Rigor related changes in mechanical properties (tensile and adhesive) and extracellular space in beef muscle

Meat Science4 (1980) 123-143

RIGOR RELATED CHANGES IN MECHANICAL PROPERTIES (TENSILE AND ADHESIVE) AND EXTRACELLULAR SPACE IN BEEF MUSCLE

R. W. CURRlE & F. H. WOLFE Department o f Food Science, University o f Alberta, Edmonton, Canada T6G 2N2 (Received: 19 March, 1979)

SUMMARY

The tensile and adhesive properties o f selected beef muscle strips undergoing rigor mortis are presented at various times post mortem. The changes in these mechanical properties of the muscle correlated well with p H and its rate o f fall. Additionally, and perhaps most importantly, the shape of the curves generated over the post-mortem ageing times correlate well with changes in extracellular space. The results are discussed and the conclusion drawn that intrafibre water must be considered as a potentially important third factor, in addition to myofibrillar contraction and connective tissue orientation, in the evaluation o f meat tenderness.

INTRODUCTION

Dikeman (1977) stated that one of the prime difficulties in correlating objective measurements of meat tenderness with subjective evaluations is the problem of interpreting the printout of the particular objective measurement and relating it to meat tenderness. In order to achieve this it is essential to understand all of the factors associated with the microstructure of the muscle that may contribute to the profile of the particular objective measurement (Stanley & Swatland, 1976; Harris, 1976). Tensile and adhesive properties of meat may be more easily related to the muscle microstructure than shear force measurements since the forces are applied longitudinally and perpendicularly to the fibre axis and do not involve a compressive factor (Bouton et al., 1975). However, the problem of relating these mechanical measurements to tenderness as determined by taste panels (Stanley et al., 1972) suggests that while the myofibrillar contraction state and collagen angle in the connective tissue network are the prime contributors, other factors may be involved. 123 Meat Science 0309-1740/80/0004-0123/$02.25 © Applied Science Publishers Ltd, England, 1980 Printed in Great Britain

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R. W. CURRIE, F. H. WOLFE

It is well accepted that pre- and early post-mortem events significantly affect the tenderness of the resultant meat (Khan, 1977). Therefore, we have measured the tensile and adhesive properties of beef muscle, during the onset of rigor, in order to gain a better insight into the factors that contribute to these mechanical properties in post-rigor muscle. During rigor development the water-holding capacity of the muscle fibre is drastically altered, resulting in a movement of water from the fibre into the extracellular space (Heffron & Hegarty, 1974). In view of the fact that water is considered to be an important factor affecting both protein structure and interaction (Warner, 1979), it seemed necessary to include extracellular space measurements in this study'.

MATERIALS AND METHODS

Animals, muscle and muscle treatment Samples of M. semitendinosus and biceps femoris obtained from steers slaughtered at Gainers Ltd, Edmonton, Alberta (graded A; aged 112 years) were utilised in this study. The muscles were maintained in two states for comparison purposes. The first state refers to those muscles removed within an hour of slaughter, brought back to the laboratory and allowed to enter rigor mortis unrestrained. These samples were labelled off-carcass (OFC) and were stored in a Labline temperature controlled room which allowed the temperature to be regulated to conform to the temperature drop (measured at a depth of 2 cm) within the carcass. At the packing plant samples were removed from the carcasses at regular intervals during the development of rigor mortis. These samples would be restrained from marked contraction due to the load placed on the muscle by the weight of the carcass and were labelled on-carcass (ONC). Measurement of ECS and expressed juice The extracellular space was determined by incubating 100-150 mg of muscle for 2½h at 18-20 °C in a Ringer Locke solution containing 0.3 ~ inulin according to the procedure of Heffron & Hegarty (1974), modified by the use of low oxygen tension. The inulin within the ECS was measured by the method of Pappius & Elliot (1956) which involved homogenisation of the blotted muscle strip in 6 ~ TCA, centrifuging the precipitated proteins and determining the inulin content using resorcinol. The procedure of Bouton et al. (1971) was followed for determining expressed juice. Mechanical measurements The tensile properties, as well as the extensibility of the muscle strips, were examined using the Instron Universal Testing Instrument (Model 1132). The muscle strips were cut parallel to the fibre axis about 5 cm long and were formed into

RIGOR RELATED CHANGES IN MECHANICAL PROPERTIES OF BEEF MUSCLE

125

dumbbell shapes (Bouton & Harris, 1972a,b) to prevent breakage of the sample at the clamp attachments. The narrowest point was cut to have a circumference (C) within 0.8-1-1 cm and was accurately measured by wrapping a flat nylon string about the muscle strip, marking it and measuring the distance between the m a r k s with a dial caliper. The cross-sectional area (A) could then be calculated by A = C 2 x 0-0795. The sample was mounted in a lucite cell consisting o f a jacketed chamber and a separate clamping device which was designed and constructed at the University of Alberta to facilitate clamping of the muscle strip outside the chamber. The test was performed in the air at r o o m temperature since the test was rapid and little change in the muscle would occur between preparation of the sample and the completion of the measurement. After the muscle strips were clamped in the stainless steel clamps (3 cm apart) the cross head was set to move at 2 c m / m i n by means of a decade reducer and the chart was set t o r u n directly proportional to the cross head (five times greater). As the cross head rose, the tensile properties of the muscle could be measured from the response of the 500 g tension load cell and the extensibility by measuring the distance the cross head moved. The cross head was kept moving until the muscle parted or exhibited a 'final yield'. Two points on the curve generated were measured (see Fig. 1). The first point was the initial deflection which is referred to as the initial yield. According to Bouton et al. (1975) the initial yield reflects a yielding of the myofibrillar structure. The second point was the breaking point or 'final yield' o f the muscle strip. The tension generated at each of the two points was calculated in grammes per square centimetre FINAL YIEI.D

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Fig. 1.

A typical profile of the curve generated by the Instron testing instrument and the points

measured. (This was a plot of a curve generated near rigor.)

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R. W. CURRIE, F. H. WOLFE

and the extension of the muscle was measured directly from the chart where it was expressed as fives times the actual muscle strip extension in centimetres. The adhesive properties of the muscle were measured in the same way as those described above except that the muscle strips were cut so that the forces developed were perpendicular to the fibres and the circumference was increased to be within the range 0-9-1-3 cm. Care was taken in the preparation of the muscle strips to avoid the major perimysial sheets o f muscle mentioned by Rowe (1977a) which can result in significant within-sample variability. All the mechanical measurements were run in triplicate at each ageing period.

Photography of muscle strips stretched to initial yield In an attempt to observe the appearance of the muscle fibres at initial yield, muscle strips of approximately the same dimensions as those for the tensile tests were tied at both ends with silk surgical suture at a distance of 3 cm apart and stretched to the same extension as that needed to observe the initial yield in the Instron. The stretched muscle strip was fixed for 2 h in Karnovsky's fixative (paraformaldehyde crystals were used to prepare the fixative fresh at each use). The fixed muscle fibres were disrupted by homogenisation in 50mM cacodylate buffer and were photographed using an Olympus Model BH microscope equipped with phase contrast optics.

RESULTS

The time post mortem that specific changes in pre-rigor muscle were observed varied between carcasses. This is no doubt related to the many different factors associated with slaughter conditions (Khan & Lentz, 1973; Khan, 1977). Currie & Wolfe (1979) observed that muscles attaining an ultimate pH of 5.4-5.5 undergo biochemical and physical changes that are closely mirrored by pH fall. Similarly, in this study p H fall appeared to provide a c o m m o n factor to which specific points of change in the prerigor muscle could be related. F o r this reason most of the measurements will be presented along with p H fall in order to assist in correlation and comparison of results. The changes in the tensile strength of the muscle as it approached rigor are presented in Fig. 2. A plot of the tension at initial yield versus time post mortem revealed that the force to initial yield gradually increased to a peak at a point when the muscle was in the region of pH 5-85-5.95. Following this, a decrease in the tensile strength of the muscle was observed until the minimum values were recorded, coincident with rigor maximum. Samples analysed beyond rigor maximum indicated some variability in the rise o f the initial yield. The tensile strength of the muscle at final yield (Fig. 2) required greater force than the initial yield for failure, but mirrored the profile of the initial yield.

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The extensibility of the muscle, presented in Fig. 3, showed a similar profile to that of the tensile properties shown in Fig. 2. The extensibility of the muscle was greatest when the muscle was at pH 5.85-5.95 and is supported by the observation that the final yield or breaking point profile was also the highest at this point. After this the muscle rapidly became less extensible with a sudden drop in the extension needed to exhibit an initial yield. However, the extension needed for the final yield did not drop to as low a value as the initial yield extension. Even some of the post-rigor samples did not lose the ability to extend once the initial resistance to stretch was overcome. This supports the results o f Hegarty (1972) who showed that mouse muscle in rigor was partially extensiblel Other post-rigor samples which we examined did become very inextensible and such a sample is also presented in Fig. 3. The adhesive strength of the pre-rigor samples (Figs. 4 and 5) changed with time, but a marked contrast between samples undergoing slow rigor development and fast rigor development was evident. Both types of muscle samples showed an increase in

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their adhesive properties when the pH had dropped to the region of 5-85-5.95 (indicated by a rise in the tension needed to exhibit both initial and final yield (Figs. 4 and 5). The adhesive strength of both samples also dropped as the pH continued to fall but near the point of rigor maximum the response to the stretch was completely different. The muscle strips from samples experiencing a slow pH fall (Fig. 4) remained minimal throughout rigor whereas samples undergoing rapid pH fall(Fig. 5) showed a new increase in strength, peaking at rigor. After rigor maximum the tension again dropped to lower values. In Figs. 6 and 7 only the extension to initial yield is presented since the profile at final yield is identical except that it is of greater magnitude. Both figures indicate that the extensibility of muscle undergoing slow and fast pH fall rises to a peak at rigor maximum. Although the bimodal curve is common to both sample types it was also observed that the extensibility was greater for the samples undergoing slow pH fall (compare Figs. 6 and 7).

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Fig. 8(a). A light phase contrast micrograph o f a muscle fibre (pH 6-25) fixed at initial yield (magnification 770 x ). The myofibrillar units within the fibre appear to be out of register.

Fig. 8(b). A light micrograph o f a muscle fibre (pH 6.10) fixed at initial yield (magnification 850 x ). More of the myofibrillar units within the fibre appear to remain in register rather than slip past one another as in Fig. 8(a).

132

Fig. 8(c).

R. W. CURRIE, F. H. WOLFE

A light micrograph of a muscle fibre (pH 5.95) fixed at initial yield (magnification 610 x ). Regions of the fibre are extended and the myofibrillar units appear in register.

The phase contrast micrographs of muscle at the initial (tensile) yield presented in Figs. 8 to 10 (selected areas for detailed examination) help to explain the profiles observed in Figs. 2 and 3. It is doubtful if these photographs represent the precise state of the muscle at initial yield since the time between stretching of the muscle and fixation would allow for some equilibration of the effects of stretch. However, an examination of the photographs does indicate a considerable difference between the early pre-rigor samples and those approaching rigor. The approximate pH of the muscle is indicated in the legends to these photographs so as to correlate the photographs with the profiles in Figs. 2 and 3. Figure 8(a) depicts the state of the muscle following stretch in the early pre-rigor state. The sarcomeres are clearly stretched and units ofmyofibrils within the fibre are out of register with one another (i.e. not all A-bands and Z-lines are aligned across the fibre). In Fig. 8(b) the myofibrils towards the interior of the fibre are stretched but in comparison with those shown in Fig. 8(a) are more likely to stay in register before myofibrillar units slip past one another. Figure 8(c) depicts the state of the fibre almost at the peak of the tensile profile. The sarcomere lengths become very long ( > 4-0 ~t) with all the myofibril units tending to remain in register (see arrow). As a result, instead of the myofibril units slipping past one another as in the previous photographs, they tend to break and be totally disrupted. Figure 9(a) presents the fibre at initial yield after the muscle begins to lose its extensibility. It is interesting to note that in certain regions of the fibre (see arrow 1) we can see normal sarcomere

RIGOR RELATED CHANGES IN MECHANICAL PROPERTIES OF BEEF MUSCLE

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"Fig. 9(a). A light micrograph o f a muscle fibre (pH 5.75) fixed at initial yield (magnification 840 x ). Extension of certain regions o f the fibre (arrow 1) are presented, while other areas are free to extend (arrow 2).

Fig. 9(b). A light mierograph o f a muscle fibre (pH 5.65) fixed at initial yield (magnification 720 x ). Breaks across the fibre can be seen, suggesting actin-myosin interactions have been broken.

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R. W. CURRIE, F. H. WOLFE

Fig, 9(c). A light micrograph of a muscle fibre (pH 5.5) fixed at initial yield (magnification 830 x ). Breaks across the fibre appeared sharper in most instances (than the breaks in fibres at pH 5.65), involving only a few sarcomeres.

Fig. 10(a). A light micrograph o f a muscle fibre (pH 5.6) fixed after stretching the fibre beyond initial yield (magnification 580 x ). It appears that the fibre will continue to extend where the breaks had occurred at initial yield.

RIGOR RELATED CHANGES 1N MECHANICAL PROPERTIES OF BEEF MUSCLE

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Fig. 10(b). A light micrograph o f a muscle fibre (post rigor) fixed at initial yield (magnification 860 × ). Breaks do n o t appear across the fibre but occur randomly, giving a very disordered appearance.

Fig. 10(c). A light micrograph of a muscle fibre (an on-carcass sample removed post rigor) fixed at initial yield (magnification 900 × ). Breaks appear to extend across the fibre. These fibres have a greater extensibility at final yield.

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R. W. CURRIE, F. H. WOLFE

lengths, which implies that the actin-myosin interactions are only minimumly disrupted by stretch whereas other regions (see arrow 2) freely extend until they are disrupted and exhibit the initial yield, suggesting a weakness in the actin-myosin interactions. Subsequent photographs (Figs. 9(b) and (c)) reveal that more of the fibre is affected by strong actin-myosin interactions with the result that fewer regions of the fibre will extend as rigor maximum is approached. Some of the sarcomeres of myofibrils will yield, resulting in what appear to be breaks (see arrows) at rather regular intervals along the fibre. If the muscle is extended beyond the initial yield (and before the final yield) the regions o f the fibre that have yielded will continue to extend, as shown in Fig. 10(a), resulting in regions of extended sarcomeres (see arrow), interdispersed between regions of contracted unyielding myofibrils. 65

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Fig. 11. Percentage change in extracellularspace (ECS) versus time post mortem of beef muscle. The standard deviation of the points plotted is approximately _+6 ~ of the mean. Finally, post-rigor samples stretched to initial yield had two different appearances. The first is seen in Fig. 10(b) where extension results in the totally disrupted fibre shown in the photograph and the second is shown in Fig. 10(c) where extension produced breaks and regions of extension at initial yield. In Fig. 11 a plot of the changes in extracellular space (ECS) during rigor development results in a remarkable profile which reflects the movement of intrafibre water to and from the extracellular space. This bimodal profile was observed for all samples although the precise values varied between carcasses. The TABLE 1 EXPRESSED

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RIGOR RELATED CHANGES IN MECHANICAL PROPERTIES OF BEEF MUSCLE

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ECS increased until the pH of the muscle reached about 5.85--5.95 and then dropped to minimal levels before rising again to peak near rigor. An examination of the ECS, post-rigor, indicates that there is a reduction in the ECS with post mortem ageing. This would suggest that some water has been resorbed into the fibril. This is not likely to be due to an increase in bound water of the contractile proteins since the expressed juice (Table 1) does not show a reversal in the amount of water lost from the tissue during ageing, but rather an increase.

DISCUSSION

I.n this discussion it will be suggested that intrafibre water is a significant contributing factor to the tensile and adhesive properties of muscle and must be considered in addition to the contraction state and the collagen angle in the connective tissue network if a meaningful relationship between these mechanical measurements and muscle tenderness is to be achieved. The recent papers by Stanley & Swatland (1976) and Carroll et al. (I 978) demonstrate an aligning and tensioning of the connective tissue components during the stretching of muscle strips. However, an interesting and important observation made by Stanley et al. (1972) was, in their own words, 'the puzzling finding that elongation of muscle strips correlated positively with taste panel evaluations of tenderness and negatively with sarcomere lengths'. This was puzzling since shortened sarcomeres are generally related to tough meat. It would seem that an additional factor was contributing to muscle tenderness and elongation. It is our view that the physical state of the fibre, which is greatly influenced by the intrafibre water (whether bound or not), will determine when and how rapidly the tension is taken up by the connective tissue. The importance of intrafibre water may be suggested by the following hypothesis. In early post-mortem muscle the pH is high, the water-binding capacity of the contractile proteins is high (Table 1), the intrafibre water content is high, as indicated by the low ECS shown in Fig. 11 and the ATP levels are high. As a result, the tensile and extensibility properties (Figs. 2 and 3) of the early post-mortem muscle are relatively low. The photograph in Fig. 8(a), of an early post-mortem fibre at initial yield, indicates that the myofibrillar structure has been altered. Not only have the sarcomeres lengthened but myofibrillar units have slipped past one another and are out of register. The reasons that these observations occur at relatively low tensions and extensions are probably twofold. First, the sarcomeres will lengthen easily due to high ATP levels and, secondly, the slippage of the myofibrillar units may be aided by the high levels of intrafibre water which might reduce the adhesive forces between the myofibrils. As the pH drops the proteins begin to lose their water-binding capacity to some extent (Table 1), but additionally Heffron & Hegarty (1974) have indicated that the ECS becomes hyperosmotic and contributes to a movement of water from the fibre

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R. W. CURRIE, F. H. WOLFE

into the ECS. This observation is confirmed by the ECS measurements in Fig. 11 that show a continual increase in ECS until about pH 5-9. The effect of this phenomenon on the tensile and extensibility profiles is indicated in Figs. 2 and 3. Both the tension to initial and final yield (Fig. 2) and extensibility (Fig. 3) increase as the ECS increases. An examination of the photographs in Figs. 8(b) and 8(c) reveals an increase in the sarcomere lengths as pH 5-9 is approached. The most interesting aspect of these photographs is the extremely long sarcomere lengths attained at the initial yield point, while the A-bands and Z-lines remain in register. It appears that the myofibrils were forced to remain in register as long as structurally possible rather than slipping past one another as shown in Fig. 8(a). This observation could be explained by increased adhesive forces between the myofibrils within the fibre as a result of lower intrafibre water content. The fact that a hypertonic environment could have a significant effect on the tensile profiles of muscle is supported by Hill (1968) where he observed a rise in tension to high values when muscle was stretched in hypertonic external solutions. After the pH drops below the region of 5-95-5.85, the extensibility of the pre-rigor muscle at the initial yield is drastically altered. Currie & Wolfe (1979), in their examination of the significant physical and biochemical changes in pre-rigor muscle, observed that the initiation of isotonic contraction depends upon the load on the muscle and is related to pH. They suggest that at lowered pH values the amount of C a 2÷ released from the sarcoplasmic reticulum or other C a 2+ containing organelles is ~sufficient to alter the troponin complex of enough sites to initiate actin-myosin interactions and contraction. At pH 5.95 sufficient sites have been opened to allow a considerable number of actin-myosin interactions, resulting in an abrupt drop in tension and extensibility at initial yield, as observed in Figs. 2 and 3. The photographs in Figs. 9(a) to (c) clearly indicate regions of unyielding sarcomeres that must be a result of strong actin-myosin interactions. The brief drop in ECS noted in Fig. 11 after pH 5-95 may not be a significant factor in decreasing the extensibility and tension at initial yield in the tensile measurements, as the actin-myosin interactions are probably stronger than the intermyofibrillar adhesive forces which are affected by intrafibre water concentration. The extension to the final yield (Fig. 3) remained high during the remainder of rigor development. This would suggest that once the bonds have been broken the muscle is relatively free to extend with little tension required. Variations in the extensibility profile (which are found) could be attributed to the intrafibre water concentration which, if high, would allow the fibre to be extensible with little tension. However, if the intrafibre water concentration is low the fibre may have a lower extensibility before disruption and the loading of the non-contractile components of muscle. Figure 10(a) shows clearly that regions of the fibre that have yielded will extend and will continue to do so until they are disrupted similar to the disruption observed at the initial yield in muscle near pH 5-95-5-85. Interestingly, the extension

R I G O R R E L A T E D C H A N G E S IN M E C H A N I C A L P R O P E R T I E S OF BEEF M U S C L E

139

to final yield at this point in time is only slightly below the extension profile of the initial yield at its peak (Fig. 3). This supports the thought that the disruption at final yield for muscle near or at rigor for extendible muscle strips is virtually the same as the disruption observed at initial yield near the peak at pH 5-95-5-85. It is possible that the disruptions observed in both instances are due to a rupture of the 'gap filaments' (Locker et al., 1977). The fact that the tension is not as great for the final yield at rigor as the initial yield at the peak between p H 5.85-5-95 would suggest that the A T P levels in early postmortem muscle are sufficient to generate a force to contract as the muscle is stretched. This may be due to some Ca 2 + release as a result of the physical distortion or damage to the sarcoplasmic reticulum. This does not detract from the importance of the intrafibre water content since the first measurements have relatively low initial and final yields when the A T P levels are the highest. If the force to contract were the only factor to consider in explaining why the initial yield is higher in pre-rigor than in post-rigor muscle, there would have been no reason for the peak which was observed at p H 5.85-5.95. The earliest post-mortem muscle strips would have had the same or higher initial and final yields as the peak observed in this study. Since A T P levels are very low at or near rigor, once the actin-myosin interactions are broken the most significant factor affecting ease of extension and tension development before tensioning of the connective tissue would seem to be the intrafibre water content. The final yield measurements would reflect variations in this factor. The final yield extensibility corresponding to 8-10 h post rigor showed the most significant variation between carcasses. It is at this time that the movement of the water between the fibre and the extracellular space has slowed (Fig. 11). Some movement back into the fibre is indicated, but it does not seem to be a result of increased water-binding capacity of the proteins since the expressed juice values in Table 1 continue to increase. The loss in protein water-binding capacity is probably due to the continuing low pH denaturation of sarcoplasmic and contractile proteins. The variation in tension increase at initial yield may also reflect the amount of water resorbed by the fibre. Water that diffuses between the myofibrils might somehow reduce brittleness and allow for a distortion of the myofibrils within the fibre during extension, but with increasing tension before disruption. The bimodal profile of ECS in Fig. 11 is an unexpected finding and indicates the sensitivity o f this method to changes in water activity inside and outside the fibre. The initial rise in ECS was expected but the brief drop in ECS near pH 5.95 was not. We think that the release of Ca 2 + from the SR or other Ca 2 + containing organelles within the fibre makes the intracellular region hyperosmotic, causing water to move from the ECS back into the fibre. An explanation for the rise in ECS again after this brief fall may be due to the fact that as the p H falls the isoelectric point o f the sarcoplasmic and contractile proteins is neared and some of these proteins may begin to denature, with the result that they lose their ability to bind water. The increase in loose water would result in the intracellular Ca 2+ concentration

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becoming diluted and the extracellular region again becoming hyperosmotic with the result that water moves out of the fibre into the ECS until, at rigor maximum, the ECS is the highest. Our measurements of the movement of water between the fibre and the ECS parallel the work of Chang et al. (1976). They determined the spin lattice relaxation times of water by NMR in post-mortem rat muscle and observed interesting changes in T 1 values. A plot of their changing T1 values versus time post mortem produces virtually the same profiles as we present in Fig. 11 ; however, their drop in the T 1 values following rigor maximum was more rapid than our drop in ECS. This may suggest that the location of the water in the post-rigor muscle contributes to more significant differences in the extensibility to final yield. The remarkable similarities in the profiles of the ECS (Fig. 11) and the adhesive forces of pre-rigor muscle undergoing slow and rapid pH fall (Figs. 4 and 5) support the significance of the intrafibre water content emphasised in this paper. In the early post-mortem muscle the intrafibre water content is relatively high (low ECS) and the adhesive forces are low. As the intrafibre water decreases by its movement into the ECS, the adhesive forces increase until about pH 5-95-5.85 at which point we think that the release of significant levels of Ca z ÷ from the SR reverses the movement of water back into the fibre. This, in addition to the rigidity of the fibre imposed by myosin-actin interaction, reduced the adhesive forces between myofibrils and the tension to initial and final yields drop. As rigor approaches the differences between muscles experiencing slow and fast pH drop become evident. Muscles which experience a rapid pH fall at high temperatures suffer from high drip loss (Tarrant & Mothersill, 1977). This would suggest the intrafibre water content of such muscle would be low and may explain the differences in the profiles of the adhesive measurements in Figs. 4 and 5 at rigor. Muscle experiencing a slow pH drop (Fig. 4) would not attain low pH values until the temperature is low. As a result of decreased protein denaturation, the water-holding capacity of the muscle fibres would be relatively high. The tension of such a fibre at initial and final yields would probably be low at rigor. The relatively high intrafibre water content may reduce the adhesive forces between myofibrils. The opposite effects on the adhesive properties of muscle at rigor are identified in Fig. 5. The muscle has experienced a rapid fall with greater denaturation of the muscle proteins and reduced water-holding capacity. The ECS (Fig. 11) is the greatest at rigor, suggesting that the intrafibre water content is the lowest. This may lead to greater packing and physical contact between myofibrils. The adhesive force between the myofibrils of such a muscle is high and is indicated by the tension of the initial and final yields increasing until a second peak at rigor is observed. As the intrafibre water content increases again, the adhesive forces between the myofibrils decrease and the tension drops. The adhesive extensibility profiles of muscle fibres experiencing slow or fast pH fall (Figs. 6 and 7) both have a bimodal shape. The fact that the extensibility of pre-rigor muscle undergoing slow pH fall rises to a peak at rigor (Fig. 6) but the adhesive

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forces are minimal at that time (Fig. 4) may suggest that the high intrafibre water content allows the myofibrils to deform under stretch with little tension. The fact that the extensibility was found to be greater with these samples (Fig. 6) than samples experiencing rapid pH fall (Fig. 7) indicates that the fibres which had experienced rapid pH fall are more rigid due to high adhesive forces (Fig. 5) and will be disrupted by stretch rather than conforming to the load. Although the studies of Rowe (1974, 1977a,b), Stanley & Swatland (1976) and Carroll et al, (1978) have shown that the orientation of the collagen fibres in the connective tissue network aid in the explanation of much of the mechanical behaviour of muscle fibres, we do not believe that a two-component system (myofibrillar contraction state and connective tissue orientation) can provide a complete picture. We prefer a three-component system which, in addition to the above two factors, would include intrafibre water content, to more clearly explain the relationship between the mechanical properties of muscle and tenderness. The significance of the intrafibre water to the toughness of meat is supported by the work of Bouton et al. (1973). They have shown ttiat as the ultimate pH values of muscle approached pH 7-0 the contribution of the myofibrillar contraction state to the toughness of meat decreased until, at pH 7.0, the myofibrillar contraction state was no longer important. This suggested that the toughness, even in cold-shortened meat, could be compensated for by the increased hydration of the myofibrillar proteins. Additionally, Hamm (1977) has indicated that the degree of immobilisation of water that is not bound in the network ofmyofibrillar proteins is important to the tenderness of meat. Since this water cannot be measured by the ndrmal techniques for determining water-holding capacity (WBC), he has stated that the measurement of WBC is unsuitable for determining tenderness of muscle because a significant relationship between WBC and juiciness is often not evident. Perhaps the ECS measurement as outlined in this paper is related to the degree of immobilised water in the fibre and may be related to muscle tenderness. The major reason for including intrafibre water as a third component to consider in evaluating mechanical measurements is the fact that we have made observations in this study which cannot be explained by the two-component system only. As a result of obtaining samples 'on' and 'off' carcass, we have obtained both contracted and uncontracted fibres. The sarcomere lengths of ONC samples averaged about 2.45/~ whereas the OFC samples averaged about 1-7 # (Currie & Wolfe, 1979). For tensile measurements the generally accepted view of the relationship of sarcomere length to connective tissue orientation should have lead to the OFC samples (short sarcomere lengths) demonstrating greater extensibility and a lower breaking strength than ONC samples which should have shown lower extensibility and a high breaking strength (Bouton et aL, 1975). The majority of the final yield determinations followed the pattena described by the above authors but there were numerous exceptions. For example, some OFC samples that experienced a rapid pH drop showed only minimal extensibility rather than the high extensibility predicted from the two-component system. It is possible that the low intrafibre water content

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due to rapid pH fall would have increased the adhesive forces between the myofibrils so that no distortion could occur in response to load. The load would have been borne by the actin-myosin interactions which would be disrupted with minimal extension. Another example which seems to implicate intrafibre water as an important factor in explaining the observed mechanical properties of muscle is the change in the adhesive force of muscle experiencing a rapid pH fall (Fig. 5). In adhesive measurements it is generally believed that fibres of short sarcomeres would be stronger than fibres of long sarcomeres (Rowe, 1977b). The increase of the adhesive forces at rigor was independent of the samples being ONC or OFC and hence independent of sarcomere lengths. The major factor was the rate of pH drop and the intrafibre water content. Additionally, it would have been expected that the extensibility and adhesive force of the samples would not have dropped post rigor if the sarcomere length--and thus the connective tissue orientation--were the only contributing factor. It could be argued that the movement of the water into and out of the fibre would alter the connective tissue angle. This may be true, but the opposite effect would be expected. Movement of water into the fibre could expand the fibre and increase the angle between the collagen and muscle fibres and thus increase the strength of the fibre (Rowe, 1977b). The opposite is observed in this case since water moves out of the fibre into the ECS and the adhesive forces rise. This, in our opinion, implicates the drop in intrafibre water as a more important factor than the collagen angle for the 'at rigor' samples. In summary, the experimental evidence implicates intrafibre water, which is significantly affected by the osmotic pressure within the fibre and the ECS, as an important third component to consider in relating the mechanical properties of muscle to subjective evaluations of meat tenderness.

ACKNOWLEDGEMENTS

This research was conducted under the Agriculture Canada Meat Research Contract Programme No. 02SW.01531-6-1172. The authors wish to thank Steve Yuen, Joy Lee, Judy Nuss, Avi Golan and Peter Nagainis for their assistance in performing these time-consuming experiments. Some of these results were presented at the 21st CIFST Annual Conference, Technical Session 7, 28 June 1978, Edmonton, Canada.

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