of 80°C and above, which cause the disintegration of the thin and thick filaments (Locker et al., 1977; Locker & Wild, 1982). These results led Locker to propose a theory attributing meat toughness to gap filaments (Locker, 1982). Using refinements of the struc- tural investigation approach developed here, it may in future be possible to establish the contribution of gap filaments to meat toughness.

ACKNOWLEDGEMENT

This research was funded by a LINK scheme grant from the Ministry of Agri- culture, Fisheries and Food (MAFF) and the Meat and Livestock Commission (MLC).

REFERENCES

Bouton, P. E. & Harris, P. V. (1972). J. Food. Sci., 37, 218. Cover, S., Ritchey, S. J. & Hostetler, R. L. (1961). J. Food Sci., 27, 476. Currie, R. W. & Wolfe, F. H. (1980). Meat Sci., 4, 123. Davey, C. L. & Gilbert, K. V. (1974). J. Sci. Food Agric., 25, 931.

234 G. Mutungi, P. Purslow, C. Warkup

Harris, P. V. & Shorthose, W. R. (1988). In Developments in Meat Science 4, ed. R. Lawrie, Elsevier Applied Science, London, p. 245.

Hostetler, R. L. & Cover, S. (1961). J. Food Sci., 26, 535. Lewis, G. L. & Purslow, P. P. (1989). Meat Sci., 26, 255. Locker, R. H. (1982). Rec,p. Meat Conf. Proc., 35, 92. Locker, R. H. & Carse, W. A. (1976). J. Sci. Food Agric., 27, 89 I. Locker, R. H., Daines, G. J., Carse, W. A. & Leet, N. G. (1977). Meat Sci., 1, 87. Locker, R. H. & Leet, N. G. (I.O75). J. Ultrastruct. Res., 52, 54. Locker, R. H. & Wild, D. J. C. (1982). Meat Sci., 7, 189. Martens, H. & Void, E. (1976). 22nd Eur. Meet. Meat Res. Work. Malmo, J9. Purslow, P. P. & Trotter, J. A. (1994). J. Musc. Res. Cell Motil., 15, 299. Schmidt, J. G. & Parrish, F. C. (1971). J. Food Sci., 36, 110. Stabursvik, E. & Martens, H. (1980). J. Sci. Food. Agric., 31, 1034. Trotter, J. A. (1990). J. Morphology, 206, 351. Trotter, J. A. (1993). Acta Anat., 146, 205. Trotter, J. A. & Purslow, P. P. (1992). J. Morphology, 212, 109. Wang, H., Doty, D. M., Beard, F. J., Pierce, J. C. & Hankins, O. G. (1956). J. Anita. Sci.,

15, 97. Winger, R. J., & Pope, C. G. (1981). Meat Sci., 5, 355. Wright, D. J., Leach, I. B. & Wilding, P. (1977). J. Sci. Food Agric., 28, 557.