Food Structure

Volume 8 Number 1 Article 17

1989

The Structural Basis of the Water-Holding, Appearance and Toughness of Meat and Meat Products

Gerald Offer

Peter Knight

Robin Jeacocke

Richard Almond

Tony Cousins

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Recommended Citation Offer, Gerald; Knight, Peter; Jeacocke, Robin; Almond, Richard; Cousins, Tony; Elsey, John; Parsons, Nick; Sharp, Alan; Starr, Roger; and Purslow, Peter (1989) "The Structural Basis of the Water-Holding, Appearance and Toughness of Meat and Meat Products," Food Structure: Vol. 8 : No. 1 , Article 17. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol8/iss1/17

This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. The Structural Basis of the Water-Holding, Appearance and Toughness of Meat and Meat Products

Authors Gerald Offer, Peter Knight, Robin Jeacocke, Richard Almond, Tony Cousins, John Elsey, Nick Parsons, Alan Sharp, Roger Starr, and Peter Purslow

This article is available in Food Structure: https://digitalcommons.usu.edu/foodmicrostructure/vol8/iss1/17 FOOD MICROSTRUCTURE, Vol. 8 (1989) , pp. 151- 170 0730- 5419/89$3 .00+. 00 Scanning Microscopy International, Chicago CAMF O'Hare) , IL 60666 USA

THE STRUCTURAL BASIS OF THE WATER-HOLDIN G, APP EAR AN CE AND TOUGHNESS OF MEAT AND MEAT PRODUCTS

Gerald Offer. Peter Knight, Rob1 n Jeacocke, Richard Almond, Tony Cousins. John Elsey, Nick Parsons. Alan Sharp , Roger Starr and Pete r Purslow

Mus cle Biology Department, AFRC Inst1tute of Food Research - Bristol Laboratory Langford , Bristol, BS18 7DY, UK

Abstract Introduction

A structural approach greatly clarifies which In Western societies a larger amount is spent components of meat are responsible for its on meat and meat products than on any other food. tenderness, water-holding and appearance, and the and meat foods present a part i cu l a rl y wide events occurr1 ng during processing. variety of problems and challenges . Structural In living muscle, water is held in the spaces investigations on meat are very helpful because between the thick and thin filaments. Changes in by identifying the spatial arrangement of the content and distribution of water within meat components. they can identify the component originate from changes in this spacing. responsible for a problem affecting meat quality Myofibrils shrink laterally post mortem . The and greatly clarify the mechanism responsible. fluid expelled accumulates between fibre bundles These problems are caused by rather subtle and between fibres and is drain ed by gravity c hanges in the colloidal character of meat and, forming drip. In pale, soft and exudative meat, although much is known about the factors shrinkage of the myosin heads on denaturation producing them, the exact nature of these changes increases myofibrillar shrinkage. In salt has until recently eluded a precise description; solutions used in meat processing, myofibrils only now are meat scientists beginning to explain swell laterally taking up water, probably by an them. We are fortunate in being able to draw on entropic mec hanism. a very large amount of structura l and biochemical The co lour of meat is determined by light infonnation provided by basic biological research scattering as well as by absorption . The light on muscle and connective tissue. We are also scatterers in meat are observed by scanning fortunate 1n that muse 1e ce 11 s are very regular confocal light microscopy to be smaller than 1 ~m and this facllltates both the study of their and concentrated in the A-band regions. 1t is structure and thinking about mechanisms . proposed that 1 ight scattering arises from the In this brief review we have selected three gaps between myofibri 1 s. topics, water-holding, appearance and texture . The structur es responsible for the toughness All levels of structure are informative, from of cooked meat have been studied by observing its single protein molecules up to coarser features fracture behaviour. The perimysial connective that can be seen by eye . Correspondingly, a tissue determines the breaking strength when the number of complementary structural techniques is meat is pulled apart transversely. In proving valuable, including X-ray diffraction, longitudinal tensile tests, the initial event is scanning and transmission electron microscopy, the debonding of fibre bundles from the phase- cont rast, fluorescence, pol ar1 sing, dark­ perimysium so that they contribute independently field and scanning confocal light microscopy . to load-bearing. At greater ex t ensions. it seems likely that fibre bundles progressively fail The Structure of Mus cle leaving perimysial strands as the last structure to break. With aged muscle, the muscle fibres Before considering these three quality probably fail at smaller extensions and therefore attributes, we will first briefly review the contribute less to the breaking strength. structure of muscle (for detailed recent reviews see Squire and Vi bert, 1987). Surrounding each Initial paper received February 5, 1989 muscle is a thick connective tissue sheath, the Manuscript received May 28, 1989 epimysit.rn, which is continuous with the tendon Direct inquiries to G. Offer (Figure 1}. The muscle is divided tnto muscle Telephone number: 44 -934 852 661 fibre bundles by the perimysial connective tissue network. The coarse or primary perimysial network, which is very obvious to the eye, is Key Words: Muscle, meat, light microscopy, further sub~divided by thinner sheets of confocal microscopy, electron microscopy, water­ perimysial connective tissue. typically defining holding, appearance, colour. toughness. texture. a bundle of muscle fibres of the order of 1 1m1

151 G. Offer et al.

penmys10l network

muscle fibre

endomys1al network

1 0 1 ~ ~~~~tuJ ina 1Trsaencs;iiosns t hnro~ g~ctfr0:g ~~~'i~~~lp~u~~ l: 1 1 9 3 0 0 showing the striated appearance. The lower ~~;\;tea 1 .mus~~~rs:ho~~~~c \u:ea c~~n e c t ~~:t \ tss~~ diagram schematically shows how the striations of tracts of muscle. Top, whole muscle; bottom, a the myofibrils are caused by the orderly packing fibre bundle. (Adapted f r om Purslow (1987) by of thick and thin filaments. The A-band is 1.6 ~ m permission of von Nostrand Reinhold Company) . long . {Rep r oduced by pennission of Or Craig and the Longman Group) .

are separated f r om one anothe r by t he endomys ial connect i ve ti ssue network. {Figure 1) . The musc l e fibres a re multi nucleated cells, typically 10 to 100 ~ m wide and as much as several centimetres long . They are filled with myofi bri 1 s, the contractile apparatus of muscle . These are long , thin ( 1 to 2 ~m), roughly cylindrical organelles packed side by side {Figure 2) . In white f ibres , whi c h are used for bursts of activity, few mitochondria are present and the myofibrils are separated only by the calcium-stor ing membrane-lined channels of the sarcoplasmic reticullxn . ln red fibres, which are used for sustained or repetitive activity, mitochondria also intrude between adjacent myofi bri 1 s . Myofibrils have a regular striated appearance and, because they are packed in register, the whole muscle f i bre appears striated (Figure 3) . The wi de protei n- dense bands are cal l ed A-ba nds and each has a l i ghter zone, t he H-zone , i n i t s centr al part. In t he middle of t he A-band i s a ~. Scanning el ectron mi crograph of an dense l i ne, t heM-l i ne . The lighter bands a r e ~ cut surface of beef sternomandibular1s called I- bands and each is bisected by a Z-d i sc. muscle. The fibre direction is from left to The stri ations of the myofibril arise because the right . In the two fibres near the surface the myofibril is made up of overlapping arrays of two enclosing endanysial sheaths have been largely kinds of filaments, thick and thin , which fonn a removed revealing the closely packed arrangement repeating pattern {Figure 3}. In transverse of myofibrils within them. Ba r = 20 ~m . sections through the region of overlap, the filaments may be seen to lie in a well-order ed hexagonal lattice (see Figure 4). Each thick across. The collagen fibres in the perimyshrn filament , which is 1.6 lAm long, 1s made up are crimped and arranged in a criss-cross lattice principally of about 300 molecules of the protein (Rowe, 1974). In muscle at rest length, the myosin . The myosin molecule has a long tail collagen fibres in the pe r imysium are arranged at (156 nm x 2 nm wide) at one end of which two an angle of about + 55° t o the muscle fibre axis curved pea r- shaped heads ( 19 nm long) are but asstllle larger Or smaller angles as the muscle flexibly attached (see Figure 6). In the thick shortens or lengthens . I ndividual muscle fibres fi 1 ament t he tai 1 s pack together to form t he

152 Water-hold ing, appearance and toughness of meat

shaft, the tails in the two halves pointing in opposite directions. This leaves the heads on the surface where they can interact with the thin fi 1 ament (see Figure 6c) • The thin fi 1 ament is made up principally of about 400 molecules of actin, the precise number depending on the species. Each actin molecule is dliTlb - bell-shaped and can bind one my as in head . The actin molecules pack. in a helical manner with the long axis of each dumb-bell perpendicular to the filament axis . Ot her proteins , t r opomyosin and tropon in, bind to the actin filament and confer calchrn-sensitivity on the filament . Muscle contraction is triggered by the release of calcium ions from the sarcoplasmic ~- Hypothesis to explain the origin of reticulum in response to a ne r vous impulse. The ~in the water-hal ding of raw meat. A calcil.ITI ions bind to the thin filament switching myofibril is seen in transverse section in a it on in such a way that myosin heads can attach shrunken state on the left-hand side and a to the thin filament and generate force, probably swollen state on the right-hand side. The by undergoing a change of shape. Shortening of diagram is simplified and does not depict the the muscle occurs by successive cyclic depolymeri sat ion of the thick filament that interactions of the myosin heads resulting in occurs in high salt concentrations. (Reproduced increased overlap of thick and thin filaments. from Offer et al. (1984), by permission of the The energy for contraction comes from the Royal Society of Chemistry). hydrolysis of ATP catalysed by the myosin heads. After the death of an animal, the muscle continues to hydrolyse ATP at a slow rate . For a Conversely , when meat ts treated during time, the levels of ATP are maintained by the processing with NaCl, or NaCl together with breakdown of glycogen to lactic acid, resulting polyphosphates, water is taken up . Poly­ in a fall in pH from 7 to around 5 .5 . Eventually phosphates are added for three reasons . Firstly, the glycogen stores are depleted and the ATP they reduce the concentration of NaCl required concentration falls to zero. At this time the for water uptake. Secondly , they help solubili ze heads attach strongly to the thin filaments (see myosin which forms a sticky coat on the surface Figure 6b) causing the muscle to be rigid and of meat pieces and sets as a gel on heating. stiff. This condition is known as the rigor thereby binding meat pieces together (see Jo11ey state. and Purslow, 1988). Thirdly, polyphosphates greatly reduce the loss of water on cooking . Water-Holding of Heat Since myofibrils occupy about 80\ of the volume of living muscle fibres, most of the water Lean meat iiTIJlediately after slaughter in the muscle cell is present in the myofibrils contains about 75'1 water. But, depending on the in the spaces between thick and thin filaments properties of the meat and how it is treated. it (Figure 4). Only a small fraction of this water may gain or 1ose water, (see reviews by Ha11111, (perhaps about a tenth) is actually at any 1960, 1986; Offer and Knight, 1988a ,b). This is instant bound to protein ( see Offer and Knight, important economically since meat is sold by 1988a). The simplest hypothesis to explain weight . The content of water and its changes in the water held by meat is to suppose distribution within the meat also determines its that they have their origin in changes in the quality . For example, it is possible to have two volume of the myofibril s. In the raw state, pieces of meat with initially the same water where the filament lattice is regular, we suppose content, one dark and dry in appearance, and the that uptake of water occurs by the entry of water ot her pale and rapidly exuding water (Wismer­ into the myofibrils as they swell laterally by an Pedersen, 1959) . expa nsion of the ~ilament lattice. Co nv ersel y, Water can be lost from raw meat by we suppose that 1ass of water occurs by the evaporation from the surface, or by the exudation expulsion of water from the myofibrils as they from the cut surfaces of drip. This is a shrink laterally when the filaments get closer concentrated solution of intracellular muscle together (Figure 4) • proteins, including myoglobin, which gives it its red colour . The amount of drip is increased in Drip Loss pa 1e, soft and exudative ( PSE) meat . The PSE X-ray diffraction has the dual advantages of condition results from the denaturation of being non-invasive and being capable of proteins caused by the attainment of a low pH determining the centre- to-centre spacing between while the carcass is still warm (Wismer-Pedersen, neighbouring thick filaments with a high 1959). This can occur if the rate of breakdown precision . Figure 5 shows the filament lattice post mortem of glycogen to lactic acid is fast, spacing in beef rectus abdominis muscle as a the final pH is particularly low , or chilling is function of time post mortem. The spacing does slow . Meat loses even more water {up to 40% of not change much with time initially, but at rigor the weight of the meat) on cooking and there ts a onset there is a rapid decline to a new lower correspondingly large shrinkage. spacing. about 4.4% smaller than the original

153 G. Offer et al.

due to the attactvnent of myosin heads at rigor onset (see Offer and Knight, 1g88b) (Figure 6a ,b). Myofibrillar shrinkage 1s faster in pig 43 ···. muscles than in beef corresponding to the shorter times to rigor onset in pig (Knight, unpublished .. results). Preliminary results show that in PSE Lattice Spacing .. meat the myofibrils shrink about twice as much as (nm) in normal meat accounting for the higher drip in this state (Knight, unpublished results). What 42 is responsible for this greater s hrinkage? Penny (lg67a) and Stabursvik et al. (1984) showed that myosin was denatured in PSE muscle. We have .. .. recently found by negative staining that if ... myosin is exposed to conditions (pH 6 .0, 35°C for 5 or 10 minutes) similar to those experienced in ·····.··· PSE meat and which cause substantial loss of myosin ATPase (Penny, lg67b), the head length decreases fran 19 nm to 17 rvn (Sharp, Walker and Time after Death (h) Offer , unpublished results) . This is sufficient to draw the thick and thin filaments even closer 5 1 together at rigor onset and gfve rise to ~~~~\~9 ·in c~:~les r~~tu;he a~~~~~~~ :~;~~: increased expulsion of water (Figure 6c). post mortem detennl ned by X-ray diffraction. Calcul ations show that this smal 1 change in head (Reproduced from Offer et al. (lgag) by length is sufficient to account for the increased permission of Marcel Dekker) . myofibri 11 ar shrinkage and therefore the increased exudation in the PSE state . We can now ask where the water expel\ ed from the myofibri1s accumulates. Ideally, to observe this, we need a structural technique with a moderately high resolution (say 5 ~m), which could non-invasively image water compa rtments in a whole muscle. Perhaps in the future nuclear magnetic resonance imaging may prove helpful, but in the shorter tenn we have sludit!d water compartments in sections of muscle fixed at various times post mortem and embedded in paraffin wax (Of fer and Cousins, unpublished II II 'I ;~ Ill H111111 results) . We find that in beef sternomandibularis muscle at 2 h post mortem there are no large channels in the meat; the fibres fill the endomysia\ network and the fibre ,,,, 111111 nIll t!111111 bundles fill the perimysial network (Figure 7a). At 6 h post mortem , however, gaps of variable width appear between fibre bundles (Figure 7b}. At this stage there are few gaps between fibres. Finally, after 24 h post mortem, gaps also appear between fibres (Figure 7c) , as was first shown by Heffron and Hegarty (lg74). ~- Lateral shrinkage of a myofibril Evidently the fibres shrink as their post mortem . (a} Living muscle. The myosin constituent myofibrils shrink and the water that heads are detached and the filaments are widely i s left behind accumulates first around the spaced. {b) Norma l rigor muscle. The myosin perimysial network and later around the heads have attached to the thin filaments drawing endomysia\ network, giving rise to two the filaments closer together. (c) PSE rigor extracellular water compartments. muscle. Shortening of the myosin heads prior to Finally , we can ask whi c h water compartment rigor has caused the filaments to be drawn still is the source of drip, along what channels does closer together at rigor onset. At the bottom of it flow to the surface, and what force drives it the figure two myosin molecules are shown on the out? A slice of meat held with the fibres same scale, one normal an d the other denatured vertical produces much more drip on the lower under PSE conditions. The heads of the latter than the upper surface (O ffer, lg84a) . are shorter . Simil arly. if the slice is placed with the fibres horizontal, the lower part of the cut surface can be seen to be much wetter than the upper. If a (Knight, unpublished results). This corresponds piece of meat is placed in an organic solvent of to a 9% decrease in the cross-sectional area, and the same density as meat so that hydrostatic therefore volume , of the myofibr11s. The pressures due to gravity inside and outside the decrease is partly due to a fall in pH and partly meat are equalised at every level, drip is

154 Water-holding, appearance and toughness of meat

producing a small hydrostatic pressure on that region . If the column is small , say 1 em diameter, the solution passes through the meat parallel to the fibre direction wa shing out myog lobin and other soluble proteins fr001 a cylinder of the meat , and a circle on the opposite face of the meat appears nearly colourless (Starr , Almond, Knight and Offer, unpublished results). If the solution contains fluorescently- tagged bovine serum albumin and the opposite face is observed with a binocular m1 croscope equipped with fluorescence optics, the first appearance of fluorescence is quite local and confined to the primary perimysial network (Figure 8b) together with regions of the secondary perimysial network opening from it. With time, fluorescence emerges over a larger b area but still is confined to the perimysial network (Figures Be-f). This confirms the existence of continuous longitudinal channels through the meat between the fibre bundles. We suppose that drip arises predominantly by the action of gravity draining the fluid in these channels to the cut surfaces .

Meat Processing We have tested the hypothesis that water uptake in meat processing is due to lateral expansion of myofibrils. This was done by observing the effect on individual myofibrils of successive irrigations of salt (Offer and Trinick, 1983; Knight and Parsons, unpublished results) by the technique of Hanson and Huxley (1955). Phase-contrast light microscopy allows the myofibrils to be viewed in their hydrated state without staining. In our earlier work the myofibrils were attached to cover-slips but in ~· Light microg raphs of transverse more recent work they have been suspended between sect1ons of beef sterncxnandibularis muscle the bars of an electron microscope grid . When showing the fonnation post mo rtem of fluid-filled rabbit or beef myofibrils are treated with a channels. (a) 2 h post mortem (b) 6 h post series of NaCl solutions of increasing mortem (c) 24 h post mo rtem. Sections were concentration and buffered to pH 5 . 5 to simulate stained by Gomori' s reticulin method and counter­ rigor conditions , lateral swelling of the stained with 1% Ponceau 2R in U acetic acid . myofibrils starts in 0.6 M NaCl and is (Reproduced from Offer et al. (1g8g) by accompanied by extraction of myosin from the permission of Marcel Dekker). centre of the A-band. approximately the H-zone {Figure 9). Further swelling occurs in 0 .8 and 1 M NaCl . The response of different myofibri1s greatly r educed (Starr and Offer, unpublished is somewhat variable, but the average swell ing in results). All this suggests that drip is fanned 1 M NaCl is 90% + 52% for rabbit myofibrils and mainly by g ravity draining fluid from channels g4% + 60% for-beef myofibrils (Knight and with flexible walls. Observati on of 3 freshly Parsons, unpublished results). This is more than cut surface strongly suggests that drip wells up sufficient to explain the uptake of water during from gaps between fibre bundles, and that the meat processing . fluid in these wide channels is the source of the If pyrophosphate is included in the drip (Offer, 1g84a) . It could, however, be irrigation medium, extraction of myosin occurs at argued that drip might form uniformly at the cut lower NaCl concentrations and the extraction is surface and run into the crevices between fibre more ccxnpl ete. In the presence of 10 mM pyro­ bundles to give the observed appearance. It phosphate and 1 mM MgC1 , very little happens 2 caul d also be argued that the gaps seen between when the myofibrils are irrigated with 0.3 M fibre bundles are not continuou s along the fibre NaCl, but in 0 .4 M NaCl the A-band is completely direction or even that they are an artefact due or nearly ccxnp 1ete 1y extracted COITITlenc i ng from to fixation and dehydration. both edges eventually leaving stri ngs of To check for the presence of longitudinal !-segments (Offer and Trinick, 1983) . In the channels, we have applied a cylindrical colliTln of presence of pyrophosphate. swelling is markedly 0.9% Na Cl (normal saline) to one surface of a less than that produced in NaCl alone {42\ in thick transverse slice of either beef rabbit, 14% in beef) (Knight and Parsons, sternomandibularis or semitendinosus muscle, unpublished results).

155 G. Offer et al.

8 5 0 1 0 0 ;1 ~~~:sc e~tly~~~~~~~ras~~~~ ~lfbu!~~g~~~d~~:~ acph:t,need \a t~~~u~~r~aecaet ·of : 1 ~~ c~ t~:~~v~:sea s 1 i ~~\ h r ou~~ beef semitendinosus in the rigor state and the emergence of this solution on the opposite face followed with time. (a) Opposite face observed by visible light showing perimysial network. {b-f) observed with fluorescence optics (b) 8 minutes (c) 16 minutes (d) 20 minutes (e) 24 minutes (f) 32 minutes after application of hydrostatic head .

When meat is processed, it is often treated lower salt concentrations, this is not so. As with small volumes of very concentrated brines so the concentration of neutral salts like NaCl that Initially myofibrils may be exposed to high increases, initially there is an increasing concentrations of NaCl. During curing, for tendency for protein assemblies to dissociate example, near the sites of brine injection there ( the 'salting- in' range) but at higher will initially be a very high salt concentration concentrations this tendency diminishes (the which progressively diminishes, whereas further 'salting-out' range) . Callow (1932) found that from these sites the concentration of NaCl the water uptake by meat in brines was maximal at increases progressively from zero. It is 1 M NaCl and no water was taken up at therefore important to know how myofibril s concentrations above about 4 M NaCl. respond to very high NaCl concentrations. While Correspondingly, myofibrils do not swell and show naively one might think that higher NaCl 1 ittle change in structure when treated with 5 M concentrations would have a greater effect than NaCl buffered to pH 5.5. although at 1 ower

156 Water-holding, appearance and toughness of meat

.••...... b...... 0·2 ...... N ...... d .0·4...... -' 0·5 ...... ' ••••••••••••• 0·8 1·0 .... Figure 10. Variability in response of myofibrils ~treatment. A field of rabbit plantaris 1 myofibrils is shown (a) before and (b) after ~~f~~~o~;. s;he~ sia~ge ~:;f~~~\~ ~~o:~~~;~~r~~g~~~~ 0 by phase-contrast microscopy. {a) After ~~~~~~~~~ha~~t.h p~ ·~:5 .M ~~c~~sl o~M t~ic~}dfi~ ri ~~ preparation in pH 7 preparation medium. {b) shown, the A-bands are extracted but the arrowed myofibril is resistant to this salt ~6t~~ ~~~\ ~~tiancget:~~~ ~H \ .M5 • NaCi~) 1_ '(~ /~~~~; concentration. Bar indicates 10 ~.~m. further 3 minute irrigations with NaCl at the molar concentrations shown on the left hand side together with 1 mM MgC1 , 10 mM sodium acetate, pH 5.5, some myofibrils are extracted and swell, 2 pH 5.5. Sarcomere 1 eng-th 2.3 flm . (Reproduced others are only slightly extracted and do not from Offer and Trinick (1983) by permission of swell (Figure 10) (Knight and Parsons, Elsevier Applied Science). unpublished results). This is at least in part due to the difference between fibre types. Myofibrils prepared from muscles with a high concentrations (e.g., 1 M) they swell and the proportion of white fibres respond to a 1ower A-band is partly extracted (Knight and Parsons, salt concentration than those from muscles like 1g88). the soleus with a high proportion of red fibres. We may conclude that during the curing By antibody labelling of myofibrils from a muscle process the sites where swelling and protein such as rabbit plantaris, which comprises a solubil isation first occur are not immediately mixture of fibre types, we have shown that adjacent to the injection sites but at some myofibrils from fibres containing slow myosin distance from them. The difference in the time­ require a higher concentration of salt for course of changes in NaCl concentrations in extraction than myofibril s from fibres containing different parts of the meat is 1 ikely to be the fast myosin {Knight and Parsons, unpublished source of the approximately periodic stripes results). The suitability of meat for processing often present in bacon slices (Voyle et al., may therefore be influenced by variation in the 1986). content of fibre types from muscle to muscle and We commented above on the variability of the animal to animal. response of myofibrils to salt. In a field of Mechanism of Swelling. In a previous myofibril s prepared from rabbit p l antari s muse 1 e publ1cat1on we supposed that chloride ions irrigated with 0.45 M NaCl plus pyrophosphate at binding to the filaments increased their negative

157 G. Offer et al. charge, causing increased long-range electro­ (a} static repulsive forces and therefore swelling (Offer and Trinick , 1983}. However, at high salt concentrations, the excess of coun terions balancing the negative charge on the filaments is localised so closely to the filament surface that the charge on the filaments would be effectively screened. This screening effect would more than outweigh the effect of inc reased charge due to Cl- bi nding (Ledward, 1983 ; Offer , 1984b; Offer et al . , 1989} . In collaborati on with Dr B Mil l man and Dr 8 Nickel of Guelp h University, we have proposed a new kind of hypothesis in whi ch salt-induced swelling is entropically rather than (b) electrostatically driven (Offer et al., 198g} . Th is is illustrated in Figur e 11. It is well kno wn that moderately high salt co ncentrations , , _! (0 .5 to 1 M} depolymeri se thick fi 1 aments into myosin molecules. In the region where thick and ---= ~ thin filaments overl ap. these myosin molecules would tend to remain attached to actin filaments (Figure lib}, although in the H-zone they would ->- be free to diffuse away . The myosin tails, being ¥ s- b flexibly attached to the heads, would tend to explore space but this motion would, we suppose, in the unswollen lattice be severely restri cted by the presence of neighbouring thin filaments and the myosin molecules attached to them (Figure (c ) 1 llb}. If the latti ce swe lls (Figure llc} the tails would have greater freedom of motion and therefore hig he r entropy . Si nce systems tend to move to a state wh e re they have highest entropy . there would be a marked t endency for the lattice to expand, whi ch, compared with the electrostatic swelling pressure, would diminish only slowly with expansion. On this hypothesis, depolymerisation of the thick filaments liberates the myosin tails necessary for the driving force to be developed . By contra st, dissociation of the myosin from actin would reduce the swelling pressure by reduci ng the amount of bound myosin. This effect would be particularly great if there were an ~- _!_l. Mechanism of swelling of myofibril s excess of br i ne such as in the irrigated ~ ( a ) Longitudinal section through the myofibrils, since much of the myosin would then overlap region of a myofibril showing two thin escape . filaments and one thick filament with its myosin The effectiveness of polyphosphates in heads attached to the thin filament. (b) As (a} promoting water uptake and pr otein solubilisation but showing the depolymeri sation of the thick in meat pr ocessing is in part due to the small filament as a result of treatment with NaCl increase in pH alkal ine pol yphosphates produce before swell i ng has occu r red . The motion of the (Lewis et al ., 1g8 6 ; Trout and Sc hmi dt , 1986) but myos i n t ails i s rest r icted. {c) Aft er swell ing t here is also a specifi c effect (Bendall, 1954) . of t he f il amen t l attice, the myosin tails are It is very well establ i shed t hat pyrophosphate in able to move t hr ough a larger ang l e . (Reproduced the pr esence of magnes i um i ons weakens the f r om Offer et al. (1989} by permission of Marcel association of actin and myosin heads ( see Offer Dekker}. and Knight, 1988a) . It is also known that pyrophosphate assists chlor ide in causing the depolymerisation of thick filaments (Harrington especially in systems, li ke irrigated myo fibrils , and Himmel farb , 1972). This gives us a basis for where myosin would be lost. In systems where the under standing the effects of pyrophosphate . brine :meat ratio is much smaller, such as occurs Because depolymerisation is pranoted by in meat processing, the swelling with pyro­ pyrophosphate, extraction of myosin and swelling phosphate would be greater since more myosin occur at a lower concentration of NaCl than in would remain bound to actin . its absence . But because pyrophosphate promotes The importance of myosin to swelling has been dissociation of myosin from actin, maxim1.111 tested by irrigating myofibrils, fr001 which swelling in the presence of pyrophosphate would myosin has been previously extracted, with a be expected to be lower t han in its absenc e. solution of myosin . The thin filaments bind the

158 Water-holding. appearance and toughness of meat

myosin and the myofibrils swell substantially. meat and its water-holding characteristics (Hamm. Thus the hypothesis appears plausible and we will 1g6Q), although drip loss and light scattering do be testing it critically. not accurately parallel one another (Warriss and Effect of the EndomysilJI1 on Swelling . Having Brown, 1gs7) . We have supposed that the 1 ight discussed the sweii1ng of myof1bnis in salt scattering property of muscle resides in the solutions, we can now consider the swelling of myofi bri 1 s and depends on their degree of whole fibres, and consider the influence of the expansion (Offer and Trinick, 1983). Recent connective tissue. The degree of swelling of experiments have enlarged and modified this view . dissected muscle fibres in salt solutions is in Jeacocke (1g84) showed that the our hands very variable from fibre to fibre. 1 ight-scattering ability of muscle increases Wilding et al . (1g86) had shown that, If the substantially at rigor onset, presumably due to endomysial sheath surrounding a fibre i s damaged the shrinkage of myofibr1ls occurring at this at one point , much more swelling takes place tirne. He also showed that the light-scattering there and they concluded that the endomysium acts ability is 1 ittl e affected by treatment of muscle as a mechanical restraint to swelling . It seemed with detergent. suggesting that membranes and possible that the variability we observed was due membrane-lined organelles contribute little to to some fibres being dissected with an endomysial the 1 ight- scattering by muscle. sheath and some without. Whether or not a fibre Dark field microscopy provides a rather has a sheath can be detennined by dissolving out direct way of investigating light scatter since muscle proteins from the muscle fibre with SDS. the image is constructed from 1 ight scattered by After irrigation of a sheathed fibre, the the object. Dark-field images of isolated endomysial sheath is l eft. but with an unsheathed myofibril s in suspension indicate that the fibre the entire preparation dissolves. We found A-band , and to a lesser extent the Z-disc, are that the sheathed fibres swelled only a 1 ittle in bright sources of scattered light (Jeacocke, salt, and the unsheathed fibres swelled much more unpublished results). The intensity within the ( Knight et al., 1g8g; Offer et al., Jg8g). This A-band is not unlfonn; the intensity distribution reinforced the conclusion of Wilding et al. that is consistent with an origin in 1 ight scattered the endomysium could act as a mechanical principally at the surfaces between the A-band constraint and also showed that fibr es can be and the suspending medium and at the A-I junction dissected with or without a sheath. (Figure 12a). In general, light scattering We further found that the remarkable increase occurs at interfaces between two phases that have in swelling over a narrow period of time post different refractive indi ces. The observations mortem observed by Wilding et al. could be on myofibrils are therefore explained by the entirely explained by the change with time of the steps in refractive index occurring at their ease of stri!J~.dny of the endomysium; there was no surfaces and at the A- I intertaces. Light other effect of time (Knight et al., 1g8g). scattered from a suspension of myoflbrils is Stanley ( Jg83) showed that emptying of the maximal at a pH of about 5 and decreases at 1 ower contents of musc le fibres when exposed to calcil.l11 or higher pH• s (Jeacocke, unpublished results). ions requires an extended period of conditioning. This is consistent with light scattering All this suggest s that the optimum time post depending on myofibrillar volume which would be mortem for processing of meat may depend on expected to be minimal at pH 5 , the isoelectric proteolytic weakening of the connection between point of myosin and actin. muscle fibres and connective tissue allowing In muscle the 1 ight scattering properties of stripping of the endomysial sheath during myofibrils are modified because they are packed conrni nut ion, and therefore greater myosin alongside one another in register with a small extract ion and water uptake . but variable gap between neighbours {Figures 12b, c ,dl . If neighbouring myofi bri 1s touched (Figure Appearance of Meat 12b • there would be no step in refractive index at their junction and therefore no light The colour of meat is detennined not only by scattering at this interface. A small gap, say the quantity and oxidation state of the pigment greater than a tenth the wavelength of 1 ight myoglobin, but by the 1 ight scattering properties (Figure 12c), would be sufficient to create of the meat (Macoougall, 1g82) . In samples with substantial scattering and the degree of scatter a high light-scattering ability , such as PSE would increase with the width of these gaps. The meat, light does not penetrate far into the meat step in refractive index would be greatest in the before being scattered; hence there is relatively A-band and hence light scattering would be little absorption by myoglobin and so the meat greatest at the intermyofibri 11 ar gap between appears pale. In contrast, dark, firm and dry A-bands. (DFD) meat , fanned when the final pH is high, say The confocal scanning 1 ight microscope >6.0, scatters light only to a small extent. produces r eflectance images of skeletal muscle Incident light is therefore able to penetrate the which differ substantiall y from those produced by meat for a substantial depth and is strongly conventional transmission light microscopy absorbed by myoglobin . Such meat therefore (Jeacocke, unpublished results). The particular appears dark . We shall now discuss the advantages of this technique is that artefacts structural features responsible f or this 1 ight due to fixation, dehydration and embedding are scattering. avoided and that the optical section providing It has long been appreciated t hat there is a the image can be as thin as 0 .8 ~ m, 1ess than the broad relationship between the appearance of the diameter of a myof ibri l. This allows the light

159 G. Offer et al.

a Il l Il l Il l

Figure 13. Scanning confocal light micrograph of part of a fibre within a fibre bundle fr001 rabbit psoas muscle in rigor showing 1 ight scattering features. The figure shows a l ong itudinal optical section (app r oximatel y 0 . 8 ~m thick) taken on a Lasersharp MRC-500 1 nstrument wlth a 60 x 1.4 NA oil-immers ion objective and operated in the reflectance mode. Bar z 20 u m.

same periodicity as the sarcomere. Comparison between reflectance and f1 uorescence confocal images of skinned muscle in which the myosin ha s been labelled fluorescent 1y suggest that each speckl ed band coincides axially with the central region of an A-band . The speckled appearance varies along the length of a fibre and also between fibres. In PSE meat the speckles are 1arger and there appear to be more of them . The speckled appearance is not substantially al tered by treatment with detergents, suggesting that ~t· scHJf~! ~~~~s i~xp~:!~i.ng {:) ~=~~~t~~ membrane- bound organelles , such as mitochondr ia myofibril. (b-d) Neighbou r ing myofibrils in and the sarcoplasmic reticulllll, are not directly muscle. In (b) the adjacent myofibrils touch. responsible for the scattering. In (c) ther e is a small space between Assum ing that the speckled character is not neighbouring myofibrils. In (d) structures an artefact of coherent light illumination, we l inki ng myofibri1s are also shown but not the may conclude that l i ght scatt ering from muscle is transverse tubules and sarcoplasmi c reticu lum pred001in antly not from molecular features {for whi ch are also present in the gaps between example, cross-bridges) but rather from much myofibril s. larger structures. It seems possibl e that the speckled appearance arises f r om variations in the closeness of packing of adjacent myofibrils . On scattering featu res in a thin layer within a this basis the light scattering sources would sample of meat to be vi ewed without superposition corr espond to small regions where the gap between effects from structures ab ove and below the adjacent myofibrils was wider than elsewhere. section . Figure 13 shows the appearance of a Adj acent myofibrils are joined together at Z-disc thin optical section within a fibre bundle fran ( and M-1 ine ) level by proteins such as desm in rabb1t psoas muscle . The most obvious feature of (G ranger and Lazarides , 1978) (Figure I2d). The this image is the highly speckled appearance. speckles may therefore arise f r cwn l ight 9na11, bright sou r ces of 1 ight substantially scattering at the boundaries of intennyofibr ill ar sma ller than 1 IJ m in width are distributed ccmpartments bounded by these structures . It is throughout the sect ion but are particularly at present unclear whether the T-tubules and the numerous along transverse bands about 1 ~ m wide. tenni nal cisternae of the sar copl asmi c reticultiTI , These bands often extend right ac ross the fibre whi ch are present as a collar around each withou t dislocations and are arranged with the myofibril at the ends of the A-bands, play any

160 Water-holding, appearance and toughness of meat

role in producing light scattering, for example stretched to twice its original length, does the by acting as spacers between myofibrils. stiffness rise steeply due to stretching of the Further work. 1s required to test these not 1ons perimysium (Davey and Dickson, 197D). Cooking and to detennine the precise nature of the dramatically changes the shape of the load­ scatterers. extension curve . After cooking at 8o•c, the initial stiffness is much higher but after loads Toughness of approximately 1 to 1 .5 kg/em 2 and extensions of 5 to 20% (the yield point), the stiffness The most important attribute of the eating falls markedly (Bouton et al., 1975; Locker et quality of meat is its tenderness. When we c hew al., 1983). The breaking strength of meat cooked meat, it i s pulled apart, and the difficulty with at 70 or SO'C is appreciably greater (1.5x) than which this is achieved is perceived as the that of raw meat (Bouton et al ., 1975; Locker et sensation of toughness . A variety of mechanical al ., 1983), whereas breaking extensions have been tests has been applied to meat , but tensile reported to decrease slightly on cooking (Locker tests, in a direct i on either parallel or et al., 1983) or to increase (Bouton et al., perpendicular to the fibre axis, are the simplest 1975). Cold- shortened samples have a far greater to interpret ( 8outon et al ., 1975). The breaking extension than an unshortened control, longitudinal breaking strength of raw meat and stretched muscles have a smaller breaking carrel ates reasonably well with the sensory extension {Bouton et al., 1975) . For meat cooked perception of toughness of cooked meat (Stanley at 8o·c the peak force is 1 argest for cold­ et al., 1972) although no such comparison seems shortened samples, followed by stretched samples to have been done on the longitudinal breaking and 1 s 1 east for unshortened controls. strength of cooked meat . If cooked muscle is stretched to about 65% A particularly useful approach is to examine extension, then the tension is removed, the strip the structural events occurring when samples of returns almost to its original length (Locker et meat are extended to breaking point (Carroll et al., 1983). However, on reloading the strip, the al., 1978; Purslow, 1985. 1987). In a tensile load-extension curve is markedly different: the test, the progressive breakdown of the structure load required for small extensions is much leading to its final fracture will depend not smaller, although as would be expected, the load onl y on the stiffness , breaking st r engt h and required to return to a 65% extension is the same breaking strain of its com ponents, but on their as for the first run. The form of the curves is spat 1a 1 arrangement and the con ne ctions between consistent with elastic structures progressively them. failing at extensions right up to 65% and not When a transverse s 1 ice of cooked meat is only at or near the yield point. pulled apart in a direction perpendicular to the Ageing for 7 d at z•c prior to cooking almost muscle fibre axis, the first event seen is the halves the initial stiffness without affecting opening up of cavities throughout the slice in the stiffness at higher extensions (Locker et regions between fibre bundles {Purslow, 1985, al., 1983). The breaking strength is slightly 1987) (Figures I4a,b). Histological reduced. After cooking for 3 h at 10o•c, which investigation shows that the site of fracture causes the sol ubil isation of a substantial 1 ies between the perimysial network. and the fraction of the collagen as gelatin, the yield endomysia of fibres on the surface of a bundle point survives but the longitudinal breaking {Purslow, 1987). A striking demonstration that strength is greatly reduced (Locker et al ., the cleavage pathway is between perimysium and 1983) . This suggests that connective tissue endooysiliTI is obtained by viewing the cleaved contributes substantially to the breaking surfaces by scanning electron microscopy strength but is not implicated in the structural (Purslow, 1987) . Evidently the weakest component changes responsible for the yield point. of the cooked meat, and therefore the first to The precise sequence of structural events break., is the junction between the endomysium and that occurs as the cooked meat is stretched is perimysium. As the load is further increased, not yet clear , although we do know many of the some of the cavities joi n up in a fracture path elements of the overall picture. At the which runs along the boundaries between fibre macroscopic level, the first clearly observable bundles, with strands of perimysium, presumably event upon extending unaged cooked meat is the originating from nodes of the perimysial network, separation (debonding) of fibre bundles from each bridging the gap at intervals and carrying the other (Purslow, 1985, 1987) (Figure 15b), showing load (Figure 14b ,c) . When a still higher load is that the endomysial -perimysial junction is a weak applied, further extension occurs and these component in this testing direction, as well as perimysial strands rupture (Flgure 14c,d). Thus, in the transverse direction. in this transverse direction . the breaking The result is that each fibre bundle and the strength (maximum stress) of the meat is simply perimysial network become isolated from one determined by the amount and strength of the another and independently bear the tension. The perimysi t.m, the 1 ast structure to break . fibre bundles and perimysil.m can then be regarded We shall now consider what happens when as elements acting in parallel. In this case, strips of raw and cooked meat are subject to the element with the least extensibility (least tensile testing parallel to the muscle fibre breaking strain) is the first to break and the direction . In raw meat, after an initial stiff load is then thrown onto the remaining elements . phase for small extensions, the material becanes Structures progressively break. down and the most more compliant, and only when the meat is extensible component finally remaining detennines

161 G. Offer et al.

~· Fracture behaviour of a transverse slice of cooked meat when H is pulled apart in a ~perpendicular t o the fibre axis. The stippled dark grey areas represent the fibre bundles and the dark lines show the perimysial network . (a) Prior to the application of tension the fibre bundles are joined to the perimysium by tenuous connective tissue threads . {b) After extension , cavities develop due to the pa r tial separation of fibre bundles and perimysilJll . (c) On fur ther extension, a fracture pat hway connects some cavities but is bridged by perimysial strands. (d) Complete fracture .

the load and extensi on at fail ur e . Let lSc), finally leaving perimysial strands as the us consider the ex tensibll ity of these two last structures to break (Ca rroll et al., 1978 elements . Mus cle fibres isolated from cooked (F igure 15d). Howe ver, 1t i s not kno wn whether mu scl e can be stretched by greater amounts before peak 1 oads have been passed at the stage when they break than can raw fibres, in some cases t o only perimysial strands remain. In other words mor e than 100%, although considerable variability it is not yet c lear what contribution fibre is obs erved (Wang et al., 1956; Hostetl e r and bundles make to the longitudinal breaking Cover, 1961; Jeacocke, unpublished experiments) . str ength (maximum stress). Ageing the meat decreases the extensib1l ity of This kind of model can expl ai n in general the cooked fibres. Perimysium isolated from tenns the differences between the 1 eng it ud ina 1 cooked muscle is also very extensible . Breaking and lateral breaking strengths of meat and the extensions are very vari able, but in some cases effect of ageing . In unaged meat the exceed 150~ (Lewis and Purslow, unpublished longitudinal breaking strength of cooked meat is results). mo r e t han ten times higher than the later al Thus when a cooked muscle strip continues to breaking strength (Bouton and Harris, 1972; be stretched longitudinally, after t he point Purslow, 1985). This can be under stood if in where debonding of fibre bu nd les frcrn the unaged meat the fibr e bundles make a substantial perimysium occurs , both the perimysial network con tribution to the longitudinal breaki ng and the fibre bundles will contribute to the strength and none to the transver se breaking load-bearing at substanti al extensions . It seems stre ngth. As meat is aged, the lateral breaking most li kely that fibre bundles progressively fail strength is unaltered (Bou t on and Harris, 1972 ; as independent units (Purslow, 1985) (Figure Purslow, unpublished exper iments). This

16 2 Water-holding, toughness and appearance of meat

a c

' $ >:- s , ''; -a > 'ttt>< ~ a;

b d

), ·'<'•'(

. a 1 p· '

f_i gu_re_l_~. Fracture behaviour in a strip of cooked, unaged meat when pulled apart in a direction ~to the fibre axis, The grey areas represent fibre bundles. The heavy lines represent the perimysium and the fine diagonal 1 ines represent fine connective tissue strands between perimysium and endomysium. (a} Prior to the application of tension. (b) After extension, separation of fibre bundles and perimysium occurs. (c) On further extension, fibre bundles break. (d) Complete fracture.

indicates that although the perimysium may be regulatory proteins form a gel. When unaged subtly altered during conditioning (Stanton and cooked meat is stretched to breakage, Locker and Light, 1987), its strength after cooking is Wild {1982) considered that there was no visible unchanged. However, the longitudinal breaking damage to the integrity of the myofibril. At strength declines considerably during ageing extensions up to 30%, the I-band increases in (Bouton and Harris, 1972; Purslow, unpublished length, but at extensions up to 60% the A-band experiments). This can be understood if the also stretches. Unfortunately no infonnation is ;elative contribution of the fibre bundles to the available for larger extensions. It appears that longitudinal breaking strength declines with age. up to 60% extension at least, the length changes This may come about because when the meat is in the muscle strips are accommodated by a aged, fibre bundles tend to fail at smaller uniform, proportional increase in the length of extensions and therefore contribute less to the all sarcomeres. However, the point does not seem maximum stress. to have been rigorously tested. Sarcomeres from It is of considerable interest to know what muscle that had been stretched to breaking point happens in the myofibrils when a cooked fibre is and allowed to recover appear similar to those stretched. In muscle cooked at temperatures from rest length muscle (Locker and Wild, 1982) above 60°C, the thick filaments fuse to form an but the available resolution may be inadequate to apparently amorphous A-band (Schmidt and Parrish, detect breaks in the structure. On stretching up 1971) probably consisting of a myosin gel to 30%, filaments of unknown origin, possibly gap ( Ishioroshi et al ., 1983; Hermansson and Langton, filaments, appear in the I-band. At 50% 1988) in which gap filaments are embedded. The extension, the stretched A-band develops a thin filaments in the I-band lose their identit.y speckled appearance due to fragments superimposed on cooking above 60°C (Scl"midt and Parrish, 1971) on an array of fine filaments, which have been but it is not yet clear whether the actin and assumed to be gap filaments, although this

163 G. Offer et al. remains to be proved. Muscle as Food, Bechtel PJ (ed), Academic Press, When cooked beef muscle that had been aged 7d Orlando, 135 - 199 . at 2•c is stretched to the breaking point, the Hanson J, Huxley HE ( 1955) The structural extension occurs by the stretching of the 1-bands basis of contraction in striated muscle. Symp . alone; the A-bands are unaltered (Locker and Soc . Exp . Biol. 9, 228- 264. Wild, lg82). Harrington WF, Himmel farb S ( 1972) Effect At present , it is therefore far from clear of adenosine di- and triphosphates on the what structural events are responsible for the stabi 1 i ty of syntheti c myos 1n fi 1 aments . yield point, what s tructures in the A and I bands Biochemistry II, 2945-2952 . are load-bearing and where in the sa rcomere Heffron JJA, Hegarty PVJ ( 1974) Evi dence failure occurs. It is also unclear when the for a relationship between ATP hydrolys is and fibre con t ents fail, whether structural changes in extracelluar s pace and fibre diameter con tinuity is provided by the endomy sium; Str eet during rigor development in skeletal muscle. and Ramsey ( lg65) show that, at l east in r ed Comp. Biochem . Physiol. 4gA, 43-56. muscle fibres. following rupture of the fibre, Hermansson A- H, Langton M (1988) Filament ous the endomysium is capable of maintaining the structures of bovine myosin in diluted integrity of the fibre at nominal loads of suspensions and gels . J. Sc i. Food Agric. 42, 3.7 kg/em 2 , a value close to the breaking 355-369 . - strength of cooked meat. Hostetler RL , CoverS (1961) Relationship of Clearly more information is needed on the extensibility of muscle fibres to tenderness of relative contributions of fibre bundles and beef . J . Fd Sci. 26, 535-540 . perimysil.lll to the stress-strai n curves of muscle Ishioroshi M-;-samejima K, Yasui T (lg83) aged for d1 fferent times, stretched or shortened Heat- induced gelation of myosin filaments at a to different extents and cooked under different low salt concentration . Agric. Biol . Chern. 47. conditions. We especi ally need to establish at 280g - 2816 . - what point different structural elements fail in Jeacocke RE (1984) Light scattering from a longitudinal tensile test. muscle during the onset of rigor mortis. Proc . The structural approach therefor e is valuable 30th European Meet . Meat Res . Workers, Bristol, in focussing attention on the components that 104 -107. determine the strength of the meat and enables Jolley PO, Purslow PP ( 1988) Refo rmed meat the we alth of biochemical information o n the products fundamental concept s and new nature of t he collagen making up the connective developments . In : Food St ructure - its Creation ti ssue, and in particular its cross-linking, to and Evaluation. Mitc hell J, Blanshard JMV be int eg rated into the mechanical pi c ture . It is (eds) , Butterworths, London, 231-264. also useful in providing a framewor k within which Knight P, Parsons N (1988) Action of NaCl to investigate the effects of other processes and polyphosphates in meat processing: responses which affect toughness , such as cooking , ageing of myofibril s to concentrated salt solutions. and cold-shortening. Meat Sci . 24, 275- 300 . Knight?, Elsey J , Hedges N (1989) The role References of the endomysium in the salt-induced swelling of muscle fibres. Meat Sci. in press . Bendall JR (1954) The swelling effect of Ledward OA (1983) Letter to the editor. polyphosphates on lean meat. J . Sci . Fd Agric. Meat Sci . 9, 315-316 . 5, 468-475. Lewis-OF, Groves KHH, Holgate JT (1986) - Bouton PE, Ha rris PV (1972) The effects of Action of polyphosphates in meat products. Food some post-s\ aughter treatments on the mechanical Mi crostruc . 5. 53-62 . properties of bovine and ovine muscle . J , Fd Locker lUi, Wild DJC ( 1982) Hyofi bril s of Sci. 37, 539 - 543 . cooked meat are a cant i nuum of gap fi 1 am ents . 8outon PE, Harris PV, Shorthose WR (lg75) Me at Sci. 7, 189-196 . Possible r elationships between shear, tens1le and Locker- RH , Wild DJ C, Daines GJ ( 1983) adhesion properties of meat and meat structure. Tensile pr operties of cooked beef in relation to J. Text . Stud, 6, 297-314. rigor tempe r ature and tenderness . Meat Sci . 8, Callow EH-(1932) The theory of cu ring, 283-299. - Report Food Invest. Board 1931, 144-147. MacDougall DB ( 1982) Changes in the colour Carroll RJ , Ro rer FP, Jones SB . Cava naugh JR and opacity of meat. Food Chemi stry 9 , 75-88. (lg78) Effect of tensile stress on the ultra­ Offer G, Trinick J (1983) On the mechanism st ructur e of bovi ne muscle. J . Fd Sc1. 43, of water- ho l ding in meat : the swelling and 1181-1187. - shrinking of myofibrils. Meat Sci . 8, 245-281. Davey CL , Oi ckson HR (1970) Studies in meat Offer G (1984a) Progress in the tenderness. 8 . Ultra-structural changes in meat biochemistry . physiology and structure of mea t. during aging. J . Fd Sci. 35, 56-60. Pr oc. 30th Eur opean Meet. Meat Res . Workers. Gr ange r BL, Lazarides E (1978) The Bristol, 87 - 94 . existence of an insoluble Z-disc scaffold in Offer G ( 1984b) The nature of the repulsive chicken skeletal muscle. Cell 15, 1253-1268. forces between filaments in muscle and me at: a Hamm R ( 1960) 8iocnemi stry of meat reply to Dr Ledward. Meat Sci. 10, 155-160. hydration . Adv. Food Res. 10, 355-463 . Offer G, Restall 0 , TrfiiTck J (1984) Hamm R (1986) Functional properties of the Water- holding in meat. In : Recent Advances in myofibrillar system and their measurements. In : Chemistry of Meat. Bailey AJ (ed), Royal Society of Chem istry , London, 71-86 .

164 Water-holding, appearance and toughness of meat

Offer G, Knight P (lg88a) The structural 1416-1423. basis of water-holding in meat. Part 1. General Voyle CA , Jolley PO, Offer GW (lg86) principles and water uptake in meat processing. Microscopical observations on the structure of Oeveloj)'llents in Meat Science - 4, Lawrie R (ed), bacon. Food Microstruc. 5 , 63-70. Elsevier Applied Science , London, 63-171. Wang H. Doty OM, Beard-FJ, Pierce JC, Hankins Offer G, Knight P (lg88b) The structural OG (lg56) Extensibility of single beef muscle basis of water-holding in meat. Part 2. Drip fibers. J. Anim. Sci. 15, g7-108 . losses. In : Oevelo~ents in Meat Science- 4, Warriss PO , Brown SN (lg87) The Lawrie R (ed) , Elsevier Applied Science, London, relationships between initial pH, reflectance and 173-243. exudation in pig muscle. Meat Sc i. 20, 65-74. Offer G, Elsey J, Parsons N, Cousins A, Wild ing P, Hedges N, LillforaPJ (lg86) Knight P (lg8g) Molecular and cellular Salt-induced swelling of meat: the effect of mechanisms of water gains and losses in meat . In: storage time, pH, ion-type and concentration . Basic Interactions of Water and Foods, Le Maguer Meat Sci . 18, 55-75 . M, Powrie W (eds), Marcel Dekker, New York, in Wismer-=P"edersen J (lgsg) Quality of pork in press. relation to rate of pH change post mortem. Food Penny IF (lg67a) The effect of post mortem Res . £1_, 711-727. conditions on the extractability and adenosine triphosphatase activity of myofi brill ar proteins Discussion with Rev1ewers of rabbit muscle . J . Fd Technol. 2, 325-338 . Penny IF (lg67b) The influence of pH and R. Hanm: The authors explain the high drip loss temperature on the properties of myosin. Otl'"rr pork by denaturation of myoflbrillar Biochem . J . 104, 6og-615. proteins, particularly by changes in the head PurslowPP (lg85) The physical basis of moiety of the myosin molecule. On the other hand, meat texture: observations on the fracture Honikel and Kim (lg86) concluded from their behaviour of cooked bovine M. semitendinosus. results that the fast release of drip post mortem Meat Sci. 12, 3g-60 . from PSE muscle must be mainly due to changes in Purslow PP (lg87) The fracture properties the muscle cell membranes, and not more than 25~ and thennal analysis of collagenous tissues. In: might be due to denaturation of myoffbrillar Advances Meat Research, Pearson AM, Dutson TR, protei n. Do you agree that increased permeab1l ity Bailey AJ (eds) , von No strand Reinhold Co. , New of cell mem bra nes contributes considerably to the York, Vol. 4, 187-208. wateriness of PSE pork? Rowe RWD (lg74) Collagen fibre arrangement Authors : No. we are not convinced by their in intramuscular connective tissue. Changes arguments. Honik.el and K1m (1986) found that in associated with muscle shortening and their pig psoas muscles considered to be PSE in that possible relevance to raw meat toughness they had a pH < 5 .8 at 45 min post mortem, only measurements. J. Fd Technol. g, 501-508. 25% of the myosin was denatured as judged by loss Schmidt JG, Parrish FC 11971) Molecular of ATPase or by differential scanning properties of post mortem muscle. 10. Effect of calorimetry. But their argument, that wateriness internal temperature and carcass maturity on cannot be due to myosin denaturation because it structure of bovine longissimus . J. Fd Sci. 36, is so limited , is logically flawed. 110-llg. - PSE is not an all-or-none phenomenon. Drip Squire JM, Vibert PJ (eds) (lg87) Fibrous loss increases progressively with decrease of Protein Structure, Academic Press, London. pH . In studies on pig longissimus dorsi Stabursvik E, Fretheim K, Froystein T. (lg84) mu~~les, maximtlll drip loss occurred at a pH of Myosin denaturation in pale, soft and exudative 6 .0 or bel ow ( Wa rri ss and Brown, lg87), alt~augh (PSE) porcine muscle tissue as studied by the extent of denaturation, and its dependence on differential scanning calorimetry . J. Sci. Fd Agric. 35 , 240-244. ~~t~' ;~~l~ab; =~~~ct;~P~~dde~~n~h~n ;~:c~~i.llj~~ StanTey OW, McKnight LM, Hines WGS, Usborne example due to fibre type differences. Honikel WR , Deman JM ( 1972) Predicting meat tenderness and Kim excised their muscles soon after death from muscle tensi 1 e properties. J. Text. Stud. and then kept them at 35°C, The conditi ons 3, 51-68. experienced by these muscles were therefore 1 ess - Stanley OW (lg83) A review of the muscle severe than would have been encountered had the cell cytoskeleton and its possible relation to muscles remained on the carcass, wh ere in the PSE meat texture and sarcolerrma emptying. Food state the temperature can exceed 40°C at 1 h post Microstruc. 2, 99-109. mortem. It is not therefore surprising that the Stanton l:, Light N ( 1g87) The effects of degree of myosin denaturation they observed was conditioning on meat collagen . Part 1. Evidence low . By contrast, Stabursvik et al . (lg84) showed for gross in situ proteolysis. Meat Sci. 21, that in muscles kept in a PSE carcass, a larger 24g- 265 . - fraction (about 50%) of the myosin was Street SF, Ramsay RW (lg65) Sarcolemma: denatured. Transmitter of active tension in frog skeletal Honikel and Kim did not measure the drip muscle . Science 14g, lJ7g-1380 . from their muscles and were therefore not in a Trout GR, SC!iiiiidt GR (lg86) Effect of position to prove that their muscles gave the phosphate on the functional properties of maximum amount of drip that pig psoas muscles are restructured beef rolls: The role of pH, ionic capable of. In other words they did not establish strength and phosphate type . J. Fd Sci. i!_, that their muscles were in an extreme state of

165 G. Offer et al.

PSE, and therefore could not expect the degree of still warm. As far as we are aware, all cases of myosin denaturation to be maximal. PSE can be explained on the s imple hypothesis Even if it were established that, in PSE that it is due to the time-dependent denaturation muscles giving maximum drip, the degree of myosin of protein occurring under these conditions. denaturation was only about 251., this would not Of the causes given above, (2) and (4} are demonstrate that myosin denaturation was due to bad handling of animals or carcass, rather unimportant . We do not know the dependence of than a genetic defect. The high rate of myofibrillar shrinkage on the degree of myosin glycolysis in stress-susceptible pigs is well denaturation. There is no reason to think that established as a genetic defect. The cause of maximum shrinkage requires all the myosin to the low ultimate pH in (3) is not known and has denature. It could be that when only a fraction been rather little stud ied, but might be the of the heads has denatured, their tendency to result of another genetic defect, for example shorten over-rides the undenatured heads and causing an increase in the amount of muscle causes the lattice to shrink maximally. What is glycogen at slaughter. urgently needed is to determine the degree of Whi1 e paleness in meat might come about myosin denaturation and myofi bri 11 ar shrinkage simply by a reduced myoglobin level (which might for a wide range of pH values. be genetically controlled), our current With regard to m~brane damage , it is known understanding is that the combination of pale, that if a muscle is cut very soon after death and soft and exudative characteristics is caused only centrifuged, very little water is lost (Ling and by thermal denaturation of proteins. It is Walton, 1976); in other words its water-holding difficult to envisage any other set of capacity is high. Thus despite the cell membrane circumstances, geneti cally controlled or having been severed, water is well retained by otherwise, that would bring about this thermal the cell contents (by the myofibrils in our denaturation, and which would be the cause of view). This shows that breakage of the cell PSE. membrane by itself is not the cause of drip. The However, the severity of the PSE state fact that drip is a solution of sarcoplasmic resulting from a particular time-course of proteins {Howard et al., 1960; Savage, Warri ss temperature and pH might be influenced and Jolley, personal communication), suggests genetically. For example, the myosin isoforms that even in normal rigor the cell membrane present in different muscle fibre types might becomes 1 eaky. Our own experiments have shown have slightly different susceptibilities to that in PSE muscle the myofibrils have shrun k by denaturation. Apart from this, we think it an amount that can explain the increased drip and unlikely that there are genetic differences in there does not seem to be a need to invoke i!lny myosin between breeds which affect stability, but other explanation. this cannot be ruled out.

G.R . Schmidt: The authors mention that the R. Hamm: The authors propose a new hypothesis in amount of drip in PSE meat is due to a rapid ~salt-induced swelling is entropically, breakdown of glycogen post mortem, or due to slow rather than electrostatically, driven, and the chilling of the carcass. Could there be other depolymerisatlon of the thick. filaments is the properties of the muscle which are genetical l y decisive factor. It was relatively easy to controlled which could affect the water binding explain the effect of different anions and capacity and appearance of the muscle? Do these cations on the swelling of muscle and the water­ genetically-controlled factors need to be holding capacity of meat by changes in electro­ specifically associated with glycogen breakdown static interactions between protein molecules. and rate of chi 11 i ng? How can the new hypothesis explain these Authors: There are four circumstances in which differences: by different effects of the ions on PSE meat has been reported to fonn: the depolymerisation of the thick filament? Is 1) In animals that are stress-susceptible due to it possible that the association of the tail a genetic defect, there is a high rate of post moieties of myosin is caused by electrostatic mortem glycolysis. forces and that short-distance electrostatic 2) Even in stress-resistant animals, excessive effects of ions result in more or less stress at slaughter can also trigger a high rate depolymerisation of the filament depending on the of post mortem glycolysis. type of ion, rendering possible entropic 3) Certain pig carcasses have been shown to swell ing? In this case, both electrostatic and exhibit a near-normal rate of glycolysis but a entropic effects would parti cipate in the larger-than-normal extent of glycolysis, so that swelling. the final pH is low (- 5.1) (Lawrie et al ., 1958; Authors: Neutral salts have their effect on Monin and Sellier, 1985 ) . protein systems by affecting the stability not 4) If the chilling rate is excessively slow, the only of electrostatic bonds but also of hydrogen temperature in the deep muscles of a carcass can bonds and hydrophobic bonds (see von Hi ppel and remain high for long time post mo rtem Schleich, 1969). When the concentration of the ( MacOougall, 1982). The formation of PSE meat salt is raised, hydrophobic groups make when the post-mortem temperature i s kept high can increasingly un favou rabl e interactions with the be demonstrated by holding meat post mortem at solution, whereas cha rged groups or polar groups 37"C (Wisme r-Pedersen and Briskey, 1961). make increasingly favourable interactions. The The common feature of these is that the net result of increasing the concentration of the muscles experienced a low pH while the carcass is salt is first to weaken interactions between

166 Water-hal ding. appearance and toughness of mea t

protein molecules ( or subunits). This is the process where swelling and protein solubilisation salting-in effec t and one of its manifestations occur extensively at the sites of needle is to increase the solubility of the protein. For injection. This phenanenon causes the appearance example, raising the NaCl concentration to 0 .6 M of periodic stripes in bacon slices and we also at pH 5.5 will cause depolymerisation of a myosin observe this in roast beef and hams from time to filament, and thereby bring myosin into solution . time . Do the authors have recommendations for However, above a cer tain 1 imit, r aising the processors to prevent the formation of these concentration of neutral salt has the opposite periodic stripes of variable protein effect and interactions between protein molecules solubilisation in meat products? (or subunits) are enhanced . This i s the Authors: We have investigated the nature of salting-out range, where the solubility of the ~eriodic stripes in bacon and shown that protein decreases to such an extent that it may near the sites of brine injection there i s precipitate . Thus at ve ry high NaCl actually rather little change in structure: the concentrations (-4 M), myosin filaments are not myofibrils are still clearly seen and thick and depolymerised and muscle and myofib ri ls do not thin filaments are still apparent (Voyle et al., swell (Callow, 1932; Knight and Parsons , 1988) 1986). It is further from these injection sHes and it is possible to precipitate myosin at such where myofibrillar structure is grossly high salt concentrations (Edsall, 1930) . disrupted. We suggested that although the final Although this behaviour applies to all salt concentration is likely to be uniform cations and anions, they differ markedly in these throughout the meat, the difference in behaviour abilities. Some ions, for example CNS- or I-, might be due to the different time-courses of are very effective at dissociating protein salt concentration at the two locations. Near the assemblies at 1 ow concentrations and wi 11 even sites of brine injection, the salt concentration cause protein subunits to unfold . They increase will rise very fast to a high concentration and the solubility of proteins very greatly but then slowly fall to the final concentration. maximum solubility requires relatively high ion Further from the injection site, the salt concentrations and the salting-out effect may not concentration will be zero for some time after be observed at practically realisable injection and then slowly rise to the final concentrations. Other ions, such as sulphate, concentration as salt diffuses in from the are much less effective at salting-in, but injection sites. Experiments with myofibrils show achieve their maximum effect at relatively low that prior exposure to high salt concentrations concentration so that salting-out can be readily reduces both swe lling and extraction at moderate demonstrated. That is why ammonium or sodium concentrations (Knight and Parsons, 1988), sulphates are used to precipitate protei ns. The suggesting that salt-induced denaturation may relative abilities of different ions in processes have a role in the formation of the stripes . such as salting- out are expressed in the Since the periodicity of the stripes is the lyotropi c or Hofmeister series. The relative same as the interval between needles, it would effectiveness of ions, which is similar for seem reasonable to attempt to combat the problen several systems , has no obvious relationship to by using a smaller separation between needles. the s i gn or magnitude of the ionic charge suggesting that their act ion is not simply a R. Hamm: Is it possible to explain the effect of question of suppressing electrostatic ~the absence of salt , on the swelling and interactions ( von Hippel and Schleich, 1969 ) . water-holding capacity of meat only by the Professor Hamm ( 1960) showed that the same order electrostati c hypothesis, or does pH exert an applied for water uptake by muscle. We think this influence on the depolymerisation of the thick is entirely consistent with the view that salts filament? cause swelling of myofibrils by disrupting Authors : Although pH undoubtedly has a strong protein structure. On our hypothesis, the influence on the myosin molecule-myosin filament relative effectiveness of different salts in equilibrium (Josephs and Harrington, 1968), we causing swelling would be seen as being due to doubt whether, in the absence of added salt, the differences in their abilities to there is significant depolymerisation of myosin depolymerise thick Filaments. filaments in the pH region of greatest interest, It is not necessary to suppose that the pH 5.5 to 7 .0. Rather, we suppose that raising interactions between the myosin ta il s in the the pH increases the negative charge on the filament are exclusively electrostatic in filaments , thereby increasing the electrostatic character; hydrogen bonds and hydrophobic bonds force between them and expanding the lattice. may also be involved. It is possible that salt This is well shown by Matsuda and Podolsky depolymeri ses myosin fi 1 aments by increasing the (1986) . In their work, raising the pH from 5 .5 to interactions which polar, rather than charged, 6 expands the lattice considerably, but raising groups make with the medilJTl . In the sense that the pH further (to 7) had little further effect. electrostatic effects may be responsible for This l atter result can be explained on the thick filament depolymerisation, we would agree electrostati c mechan i sm by charge saturat ion that electrostatic effects may play a role in (Offer and Knight, 1988a), but would be hard to swelling, but it seems to us likely that the explain by depolymerisation. swell i ng pressure generated by the addition of We conside r that the filament lattice salts is not electrostati c . spacing of myofibrils, and therefore the water-holding capacity of meat, depends on a G.R. Schmidt: The authors ment ion the curing number of factors: pH and ionic strength affect

167 G. Offer et al. the e 1ectrostat i c repulsive force between is 5.5, whereas in meat processing the pH is filaments; the structura 1 integrity of the normally 6.0 . Would altering the mechanical myofibri 11 ar components affects the mechanical action and the pH affect the interaction between constraints on swelling; the presence of osmotic the proteins and the irrigation medium? agents such as sarcoplasmic proteins, partially Authors: In those experiments we were not trying or completely excluded from the lattice, lowers toaliPlicate precisel y the conditions occurring the chemical potential of the water outside the in meat processing, but rather by simplifying the myofibrils relative to that inside and thereby system to gain insight into the mechanisms causes shrinkage. In addition, entropic involved in water uptake and myosin extraction. mechanisms, such as we have outlined, may come We chose a pH of 5.5 to mimic the conditions in into play under conditions where the degree of fresh meat. We wanted to explore the specific molecular freedom would increase if there were an effects of NaCl and polyphosphates without expansion of the filament lattice. At present, we complicating matters by altering the pH as well. can envisage such a mechanism operating only when However, Or Sctmidt is right to emphasise the the myosin filaments depolymerise and we think small rise in pH that occurs when alkaline this would occur only in the presence of salt and polyphosphates are used and it would be desirable polyphosphate, or with acid marinades (Offer and to determine more precisely what effect this Knight, 1988a). might have. A rise in pH waul d be expected to 1 ower the G.R. Trout: In the section on water-holding, no NaCl concentration required to depolymerise the mention is made of the effect of heating and the thick filament (Josephs and Harrington, 1968), interaction between heating, pH, salt type and and hence assist both swelling and myosin concentration on the microstructural changes and extraction. their relationship to water-holding. Would you With regard to mechanical action, it is at comment on the importance of these factors in present far from clear precisely how much light of the fact that the major objective of disruption of muscle structure occurs in studying the effects of salts, such as sodium different products. At one end of the spectrum, chloride and sodium tripolyphosphate, on in a traditional ham, gross disruption of the water-holding is to determine how they affect the muscle fib re probably occurs only at the surface water-holding ability of the cooked meat product? of the meat and, in a British-style sausage, Authors: We quite agree that it is very muscle fibres probably also survive intact. In a important to understand how NaC1 and frankfurter, it seems that even the myofi bri 1 is po l yphosphates affect the water-holding ability grossly disrupted, although to what level is of cooked meat products. But meat is treated with unknown . However, regardless of the size of the these sa 1 ts in the raw state and we do need to meat piece or fragment of myofibril, the know what is happening in this system first underlying molecular interactions between the before tackling the experimentally harder problem ions of the medium and the myofibrillar proteins of the structural changes occurring when are presumably the same as with isolated salt-treated meat is cooked. proteins. We think it 1 ikely that polyphosphates in the presence of NaCl decrease cooking loss by G.R. Schmidt: The authors discuss the ageing of their action in solubilising myosin (Offer and the endomys1um and indicate that aged muscles Knight, 1g88a}. When myosin molecules are heated, empty the contents of their muscle fibres when they form a gel with good water retention {Tsai exposed to the proper ionic environment more et al., 1972). The solubilised myosin is readily than fresh muscle , If this is so, how presumably capable of permeating the entire does this phenomenon interact with the property muscle, that is to invade the !-bands of the of myosin being more extractable pre-rigor when, muscle fibres from which it was previously absent obviously, the endomys ium is still in a very and also to invade the extra-cellular space. If strong state? this is correct, when the salt-treated meat is Authors: The work we have discussed was cooked, a myosin gel wi 11 be formed throughout performed on rigor muscle. The pre-rigor state the meat and is likely to play a major role in has quite different properties. We have found water retention (Bend all, 1954; Sherman, 1961; (Elsey and Knight, unpublished experiment) that Hellendoorn, 1962; Kotter and Fischer, 1975; it i s quite easy to draw muscle fibres. free of Offer and Knight, 1988a}. We can explain the endomysi urn. from a transversely cut surface of effect of polyphosphate in the presence of NaCl pre-rigor muscle {Schoenberg and Eisen berg, on cooking loss, since polyphosphates cause 1985). but this is difficult from rigor muscle. dissociation of actomyosin and more myosin will Perhaps, therefore, a high incidence of stripping be solubilised. Greater solubilisation will also accompanies comminution pre-rigor, but this needs occur at higher pH. to be tested. We suppose that removal of the endomysium from around a muscle fibre would G.R. Scllnidt: The authors discuss extensively a promote extraction of solubilised myosin, in both prev1ous paper by Offer and Trinick ( 1983) in pre-rigor and rigor muscle, by removing an which myofibrils are irrigated with an extraction impediment to outward diffusion. medium. Generally, this research differs from The ATP present only in pre-rigor meat applied meat processing in two ways. There is no produces a high degree of dissociation of myosin mechanical action applied to facilitate the from actin, permitting a quicker diffusion of disruption of the myofibrils and the pH utilized myosin molecules once added NaCl has

168 Water-holding, appearance and toughness of 111eat

depolymerised the thick filaments . This factor Additional references will influence the outward movement of myosin even in fibres still enclosed in endomysium. Edsall JT (1930) Studies in the physical chemistry of muscle globulin . 2. On scrne G.R. Sd"mfdt: The authors discuss the changes physicochemical properties of muscle globulin that take place in muscle when myofibrils in a (myosin). J, 8iol, Chern. 89 , 289-313 . cooked fibre are stretched. The authors do Hellendoorn EW (1962)Water binding capacity mention gap filaments. What do the authors of meat as affected by phosphates. 1. Influence speculate is the contribution of titin to the of sodium chloride and phosphates on the water longitudinal strength of cooked meat? retention of corrminuted meat at various pH Authors: We discussed this imp ortant question values . Food Technol. 16, 119-124 . recently (Offer et al., 1988) . Tit in is a very Honlkel KO, Kfii1C (1986) Causes of t he large protein {molecular weight 3 million) and development of PSE pork. Fleischwirtsch. 66, titin molecules are very thin filaments about 4 349-353 0 - nm wide and up to 1 ~o~m long {see reviews by Howard A, Lawrie RA, Lee CA ( 1960) Studies Squire et al . (1987) and Offer (1987)). Recent on beef quality . 8 . Some observations on the location studies using monoclonal antibodies to nature of drip. CSIRO Oiv. Food Pres. Trans . titfn have shown that a single titin molecule Tech . paper No 15, 5- 29 . spans all the way fran the Z-disc to the M-line Josephs R, Harrington WF (1968) On the (Whiting et al ., 1989) . There is evidence that stability of myosin filaments. Biochemistry 7 , tit in is the protein canponent of the gap 2834-2847 0 - filaments, the structures spanning the gap King NL, Kurth L (1980) In: Fibrous between A and I-bands in highly stretched muscle. Proteins: Scientific, Industrial and Medical When muscle is cooked , the thick filaments Aspects, Parry DAD, Creamer LK ( eds) , Academic fuse together and the thin fi 1 aments Press, London, Vol 2, 57-66 . disintegrate, although the A and 1 bands persist Kotter L, Fischer A (1975) The influence of (Schmidt and Parrish, 1971) . Locker et al, phosphates or polyphosphates on the stability of (1977) have shown that, unlike the thick and thin foams and emulsions in meat technology . filaments, gap filaments surv ive cooking and have Flei schwi rtsch . 55, 365-368. argued that gap filaments alone provide the La wri e R.o;:- Gatherum OP, Hale HP (1958) tension-bearing elements in the cooked myofibril. Abnorma ll y low ultimate pH i n pig muscle. Nature

It has been suggested that since titin is rapidly 182. 807-808 0 degraded by proteolysis on ageing, it could not - Ling GN, Walton CL (1976) What retains water sup[lort loi!d (King and Kurth, 1980), but there is in living cells? Scienc:e 191, 293-295. no reason why a break in the primary structure Locker RH, Daines"'-"G"J, Carse WA, Leet NG should necessarily cause the titin molecules to {1977) Meat tenderness and the gap filaments. lose their secondary or tertiary structural Meat Sci. 1, 87 -103 . integrity . Myosin filaments fonn a strong gel on Matsuda T, Podol sky RJ ( 1986) Ordering of heating, so that, in the A-band region, load is the myofilament lattice in muscle fibres. J. Mol. probably borne both by the myosin gel and by Biol. 189, 361-365 . tfttn filaments. However, the structure fanned Monin G, Sellier P (1985) Pork of low fran the thin filaments on cooking is not known, technological quality with a normal rate of and it Is possible that, at the high muscle pH fall in the immediate post mortem concentrations present in muscle , the thin period: the case of the Hampshire breed. Meat filament canponents also form a gel . Thus we do Sci. 13, 49-63. not know whether or not titin is the only Offer G (1987) Myosin filaments . In: Fibrous component in the 1- band that supports tension and Protein Structure , Squire JM, Vi bert PJ (eds), thus whether titin alone determines the breaking Academic Press, London, 307-356. strength of the cooked myofibril. Offer G, Purslow P, Almond R, Cousins T, Elsey J, Lewis G, Parsons N, Sharp A, Starr R, G. R. Sctrnidt: The authors commen t that there i s a Knight P (1988) My ofibrils and meat quality. variabil 1ty of f'esponse of myofibrlls to sa lt. Proc . 34th Int. Cong . Meat Sci. Te c hnol., They attribute this differ ence to muscle fibre Sri sbane, 161-168. types . Is it possible that the protein titin Paterson 8C, Parrish FC, Stromer MH (1988) could be involved in this phencxnenon? Effects of salt and pyrophosphate on the physical Authors: Paterson et al . (1988) have found that and chemical properties of beef muscle . J . Fd ~is extracted from myofibrils under Sci. 53 , 1258-1265. conditions of salt concentration that extract Schoenberg M, Eisen berg E ( 1985) Muscle myosin and cause swelling . They have proposed cross-bridge kinetics in rigor and in the pres­ that loss of tittn may be a cause of swelling, ence of ATP analogues. 8iophys. J. 48, 863-871. so it is conceivable that variable loss of titin Sherman P (1961) The water bindTng capacity could underlie variable swelling . variable loss of fresh pork. 1. The influence of sodill11 of titin might arise either from variable chloride , pyrophosphate and polyphosphate on conditioning or to differences in fibre type. It water absorption. Food Technol, 15, 79-87 . remains to be shown whether titin and/or nebulin Squire JM , Luther PK, Trlnick J (1987) do indeed constitute constraints on swelling of Muscle myofibril architecture. In: Fibrous the myofibril, and whether extraction of tit in Protein Structure , Squire JM, Vi bert PJ (eds) , correlates with the amount of swelling. Academic Press , London, 423-450.

169 G. Offer et al.

Tsai R, Cassens RG, Briskey EJ ( 1972) The ernul sHying properties of purified muscle proteins. J. Fd Sci. 37, 286-288 . von Hippel PH, SChleich T (1969) The effects of neutral salts on the structure and confonnational stability of macromolecules in solution . In: Structure and Stability of Biological Ma cromolecules, Timasheff SN. Fasman GO (eds), Marcel Dekker, New York, 417-574. Whiting A, Wardale J, Trinick J (1989) Does titin regulate the length of muscle thick filaments? J. Mol. Biol. 205, 263-268. Wismer-Pedersen J, Bi'lsley EJ (1961) Role of anaerobi c glyco lysis versus structure 1n pork. muscle. Nature 189, 318-320.

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