Postmortem Changes during Conversion of Muscle to Meat

13 I. Introduction 391 II. Glycogen, High-Energy Phosphates, and Their Metabolites 392 III. Postmortem Changes Occurring in Conversion of Muscle to Meat 395 IV. Some Conditions Occurring in Muscle/Meat 413 V. Some Postmortem Processing Characteristics of Muscle 431 VI. Meat Flavor 433 VII. Summary 434 Literature Cited 434

Postmorte m Change s durin g Conversio n of Muscle to Meat

time as they are no longer capable of synthesizing additional high-energy phosphate compounds or of removing the breakdown products from the system. Thus, conversion of muscle to meat is delimited by the supply of glycogen, high-energy phosphates, and their metabolites present in the tissues at the moment of death. Although some respiratory activity may occur on the exterior surfaces of the muscles, oxygen for deep tissue respiration is no longer available so that the reactions taking place within the deep tissues become strictly anaerobic and are limited by cessation of respiration and by the buildup of the waste products of metabolism. Central to the process of conversion of muscle to meat under postmortem conditions is the role of the high-energy phosphate compounds and their metabolites. Thus, the biochemistry of glycogen and of the high-energy phosphates ATP, ADP, and creatine phosphate (CP) and their metabolites are briefly reviewed from the standpoint of their meta-

I. INTRODUCTIO N Postmortem changes that occur in the conversion of muscle to meat not only alter some of its biochemical and physical properties but also play an important role in improving its keeping quality and acceptability as a food. Nevertheless, there are some advantages to the use of prerigor muscle or so-called hot meat in certain products, which are also discussed in this chapter. On the whole, however, discussion focuses on the postmortem events that transpire in muscle following death and their effects on its properties as it becomes known as meat. The nature of these changes and their consequences for meat have been reviewed earlier by Greaser (1986) and Pearson (1987). At physiological death, cessation of blood circulation results in limiting the energy supply to the amount present in the muscles at that particular

391

392

13. Postmortem Conversion of Muscle to Meat

bolic conversions and their influence on the properties of meat.

II . GLYCOGEN , HIGH-ENERG Y PHOSPHATES , AND THEI R METABOLITE S ATP, ADP, CP, and their metabolites are all involved in the energy transformations that occur in muscle. Their synthesis and degradation are briefly discussed along with that of glycogen, which is their major source in muscle, especially postmortem.

A. GLYCOGE N Glycogen is the main storage carbohydrate present in animal cells, being the counterpart to starch in plant cells. It is especially abundant in liver where it may constitute as much as 10% of the wet weight. Glycogen commonly comprises about 1-2% of the wet weight in resting muscle. It is a polysaccharide of D-glucose linked together by a-1,4 linkages, with each branch point, occurring about every 8-12 glucose residues, being in the á-1, 6 linkage. Glycogen is hydrolyzed to glucose by the action of glycogen phosphorylase, which sequentially cleaves off the terminal glucose to leave a glycogen molecule with one less glucose unit in its structure. The enzyme can act repetitively on a-1,4 linkages but cannot attack the a-1,6 branch points; the action of a debranching enzyme is required before glycogen phosphorylase can degrade the remainder of the branched chain. Under intracellular conditions, in which the inorganic phosphate concentration is high and the glucose 1-phosphate concentration is low, the action of glycogen phosphorylase favors the formation of glucose 1-phosphate. Conversion of glucose 1-phosphate to glucose 6-phosphate is catalyzed by the enzyme phosphoglucomutase, which contains a serine residue that is essential for catalytic activity and becomes esterified with phosphoric acid that transits between the 1 and 6 positions on the glucose molecule. In living skeletal muscle at rest, some 30% of the total oxygen consumption of the animal body is

used by the muscle. Heavy muscular work may increase this to as much as 90%, depending on the duration and nature of the work. Under these conditions most of the glucose consumption by skeletal muscle is derived from hydrolysis of muscle glycogen. Suffice it to say that in living muscle, glycogen cannot be converted into blood glucose because glucose-6-phosphatase is absent (Lehninger, 1975). In postmortem muscle, there is likewise no conversion of glucose 1-phosphate to glucose 6-phosphate. The major role of glycogen in postmortem muscle is release of glucose, which can be used to replenish the high-energy phosphate compounds. Thus, glycogen is largely degraded and is mainly responsible for the formation of lactic acid in muscle, which accounts for the pH decline that occurs in postmortem muscle. Therefore, glycogen is ultimately responsible for the changes in the properties of muscle that accompany the drop in pH as glycolysis proceeds. These changes are reviewed later in this chapter (see Section ÉÉÉ,Â) . Â. I N T E R C O N V E R S I O N A N D DEGRADATIO N OF HIGH-ENERG Y PHOSPHATE S Electron transport and ATP synthesis by the mitochondria have already been discussed in considerable detail in Chapter 11 in connection with the sarcoplasmic proteins. The sites and mechanisms involved in oxidative phosphorylation and synthesis of the high-energy phosphate compounds ATP and ADP were also reviewed. Thus, discussion in this section focuses on interconversions of glycogen, high-energy phosphate compounds, and their metabolites. By a series of chemical reactions in the body, glucose, excess protein, and fatty acids can be utilized for synthesis of ADP and ATP (Chapter 11). By way of the citric acid or tricarboxylic acid (TCA) cycle, aerobic oxidation of glucose results in its conversion to two molecules of pyruvic acid. As shown in Fig. 13-1, food sources of carbohydrates can be utilized by being broken down to glucose to enter this pathway. Glycogen stores can also be broken down to glucose, which enters the cycle in the same way. The peptide bonds in proteins can be hydrolyzed

II. Glycogen and High-Energy Phosphates

to form amino acids, which can be deaminated and enter by way of pyruvate, through acetyl-CoA, or directly into the citric acid cycle (Fig. 13-1). Although the main pathways for the amino acids are through pyruvate (alanine, glycine, cysteine, serine, threonine, and tryptophan) and acetyl-CoA (phenylalanine, tyrosine, leucine, lysine, tryptophan, and isoleucine), a limited number of amino acids enter directly into the citric acid cycle through oxaloacetate (aspartate and asparagine) and either directly or indirectly through a-ketoglutarate (2-oxoglutarate) (glutamate, arginine, histidine, glutamine, and proline), through succinylCoA (isoleucine, methionine, and valine), or by way of fumarate (tyrosine or phenylalanine). Fats are broken down into fatty acids and glycerol, which enter the metabolic pathway via acetylCoA and dihydroxyacetone phosphate, respectively. The waste products of these reactions are N H and C 0 (Fig. 13-1), which in some cases in living muscle can be used for other synthetic processes such as reamination and building of longer carbon chain skeletons. After death, there are only a few sources of energy, such as the glycogen stores, residual ATP and ADP, and any unused CP. As long as residual ADP sources remain high, the following reaction can also occur to provide additional ATP: 3

393

also occur as proposed by Lee and Newbold (1963): IMP —> I + P 0

nucleoside 4

phosphorylase

hypoxanthine +

^

Þ

> hypoxanthine + ribose

In addition to IMP, both inosine diphosphate (IDP) and inosine triphosphate (ITP) have been found in postmortem muscle by Bendall (1973a), who postulated that they may be formed as follows: 2 ATP + 2 IMP ^ 2 ADP + 2 IDP 2 IDP ^ ITP + IMP ADP ^± IDP + N H 3

The breakdown of IDP to ITP plus IMP and of ADP to IDP plus N H are probably unimportant from the standpoint of postmortem changes. Their levels are relatively low, and they do not possess any highenergy phosphate bonds. 3

2

2 ADP

ATP + AMP

The AMP formed in the above reaction can then be deaminated to produce inosine monophosphate (IMP) by the following reaction: AMP ^ p j - f

™

I

M

p

+

N

H

?

The disappearance of AMP is highly correlated with N H production (r = 0.99), which indicates that there is a stoichiometric relationship between the disappearance of adenine nucleotides and the production of ammonia (Bendall and Davey, 1957). ADP can also react with CP to yield ATP: 3

creatine kinase

ADP + CP

± ATP + creatine

This reaction is reversible and requires the enzyme creatine kinase. Further breakdown of IMP can

C. ENZYME S CATALYZIN G AT P HYDROLYSI S IN MUSCL E There are a number of enzyme systems in muscle that require ATP as a source of energy in order to carry on cell functions. In postmortem muscle, the cells attempt to maintain ATP at physiological levels for as long a period as possible, which results in maximizing ATP formation and minimizing its hydrolysis to those essential processes. Since ATP is split at a rate of about 0.50-0.65 ì,ðéïÀ/g of muscle/ minute at 38°C (Bendall, 1973a; Scopes, 1973), these enzyme systems are active and continue to breakdown ATP in postmortem muscle. The two most important ATP-splitting enzymes in normal resting muscle are myosin-ATPase and Ca -ATPase. There are, however, three other ATPases in muscle, which include the following: (1) mitochondrial ATPase, (2) plasmalemmal N a , K - A T P a s e , and (3) plasmalemmal C a ATPase. Also present in muscle are a number of other enzymes that hydrolyze ATP as one of their normal functions, and they comprise the following combination enzyme systems: (1) phosphofructokinase-fructose-bisphosphatase, (2) phosphorylase-glycogen synthase, and (3) phosphorylase b kinase-phosphorylase a phosphatase. These enzymes and their roles in postmortem muscle have been outlined by Greaser (1986). 2+

+

+

2+

394

13. Postmortem Conversion of Muscle to Meat

Myosin-ATPase, which is located in the myosin heads, possesses the highest potential ATPase activity of any of the muscle enzymes that can split ATP. In normal resting muscle, myosin is not bound to actin (Chapter 7), with millimolar concentrations of M g strongly inhibiting the rate of ATP hydrolysis (Taylor, 1979). Nevertheless, there is a continual but slow splitting of ATP to form ADP and inorganic phosphate. The high concentration of myosin in muscle (—20% of the total protein) results in muscle having a relatively large amount of myosin-ATPase activity. Thus, myosin-ATPase probably accounts for most of the ATP breakdown, both in resting and in postmortem muscle (Greaser et al, 1969a,b; Bendall, 1973a,b). The effect of temperature on ATP turnover closely parallels the temperature dependence of myosin-ATPase (Table 13-1). Myosin-ATPase activity is stimulated some 300- to 500-fold if the level of cytoplasmic C a is increased from concentrations below 10" Ì to 10" -10- Ì or above (Bendall, 1973a, 1978). This increase in C a concentration commonly occurs in muscle during contraction and catalyzes the interaction of myosin and actin, with M g stimulating myosin-ATPase activity. Thus, cytoplasmic C a may dramatically accelerate myosin-ATPase activity and stimulate ATP breakdown. This, no doubt, is involved in the development of rigor mortis. 2+

2+

8

6

5

2+

2+

2+

2+

The Ca -ATPase, which is bound to the sarcoplasmic reticulum (SR) membrane in muscle, removes calcium from the cytosol during the rest cycle of an ATP-dependent transport process. Since the muscle membranes are not completely impermeable, there is slow leakage of C a into the cytosol. Thus, Ca -ATPase catalyzes the breakdown of ATP to supply energy for pumping the C a back 2+

2+

2+

Tabl e 13-1

across the membranes in order to maintain resting physiological levels of C a and to assist in prevention of contraction. Maintenance of the low concentration of C a (<10~ M) in the cytosol, therefore, requires the cooperative action of C a ATPase to furnish energy by breakdown of ATP to support the calcium-pumping action of the SR (Peachey and Franzini-Armstrong, 1983). The contribution of Ca -ATPase to total ATPase activity, however, is probably small in terms of ATP turnover. Nevertheless, since the SR controls the level of cytosolic C a , the Ca -ATPase and myosinATPase clearly function in a cooperative way. Mitochondrial ATPase provides a source of energy through hydrolysis of ATP under anaerobic conditions. The specific activity of this enzyme in isolated mitochondria at temperatures above 30°C is very high, which suggests that under these conditions it could account for a major proportion of ATP turnover. The work of Haworth et al. (1981) with cardiac muscle cells indicated that mitochondrial ATPase can cause a significant drain on energy sources in the quiescent state. On the other hand, the temperature dependence of mitochondrial ATPase is much greater than ATP turnover for intact muscle, thus indicating that it plays only a minor role in total turnover. The N a , K - A T P a s e functions in transporting sodium out of and potassium into the muscle cell. Thus, it maintains membrane polarity (Robinson and Flashner, 1979; Wallick et al., 1979). It is bound to the plasmalemma and can furnish energy for exporting C a if too much is leaked into the cytosol. Plasmalemmal Ca -ATPase can also function in the same way. Both of these enzyme sources are available only at the membranal site, however, 2+

2+

8

2+

2+

2 +

+

2+

+

2+

2+

Effect of Temperature on ATP Turnover Rate ATP Turnover (%) at Temperature (°C)

Rabbit psoas Beef sternomandibularis Myosin-ATPase a

38

35

30

25

20

15

10

2

100* 100* 100*

83.3 83.3 84.0

61.4 61.4 62.9

49.3 49.3 46.3

41.7 44.2 33.8

36.4 39.4 24.4

32.4 35.8 17.4

28.0 38.5 9.8

Fro m Bendal l (1973a) , wit h permission .

* Value s normalize d t o 100% at 38°C .

III . Postmortem Physicochemical Changes

so are somewhat limited in their effects on ATP turnover. The combination enzyme systems, which include phosphofructokinase-fructose-bisphosphatase, phosphorylase-glycogen synthase, and phosphorylase b kinase-phosphorylase a phosphatase, are known to have ATPase activity (Scopes, 1974a). Their activity is probably minimal, though, since conditions activating one enzyme of the pair tend to be inhibitory to the other member. This does not, however, indicate that they are unable to hydrolyze ATP in postmortem muscle. Explanation of ATP turnover rates in intact muscle is difficult to understand from results of studies on isolated ATPases. Nevertheless, the greatest amount of ATP hydrolysis seems to be associated with the activity of myosin-ATPase in the resting state plus that of C a activation of the same enzyme on complexing with actin. The latter situation results in rapid ATP breakdown. The observed range in ATP turnover rates from very slow to extremely fast differs by only about a factor of 10, so even a small increase in cytosolic C a concentration could account for the maximum rate of ATP hydrolysis. 2+

2+

395

Higher temperatures speed up glycolysis and hasten onset as does struggling at the time of death. Stimulation of respiration, on the other hand, accelerates aerobic metabolism and delays rigor development (Lawrie, 1979). Thin strips of muscle have been reported by Lawrie (1979) to produce ATP in an oxygen atmosphere so efficiently that not only is rigor mortis delayed but CP can be resynthesized in amounts above its original concentration. The loss of extensibility associated with rigor mortis has been related to formation of actomyosin, which after death soon forms the irreversible rigor actin-myosin complex as explained in Chapter 7. Failure of the actomyosin complex to separate into myosin and actin after the onset of rigor, as it does on relaxation following muscle contraction, is due to the absence of ATP as originally shown by Erdos (1943). These observations have been reinforced and expanded by Bate-Smith and Bendall (1947, 1949; Bendall, 1951). It should be pointed out that ATP can still be present in muscle after rigor development as the decline in pH can poison the ATPases and prevent ATP hydrolysis, thus blocking the action of ATPase in separating the actomyosin complex. Effec t of Rigo r Morti s on Mea t Tendernes s

III . POSTMORTE M CHANGE S OCCURRIN G IN CONVERSIO N OF MUSCL E TO MEAT In conversion of muscle to meat both physical and chemical changes occur. Although these alterations are interrelated, they are discussed independently. A. PHYSICA L CHANGE S At the time of death the muscle is flaccid and highly extensible. Within a few hours, however, it becomes inextensible and relatively rigid, a phenomenon known as rigor mortis. This process takes place at fairly well recognized periods of time following death so it is widely used in helping to establish the time of death in forensic medicine. A number of factors can influence the time of onset of rigor mortis such as temperature, stimulation of respiration, or struggling at the time of death.

Early work on the influence of rigor mortis on meat tenderness resulted in conflicting conclusions. Paul et al. (1944) indicated that beef was least tender if roasted immediately after slaughter but became more tender if cooking was delayed. On the other hand, Ramsbottom et al. (1945) reported that deepfat fried beef steaks were maximally tender if cooked immediately following slaughter and increased in toughness on delaying cooking for 24-48 hours. Table 13-2 shows that steaks fried in deep fat within 1 hour of death had the lowest shear force values and reached maximum shear force readings at 24-48 hours postmortem, after which tenderness increased on subsequent holding (Paul et al., 1952). However, larger roasts cooked in the oven at an internal temperature of 63°C immediately after death (<1 hour postmortem) were toughest at 0 time, with decreases in shear force being recorded as cooking was delayed. These results suggested

396

13. Postmortem Conversion of Muscle to Meat

Tabl e 13-2 Effects of Postmortem Cooking Times and Size (Roasts versus Steaks) on Shear Force Values for Beef fl

Shear Force Values

6

Biceps femoris

Semitendinosus

Cooking Time Postmortem (hours)

Roasts

Steaks

Roasts

Steaks

0 5 12 24 48-53 144-149

9.05 9.04 8.55 7.11 7.98 5.48

5.67 7.72 8.66 14.00 7.77 7.55

8.08 6.87 6.58 6.00 4.45 3.70

4.96 6.86 6.73 6.68 5.60 4.47

a

Dat a recalculate d fro m tha t of Pau l et al. (1952).

* Shea r value s recalculate d a s k g require d t o shea r a cor e 1.25 c m in diameter .

that prerigor meat is tender, and if cooked in thin pieces at high heat so as to achieve rapid heat penetration is maximally tender, as suggested by Ramsbottom et al. (1945). When roasted in large pieces, however, heat penetration is delayed and the meat goes into rigor mortis before cooking is completed, making it tough. Cia and Marsh (1976) have theorized that the tenderizing effect of rapid cooking of prerigor muscle is due to shattering of the myofibrils caused by supercontraction during the heating process. Although the supercontraction theory of tenderization during cooking of prerigor meat would explain the reason that shortening caused by cooking does not make meat tough in contrast to cold shortening (see Section IV,A), the fact that actin and myosin are not locked into the actomyosin complex in prerigor meat should be adequate to explain the greater tenderness in rar idly cooked prerigor meat. Furthermore, high temperature cookery tends to have a deleterious effect on tenderness. Further research is required to clarify the reason that rapidly cooked prerigor meat is more tender than similarly cooked immediately postrigor meat.

B. C H E M I C A L C H A N G E S Pyruvic acid is oxidized to C 0 and H 0 by way of acetyl-CoA entering the citric acid cycle and its 2

2

associated phosphorylation (see Fig. 13-1). In living muscle, a total of 12 mol ATP is formed for every mole of acetate utilized in the cycle. This is an aerobic reaction and so ceases soon after death when the residual oxygen becomes depleted. Thus, glycogen reserves and the high-energy phosphate compounds are utilized in an attempt to maintain normal muscle cell processes. Therefore, these compounds are discussed from the standpoint of their levels in muscle at the time of death and the consequences of their breakdown following death. Concentrations of the high-energy phosphate compounds, glycogen, and their metabolites change rapidly on biopsy or on the struggling accompanying death. This makes it difficult to obtain true resting values for these compounds, which can only be obtained by anesthetization with M g S 0 or curare to block nerve impulses. In this way the effects of electrical stunning or the localized stimulation arising from sampling can be avoided to give more accurate resting values, which are discussed below. Resting values are considered first since in a large part they influence the final levels in meat. 4

1. Glycoge n Level s As mentioned it is difficult to obtain resting values for glycogen and other metabolites, which was shown for initial glycogen levels in chicken breast muscle by deFremery (1966). He demonstrated that anesthetized birds had about one-third higher glycogen concentrations than birds killed by stunning without struggling, while those that struggled had only about one-third as much glycogen as the unanesthetized birds that did not struggle. The data shown in Table 13-3 demonstrate that anesthetization before killing resulted in 8.4 mg of glycogen per gram of muscle as compared to 4.1 mg/g for birds that were slaughtered in the normal manner. These data clearly illustrate the importance of avoiding stimulation of glycolysis during the stress of slaughtering, since glycogen had virtually disappeared by 10 minutes postmortem in the unanesthetized birds. Glycogen normally comprises about 1% of the muscle weight according to Lawrie (1979). Table 13-4 gives resting values of muscle glycogen concentrations for various species, which range from about 45 ìðéï ß (glucose equivalents) per gram for sheep pectoral muscle up to 65 ìðéïÀ/g for longissi-

III .

Postmorte m Physicochemica l Change s

FOOD S

CARBOHYDRATE S GLUCOS E

I

PROTEIN S

GLYCOGE N — ( P(PI HOSPHORYLASE t—

FAT S

)

GLUCOSE-I- P AMIN O

J—

ACID S

(PHOSPHOGLUCOMUTASE )

GLUCOSE-6- P IATP . ADP

(PHOSPHOGLUCOISOMERASE (PHOSPHOFRUCT O

4

FRUCTOS E

GLYCERALDEHYDE-3-P *

i

DIPHOSPHOGLYCERI C

t

KINASE ) E

MUTASE )

^

TRIGLYCERID E

ACI D

I—(PHOSPHOGLYCERAT 2-PHOSPHOGLYCERI C

GLYCEROL-P^GLYCEROL

ACI D

I—(PHOSPHOGLYCERATE 3-PHOSPHOGLYCERI C

J —

KINASE )

1,6-di-P

DIHYDROXYACETON E

"-I 1,3

)

FRUCTOSE-6- P

I FATT Y

ACID S

ACI D

(ENOLASE )

PHOSPHOENOLPYRUVI C J — (PYRUVAT E PYRUVI C

ACI D

KINASE )

ACID -

Figur e 13-1 Pathwa y b y whic h f o o d s ente r th e energ y c y c l e s in livin g muscle . Afte r deat h glucos e ca n ente r th e c y c l e onl y b y breakdow n of g l y c o g e n .

397

398

13. Postmortem Conversion of Muscle to Meat

Tabl e 13-3 Changes in Glycogen Concentration in Chicken Breast Muscle Associated with Type of Slaughter a

Glycogen Concentration (mg/g fresh weight) Time Postmortem

Shrimpton (1960)

deFremery and Line weaver (1962)

4.1* 2.7 0.1 0 0 0.3 0.2

8.4 3.8 3.7 3.3 1.4 1.0 0.2

Live 1-3 minutes 10 minutes 30 minutes 2-2.5 hours 4-4.5 hours 24 hours a

C

Fro m d e F r e m e r y (1966).

b

Nonanesthetized .

c

Anesthetized .

mus dorsi (LD) muscle from certain strains of pigs. Bendall (1973b) reported that beef sternomandibularis muscle contained approximately 45 ì,éçïÀ/g, while Bodwell et al. (1965a) found that beef LD muscle contained approximately 56 ìçéïÀ/g at 0 hour (<10 minutes) postmortem. Howard and Lawrie (1956) reported comparable initial glycogen levels in beef LD muscle, with about 52 ì,ðéïÀ/g. Bodwell et al. (1966) also reported the initial glycogen levels in pig LD muscle to be about 57 ìðéïÀ/g, although there was a considerable amount of varia-

tion among different pigs. The high amount of variability in the glycogen levels of pig muscle observed within 10 minutes postmortem by Kastenschmidt et al. (1968) can be explained by variations between breeds, which they have classified as fast-glycolyzing (Poland China) and slowglycolyzing (Chester White and Hampshire) muscles. Initial (within 3 minutes of death) glycogen levels were about 106, 35, and 23 ìðéïÀ/g of muscle for Hampshire, Chester White, and Poland China pigs, respectively. Results of this study indicate the great amount of variability that can occur in initial glycogen levels in pig muscle, in which glycogen appears to be much more labile than is the case for other species producing red meat. The initial glycogen concentrations given above indicate that a considerable amount of variation in residual glycogen levels exists in the same muscle for different animals immediately after slaughter. Thus, the values given are only rough approximations of glycogen levels that would be expected in the same species. The variability in values merely reflects the influence of a great many factors on glycogen levels at the time of or soon after death, among which are breed, stresses of various types and durations, the initial concentration of glycogen in the tissues, and differences in the rate of glycolysis for different muscles. Changes in glycogen concentrations in beef LD muscle (Table 13-5) show that levels dropped quite rapidly postmortem. From its initial value of about 56 ì,ðéïÀ/g, the glycogen level reached about 42, 30,

Tabl e 13-4 Resting pH Values and Concentrations of High-Energy Phosphate Compounds, Glycogen, and Their Metabolites in Muscles from Various Species 0

Parameter

Rabbit Psoas

Beef Longissimus Dorsi

Pig Longissimus Dorsi

Sheep Pectoral

Initial pH Total creatine* Creatine phosphate* ATP* ADP* Glycogen (glucose equivalents)* Initial lactate*

7.10 42.0 23.0 8.1 1.1 —60 13.0

7.08 42.0 19.0 5.7 0.9 -50 16.0

7.18-7.30 44.0 18.0-19.0 6.6-6.8 1.0 -55-65 6.0-11.2

7.18 34.0 13.1 5.9 0.9 -45 9.4

a

Fro m Bendal l (1973b) , wit h permission .

* Value s e x p r e s s e d a s ìðéïÀ/ g of m u s c l e .

III . Postmortem Physicochemical Changes

Tabl e 13-5

399

Values for pH and Chemical Constituents in Beef Muscle at Various Times Postmortem

Time Postmortem (hours)

pH

Initial

Glycogen

b

Glucose'

Lactic Acid

ATP

d

a

ATP'

CP

TSP/

Ortho Ñ 2 2 ,.1

6 .9 9

5 6 .7

7 ,, 9

1 3 ,. 1

6. 4

10. 9

9. 1

5 4 ,. 9

6

6 .5 7

4 1 ,.6

6 ,.3

4 4 ,.8

5. 0

10. 0

2. 0

5 5 ,. 2

2 3 ,.2

12

5 ,. 9 6

3 0 ,.4

1 2 ., 2

5 8 ,.0

3. 9

5. 3

1. 5

5 4 ,. 9

2 5 ,.9

5 3 ,. 6

2 1 , .6

5 4 ,.2

2 7 ,.5

5 3 ,.6

3 0 ,.3

24

5 ,. 7 4

1 0 ,. 1

1 8 .. 1

7 1 , .2

1. 7

0. 0

48

5 ,. 5 7

1 0 ,. 0

1 5 .. 9

8 2 ,.4

1. 1

—

— —

72

5 ,. 4 6

—

—

—

1 2 ,. 7

1 2 .. 1

8 0 ,,9

96

5 ,. 3 6

10 2

5 ,. 4 2

12 6

5 ,. 5 0 c J.

—

—

—

—

—

—

28 8

5 ,. 5 4 c J. 5 ,. 5 9

9 .. 9

1 6 ., 8

8 2 ,.7

48 0

5 ,. 4 6

1 ., 4

1 7 ., 9

8 4 ,,6

—

—

—

15 1 17 3 19 8

5 4 ,. 9

3 3 ,.6

5 4 ,.2

3 5 ,,5

a

Fro m B o d w e l l et al. (1965a) . All value s ar e averag e observation s on L D muscl e fro m five c a r c a s s e s an d ar e e x p r e s s e d in ìðéï ß (diatom s for T S P an d orth o P)/ g fres h tissue . * G l y c o g e n is e x p r e s s e d in g l u c o s e equivalents . c

Tota l reducin g sugar s e x p r e s s e d a s g l u c o s e .

d

Value s obtaine d b y aci d hydrolysis .

' Value s obtaine d b y e n z y m a t i c a s s a y . /

Tota l acid-solubl e phosphate .

10, and 10 ìðéïÀ/g at 6, 12, 24, and 48 hours postmortem, respectively. After 288 hours, the aged muscle still contained as much as 9.9 ìðéïÀ/g. Thus, an approximately 5.5-fold decline in glycogen concentration occurred during the first 24 hours after slaughter. Howard and Lawrie (1956) observed that initial glycogen values for beef LD muscle were about 52 ìðéïÀ/g but declined to 8.3 ìðéïÀ/g by 24 hours, a 6.25-fold decrease. The relatively slower decline in glycogen levels in beef muscle can be contrasted to that of pig muscle in which Kastenschmidt et al. (1968) observed a decrease of 30-50% by 10 minutes postmortem. Several workers (Sayre et al., 1963a,b; Beecher et al., 1965a,b; Bodwell et al., 1966; Kastenschmidt et al., 1968) have observed that glycogen undergoes an extremely rapid breakdown in muscles of some pigs, reaching low levels within 3-5 hours after death. This phenomenon has been shown to be related to development of pale, soft, and exudative (PSE) muscle in the pig. This topic is discussed later in this chapter (Section IV,F).

The amount of glycogen remaining when the muscle goes into rigor mortis falls in the range of 2 30% of resting levels and depends on the original resting concentration and the rate at which phosphorylase and the other glycolytic enzymes become inactive and are no longer capable of degrading glycogen to glucose. The development of low pH as a result of lactic acid production during anaerobic glycolysis is a major factor in the inactivation of phosphorylase and the other glycolytic enzymes. Thus, muscles that undergo rapid glycolysis may have as high a level of residual glycogen as those that hydrolyze it more slowly, since the enzyme systems may be inactivated earlier owing to the rapid drop in pH associated with early production of lactic acid. 2. High-Energ y Phosphat e Compound s an d Thei r Intermediate s ATP, ADP, and CP concentrations in muscle from various species are shown in Table 13-4. Resting

400

13. Postmortem Conversion of Muscle to Meat

ATP levels fall within a range of 5.7-8.1 /xmol/g of muscle, whereas ADP concentrations vary from 0.9 to 1.1 /xmol/g. CP varies from 13.1 to 23.0 /xmol/g of muscle, making up one-third to about one-half of the total creatine content in muscle (Bendall, 1973b). Bod well et al. (1965a) reported that the initial CP levels in beef muscle are about 9 /xmol/g (Table 13-5), while ATP concentrations amount to about 10.9 /xmol/g of muscle. Thus, initial values for CP are higher for pig than for beef muscle, but ATP values are somewhat higher in beef muscle. Bendall and Davey (1957) concluded that ATP and ADP values can only be accurately determined during the immediate postmortem period and early stage of rigor when CP and glycolytic resynthesis maintain ATP at fairly constant levels. Total creatine concentrations in resting muscles fall in a range of 34-44 /xmol/g (Table 13-4). Scopes (1973) reported that the highest ratio of phosphorylated creatine (CP) to total creatine is approximately 65% even though it should theoretically be about 90% phosphorylated based on the creatine kinase reaction and the substrate concentrations present in muscle. CP values for resting muscle fall within the range of 13.1-23.0 /xmol/g (Table 13-4). Predominantly white pig muscles, such as the LD and the white portion of the semitendinosus, have CP levels of about 20 /xmol/g, whereas red pig muscles have only about half that much CP (Bendall, 1975). Initial postmortem CP levels in poultry are influenced greatly by the method of slaughtering, with initial concentrations in breast muscle from birds anesthetized before slaughtering being about 0.25 mol/mol total acid-soluble phosphate (TSP) as compared to about 0.05 mol Cp/mol TSP in unanesthetized birds (deFremery, 1966). Thus, about 80% of the initial resting level of CP in chicken breast muscle is dissipated by brief electrical stunning. The data on the resting levels of CP in pig muscle are highly variable, but they generally appear to be more like those of the chicken than the bovine, declining rapidly on any stimulation. Table 13-6 reveals that ATP in beef muscle at 48 hours postmortem dropped to about 17% of its initial value. CP, on the other hand, fell to about 17% of its initial value at 12 hours postmortem and had disappeared by 24 hours, which is in agreement with the results of Marsh (1954) for beef muscle. Howard and Lawrie (1956) observed that the onset

Tabl e 13-6 Levels of ATP and CP and pH Values for Beef Muscle during the Initial 48 Hours Postmortem a

Time Postmortem (hours) Initial 6 12 24 48 a

pH

ATP (% of initial level)

CP {% of initial level)

6.99 6.57 5.96 5.74 5.57

100.0 78.1 60.9 26.6 17.2

100.0 22.0 16.5

— —

Dat a fro m B o d w e l l et al. (1965a) .

of rigor mortis in beef muscle held at 37°C occurred when about one-half to two-thirds of the initial ATP had disappeared. This result is in relatively good agreement with that of Bendall and Davey (1957) who reported that the onset of rigor in rabbit muscle at 37°C took place when one-half of the ATP was depleted. When the muscle was held at room temperature, however, it was observed that the onset of rigor did not occur until three-fourths of the ATP was depleted. Table 13-7 shows the initial concentration of a number of glycolytic intermediates in beef and pig muscle. The concentrations of the hexose phosphates, á-glycero l phosphate, and lactate are much higher than the other glycolytic intermediates, both initially and at 3 (pig) and 24 (beef) hours postmortem. Follett et al. (1974) observed that the concentration of hexose phosphates in beef semitendinosus muscle doubled between 0 and 8 hours postmortem. The large increase in hexose phosphate concentration indicates that phosphofructokinase activity may be the rate-limiting step in glycolysis as suggested by Newbold and Scopes (1967). 3 . Breakdow n Product s of High-Energ y Phosphate s AMP, which is the product of ATP and ADP hydrolysis, comprises only about 0.2-0.3 ìðéïÀ/g in immediate postmortem muscle (Bendall, 1973b). In beef sternomandibularis muscle, the concentration of AMP may increase by two- to threefold in the first 10 hours postmortem but by 24 hours has usu-

III. Postmortem Physicochemical Changes

401

Tabl e 13-7 Concentrations of Glycolytic Intermediates in Beef and Pig Muscle at Various Times Postmortem a

Beef

Glucose 1-phosphate Glucose 6-phosphate Fructose 6-phosphate Fructose bisphosphate Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate 3-Phosphoglyceric acid 2-Phosphoglyceric acid Phosphoenolpyruvate á-Glycero l phosphate Lactate

b

Pig'

1.5 hours

24 hours

4.5

4.7

0.08

0.04

2.6 32.0

1.2 98

0 hours

3 hour;

0.3 4.5 0.7 5.8 0.2 0.1 0.5 0.06 0.11 2.2 40

0.4 6.5 1.8 0.2 ~0 ~0 0.1 0.03 0.03 1.8 90

a

Fro m Grease r (1986). V a l u e s e x p r e s s e d a s ìçéïÀ/ g of m u s c l e .

b

Dat a fro m N e w b o l d an d S c o p e s (1967). Sternomandibulari s muscle s hel d at 15°C.

c

Dat a fro m K a s t e n s c h m i d t (1970). L o n g i s s i m u s dors i m u s c l e s (slow glycolyzing ) left on th e carcas s unti l tim e of sampling .

ally declined to its original level (Newbold and Scopes, 1967). In pig LD muscle, the concentration of AMP remains fairly constant at different times postmortem. Inosine nucleotide concentrations in resting muscle amount to about 0.5 /xmol/g according to Bendall (1973b). Most of this consists of IMP, with its concentration being almost directly related to the amount of ATP being hydrolyzed. Bendall and Davey (1957) showed that ATP and IMP levels in rabbit muscle at 10 minutes, 10 hours, and 24 hours postmortem amounted to 6.8, 1.7, and 0.7 ìðéïÀ/g and 0.8, 6.6, and 8.3 ìðéïÀ/g, respectively. IMP concentrations increase to about 5 ìðéïÀ/g at 12-24 hours postmortem in beef muscle (Disney et al., 1967; Newbold and Scopes, 1967), with pig LD muscle reaching similar levels (Tsai et al., 1972). Dannert and Pearson (1967) found that the concentration of IMP did not differ significantly between three muscles (LD, biceps femoris, and semimembranosus) of the pig at 48 hours postmortem, falling in a range of 2.98-3.23 ìðéïÀ/g. Several researchers (Kazeniac, 1961; Zhivkov, 1963; Terasaki et al., 1965) have reported that chicken breast muscle has higher concentrations of IMP than leg and other dark-colored muscles. The IMP content of the dorsal muscle (light colored) of rainbow trout was

found to be considerably higher than that of the lateral red muscle (Saito et al., 1959). Dannert and Pearson (1967) found that beef LD muscle reached its maximum concentration of IMP in 12-24 hours postmortem but declined on further aging through 28 days, at which time it contained only 0.75 ìðéïß / g. Rhodes (1965) observed that IMP was almost completely degraded in some beef cuts by 30-40 days of aging, but other beef and lamb cuts still contained appreciable amounts. An interesting aspect of IMP is its extremely low level in heart muscle; it amounts to only 0.13 ìðéïß / g of pig cardiac muscle (Dannert and Pearson, 1967). This has also been shown to be the case in rats (Visioli et al., 1964) and rabbits (Imai et al., 1964) and indeed in other species, since the enzyme AMP-deaminase, which hydrolyzes AMP to IMP, appears to be absent in cardiac muscle. This, of course, accounts for the low concentration of IMP in heart, which is discussed later from the viewpoint of its effects on meat flavor (Section VI). Another breakdown product of the high-energy phosphates is inorganic phosphate, which is released on cleaving of the high-energy phosphate bonds in ATP, ADP, and CP. Breakdown of IMP also contributes an inorganic phosphate for every molecule hydrolyzed. Thus, inorganic phosphate

402

13. Postmortem Conversion of Muscle to Meat

(Pi) should increase as long as high-energy phosphates are being broken down by anaerobic oxidation. Bodwell et al. (1965a) reported the initial level of Pj in beef LD muscle to be about 22 /xatoms/g, which had increased to a level of nearly 36 ^atoms/ g by 20 days postmortem (Table 13-5). Levels of Pi in lamb LD muscle doubled between 1 and 24 hours postmortem (Pearson et al. 1973b). Similar patterns for Pj concentrations have been shown for the pig by Kastenschmidt (1970). The data available clearly show that the levels of high-energy phosphates and IMP are closely related to the concentrations of Pj found in postmortem muscle, regardless of the species, apparently reflecting their breakdown to Pj. Newbold and Scopes (1971a) have demonstrated that mincing increases metabolism in beef muscle, with the concentration of lactic acid increasing from about 18 ìçéïÀ/g at 10-15 minutes postmortem to approximately 36 /xmol/g at 400 minutes. Adding of 50 mM Pj to the minced muscle stimulated resynthesis of ATP without altering ATPase activity. Addition of Pj resulted in an initial concentration around 33 ìðéïÀ/g for Pj, which increased to only about 36 ìçéïÀ/g at 400 minutes postmortem. Initial IDP concentrations in resting muscle are extremely low or undetectable but increase to 0.40.8 ìçéïÀ/g between 3 and 72 hours postmortem in pig LD muscle (Tsai et al., 1972). Bendall and Davey (1957) have detected ITP in rabbit muscle, while Tsai et al. (1972) also identified it in pig muscle; however, in both cases the concentrations were lower than that of IDP. The levels of inosine and hypoxanthine are normally zero in resting muscle (Rhodes, 1965) since they are formed by degradation of IMP, which as already indicated is very low in normal resting muscle. Production of hypoxanthine may reach 20-40% of the resting ATP concentration by 24 hours postmortem for beef (Howard et al., 1960; Disney et al., 1967), with the level continuing to increase slowly during up to 60 days of aging at 2°C (Rhodes, 1965). Beef LD muscle was shown to contain only about 1 ìðéïÀ/g of inosine and an approximately equal amount of hypoxanthine at 5-6 days postmortem by Hamm and van Hoof (1974). In the case of the pig LD muscle, however, inosine plus hypoxanthine rose from less than 1 ìðéïÀ/g at 3 hours postmortem to approximately 2 ìðéïÀ/g by 72 hours (Tsai et al., 1972). y

Although the sum of total adenine and inosine compounds are relatively constant over time postmortem (Bendall and Davey, 1957), they are not always found in the same relative distribution, indicating variability in their postmortem degradation patterns. Hypoxanthine has been shown to be useful as an index of spoilage in fish muscle (Dawood et al., 1986) but has not been found to be useful for detection of spoilage in red meats and poultry. This would suggest that breakdown of the adenine nucleotides is not as fast or as direct in the red meats or poultry as is the case for fish. The concentration of nicotinamide adenine dinucleotide (NAD) also changes in postmortem muscle. Normal resting muscle contains NAD at about 0.5-1.0 ìðéïÀ/g, with these levels gradually declining during postmortem aging. In deep portions of beef semimembranosus muscle, the levels of NAD decline to about 35% of their initial concentrations by 8 hours postmortem and then remain at these levels up to 36 hours of aging (Follett et al., 1974). In muscles removed from the carcass and held at temperatures of 15°C or lower the decline is smaller, with final levels of NAD being 66-75% of the initial value. Hatton et al. (1972) found that the NAD concentration in beef biceps femoris muscle declined from 0.8 to 0.5 ìðéïÀ/g between 15 minutes and 24 hours postmortem. The NAD concentration in pig muscle declines by about one-third in the first 3 hours following death (Kastenschmidt, 1970), while the level of NAD in lamb muscle drops by about 50% during the first 24-48 hours postmortem (Atkinson and Follett, 1973). Degradation of NAD is also accelerated by mincing or grinding of muscle (Newbold and Scopes, 1971a) as well as by homogenization (Severin et al., 1963). These processes appear to exert their effects by acceleration of glycolysis, which is reviewed in Section III, C of this chapter.

4. Lacti c Acid Virtually all living cells are capable of partially oxidizing glucose under anaerobic conditions. This results in a net yield of two molecules of ATP for each glucose molecule that is converted to lactic acid: Glucose + 2 ADP + 2 Pj ^ 2 lactate + 2 ATP + 2 H Q 2

III . Postmortem Physicochemical Changes

Thus, glycolysis provides a means for rapidly obtaining ATP under anaerobic conditions, which can occur in muscle during times of stress. This reaction can also occur after death, with the breakdown of glycogen providing a source of glucose to drive the reaction forward. Once the glycogen is dissipated, however, this reaction ceases. Although one would expect lactate levels to be very low or absent in resting muscle, lactate is present at concentrations of approximately 6-16 ìðéïÀ/g (Table 13-4). Lactate increases rapidly following death in a stoichiometric relationship to glycogen, the high-energy phosphate compounds, and their metabolites (Bodwell et al., 1965a, 1966). Bodwell et al. (1965a) calculated that 46.5 ìðéï ß lactic acid/g muscle was required for every pH unit decrease in beef LD muscle. Lactic acid levels for beef LD muscle generally fall within the 6-16 ìðéïÀ/g resting concentration range as shown in Table 13-4 (Bodwell et al., 1965a), whereas initial values for pig muscle are usually about 30-40 ìðéïÀ/g (Bendall et al., 1963; Kastenschmidt et al., 1964). Since the values for pig muscle were obtained from immediately postmortem unanesthetized muscle, it is possible that lower concentrations actually exist in normal living pigs. As indicated earlier, pig muscle appears to be easily stimulated, making it difficult to obtain true resting values. Muscles of domestic chickens and birds are known to undergo even faster rates of glycolysis; deFremery and Pool (1960) showed that chicken breast muscle goes into rigor within 2-4.5 hours postmortem (Table 13-3). Similar changes have been reported for turkey muscle by Dodge and Stadelman (1960). Disney et al. (1967) have demonstrated that the lactate levels in beef carcasses (semimembranosus muscle) increase from 26 ìðéïÀ/g at 1 hour postmortem to 38, 56, 73, and 77 ìðéïÀ/g at 3, 6, 13, and 24 hours postmortem, respectively. Similar increases in lactic acid concentrations were shown to occur in beef LD muscle by Bodwell et al. (1965a), with the level increasing from an initial value of approximately 10 to about 85 ìðéïÀ/g muscle at 24 hours postmortem. Newbold and Scopes (1967) found quite similar concentrations of lactic acid in beef sternomandularis muscle. Kastenschmidt (1970) observed that lactate levels in pig LD muscle increased much faster, reaching a concentration of 80 ìðéïÀ/g by 3 hours postmortem. However, Kas-

403

tenschmidt (1970) also reported that some breeds of pig had slow-glycolyzing muscles and accumulated lactic acid much more slowly.

5. p H Change s Table 13-4 presents resting pH values for muscle from several species of animals, which vary from 7.08 to 7.30. Even the higher value is lower than the normal pH of 7.4 reported for living muscle by Bate-Smith (1948), who further indicated that the buffering effect of bicarbonate on the release of C 0 and the breakdown of CP with production of Pj gives a net pH of about 7.6 for living muscle. As the pH declines in muscle, lactic acid per se also acts as a buffer (Bate-Smith, 1948). The amphoteric effects of the muscle proteins also come into play as the pH becomes more acidic, which helps to buffer further pH changes. The stimulation of slaughtering and/or of excising muscle samples causes a marked drop in the pH of unanesthetized muscle. pH measurements for beef muscle taken within 10-15 minutes postmortem usually fall within the range 6.9-7.0 but decline to about 5.5-5.6 by 48 hours postmortem as shown in Table 13-5 (Bodwell et al., 1965a). Similar values for beef muscle were obtained by Cassens and Newbold (1966). Mutton muscle has been reported to have a pH of about 6.9-7.0 when measured at less than 30 minutes after death (Pearson et al., 1973a,b). Similar pH values for lamb muscle at 4080 minutes after death have been reported by Marsh and Thompson (1958). Hallund and Bendall (1965) observed that the pH in pig muscle was 6.66.8 at 10-15 minutes postmortem. These results suggest that the pH for the LD muscle declines in a curvilinear fashion from its initial value until it reaches its final 48-hour pH of about 5.4-5.7 (Greaser, 1986). Although most information on muscle pH of meat-producing species has been determined on the LD muscle, other muscles appear to follow a similar pattern. There is, however, some variation in the pH of different muscles from the same animal. Conditions may also occur that alter the usual pattern of pH change, such as exhaustion, other conditions causing stress, and prerigor muscle stimulation. The effects of stress are, however, quite 2

404

13. Postmortem Conversion of Muscle to Meat

beef semimembranosus muscle requires 24-48 hours to reach its ultimate pH at 1.5 cm from the surface but needs only 12 and 6 hours at 6 and 8 cm, respectively. The rate of pH decline is also species dependent: the pH of pig muscle changes at 0.64 units/ hour at 37°C (Hallund and Bendall, 1965), while

variable depending on its nature and duration. For example, stress to exhaustion will produce little or no change in muscle pH, whereas less extensive stress may actually accelerate glycolysis and greatly speed up the pH drop. The distance of the sample from the muscle surface may also influence pH readings, with Tarrant (1981) indicating that

L DORS I

I

OL

2 0 0 TIM E

IN

3 0 0

ULTIMAT E

MINUTE S

DIAPHRAG M

(HORSE )

HEAR T

(HORSE )

SEMIMEMBRANOSU S

(OX )

(OX)

(OX)

TIM E

IN

MINUTE S

Figur e 13-2 Effect of species (a) and muscle (b) differences on the rate of postmortem pH fall at 37°C. Zero time equals 1 hour postmortem. Reprinted with permission from Lawrie, "Meat Science." Copyright © 1979, Pergamon Books Ltd.

III. Postmortem Physicochemical Changes

beef, sheep, and rabbit muscle pH decline more slowly (about 0.27-0.40 units/hour) at the same temperature (Greaser, 1986). The rate of glycolysis is also influenced by a number of factors besides species which are discussed later. Figure 13-2 shows the effect of species and type of muscle on postmortem pH. The plot demonstrates that the drop in muscle pH is faster in pig LD muscle in comparison to beef LD, which is intermediate, and slowest in horse LD muscle. The differences between pH values are most noticeable during the early postmortem period (first 3 hours). By 5 hours postmortem, the pH for all three species were essentially the same. As shown in Table 13-3, postmortem muscle glycogen levels in the chicken had declined to extremely low levels by 2-4.5 hours postmortem, which indicates that the muscles are in full rigor and have achieved pH values near that found at 24 hours. The pH plots for various muscles are also given in Fig. 13-2 and demonstrate that different muscles from the same species have different postmortem pH curves. For example, the curves are quite different for the beef (ox) semimembranosus, LD, and psoas muscles. The psoas reached pH 5.8 at 2.5 hours, whereas the LD and semimembranosus required 3.7 and 5.4 hours, respectively, to reach the same pH, which is frequently taken as the onset of rigor. The diaphragm and heart muscles of the horse show even greater differences in their pH curves. It may be that the higher pH values in the diaphragm muscle are related to its oxidative characteristics and its thinness, both of which would contribute to oxidative metabolism and maintain its pH at higher levels. Mincing or grinding also speeds up development of acidity in muscle and thus accelerates the pH drop (Fig. 13-3). Although ultimate 24-hour pH values were essentially the same for the unminced control and the minced samples, only about onethird as much time was required to reach the ultimate pH in the minced samples. Grinding of lamb muscle has been shown to decrease the initial pH from 6.82 to 5.63 by 6 hours as compared to pH 6.46 for the excised but unground control (Pearson et al., 1973b). Addition of C a and epinephrine accelerated the drop in pH over that of mincing alone, although the decline appeared to be associated with some mechanism other than conversion 2+

Ï

IOO 200 300 MINUTES AFTER START OF MINCING

405

400

Figur e 13-3 Influence of mincing or grinding on postmortem pH: • , unminced control muscle; · , minced samples. From Newbold and Scopes (1971a).

of phosphorylase b (inactive) to the a (active) form. Newbold and Small (1985), in agreement with these results, found that conversion of phosphorylase b to the a form is only transitory in nature and probably does not contribute appreciably to the rapid pH decline that results from electrical stimulation.

C. FACTOR S INFLUENCIN G P O S T M O R T E M GLYCOLYSI S A number of factors that influence postmortem rate of glycolysis have already been discussed, such as breed and muscle differences, and so are not reviewed again. In this section, however, temperature and some other factors are discussed. 1. Postmorte m Temperatur e Muscle temperature has a marked effect on the rate of postmortem glycolysis. This is demonstrated by the data plotted in Fig. 13-4, which illustrates that high postmortem temperatures accelerate glycolysis (in this case measured as the drop in pH), whereas low temperatures retard the rate and hence lessen the number of hours needed to achieve a pH of 5.8. This is not surprising since higher temperatures are known to speed up the rate of chemical reactions, and thus their effects in

406

13. Postmortem Conversion of Muscle to Meat

Time (hr)

Figur e 13-4 Effect of environmental temperature on postmortem rate of pH decline in beef LD muscle. From Marsh (1954).

speeding up glycolysis in postmortem muscle would be expected. The effects of temperature on postmortem pH are best illustrated by examining the results of Bodwell et al. (1966) for pig LD muscle (Table 138). One side of 13 pork carcasses was placed at -29°C immediately after slaughtering and dressing (—30 minutes), whereas the other side of each carcass was held at 37°C. Initial pH values averaged 6.54, but after 3 hours at -29°C the pH remained high (6.24) while the carcasses held at 38°C showed a rapid decline to pH 5.48. Thus, high postmortem holding temperatures were shown to accelerate gly-

Tabl e 13-8 Effect of Postmortem Temperatures on pH and Quality in Porcine LD Muscle a

pH Value

Muscle Quality Score*

Time Postmortem

-29°C

38°C

-29°C

Initial (<30 minutes) 3 hours 48 hours

6.54 6.24 5.57

6.54 5.48 5.38

—

—

0.06

0.73

a

38°C

Dat a recalculate d fro m Bodwel l et al. (1966).

* Qualit y w a s evaluate d for pale , soft , an d exudativ e muscl e usin g th e followin g scale : 0 = n o n e , 1 = slight , 2 = moderate , an d 3 = e x t r e m e .

colysis, whereas low temperatures delayed the glycolytic changes. After 48 hours postmortem, the carcasses held at both temperatures were in full rigor (Table 13-8). 2. Specie s Effect s Species also has a marked effect on postmortem glycolysis, with the rate of pH decline indicative of glycolysis varying greatly between muscles of different species. The pH drop in muscle from the pig declines by 0.64 pH units/hour, while beef, sheep, and rabbit muscles have pH declines of about 0.270.40 pH units/hour at a temperature of 37°C. Bendall (1973b) has pointed out that the rate of glycolysis is also pH dependent. Beef semimembranosus muscle declines by only 0.13 pH units/hour between pH 7.0 and 6.7, but the rate of decline becomes 0.25 pH units/hour at pH values between 6.6 and 5.8 after correcting to 38°C (Bendall, 1978). It is quite possible that the species variations in glycolytic rates are at least in part related to the effects of pH, since the pH of pig muscle drops more rapidly than that of beef and sheep. It is more probable, however, that some other factor is directly related to and causes the rapid pH decline. Bendall (1978) has suggested that the glycolytic rate may be the result of varying concentrations of intracellular free C a , which may arise from the mi2+

III . Postmortem Physicochemical Changes

toehondria as they become anaerobic following death. The latter aspect has been examined by Cornforth et al. (1980) and is reviewed later in relationship to the influence of mitochondria and SR on C a release and their influence on cold shortening in red and white muscles (Section IV,A,3). 2+

3. Variatio n betwee n Differen t Muscle s The beef, sheep, and pig LD muscles are more active during the death struggle than the beef sternomandibularis, triceps brachii, and biceps femoris muscles or the pig vastus intermedius (Bendall, 1978). Glycolysis in the active muscles is greatly slowed down by immobilization of the live animals with myanesin or massive doses of MgSC>4 solution. This has been shown to be especially true in the case of rabbit psoas muscle, which may have initial (10 minute) pH values of 6.6 or less. On the other hand, slow glycolysis in beef triceps brachii is indicated by the high initial pH values of 7.1 and above. The pH at depletion of one-half of the ATP stores is 6.04 for beef LD muscle but 6.31 in the same muscle for Large White pigs and 6.58 for vastus intermedius muscle of the same pigs. However, the pH at one-half ATP in the LD muscle of Pietrain/Hampshire pigs, which are susceptible to the PSE condition, is only 6.1 (Bendall, 1979). The final postmortem pH (at 24 hours) can vary from 6.23 in the red vastus intermedius muscle of the pig to 5.4 in the large hindquarter muscles of electrically stimulated beef carcasses. This clearly demonstrates the effects of electrical stimulation on glycolysis. Thus, different muscles have variable glycolytic rates. Exponential relationships exist between CP levels and both lactate concentrations and pH values, but the relationships have very different rate constants for various muscles (Bendall, 1979). The slope constants can vary by as much as threefold and are related to whether the muscle is quiescent or active during slaughtering, being large in quiescent muscles and small in active muscles. In quiescent muscles during the latter stage of glycolysis, free ADP levels increase to about one-tenth of initial ATP levels, while in active or stimulated muscles free ADP concentrations at the same stage of glycolysis increase to only about one-hundreth of initial ATP values. ATP levels in quiescent muscles

407

begin to decline only when CP reaches low levels, whereas in active muscles the decline in ATP begins while CP levels are some 3 times greater. It is quite possible that the faster glycolytic rates in some muscles as compared to others are related to the relatively higher proportion of phosphorylase a (Bendall, 1979). Scopes (1974b) has suggested that electrical stimulation results in more phosphorylase a activity, which accelerates glycolysis by mobilization of glycogen. In a later study, however, it was shown that the increase in the amount of phosphorylase a relative to the b form was only transitory on electrical stimulation of muscle (Newbold and Small, 1985). 4. Effect s of Stres s The levels of high-energy phosphate in muscle are greatly affected by preslaughter stress. The PSE, PSS, and DFD conditions in the pig are related to preslaughter stress and are a consequence of alteration in the levels of high-energy phosphates and their metabolites (Briskey, 1964; Sybesma and Eikelenboom, 1978), all of which are related to the rate of glycolysis and are discussed later in Sections IV,F, IV,G, and IV,H. Fast-glycolyzing muscles are not only present in the pig (Lawrie, 1960; Kastenschmidt, 1970) but have also been observed in the bovine, although less frequently than in pig muscle (Hunt and Hedrick, 1977; Fischer and Hamm, 1980). Changes in CP and ATP concentrations in fastglycolyzing and normal beef muscles were followed by Fisher and Hamm (1980). Results demonstrated that ATP and CP levels in fast-glycolyzing muscles arise not only from more rapid glycolysis but also from glycolysis occurring at an earlier time postmortem than in normal muscle. Both of these highenergy phosphate compounds were present in fastglycolyzing muscles at lower initial concentrations (30 minutes). The lower initial pH values in the fastglycolyzing muscles were associated with lower levels of muscle glycogen, a lower water holding capacity, a paler color, and higher concentrations of lactic acid than was the case for normal muscle. By 1 hour postmortem, CP was almost absent and ATP levels were very low, indicating a rapid rate of breakdown of the high-energy phosphates. The changes in quality in fast-glycolyzing beef muscle,

408

13. Postmortem Conversion of Muscle to Meat

however, are less severe than in PSE pork (Fischer and Hamm, 1980). Stress as a factor in determining muscle quality has been related to the rate of glycolysis. This has been shown to be the case in development of PSE pork, where stress greatly accelerates the rate of glycolysis and is responsible for the pale, soft, and exudative muscle (Briskey et al, 1959; Say re et al., 1961, 1963a,b,c; Briskey, 1964; Sair et al, 1970). Stress was recognized as causing depletion of glycogen stores and as being a major contributing factor to development of spoilage in bacon by Callow (1936). The consequences of exhaustion on glycogen stores in the dog were described by Bernard (1877), who pointed out that exhaustion resulted in development of alkaline rigor without any remaining ATP reserves. The problem of exhaustion is most often encountered in game animals in connection to exhaustive exercise during the hunting season, but it also can occur under conditions of starvation. Low levels or the absence of glycogen is a problem in DFD pork and in dark cutting beef, both of which are discussed later (Sections IV,G and IV,E).

D . STRUCTURA L A N D TENSIO N CHANGE S DURIN G DEVELOPMEN T O F R I G O R MORTI S The most easily observed structural change during development of rigor mortis is the transformation of the soft, pliable, and naturally elastic prerigor muscle into a rigid and quite inextensible tissue. The development of rigor mortis can be measured by following the degree of stretching of muscle strips while alternately loading and unloading them at regular intervals over postmortem time (BateSmith and Bendall, 1949; Briskey et al, 1962). Prerigor muscle will stretch to 120% of its resting length under a load of 50 g/cm , but after completion of rigor stretches to only 101% under the same load (Bendall, 1973a). Increasing the load by 10fold in postrigor muscle results in only a 2% increase over resting length. Figure 13-5 shows the effects of temperature and stress on development of rigor mortis. The effect of glycogen depletion by insulin injection, which imitates stress and results in low glycogen stores, is 2

clearly shown in Fig. 13-5d. The effects of struggling at the time of death in hastening development of rigor (Fig. 13-5c) is also contrasted with that of anesthetized muscle (Fig. 13-5a), which required about fourfold longer to go into rigor. Equally dramatic is the rapid development of rigor at high temperature (Fig. 13-5b), with muscle held at high temperatures developing full rigor in about one-half the time required for that of anesthetized muscle. The effects of postmortem temperature on development of rigor mortis is further verified by Locker and Daines (1975), who found that beef sternomandibularis muscle developed full rigor in 7 hours at 37°C but required 10, 10, 12, and 24 hours to reach the same stage at 34, 28, 24, and 15°C, respectively. The loss of extensibility that occurs in postmortem muscle has been shown to be related to a decrease in the ATP concentration (Bate-Smith and Bendall, 1947; Bendall, 1951). There is an approximately 50% decline in muscle elasticity by the time the resting ATP level drops by about one-half of its initial value (Bendall, 1951). Although MgATP levels above 0.1-0.2 m M are adequate to prevent rigor development in glycerinated muscle fibers (Izumi et al, 1981), in intact muscles elasticity begins to decrease while ATP concentrations are still 2-4 mM. The decline in elasticity beginning at this high level of ATP suggests that there are wide differences in the ATP concentrations in individual muscle fibers, which means ATP is absent in some fibers so that they are no longer extensible. Apparently, ATP is absent in enough of the fibers to account for the decrease in extensibility that occurs when ATP declines to about one-half of its resting concentration. Furthermore, if glycolysis has produced enough lactic acid to inactivate the glycolytic enzymes, ATP can no longer be produced from glycogen and serve as a plasticizer to prevent the interaction of the myosin heads with actin so they become firmly locked into the actomyosin complex. Muscles that are unrestrained (or under light load) will shorten as they develop rigor mortis. However, on restraining muscles by maintaining a constant length, tension is developed. Temperature has a marked effect on the amount of tension generated, but its effect is species dependent. In beef muscle, tension development is maximum at 12°C, less at 37°C, but is very little at 16-25°C (Busch et al, 1967; Cassens and Newbold,

III . Postmortem Physicochemical Changes

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Figur e 13-5 Changes in muscle extensibility over prerigor time for postmortem rabbit psoas muscle. Muscles were alternately loaded and unloaded every 8 minutes until rigor was complete, (a) Well-fed animal anesthetized with myanesin, muscle held at 17°C. (b) Same as (a) except muscle was held at 38°C. (c) Muscle of animal that struggled at death, (d) Muscle from animal in which glycogen was depleted by insulin injection, (e) Enlarged tracing of early stage of rigor. Arrows on top indicate load on, arrows at bottom, load off. From Bendall (1973b).

1967a,b; Nuss and Wolfe, 1981). In pig and rabbit muscle, however, maximum tension development occurs at 37°C, with less tension being developed at all lower temperatures (Busch et al., 1972). Tension declines following completion of rigor mortis (Jungk et al., 1967), which may in part explain the softening that occurs in muscle with postmortem time. No doubt some of the softening of muscle during postmortem holding is due to enzymatic proteolysis, especially during prolonged aging. Initiation of shortening under light loads is known to be pH dependent at higher temperatures. Currie and Wolfe (1979) observed that muscle shortening began at pH 6.15-6.35 with loads of 5 g/ cm , but at loads greater than 25 g/cm muscle did not shorten until the pH dropped to 5.75-5.85. All muscles will shorten when the pH reaches 5.75, with the degree of shortening being dependent on 2

2

residual ATP levels and muscle load (Currie and Wolfe, 1979, 1980). Shortening during rigor development is reflected by a decrease in the sarcomere lengths of postrigor as compared to prerigor muscle (Asghar and Yeates, 1978). Maximum force development amounts to less than 5% of that generated during contraction in living muscle (Bate-Smith and Bendall, 1947), apparently because only a small fraction of the fibers actually undergo contraction during rigor mortis (Bendall, 1966). Completion of rigor is accompanied by maximum tension development (Schmidt et al., 1970a,b). Rigor shortening can be blocked by injection of C a chelators (Weiner and Pearson, 1966), apparently by preventing the interaction of actin and myosin (Weiner and Pearson, 1969). A relationship between the degree of muscle contraction and meat tenderness was observed by 2+

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13. Postmortem Conversion of Muscle to Meat

Locker (1960a). This was confirmed by later work showing a connection between sarcomere length and meat tenderness (Herring et al., 1965a,b), which led to development of hanging of beef carcasses by the aitchbone (obturator foramen) instead of by the Achilles tendon (Hostetler et al., 1970, 1972,1975, 1976). Hanging the carcass by the aitchbone results in restraining of the large muscles of the loin and round so that they cannot shorten during development of rigor. This maintains the sarcomeres in the stretched position so that fewer of the myosin heads interact with actin and become locked into the contracted state. Hanging the carcass by the obturator foramen has become known as the Tender-Stretch procedure and is discussed further under prevention of cold shortening (Section IV,A,4,b). Histological examination of muscles undergoing rigor mortis reveals that there is some shortening (Johnson and Bowers, 1976). However, the amount of shortening found in cooked muscle in full rigor does not appear to be adequate alone to account for the increased toughness in comparison to that of rapidly cooked prerigor muscle (Paul et al., 1952). Furthermore, increased shortening of prerigor muscle prior to cooking causes an increase in toughness, but shortening to the same extent during cooking of postrigor meat does not greatly influence tenderness (Locker et al., 1977). This suggests that the important factor in the resistance to chewing or shearing of meat is the position at which actin and myosin interact and form actomyosin. In other words, if actomyosin is formed so that only a few of the myosin heads and actin filaments interact, such as is the case in relaxed postrigor meat, the resistance to shear is less. On the other hand, if the fibers are contracted when they go into rigor, there would be more interaction of the myosin heads with the actin filaments, the end result being greater toughness of the prerigor contracted muscle. In postrigor cooked meat, however, shortening does not result in more interaction of the myosin heads with actin filaments but is merely a physical phenomenon that is not accompanied by any further interaction; hence, it does not greatly affect tenderness. Hydrolysis of both the myofibrillar and connective tissue proteins may also be involved in tenderization during the cooking of meat. Locker et al.

(1977) has proposed that the gap filaments, first described by Huxley and Hanson (1954) and later given that name by Sjostrand (1962), may play an important role in meat tenderness. This theory suggests that the gap filaments form a core in each thick filament and emerge at one end to pass between the thin filaments through the Z-line and then between the thin filaments of the adjacent sarcomere and into the thick filaments where they terminate. Although Locker et al. (1977) first suggested the gap filaments were composed of titin, the theory has since been revised to propose that nebulin and other N-line proteins may be constituents (Locker and Wild, 1984). Horowits et al. (1986) have provided evidence that titin or nebulin (or both) gives axial continuity for production of resting tension on stretching of muscle and also tends to keep the thick filaments in register during force generation. This was shown by subjecting skinned muscle fibers to low doses of ionizing radiation to degrade titin and nebulin, after which the ability of the skinned fibers to respond to stretch and active tension on addition of C a was greatly reduced. This is illustrated by the proposed model in Fig. 13-6 that shows the breakage of titin and/or nebulin in gap filaments under Ca -generated tension. Thus, the effects of postmortem degradation on the gap filaments may be important in meat tenderness. 2+

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1. Effect s of A T P an d Calciu m Ion s on Rigo r As mentioned earlier, rigor does not occur until approximately one-half of the ATP is depleted. The effect of ATP in preventing development of rigor is illustrated in Fig. 13-7, which shows that addition of ATP results in relaxation. The addition of excess C a , on the other hand, causes contraction of the muscle fiber bundle, which is a condition analogous to development of rigor (Bendall, 1963). Figure 13-7 also demonstrates that blockage of the effects of C a is essential for relaxation of muscle as shown by the addition of EDTA. The addition of EDTA and MgATP produces relaxation, which is similar to the conditions existing in prerigor muscle. Huxley (1960) has pointed out that depletion of ATP leads to formation of fixed links between the actin and myosin filaments. The cross-bridges formed cannot be broken in the absence of ATP (Nauss and 2+

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III. Postmortem Physicochemical Changes

a

Figur e 13-6 Proposed model in which each end of the thick filament is linked to the nearest Z-disk by elastic filaments composed of titin and/or nebulin. (a) Control showing intact inelastic elements on stretching with C a generating tension, (b) Irradiated sample in which elastic elements are degraded and break under Ca -generated tension. From Horowits et al. (1986). 2+

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Figur e 13-7 2+

Effects of ATP and C a on rigor mortis as shown by using washed glycerinated muscle fiber bundles to show the essential features of contraction, relaxation, and rigor mortis. The bundle is about 0.15 mm thick by 1 mm wide by 2 cm in length. The initial load was 800 g/cm . An additional load of 300 g/cm was put on ( j ) or taken off ( | ) as indicated. MgATP, Addition of 2 mol MgCl plus 2 mol ATP in 0.10 Ì KC1; MgATP + EDTA, addition of MgATP as above plus 0.2 mol EDTA (ethylenediaminetetraacetic acid); C a , addition of CaCl to give a concentration of 0.2 M; zigzag line, washing of fibers with 0.1 Ì KC1; full curve, changes in length; dashed line, changes in ATPase activity. From Bendall (1963). 2

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13. Postmortem Conversion of Muscle to Meat

Davies, 1966). Thus, in the absence of ATP, the stiffness characteristic of rigor mortis is maintained by continuous tension exerted by the cross-bridges between myosin and actin filaments. Figure 13-8 illustrates the relationship between some chemical and physical changes that occur in muscle during development of rigor mortis. These changes include a decline in ATP and CP concentrations and a related decrease in pH. It is evident that CP levels fall rapidly beginning immediately after death. ATP concentrations, as reflected by acid-labile phosphorus levels, remain relatively constant until CP reaches quite low levels. When CP declines to quite low concentrations (from about 6 to about 1 ìðéïÀ/g), ATP levels begin to drop rapidly. The interesting point is that as ATP concentrations decline by about one-half, muscle extensibility begins to decrease rapidly and the muscle becomes highly inextensible within a short period of time (Fig. 13-8). The decline in pH is relatively constant until the ATP levels become fairly low. At this point the muscle is quite inextensible and is locked into full rigor.

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2. Instrument s for Measurin g Muscl e Tensio n A number of devices have been developed for measuring the amount of tension developed in muscle during the onset of rigor mortis (Bate-Smith and Bendall, 1949; Briskey et al, 1962; Partmann, 1963). Jungk et al (1967) have also developed a different type of instrument for following changes in isometric tension and shortening of muscle strips during postmortem holding of meat. More recently, Locker and Wild (1982a) have developed an instrument that they call the "yieldmeter" which makes tension traces. The yieldmeter has been shown to be useful for following postmortem changes in the yield point of raw muscle by registering maximum tension generated at a low rate of extension. Plots indicate that less tension is developed by aged than by immediately postmortem muscle, even after relatively short periods of aging. This novel device can be used on raw meat (Locker and Wild, 1982b), unlike the shear devices which are only useful for cooked meat. Electron micrographs of aged meat subjected

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Figur e 13-8 Chemical and physical changes in muscle during development of rigor mortis. Values are taken from bovine sternomandibularis muscle held at 35°C. Extension was recorded by an apparatus similar to the one used by Bate-Smith and Bendall (1949) with a load of about 60 g/cm and a loading-unloading cycle of 8 minutes postmortem. From Newbold (1966). 2

IV. Some Conditions Occurring in Muscle/Meat

to yield point analysis suggest that aging weakens both actin filaments and gap filaments so that the Iband is readily fractured (Locker and Wild, 1982b), thereby improving meat tenderness. This is discussed further with respect to the effects of aging (Section IV,C).

IV. SOME CONDITION S OCCURRIN G IN MUSCLE/MEA T A. COL D SHORTENIN G Shortening of prerigor muscle on exposure to chilling temperatures (<10°C) was first observed by Locker and Hagyard (1963), who noted that unrestrained beef muscle shortened rapidly at 0°C but suffered minimal shortening at 14-19°C (Fig. 13-9). The maximum amount of shortening occurred below 5°C but decreased as the temperature was raised until further shortening resulted at about 35°C.

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1. Effect s of Col d Shortenin g on Mea t Tendernes s Marsh and Leet (1966) have shown that there is a clear-cut relationship between percentage shortening and shear force. Maximum toughness was shown to develop between 35 and 50% shortening. Marsh and Carse (1974) have presented evidence that at 35-40% shortening the thick filaments penetrate the Z-disks, which they suggested may interact with the actin filaments in adjacent (end to end) sarcomeres to form a continuum of myosin. They concluded that this structure is responsible for the increased toughness of cold-shortened meat. On the other hand, Voyle (1969) concluded that cold shortening increased the proportion of actively contracting fibers, which is responsible for the increase in toughness. Marsh and Leet (1966) have demonstrated that massive muscle shortening (50-60%) will also cause increased tenderness. Marsh et al. (1974) have shown that shortening beyond 40% produces an increased number of contracture bands, which they proposed causes tearing of the muscle, especially in the area of the Z-disks, and is responsible for the increase in muscle tenderness associated with massive shortening of prerigor muscle. 2. Discover y an d Significanc e of Col d Shortenin g

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Figur e 13-9 Relationship between temperature and shortening in prerigor meat. From Locker and Hagyard (1963).

The problem of cold shortening first became apparent when the New Zealand meat industry began to receive complaints from their North American markets about excessively tough lamb meat. Although the toughness was not known to be associated with cold shortening, it soon became of sufficient magnitude for the New Zealand meat industry to follow up on the complaints, which they could not understand as New Zealanders commonly ate lamb and found no problem with toughness. As a consequence, the industry investigated the complaints on lamb tenderness in the United States and indeed found them to be true. Even though many different factors were investigated, the first promising lead on the cause of toughening came through the classic study of Locker and Hagyard (1963), which demonstrated that prerigor meat shortened extensively under the influence of cold. Some of these

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facts have been reviewed by Locker et al. (1975; Locker, 1985). The first studies to demonstrate a clear relationship between cold shortening and the problem of toughness in New Zealand lamb was by Marsh and Leet (1966) and Marsh et al. (1968). The latter authors showed that prerigor lamb carcasses which were blast-frozen, a common practice in producing New Zealand lamb for export, suffered extensively from toughening. It was also shown that delaying of chilling resulted in lower shear force readings and higher tenderness scores. Thus, prevention of cold shortening could be achieved by delaying freezing until after rigor ensued. This led to development of recommendations for delayed chilling in order to circumvent cold-induced toughness, which is discussed in greater detail below in Section IV,A,4. There is little doubt as to the significance of cold shortening to the meat industry in New Zealand where the North American market was at stake. Steps were immediately taken to institute conditioning and aging of all lamb carcasses intended for those markets prior to blast-freezing. The importance of cold shortening to the meat industry in other countries, including the United States, is harder to quantitate. However, the use of electrical stimulation as a means of preventing cold shortening plus its effects in improvement of carcass grades have led to its wide acceptance in the United States (Savell, 1985; Smith, 1985). Evidence indicates that electrical stimulation is also widely accepted in other countries, suggesting that cold shortening is at least perceived as being a problem in many other countries.

1983). Third, ATP levels are critical to cold shortening, with cold-induced shortening-toughening occurring only when adequate ATP is present to support shortening; however, this will only occur when the muscle is in the prerigor state (Honikel et al., 1983). All three of the following conditions must exist for muscle to undergo cold shortening: (1) it must be in the immediate prerigor state; (2) it must be held in a cold environment (< 10°C); and (3) an adequate ATP concentration must be present to prevent locking of the actin and myosin filaments into actomyosin so that shortening (contraction) can occur. One other factor is also involved in cold shortening of muscle, namely, only predominantly red muscles will cold-shorten. It has been demonstrated that rabbit muscle, which is composed primarily of white fibers, does not cold shorten (Bendall et al., 1976). Pig muscle, which has a high proportion of white fibers, has been reported to suffer only marginally from cold shortening. However, James et al. (1983) found rapidly frozen (-30°C at 1 m/second airflow) pork loins and carcasses were tougher than controls (0°C at 5 m/second). Nevertheless, the problem of cold shortening is closely related to muscle fiber types, with muscles composed of predominantly white fibers suffering little or not at all from cold shortening. Thus, cold shortening is encountered mainly in the red meats, especially in beef and sheep meat. The basic underlying cause of cold shortening is related to the inability of the SR to sequester and bind excess C a under the influence of cold temperatures. At low temperatures (0-5°C), the SR has a decreased ability to bind C a (Kanda et al., 1977a,b). The mitochondria, which normally also bind C a , also have a decreased Ca -binding capacity (Cornforth et al., 1980) and spill excess C a into the intracellular space, which then triggers shortening in much the same way as it causes muscle contraction (Pearson and Dutson, 1985a). Red muscle cold-shortens since it contains more mitochondria and hence more C a , which is spilled at low temperatures. Red muscle also has a less welldeveloped SR system and, thus, has a reduced ability to bind C a at low temperature and low pH. This also adds to the C a flux in the intracellular environment and contributes to cold shortening. White muscle has fewer mitochondria and a bet2+

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3. Mechanis m for Col d Shortenin g

2+

Several factors are involved in cold shortening. First, it is known that the muscle must be in the prerigor state since postrigor muscle does not shorten under the influence of cold temperatures (Locker and Hagyard, 1963). It also has been demonstrated that the response to cold decreases with increasing postmortem time (Marsh et al., 1968). Second, temperature is known to be critical, with maximum shortening occurring near the freezing point (Locker and Hagyard, 1963) and temperatures of 10°C or above being sufficiently high to prevent the cold-shortening effect (Honikel et al.,

2+

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I V . S o m e Condition s Occurrin g in Muscle/Mea t

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ter developed SR system, so can bind more C a and hence does not suffer from cold shortening. Still another factor is the greater amount of ATP available in white versus red muscles, which also helps to prevent cold shortening by providing energy for reaccumulation of C a by the SR and to some extent by the mitochondria (Cornforth et al., 1980). These mechanisms are explained in greater detail by Pearson and Dutson (1985a). Cornforth et al. (1980) have explained cold shortening in red muscle as follows. (1) Under the influence of cold temperatures the anoxic mitochondria have a decreased ability to bind C a and spill the excess into the intracellular space as proposed by Buege and Marsh (1975). (2) The SR, which is much less well developed in red than in white muscle, quickly becomes saturated with C a and spills the excess into the intracellular space where it is able to initiate shortening and cause meat toughness. This is supported by the work of Weiner and Pearson (1966, 1969) and of Davey and Gilbert (1974). Similar shortening can be initiated in prerigor muscle by microinjections of C a and will not only cause shortening but also increase toughness as compared to untreated controls (Weiner and Pearson, 1969; Pearson et al., 1973b). 2+

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4. Preventio n of Col d Shortenin g A number of methods for avoiding cold shortening and the related cold toughening have been utilized. Among these are conditioning and aging, the Tender-Stretch method, and electrical stimulation. Other methods that show some promise but have never been exploited include carbon dioxide anesthesia, altered carcass posture, extremely rapid freezing followed by slow thawing, and a clamp for placing the LD muscle under tension. Unsuccessful attempts have also been made to prevent cold shortening by epinephrine injection (Pearson et al., 1973b). The various procedures of eliminating cold shortening are briefly reviewed. a. Conditioning and Aging The earliest procedure used for prevention of cold shortening was conditioning and aging which was developed to circumvent cold shortening in New Zealand lamb. The method calls for holding the carcasses in a conditioning-aging room until they have gone into

415

rigor mortis (Marsh et al., 1968). The temperature and time specifications were developed for the industry with the times varying with the temperature, that is, longer times were required at lower temperatures. Generally, temperatures of 15-16°C were used for about 16-24 hours. Conditioning and aging will thus prevent cold shortening and the accompanying cold-induced toughness. The conditioning and aging process has been criticized, however, since it disrupts the normal plant throughput and requires extra handling. Although there is little or no evidence of any microbiological problems resulting from the process (Locker et al., 1975), it also has been criticized as being a possible public health hazard. Thus, other methods of preventing cold shortening were sought to replace conditioning and aging; this method has now been almost if not completely replaced by electrical stimulation. b. Tender-Stretch The Tender-Stretch method is based on earlier work on preventing cold shortening by altered posture (Herring et al., 1965a, 1967). It was developed to increase the amount of tension placed on the LD and the main muscles of the round or leg. The procedure is based on the work of Hostetler et al. (1970, 1972, 1973, 1975), who showed that hanging of carcasses (beef and lamb) through the obturator foramen (aitchbone) placed tension on the main muscles used for steaks and, thus, circumvented cold shortening. The method has also been utilized in Australia. It requires some modification in rail arrangements and also alters the shape of the round or leg muscles, however, and the development of electrical stimulation has decreased interest in the use of this procedure. c. Electrical Stimulation Although electrical stimulation was first patented by Harsham and Deatherage (1951) for improving meat tenderness, it was not accepted as being of any practical value in preventing cold shortening and toughening since the phenomena were not yet recognized. Its modern use traces back to its value for prevention of cold shortening as reported by Carse (1973). Since that time a number of researchers (Chrystal and Hagyard, 1975, 1976; Bendall, 1976; Bendall et al., 1976; Davey et al., 1976; Smith et al., 1976; Savell et al., 1977, 1978a,b,c; Savell, 1979) have reported on the effectiveness of electrical stimulation in pre-

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venting both cold shortening and/or cold-induced toughness. This work has been reviewed by research leaders in the field in a book edited by Pearson and Dutson (1985b). Electrical stimulation owes its effectiveness in preventing cold shortening to the dissipation of ATP and the other high-energy phosphate compounds caused by muscle contraction elicited by electrical shock. Although ATP and other high-energy phosphates are reduced in concentration as a result of electrical stimulation, the major beneficial effect is due to the rapid fall in postmortem pH and its attendant hastening of rigor mortis (Carse, 1973; Davey et al., 1976; George et al., 1980). Many different voltages, pulse rates, and durations have been used in electrical stimulation with quite satisfactory results (Bendall, 1980). Higher voltages appear to be preferable, especially if stimulation is delayed following slaughtering. Chrystall et al. (1980) have demonstrated a decline in the nervous response with postmortem time so that higher voltages are needed to achieve the same effects if electrical stimulation is delayed. It has been suggested that electrical stimulation results in conversion of phosphorylase b (inactive) to phosphorylase a (active form) and thus stimulates the breakdown of glycogen. Newbold and Small (1985) found that there was a transient increase in phosphorylase a on electrical stimulation, but little or no phosphorylase a remained after 35 minutes postmortem. Thus, activation of phosphorylase a does not appear to account for the drop in pH since electrical stimulation did not prolong the increased concentration of the active enzyme. It is possible that the release of C a by electrical stimulation is adequate to support glycolysis and accounts for the related decline in muscle pH. Results indicate that stimulation triggers myofibrillar ATPase activity and accounts for the transient conversion of phosphorylase b to phosphorylase a through stimulating phosphorylase kinase; however, once released from the SR, C a appears to be adequate to stimulate glycolysis, and the inactive phosphorylase b again predominates (Newbold and Small, 1985). Regardless of the exact mechanism by which electrical stimulation operates, it is effective in preventing cold shortening. It is now used by New Zealand freezing works for most if not all of the 2+

2+

lamb carcasses exported to North American markets. It is also widely used on beef and lamb carcasses slaughtered in the United States and Canada. Savell (1979) has reported it to improve beef tenderness by 20 and 21% for sensory tenderness and shear values, respectively. Although cold shortening is responsible for most of the toughening in prerigor meat exposed to cold temperatures, electrical stimulation improves tenderness above and beyond that expected from prevention of cold shortening alone. Smulders and Eikelcnboom (1986) have recently verified this in veal carcasses by showing that electrical stimulation improved tenderness even though there was no measurable muscle shortening on exposure to cold (2°C). The improvement in tenderness may be due to the release of lysosomal enzymes (Dutson and Yates, 1978; Dutson et al., 1980a,b) or be due to stretching and tearing of the myofibers during the process of electrical stimulation (Sorinmade et al., 1982). Electrical stimulation also has some beneficial cosmetic effects, such as improving muscle color and brightness, reducing heat ring, and improving U.S. carcass grades, mainly as a result of brighter and more uniform muscle color (Savell, 1979). It has also been reported to improve meat flavor and extend retail caselife (Savell, 1979). These factors have all contributed to the widespread acceptance of electrical stimulation by the meat industry. d. Miscellaneous Methods Investigated for Preventing Cold Shortening Although attempts have been made to accelerate glycolysis by injection of epinephrine, and thereby prevent cold shortening, this technique has been unsuccessful. Apparently, the stress of slaughtering is enough to stimulate epinephrine to its maximum so that preslaughter injection is ineffective in further speeding up of glycolysis (Pearson et al., 1973a). It is possible, however, to slow down glycolysis by preslaughter injection of â-adrenergic blockers, such as propanolol and reserpine (Pearson et al., 1973a). Other procedures for blocking cold shortening that have been more successful include altered carcass posture (Davey and Gilbert, 1973), a muscleclamp device for placing the LD muscle under tension (Stouffer et al., 1971), and C 0 anesthesia (Pearson et al., 1973b). Altered carcass posture has been shown to improve meat tenderness by placing 2

IV. Some Conditions Occurring in Muscle/Meat

the major muscles in the round and loin/leg under tension (Herring et al, 1965a,b, 1967; Hostetler et al, 1970, 1972, 1973; McRae et al., 1971, Stouffer et al, 1971; Buege and Stouffer, 1974). Neither altered posture nor the tensioning procedures are easily adaptable to on-line operations, are not readily applicable to large numbers of carcasses, and thus have not been widely used (Buck and Black, 1967). Results of both altered posture and the muscle tensioning device have shown that the treatments produce longer sarcomeres and more tender meat than unrestrained and/or untensioned controls. However, the difficulties in application have limited their usefulness, and with development of electrical stimulation they have not been further exploited. Carbon dioxide anesthesia has also been shown to accelerate glycolysis in sheep carcasses, resulting in pH readings of 5.7-5.8 within 4-5 hours postmortem (Pearson et al, 1973b). The mechanism by which this procedure prevents cold shortening has

417

not been elucidated, but it is probably related to the release of C a from the SR and stimulation of the rate of glycolysis (Mullenax and Dougherty, 1964). Since electrical stimulation was shown to be useful in preventing cold shortening at about the same time (Carse, 1973), this method has never been used except under experimental conditions. 2+

B. THA W RIGO R Thaw rigor or thaw contracture are terms used to describe the shortening that takes place on thawing of meat that has been frozen while still in the prerigor state. It is usually characterized by massive shortening as shown in Fig. 13-10 (Forrest et al, 1975). Although the phenomenon of thaw rigor has been recognized for many years, its practical significance and relationship to drip losses and meat tenderness was first described by Sharp and Marsh (1953) during the freezing and thawing of whale meat for human consumption.

Figur e 13-10 Effect of thaw rigor on muscle shortening. The muscle at top was frozen immediately postmortem and thawed rapidly; it is only 42% of its original length. The sample at bottom is paired muscle except it was allowed to go into rigor before freezing. From Forrest et al. (1975).

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13. Postmortem Conversion of Muscle to Meat

1. Caus e of Tha w Rigo r Thaw rigor appears to result from an extensive salt flux on thawing, which is characterized by the release of an excessive amount of C a so that the SR becomes saturated and C a spills over into the intracellular space, causing massive contraction (Bendall, 1960). The onset of thaw rigor occurs when the concentration of ATP is relatively high, about 40% (Newbold, 1966). In this respect it closely resembles cold shortening. Like cold shortening, thaw rigor results in appreciable toughness in the meat in comparison to control samples frozen after reaching full rigor. Thaw contracture also results in excessive drip loss from the tissues on thawing. The interrelationships of drip loss, muscle shortening, and the decrease in muscle extensibility are shown in Fig. 13-11 (Marsh and Thompson, 1958). There is a clear-cut relationship between pH and drip loss and percentage of shortening, which re2+

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7. 0

6. 6

flects the fact that the meat is in the prerigor state. As the pH declines, drip losses and the amount of muscle shortening both decrease, while extensibility declines. Figure 13-11 further illustrates that when the pH drops below about 6.2 drip losses are minimal; however, percentage shortening continues to decline until the meat is in full rigor, at about pH 5.6. Thaw rigor is of great practical significance to the meat industry not only because of the toughening effect but also because of the increased drip losses. This has led to the recommendation that meat not be frozen until it is in full rigor, which generally requires holding for about 24 hours at temperatures of 0-4°C. Marsh et al. (1968) have demonstrated that thaw rigor is even more damaging to meat tenderness if meat frozen prerigor is cooked from the frozen state. Meat that is thawed very rapidly has been shown to suffer less from thaw contracture than similarly frozen meat that is thawed more slowly (Davey and Garnett, 1980).

6. 2

5. 8

5. 4

PH

Figur e 13-11 Relationship of thaw shortening and drip loss expressed as percentage of initial values. The values for pH and the decrease in extensibility are indicative of the pre- and postrigor states. From Marsh and Thompson (1958).

IV. Some Conditions Occurring in Muscle/Meat

Apparently, fast thawing minimizes the salt flux into the intracellular spaces and, thus, eliminates or decreases thaw contracture.

419

trical stimulation appears to be the most acceptable method for solving the problem of thaw contracture.

2. Preventin g Tha w Rigo r The traditional method for elimination of thaw rigor is to freeze meat only after it has gone into full rigor. Although this practice will eliminate both thaw contracture and cold shortening, there are many processes where it would be advantageous to freeze prerigor meat. Consequently, several procedures have been investigated for preventing thaw contracture in meat frozen from the prerigor state. The most successful method has been use of electrical stimulation, which prevents thaw rigor in essentially the same manner as it eliminates cold shortening (Gilbert et al., 1977). Electrical stimulation will allow the hot boning of meat and immediate freezing while avoiding any problems from thaw contracture. The advantages derived from electrical stimulation so that hot boning can be incorporated into the processing system may be even more important than those achieved by the elimination of cold shortening. In addition to permitting freezing of prerigor meat intended for use in sausages, electrical stimulation also allows hot boning, cutting, and freezing of prerigor steaks, chops, and roasts (Taylor et al., 1981). There are also energy savings associated with hot boning and processing of prerigor meat (Henrickson and Asghar, 1985). Locker et al. (1975) have pointed out that thaw rigor can be minimized or eliminated by thawing very slowly in comparison to cooking from the frozen state. Apparently, slow thawing helps to minimize the accumulation of a high salt flux and decreases thaw contracture. These same workers also demonstrated that glycolysis continues in prerigor frozen meat and that a sufficiently long period of frozen storage will decrease or avert thaw rigor. Davey and Garnett (1980) have also indicated that extremely rapid thawing of meat frozen in the prerigor state helps to minimize thaw rigor development. It has also been reported that storage for more than 10 days at - 12°C aids in preventing thaw contracture in meat frozen prerigor (Davey and Garnett, 1980). Thus, problems encountered in meat quality by freezing in the prerigor state can be avoided by several procedures. Nevertheless, elec-

C. AGIN G OF MEA T The ripening or aging of meat involves holding or storage while the indigenous enzymes hydrolyze some of the muscle proteins in order to improve tenderness and flavor. Normally, aging is carried out under refrigeration in a temperature range of 0 5°C. In some cases, high temperature aging (1540°C) has also been used, but problems arising from microbiological spoilage and/or food pathogens increase at these temperatures. Ultraviolet light has been utilized to control microbiological growth and does permit high temperature aging at about 1520°C. Although the higher aging temperatures do increase the rate of enzymatic cleavage and accelerate tenderization, in practice, however, nearly all aging is carried out at cooler temperatures. Aging or conditioning of meat is not a new process and has been utilized for many years (Lehmann, 1907; Hoagland et al., 1917). Nevertheless, the exact mechanism(s) of tenderization and improvement in flavor during aging of meat is still not understood. As already indicated the changes probably are due to enzymatic hydrolysis (Whitaker, 1959; Balls, 1960), but they are far from clear (Dutson and Pearson, 1985). It has been difficult, in fact, to show that any major chemical changes occur during meat aging (Bate-Smith, 1948; Whitaker, 1959; Bodwell and Pearson, 1964). Locker (1960b) followed changes in the amino-terminal amino acids in beef muscle from slaughter through 16 days postmortem but found little evidence of proteolysis, while Bandack-Yuri and Rose (1961) obtained similar results on aging of chicken muscle. Nevertheless, meat improves in tenderness during aging, and there is circumstantial evidence that the increase in tenderness is related to enzymatic cleavage of the muscle proteins (Dutson and Pearson, 1985). 1. Rol e of Muscl e Protease s The indigenous proteases in skeletal muscle that may play a role in meat tenderization include three

420

13. Postmortem Conversion of Muscle to Meat

types grouped by their optimum pH as follows: (1) the alkaline proteases, (2) the neutral protease(s) that is activated by C a , and (3) the cathepsins or acid proteases (Obinata et al, 1981). Table 13-9 summarizes the major muscle proteases from the standpoint of their localization, muscle source, optimal pH, and substrates. Muscle contains a number of proteases that are capable of degrading muscle proteins and their breakdown products. It is significant that some of these proteases have optimal activity at high pH values, such as alkaline protease (8.5-9.0), muscle alkaline protease (MAP) (9.5-10.5), and serine protease (8.0-9.0). Lochner et al. (1980) presented evidence that slow chilling during the early postmortem period improved beef tenderness, perhaps by enhancing the activity of the alkaline and neutral proteases in muscle. Further support for this concept is also found in a subsequent study by Marsh et al. (1981), which suggests that tenderization will occur in muscle at near neutral pH while the temperature is still relatively high. Petaja et al. (1985) also showed that high temperature conditioning (37 or 42°C) for relatively short times (4 or 6 hours) materially improved meat tenderness over holding at temperatures below 10°C. Thus, there is indirect evidence that indigenous proteases are active while muscle pH and temperature are still high, which could involve both the alkaline and neutral proteases (Pearson et al., 1983). It seems likely that the role of the alkaline proteases is of minor importance since muscle pH soon drops below 7.0, but the C a activated protease (CAF) would retain considerable activity in muscle even after the pH dropped below neutral. 2+

2+

Until recently it was difficult to reconcile the fact that CAF may play a role in meat tenderness with its high C a requirement (millimolar concentrations) for activation. Under normal conditions, C a concentrations in muscle do not reach the level (1-5 mM) needed for activation of CAF (Dayton et al., 1976a,b). However, the discovery of a low-Ca -requiring form (micromolar concentrations) of CAF (Dayton et al., 1981; Goll et al., 1983) has overcome this problem, and it has been suggested that CAF could, indeed, be involved in meat tenderness. Subsequently, Koohmaraie et al. (1986) have shown that the micromolar CAF is 2+

2+

2+

probably responsible for at least part of the improvement in meat tenderness taking place during postmortem aging. There is also evidence that CAF degrades meat by removing the Z-lines in a manner similar to that occurring during aging (Dayton et al., 1976b). As the pH decreases and the muscle becomes more acid, the cathepsins (A, B, C, D, and L) may become active and cause further degradation of the protein fraction (Table 13-9). Cathepsins B, D, and L are all capable of hydrolyzing the myofibrillar proteins and/or their breakdown products so may play important roles in the meat aging process. Since aging continues even after the pH and temperature decrease, it seems probable that the cathepsins and the lysosomal enzymes play important roles in meat tenderization. These enzymatic changes are also believed to release components contributing to the full flavor of aged meat. Some of the protein breakdown products and their role in meat flavor development are discussed by Pearson et al. (1983). Bird et al. (1980) have pointed out that of all the indigenous muscle proteinases, CAF and cathepsins  and D are most likely to be involved in degradation of the myofibrillar proteins. Dutson (1983) has presented evidence showing that increasing the postmortem temperature during storage improves meat tenderness, apparently by disruption of troponin T, myosin, Z-lines, titin (connectin), and gap filaments. High pH values increase the activity of CAF, but low pH enhances the activity of the lysosomal cathepsins and increases the rate of hydrolysis of troponin T, titin, and, especially, myosin. These studies indicate that a number of enzymes with different optimal pHs may play a role in meat tenderness. There are also believed to be alterations in the connective tissue proteins, namely, collagen and possibly the ground substance, during the aging of meat that result in improvement of meat tenderness. Cleavage of the reducible and nonreducible cross-links of collagen and hydrolysis of the various glycosaminoglycans or of the microfibrillar components of elastin may also be involved in the improved tenderness of aged meat. These relationships and interrelationships are virtually unexplored and need to be investigated.

Lysosome

h

Rat liver

6-7

Rat skeletal Rat skeletal

Myosin

8.5-9.0 9.5-10.5

Muscle Source pH

Substrate

Kirschke et al. (1980)

Muscle proteins, hemoglobin Muscle proteins, casein

Optimal Reference

Abbreviations : MHC , myosin heav y chain ; LC-DTNB , light chai n solubilize d by 5,5'-dithiobis(2-nitrobenzoi c acid) ; TN, troponin ; TM , tropomyosin .

" Fro m Pearso n et al. (1983).

Cathepsin Ç

— —

Localization

Koszalka and Miller (1960a,b) Noguchi and Kandatsu (1970, 1971) Serine protease Mast cells Rat and rabbit 8.0-9.0 MHC, LC-DTNB, TN-T, TN-I, Yasogawa et al. (1978), skeletal TM, actin, titin Maruyama et al. (1981) 7.0-7.5 TN-T, TN-I, TM, Z-disks, Dayton et al. (1976a,b), Calcium-activated factor (CAF) — Chicken skeletal Ishiura et al. (1979) Cytoplasm Rabbit skeletal M-protein, C-protein, also Myofibril Rabbit skeletal á-actinin , MHC 5.0-5.4 Synthetic peptides Iodice et al. (1966) Cathepsin A Lysosome Chick skeletal Cathepsin  Lysosome Rat skeletal 5.2 Myosin, actin Schwartz and Bird (1977) Iodice et al. (1966) Cathepsin C Lysosome Chick skeletal 5-7 Derivatives of dipeptides Cathepsin D Lysosome Rat skeletal 4.0 Myosin, actin, hemoglobin Schwartz and Bird (1977) Cathepsin L Lysosome Rabbit skeletal 4.2 MHC Matsukura et al. (1981) 4.7 Actin 3.0-3.5 a-Actinin 3.7-6.7 TN-T and TN-I

Protease

Some Indigenous Proteases in Skeletal Muscle"^

Alkaline protease Muscle alkaline protease (MAP)

Tabl e 13-9

422

13. Postmortem Conversion of Muscle to Meat

2. Resolutio n of Rigo r Tenderization of meat during aging would appear to depend on enzymatic cleavage of the muscle proteins or else on resolution of rigor mortis. The role of enzymes in tenderization during aging of meat was discussed in the previous section. The question as to the resolution of rigor and its possible contribution to tenderness has largely been ignored since Bendall (1960) concluded that breaking of the actin-myosin linkages of rigor would require ATP, which is low or absent in postrigor muscle. Nevertheless, Erdos (1943) had already shown that the stiffness associated with rigor reaches a maximum at completion and then gradually declines. Other evidence for the resolution of rigor can be seen in the lengthening of sarcomeres that is evident by phase-contrast microscopy of chicken skeletal muscle (Takahashi et al., 1967) and from electron micrographs of thin sections of bovine muscle (Stromer et al., 1967). More recently, Takahashi et al. (1987) have demonstrated that calcium ions cause weakening of Z-disks during the aging of muscle, which is commonly associated with an increase in tenderness of aged beef. The postmortem weakening of the Zdisks was shown to be highly dependent on pH, reaching a minimum at pH 6.5. Addition of C a had little or no effect on hydrolysis of a-actinin during postmortem storage of muscle for 10 days at 10°C, which suggests that the weakening is unrelated to enzymatic hydrolysis. Addition of ÉÏ" Ì C a caused splitting of the Z-disks in the same way as treatment with 0.1 Í NaOH for 5 minutes. This suggests that C a solubilizes the amorphous material of the Z-disks but does not affect á-actinin , leaving two sets of Z-disks. These findings offer evidence that C a is in some way involved in weakening of the Z-disks and may contribute to the lengthening of the sarcomeres during meat aging. The discovery of a new protein called paratropomyosin by Takahashi et al. (1985a,b) appears to be the link required to explain the lengthening of sarcomeres during aging of meat and indicates that rigor is indeed resolved during meat aging. The molecular weight, amino acid composition, and properties of paratropomyosin are covered in greater detail in Chapter 5. Hattori and Takahashi (1988) have shown that paratropomyosin is translocated 2+

4

2+

from its original position at the A - I junction region of the sarcomere onto the thin filaments at a sarcoplasmic C a concentration of É Ï Ì and weakens the rigor linkages between actin and myosin. Yamanoue and Takahashi (1988) later showed that the translocation of paratropomyosin to rigorshortened sarcomeres by 10" C a resulted in lengthening of the sarcomeres. Paratropomyosin appears to actually participate in the resolution of rigor without any requirement for ATP. The proposed scenario for the action of paratropomyosin in the resolution of rigor mortis is as follows. In living and in prerigor muscle, paratropomyosin is bound at the A - I junction where it serves an apparently unknown function. After death and development of rigor mortis, the sarcoplasmic C a concentration increases to about 10" Ì owing to the inability of the membranes in the sarcoplasmic reticulum and mitochondria to accumulate C a . The increased C a concentration within the myofibrils results in the release of paratropomyosin from the A - I junction, and the protein then competes with myosin for the binding sites on actin, where it appears to be preferentially bound. The disengagement of some of the myosin-actin linkages is hence responsible for resolution of rigor without any requirement for ATP. Thus, aging of meat may actually result in resolution of rigor mortis and thereby contribute to the improvement in tenderness associated with meat aging. Nevertheless, it is doubtful if any single mechanism is responsible for all of the increase in tenderness that occurs during aging. 2+

-4

4

2+

2+

2+

4

2+

2+

2+

D . WATE R HOLDIN G CAPACIT Y OF MUSCL E Skeletal muscle contains about 75% water, with myofibrillar proteins playing a dominant role in water binding. The term water holding capacity (WHC) is used to describe the binding of water by muscle (Hamm, 1986). The tenacity with which muscle binds water differs from the immobilized or bound water to the free or unbound water. The immobilized or entrapped water is bound in varying degrees, however, with part of the water being bound so tightly that it can only be driven off by heating at 100°C for several hours. The forces re

IV. Some Conditions Occurring in Muscle/Meat

stricting the mobility of water in muscle are poorly understood but are apparently determined by the spatial arrangement of the muscle proteins. Myosin is known to have an important function in binding of water, which is closely related to its capacity to imbibe water (Szent-Gyorgyi, 1973). Offer and Trinick (1983) have explained WHC on the basis of the swelling and shrinking of the myofibrils that is associated with expansion or contraction of the filament lattice. 1. Effec t of p H on Wate r Holdin g Capacit y Water holding capacity is at a minimum around pH 5.0, which corresponds to the isoelectric point (p/) of myosin and actomyosin (Hamm, 1986). At pH 5.0, the net charge of the myosin and actomyosin molecules are at a minimum so that WHC is at its lowest. In a pH range of 5.0-6.5, the area of practical interest in meat, any alteration of pH has a great influence on the WHC. The change is related to the ionization states of histidine and, to lesser extent, glutamic acid (Hamm, 1986). The changes in WHC in the pH range of 5.0-6.5 are completely reversible, whereas at a pH above 10 or below 4.5 irreversible changes occur (Hamm, 1986). Ô—é—é—é—é—é— é—é—é—é

1 2

3 U 5 6 7 8 9 10

423

Muscle swelling and pH are closely related. The graphs in Fig. 13-12 illustrate that WHC and swelling of beef muscle are both minimal near pH 5. Raising the net charge of the protein by adding acid or base shifts the pH away from the p / of myosin and actomyosin, thus increasing the WHC and swelling of muscle by increasing the interfilament spacing. Differences in the WHC of meat among individual animals of the same species are usually closely related to muscle pH. WHC increases as the pH increases, providing the pH differs more than the range of 5.5-5.8. Water binding in sausage emulsions has been shown to increase directly with pH.

2. Effec t of Divalen t Cation s on Wate r Bindin g 2+

2+

Divalent cations, principally C a and M g , are bound in at least three ways: (1) by very tight bonds that are stable at the pi of myosin (pH 5); (2) by pHdependent bonds that are relatively strong at neutral pH (pH 7) but weaken as the pH declines; and (3) by loose electrostatic binding to negatively charged proteins (Hamm, 1986). Ions bound by 1

é

4. 0

pH

1

5.0

1

1

6.0

1

1

7.0

1

Ã

8.0

pH

Figur e 13-12 Influence of pH on water holding capacity (A) and swelling (B) of postrigor beef muscle. WHC was determined on a homogenate by the filter press method, and swelling was measured on 3-mm muscle cubes. From Hamm (1986). 3

424

13. P o s t m o r t e m C o n v e r s i o n o f M u s c l e t o M e a t

electrostatic bonds are usually exchangeable with other cations or extractable by water. The release of C a and M g by muscle during and immediately after the onset of rigor (48 hours postmortem) is the result of the postmortem pH decline, whereas the same ions bound in a pH-independent manner to the myofibrils are not released as the pH drops. Divalent cations such as C a and M g thus lower the WHC of meat by reducing the electrostatic repulsion between negatively charged groups. This tightens the muscle structure, causing shrinkage. The effect of magnesium salts on WHC is much stronger than that of sodium salts of the same ionic strength. This is because M g is bound more strongly by the myofibrillar proteins. It is also probable that divalent cations participate in proteinprotein interactions after depletion of ATP. With NaCl and MgCl , shrinkage does not occur because the chloride ions superimpose the effect of cations on WHC. On the other hand, removal of divalent cations by exchange resins or other means increases WHC. The effects of divalent cations are minimal at the pi of myosin (pH 5) but increase as the pH increases because the strength of cation binding to the myofibrillar proteins is increasing. Thus, divalent cations in muscle reduce its WHC, but sequestering or exchange of these cations increases WHC. 2+

2+

2+

2+

2+

2

3. Effect s of Earl y Postmorte m Change s on W H C of Muscl e Development of rigor mortis in bovine and porcine muscle has little effect on the WHC at the normal glycogen and ATP content, although cooking losses and expressible fluids increase slightly and continuously as the pH declines. The reason rigor does not significantly affect WHC is because as the pH declines from 7 to 5.9 (the pH of rigor) salt crosslinkages are formed with myofibrillar proteins so that the cross-linking of myosin and actin has little further effect (Hamm, 1986). Neither cold shortening nor rigor contracture at high temperature (30°C) exert any significant effect on WHC during the first 24-48 hours postmortem since there is no apparent change in muscle volume. On cooking of prerigor meat, extensive contracture occurs, but cooking losses are less than in postrigor meat (Paul et al, 1952; Cia and Marsh, 1976; Cross and Tennent, 1980; Ray et al, 1980).

Both the higher pH and the higher ATP content apparently enhance fluid retention and appear to counteract the effects of shortening. 4. Influenc e of Prerigo r Saltin g on W H C Salting of prerigor meat maintains the high WHC for several days during refrigerated storage. This " hot" salted meat has superior WHC and fat-binding characteristics in sausages although salt accelerates the breakdown of ATP (Hamm, 1977). The high WHC of hot salted meat can be explained by inhibition of rigor mortis in the fiber fragments resulting from the combined effects of high pH and salt concentration before the ATP becomes depleted. Thus, it is important that the salt be mixed quickly and thoroughly with the meat while still in the early prerigor state. The enhancement of WHC increases with salt concentration up to about 1.8%, but higher concentrations apparently cause no significant improvement in emulsion stability (Hamm, 1986). Prerigor salting of meat results in increased solubilization of the myofibrillar proteins, but presalting does not appear to irreversibly protect the proteins against loss of solubility. Although prerigor salted meat suffers from loss of myofibrillar protein solubility to the same extent as postrigor salted meat, its high WHC remains unchanged. Salted prerigor meat also maintains a high WHC during freezing and thawing. 4

E . D A R K CUTTIN G BEE F The term "dark cutter" is commonly used to describe the lean meat of beef carcasses that fails to take on the characteristic bright cherry-red color on exposure of the cut surfaces of the carcass (most often the LD muscle) to oxygen (air). The color varies from slightly dark or shady to extremely dark or nearly black depending on the severity of the condition. The dark color results in difficulty in marketing with a resultant decline in economic value. Price differentials are not commonly quoted, but on average it has been estimated that dark cutters are penalized from one-third to a full USDA carcass grade. The problem, however, is not that simple since the condition is not apparent until the carcasses are broken. This means that retailers may reject the meat and return it to the packer. As dark cutting beef cannot be identified in the live animal,

I V . S o m e C o n d i t i o n s O c c u r r i n g in M u s c l e / M e a t

the economic losses from dark cutters must be spread over all animals slaughtered, resulting in lower prices for bright cutters than is actually warranted. Aside from the economic disadvantages of dark cutting beef, the meat has a closed texture and has been reported to have an objectionable soapy taste (J. F. Price, personal communication). Even more importantly, dark cutting meat is more susceptible to microbial spoilage. Although dark cutting beef has been reported by Dransfield (1981) to be more tender than normal beef, the increase in problems from increased bacterial growth and spoilage outweigh any advantages from greater tenderness. The greater susceptibility of dark cutters to bacterial spoilage has been reviewed by Gill and Newton (1981) and by Patterson and Bolton (1981). 1. Incidenc e of Dar k Cutter s The incidence of dark cutting beef carcasses has been reported to vary from less than 1% to over 12% of all carcasses (National Live Stock and Meat Board, 1949; Munns and Burrell, 1966). Tarrant (1981) reported that dark cutting beef was a significant problem, with some 1-5% of steers and heifers, 6-10% of cows, and 11-15% of all young bulls being dark cutters. The incidence of dark cutters may be even higher under certain conditions, with LaVoi (1939) reporting that some 18% of all 4-H Club steers slaughtered at the 1938 International Livestock Exposition were dark cutters. A study on the slaughter of young bulls in Finland found that the incidence of dark cutters varied from 18 to 40% in unrested animals, while short resting times (2-4 hours) decreased the frequency, which increased again on longer holding times (Puolanne and Aalto, 1981). The percentage of dark cutting beef has also been shown to be less when adequate feed is provided and stress is at a minimum (Munns and Burrell, 1966). 2. Basi s of Dar k Cuttin g Mea t LaVoi (1939) reported that 18% of the dark cutters at the 4-H Club show referred to earlier had a nervous disposition, which suggested there may be a relationship between stress susceptibility and dark cutting beef. Mackintosh and Hall (1935) had earlier observed that dark cutting beef had a high pH

425

and failed to brighten on exposure to air. Hall et al. (1944) demonstrated that dark cutting beef not only has a high pH but also has a low oxidation-reduction potential with a lower oxygen uptake than is found for normal muscle. It was shown that dark cutting beef is characterized by low glycogen reserves and low reducing sugar levels (Hall et al., 1944). Studies by the National Live Stock and Meat Board (Romans et al., 1985) revealed that the water-extractable sugar content of normal bright colored beef muscle was 0.18%, whereas shady beef contained 0.11% and black cutting muscle contained only 0.03%. These values resulted in corresponding average pH readings of 5.58, 5.68, and 6.53, respectively. The incidence of dark cutters was reported to be higher in the autumn and winter (September to January, inclusive) and in the spring of the year (March and April) than in the summer months (Tarrant, 1981). The same general seasonal pattern of the incidence of dark cutters has also been observed in Australia and New Zealand. These peak periods indicate that cold, damp weather and lack of feed and/or shelter are contributing causes to dark cutting beef (National Live Stock and Meat Board, 1949). 3. Preventio n of Dar k Cuttin g Beef The relationship between stress and dark cutting beef was further verified in an experiment by Romans et al. (1974), who found that 4 out of 8 steers cut dark when fasted for 2 days and heavily exercised during the final 10 hours of their fast at temperatures near freezing. These results agree with a report by Hedrick et al. (1959) which showed that dark cutting beef is due to the depletion of glycogen. Prevention of dark cutting beef, thus, can best be achieved by providing feed and water when temperatures are low and the animals exposed to wet and cold weather. The National Live Stock and Meat Board (1949) concluded that the incidence of dark cutting beef can be minimized by avoiding chilling and providing good housing and adequate feed and water during the period between purchase and slaughter. Minimizing stress by avoiding mixing of strange animals, especially young bulls, also can materially reduce dark cutting beef (Puolanne and Aalto, 1981). Thus, the incidence of dark cutting beef can

426

13. Postmortem Conversion of Muscle to Meat

be greatly reduced by good management and proper handling of animals prior to slaughter. F . P A L E , SOFT , A N D E X U D A T I V E POR K 1. Th e Proble m of PS E Por k Pale, soft, and exudative (PSE) pork is characterized by being unusually light or pale in color and by having soft and watery lean along with an open structure (Briskey, 1964). The exudate renders the meat unattractive and accumulates as excess fluid in prepackaged fresh chops and roasts. Evidence suggests that selection of hogs for meatiness has also contributed to the increase in the frequency of PSE pork. It is not clear whether the increased incidence of PSE pork is due to a direct relationship between the condition and meatiness or if the association occurred by chance selection of carrier animals for breeding purposes. Although PSE muscle also occasionally occurs in beef, the incidence is much lower than in pigs and is not considered to be a serious problem (Hunt and Hedrick, 1977). The cause is probably related to stress, although this has not been fully established. The PSE condition in the pig was first described by Ludvigsen (1954), although he described the condition as being muscle degeneration. It has been reported to occur in 5-20% of all pig carcasses (Briskey et al, 1959), although its incidence may be greater in certain groups of stress-susceptible pigs. PSE muscle is closely related to the porcine stress syndrome and to dark, firm, and dry (DFD) pork (Gronert, 1980), which are discussed later. Although PSE pork suffers greater in-package shrinkage and has an unsightly, objectionable appearance, it has a more open structure and will take up curing salts more readily. It has also been shown that in addition to the common objections about the appearance, PSE pork undergoes oxidation more readily. The rapid drop in pH, however, has an added benefit in that it retards microbial growth. In fact, bacteria grow poorly on PSE pork; thus, the incidence of spoilage is less for PSE pork. 2. Basi c Cause s of PS E Muscl e The PSE conditions in pork result from an extremely rapid rate of glycolysis postmortem,

which causes the pH to drop to low levels while muscle temperature is still high (Briskey and Wismer-Pedersen, 1961). The rapid pH fall results in precipitation of the sarcoplasmic proteins on the myofibrils and makes uptake of the muscle exudate difficult (Bendall et al, 1963). The early postmortem rate of pH decline is about twofold faster for PSE muscle than for normal muscle, amounting to about 1.04 pH units decline per hour compared to 0.65 units/hour for normal muscle when held at 37°C (Bendall et al., 1963). In some cases, the ultimate muscle pH may be reached as early as 15 minutes postmortem while the temperature is still around 37°C. Table 13-10 presents a comparison between the metabolite concentrations in PSE and normal muscle (Greaser, 1986). The data show that even soon after death the glycogen concentration is much lower in PSE than in normal muscle. On the other hand, the lactate concentrations in PSE muscle are nearly double that of normal muscle, even as early as 15 minutes postmortem. Both ATP and CP concentrations are lower in early postmortem PSE muscle and by 1 hour are virtually depleted (Greaser, 1986). Hexose monophosphate levels tend to be higher in normal than in PSE muscles during the first 3 hours postmortem (Fischer and Augustine, 1977). The time required for completion of rigor is greatly reduced in PSE as compared to normal muscle and is closely related to the depletion patterns for ATP (Bendall et al, 1963; Forrest et al, 1966). Closely related to the increased glycolytic rate of PSE muscle is the loss of myofibrillar ATPase activity with postmortem time (Greaser, 1986). Myosin-ATPase activity is reduced by twofold in PSE as compared to normal muscle, and PSE myofibrils fail to contract on adding MgATP (Sung et al, 1977). Actomyosin from PSE muscle does not superprecipitate as fast as that from normal muscle, and it also appears to have a reduced troponin function (Park et al, 1975, 1977). PSE myofibrils also have been shown to contain a higher proportion of a 165,000-dalton band on SDS-polyacrylamide gels than normal myofibrils (Park et al, 1975), which is thought to be a degradation product of myosin heavy chain. There is also more saltsoluble and more heat-labile collagen in PSE than in normal muscle at 48 hours postmortem (McClain et al, 1967), although the rapid drop in pH is proba-

IV. Some Conditions Occurring in Muscle/Meat

427

Tabl e 13-10 Postmortem Concentrations of Some Glycolytic Metabolites in Normal and PSE Pork* Time Postmortem (min)

Metabolite Glycogen

c

c

Glucose

Glucose 6-phosphate Lactate

d

c

Creatine phosphate ATP

c

c

b

PSE (ì,ðéïÀ/g)*

3 180 3 180

35-100 20 2.3 4.3

23 0.8 3.3 6.8

3 60 180 3 60 180 3 60 180 3 60 180

4.5 5.0 6.5 30-40 40-60 60-80 6.0 3.0 2.0 5.5 4.5 2.5

8.5 7.0 7.5 -60 105 105 3.0 1.0 1.0 3.5 <0.5 <0.5

a

F r o m Grease r (1986).

b

Fo r g l y c o g e n , ìðéï ß g l u c o s e e q u i v / g .

c

Dat a fro m Kastenschmid t et al. (1968).

d

Dat a fro m Kastenschmid t (1970).

bly responsible for the increase in solubility of the collagen from PSE muscle. The SR ATPase activity increases over postmortem time in normal muscle but decreases in PSE muscle preparations (Greaser et al., 1969a). Calcium uptake by the SR decreases much more rapidly in PSE muscle, although there is no significant difference in Ca -binding activity between PSE and normal muscle immediately postmortem (Greaser et al., 1969a,c,d). It is possible that a reduced Ca -binding activity by the SR may trigger the rapid ATP turnover in PSE muscle. Mitochondria from PSE-susceptible muscle have been suggested to accelerate glycolysis and cause PSE muscle. The mitochondria isolated from muscle of stress-susceptible pigs have about twice the C a efflux rate as similarly isolated mitochondria from normal muscle (Cheah and Cheah, 1976, 1979). The increase in C a may activate phospholipase, which can then release fatty acids from the mitochondrial membranes (Cheah and Cheah, 1981a,b). The fatty acids may in turn inactivate the SR (Cheah, 1981), which could result in an increased 2+

2+

2+

2+

Normal famo\/g)

2+

concentration of cytosolic C a and thus increase the activity of the myofibrillar ATPase. It is not clear at this time how phospholipase would be controlled in vivo, so if this mechanism is operative in the live pig some other factor(s) must be involved in changing the mitochondrial efflux. PSE muscle responds to electrical stimulation more quickly than normal pig muscle, which is manifested by faster glycolytic rates, higher excitability thresholds, and lower contraction strengths by 10 minutes following death (Forrest and Briskey, 1967). The resting membrane potential of muscle with a rapid glycolytic rate was 21-27 versus 38-62 mV for normal muscle at 45 minutes postmortem (Schmidt et al., 1972). Membrane capacitance and resistance decline more rapidly postmortem in PSE muscle (Swatland, 1980, 1981). Gallant et al. (1979) have also shown that halothane anesthesia, which has been demonstrated to trigger malignant hyperthermia, lowers the resting potential in stress-susceptible pig muscle from -84.9 to —75.3 mV but causes essentially no change in normal muscle (-83.6 to -82.8 mV).

428

13. Postmortem Conversion of Muscle to Meat

Dense, irregular bands, as viewed under the microscope, appear more frequently in PSE than in normal pig muscle (Bendall and Wismer-Pedersen, 1962). Although these bands were originally thought to be due to precipitation of the sarcoplasmic proteins, Cassens et al. (1963a,b) demonstrated them to be contracture bands. Malignant hyperthermia-susceptible animals exhibit such contracture bands in 30.1% of the fibers in biopsy samples as compared to only 7.65% in normal animals (Palmer et al., 1977). The sarcoplasmic space between myofibrils develops earlier in PSE muscle (Cassens et al., 1963b), and I-band clumping and Zline disruption are more common than in normal pig muscle (Dutson et al., 1974). Mitochondria and SR also show signs of disruption in low quality porcine (PSE) muscle as early as 15 minutes postmortem (Dutson et al., 1974). PSE muscle fibers have a more granular appearance under the electron microscope at 24 hours postmortem (Cassens et al., 1963b; Greaser et al., 1969b), which may be due to an accumulation of sarcoplasmic proteins as sug~ gested by Bendall and Wismer-Pedersen (1962) or to the denaturation of the myofibrillar proteins as proposed by Greaser (1986). The protein solubility of PSE muscle declines more than is the case for normal muscle; PSE muscles show a rapid pH drop while muscle temperature is still high, having only about 50-75% as much soluble sarcoplasmic and myofibrillar protein, respectively, as normal pig muscle (Bendall and Wismer-Pedersen, 1962; Park et al., 1975). Creatine kinase is the sarcoplasmic protein that suffers most from denaturation (Scopes and Lawrie, 1963). The amount of phosphorylase in the sarcoplasm of PSE muscle declines by 45-60 minutes postmortem and increases in the myofibrillar fraction (Fischer et al., 1977). There does not appear to be any change in myoglobin solubility of PSE muscle (Charpenter, 1969), which indicates that the pale color results from light-colored precipitates of the sarcoplasmic proteins masking the usual red color (Goldspink and McLoughlin, 1964).

3. Preventio n of PS E Muscl e There is not any one completely acceptable method for eliminating PSE pork from stress-susceptible pigs, although minimization of stress before and

during slaughter may reduce its incidence. Even with careful management and handling, some carcasses from stress-susceptible pigs will suffer from the PSE condition. Feeding and/or short-term resting of the animals will aid in reducing the frequency of PSE pork but not eliminate it (Monin et al., 1981). The only completely successful procedure for eliminating the PSE condition is by genetic selection for stress-resistant breeding animals. Use of the halothane test can serve to screen breeding animals and has been shown to be successful, even with weanling pigs (Monin et al., 1981). The halothane test is based on the ability of the pig to recover from halothane anesthesia. Those pigs recovering quickly are stress resistant, while those that metabolize halothane more slowly and require longer periods of time to recover consciousness are classified as stress susceptible or PSE susceptible and should not be used for breeding purposes. Webb (1980) presented data showing that selection of breeding swine over four generations using the halothane test reduced the incidence of not only PSE but also PSS (porcine stress syndrome, a condition characterized by extreme stress susceptibility that commonly results in death during normal production and marketing) and DFD (dark, firm, and dry) pork. On the other hand, Monin et al. (1981) has suggested that the halothane test is useful for identifying animals that are susceptible to the PSE and PSS conditions but not for DFD pork.

G . D A R K , FIRM , A N D D R Y P O R K 1. Natur e of D F D Por k Dark, firm, and dry (DFD) pork is characterized by being dark in color, firm in texture, and dry to the touch. It is essentially the same condition in the pig as dark cutting in beef, being associated with low muscle glycogen reserves at the time of slaughter. The meat feels sticky to the touch, has a closed structure, and has a high pH. It does not absorb curing salts readily and, hence, is more susceptible to bacterial spoilage (Newton and Gill, 1981). It has been pointed out by Newton and Gill (1981) that spoilage of DFD meat occurs at much lower cell densities than is the case for meat of normal pH. Results of their studies suggest that the absence of glucose at the surface of DFD meat allows the

IV .

spoilage microflora to attack and degrade the amino acids and to form potently odorous compounds earlier in the spoilage process. Callow (1936) observed a greater frequency of spoilage during curing of DFD hams. The high pH was shown to be correlated with an increase in electrical resistance. Bate-Smith (1948) concluded that the high pH was also responsible for a closed structure, which resulted in slower penetration of the curing pickle and thus favored microbial spoilage. DFD meat, therefore, suffers from a higher incidence of spoilage not only in fresh meats but also in cured products. The problem of high pH or DFD pork has been a major consideration in production of Wiltshire sides, where the salt concentration may be high enough to prevent true microbial spoilage but produces a fiery color, sticky consistency, and tainted (spoiled) flavor, which is called ''glazy bacon" (Lawrie, 1979). DFD pork is not only subject to greater problems arising from spoilage than normal and PSE pork, but it also is less attractive in appearance and is objectionable to consumers. Unfortunately, some of the research on PSE pork utilized DFD pork instead of normal pork as controls and, thus, may be confusing. Problems created by DFD pork are probably more serious than those from PSE muscle because spoilage is a more serious problem than the light color and accumulation of exudate. 2. Preventio n of D F D Por k The incidence of DFD pork can be decreased by minimizing the amount of stress during marketing and slaughtering. Practices such as feeding, resting, avoiding mixing of strange animals, and other good management procedures, which avoid stress and maintain glycogen reserves at the time of death, will decrease the amount of DFD pork. Although genetic selection against DFD pork has been suggested for reducing its incidence, Monin et al. (1981) has concluded that DFD and PSE pork are triggered by different mechanisms. Although halothane sensitivity can be used to select against PSE muscle, this may not be true for DFD pork. On the other hand, if the DFD condition is only a more severe manifestation of stress susceptibility, the halothane anesthesia selection procedure should be useful in decreasing or eliminating DFD pork as suggested by Monin et al. (1981).

S o m e C o n d i t i o n s O c c u r r i n g in M u s c l e / M e a t

429

H . P O R C I N E STRES S S Y N D R O M E I . Natur e of PS S Although the porcine stress syndrome (PSS) condition is not normally a problem in pork carcasses since the pigs usually succumb during handling on the farm or in marketing, the condition is closely related to the PSE and DFD conditions and so is given brief consideration here. Some pigs suffering from this condition, no doubt, survive and produce meat with abnormal properties, which are described below. Pigs suffering from PSS are usually extremely stress susceptible and generally heavily muscled. Bicknell (1968) has reviewed the symptoms of this condition as well as the appearance of the muscle. An excellent discussion on the interrelationships of the hormones involved and their effects on anaerobic breakdown of glycogen and formation of lactic acid and its metabolism (see below) is given by Forrest et al. (1975).

2. Mechanis m of an d Characteristic s of PS S Muscl e Since PSS muscle is due to extreme stress susceptibility, its incidence is closely associated with the release of epinephrine and norepinephrine from the adrenal medulla, of adrenal steroids from the cortex of the adrenal gland, and of thyroid hormones from the thyroid gland. Epinephrine and norepinephrine stimulate glycogen breakdown in both liver and muscle to release glucose and lactate, respectively, which is brought about by cyclic AMP binding to protein kinase receptors to yield the active phosphorylase kinase that in the presence of C a catalyzes phosphorylation of phosphorylase b to the a (active) form. Phosphorylase a speeds up the transformation of glycogen to glucose 1-phosphate, which successively forms glucose 6-phosphate and glucose or lactate as the case may be. The catecholamines also stimulate a lipase in fat cells to catalyze the breakdown of triacylglycerols (triglycerides) to yield free fatty acids that in turn are bound to serum albumin and transported in the blood. The catecholamines also accelerate heart rate and increase blood output while simultaneously increasing smooth muscle tone, thereby raising blood pressure (Lehninger, 1975). Glycogen 2+

430

13. Postmortem Conversion of Muscle to Meat

in heart muscle, however, is not converted to blood glucose but into lactate. Both heart and skeletal muscle lack glucose-6-phosphatase so cannot form glucose. In summary, the catecholamines not only increase heart output and blood pressure but also mobilize glucose and free fatty acids in the blood where they are readily available. The hormones from the adrenal cortex assist the body in responding to stress through their role in synthesis of carbohydrates and in maintaining ionic balance in the tissues. The thyroid hormones, especially thyroxine, increase metabolic rate and make more energy available in times of stress. When stressful conditions occur, aerobic metabolism may not be adequate to meet the needs of the muscles so that anaerobic metabolism is recruited to help meet demands. This results in formation of lactic acid, which is favored by the action of epinephrine, and causes the buildup of tissue acidity. Lactic acid cannot be metabolized by white muscle fibers and so will continue to increase unless it is transported to the liver, where it can be reconverted to glycogen, or to the heart, which can utilize it directly. Failure of the muscle system to reduce tissue temperatures or to minimize acidity appears to be a contributing factor to the PSS condition and if uncontrolled can lead to death owing to generalized acidosis and/or abnormally high body temperatures. The PSS condition may be manifested in the live pig by muscle spasms and tail tremors. Necropsies usually reveal congestion of the parenchymatous organs, with the heart stopping in systole and almost no visible lumen to the left ventricle. The musculature of the heart often exhibits pale streaks, especially in the left ventricle. The LD, gluteus medius, and semimembranosus muscles are usually pale and edematous. The muscle is usually grayish to white in color and has been described by Ludvigsen (1954) as resembling chicken breast muscle. The muscles are extremely watery, have a sour odor, and the muscle bundles can be readily separated. Other muscles tend to have normal color, giving the carcass a two-toned appearance. The LD muscle is easily separated from the surrounding connective tissues and in some cases has lost all of its connections. Similar muscle qualities may sometimes be found in meat from affected pigs surviving until slaughter.

3 . Preventio n of PS S Muscl e The immediate solution to PSS muscle is to eliminate all stress, which is virtually impossible. The stress of moving short distances or even of high environmental temperatures will result in death. Fighting on mixing of strange pigs is especially stressful and should be avoided. Careful management can be helpful in alleviating stressful conditions but is at best a temporary and not always successful method. The condition appears to be genetic, however, and can be reduced by selection. Perhaps the fastest progress toward elimination of PSS can be made by using halothane anesthesia as a selection tool (Monin et al., 1981).

I . D O U B L E M U S C L I N G I N CATTL E "Double-muscled" or "doppelender" cattle have unusually thick bulging muscles, especially in the round. The condition appears to be genetic and occurs quite frequently in some breeds or strains. The term double muscling, however, is misleading as double-muscled cattle have the same number of muscles as normal-muscled cattle (Forrest et al., 1975). Double muscling in cattle is due to muscle hypertrophy, which is accompanied by both larger muscle fibers and a greater number of fibers. The increased size of the fibers is at least in part the result of a higher proportion of white to red fibers, since white fibers are larger in size as explained in Chapter 9. Double muscling appears to be of genetic origin, although the exact mode of inheritance is not fully understood as its phenotypic expression is quite variable. In comparing double-muscled and normal cattle, development of muscle and growth rate are greater but fat deposition is less in the double-muscled animals. The muscle to bone ratio is greater for the doppelender, which means that double-muscled carcasses yield a greater proportion of lean to fat than normal cattle. The meat from double-muscled animals is at least equally as and perhaps slightly more tender than that from normal animals. The fat from double-muscled animals has been reported to be more highly unsaturated versus normal animals. There has been some interest in the production of double-muscled cattle because of their greater

V. Postmortem Processing Characteristics

amount of muscling and leanness. A similar condition in the turkey has been utilized in production of the Beltsville White Broad-breasted turkey. There is some evidence, however, that cattle with extremes in conformation may also have lowered fertility and encounter more frequent calving difficulties, which have largely cooled the interest in selection for double muscling in cattle. Although the Broad-breasted turkey is unable to mate normally, this problem has been overcome by using artificial insemination. Thus, it is possible that genetic manipulation in cattle may be used to take advantage of the superior muscling and leanness of doppelender cattle.

V. SOME POSTMORTE M PROCESSIN G CHARACTERISTIC S OF MUSCL E Muscle can be used for processing in either the preor postrigor state, which can markedly affect its processing characteristics, including water binding capacity, stability against oxidation, and color. All of these factors have a marked influence on the acceptability of meat products. The muscle proteins also have important functional properties such as water binding, fat binding, emulsification of fats, stabilization of protein-fat emulsions, and other desirable attributes in meat products. Some of these properties are briefly reviewed.

PROCESSIN G O F PRE - VERSU S POSTRIGO R MUSCL E Although meat is normally used for processing after it has gone into full rigor, prerigor muscle has some distinct advantages over postrigor meat. Among these advantages are (1) a higher water binding capacity, (2) a greater ability to emulsify fat, (3) formation of a more stable emulsion, (4) a more stable red color, and (5) a lower susceptibility to oxidation. Prerigor processing of meat also offers some economic advantages, including more rapid inventory turnover and greater energy savings (Pearson and Tauber, 1984; Henrickson and Asghar, 1985).

431

Heat-Induce d Gelatio n of th e Myofibrilla r Protein s Manufacture of comminuted meat products relies on formation of a functional protein matrix, which is responsible not only for a stable emulsion but also for greater fat emulsification and improvement in water binding. These products also develop a characteristic texture and bite. Gelation in comminuted meat products is related to the physicochemical properties of the myofibrillar proteins, especially those of myosin and/or actomyosin (Yasui et al., 1982). It has been reported that actin, native tropomyosin (a complex of tropomyosin and the troponins), and myosin are important contributors to the viscoelastic characteristics of heated gels of minced vertebrate muscle (Nakayama and Sato, 1971a,b). F-actin has been shown to enhance heatinduced gel formation, which is optimal at a myosin/actin mole ratio of 2.7 where about 15-20% of the total protein is in the actomyosin complex and most of the surplus myosin is in the free form (Yasui et al., 1980; Ishioroshi et al., 1980; Samejima et al., 1982). The optimum weight ratio of myosin to actomyosin for gelation has been shown to be about 4 (Yasui et al., 1982), with the regulatory proteins seemingly playing no role in the heat-induced gelation reaction of actomyosin. The gel-enhancing effects of F-actin on heat gelation appear to be due to its serving as a cross-link between the bound and free myosin molecules (Yasui et ai, 1982). These results suggest that water binding of cured meat products is determined by the ratio of free myosin to F-actomyosin available at the time of heating to form the gel. It has been shown by differential scanning colorimetry that the behavior of myosin and its subfragments is quite different at low (0.1 Ì KC1), and high (0.6 Ì KC1) salt concentrations, with myosin at low ionic strength being more stable than at high ionic strength (Samejima et al., 1983). Heat-induced gelation of myosin in 0.6 Ì KC1 stored at either 5 or 25°C diminished with both time and temperature, completely disappearing within 2 weeks at 0°C and by 10 hours at 25°C (Ishioroshi et al., 1983). Presumably, the loss of ability to form gels is related to intermolecular aggregation of the myosin molecule. The ability of freshly prepared myosin filaments to form gels on heating seems to occur

432

13. Postmortem Conversion of Muscle to Meat

through interfilamental head to head aggregation without involvement of the tail portion of the molecule. Asghar et al. (1984) have studied the physicochemical and functional properties of myosin from red and white muscles of chickens grown on different planes of nutrition. Red and white myosin from well-nourished broilers had different properties, with red myosin having a lower specific viscosity and a higher sedimentation coefficient than white myosin. The transition monomers ^ filaments of white myosin occurred over a narrower range of ionic strength than was true for red muscle myosin. However, white myosin exhibited greater gel strength than red myosin when prepared under identical pH, ionic strength, protein concentration, and temperature. The three-dimensional structure of the gels was quite different for red and white muscle myosins. The strength of the gels from red myosin of the underfed broilers was about 50% less than that from the well-fed controls, but the strength of the white myosin gels were unaltered by the plane of nutrition. The underfed chickens also had low Ca -activated ATPase activity, but the EDTA-activated ATPase, specific viscosity, and helicity of the myosin molecule did not differ between nutritional states. Thus, red and white myosins have quite different physicochemical properties, with those of red myosin being dependent on the nutritional status. 2+

Most of the work on gelation of the myofibrillar proteins has examined myosin, actin, and actomyosin, with some research focusing on the subfragments of myosin. Samejima et al. (1984) found that isolated rabbit myosin heavy chains (MHCs) formed precipitates at an ionic strength of 0.3 or below but became soluble at higher ionic strengths. On heating low concentrations of MHCs to 65°C, however, the MHCs lost their solubility and formed aggregates, even at high ionic strengths. The strength of the gels made from MHCs was almost equal to that of gels made with intact myosin molecules at the same concentration, which indicates that MHCs are the major proteins involved in gelation of myosin. Although myosin light chains (MLCs) do not contribute to the gelling of myosin, they appear to provide stability to the gel if the pH is adjusted above the optimum. Addition of actin to the MHC system weakened the gel strength in di-

rect proportion to the amount added. This work agrees with that of Siegel and Schmidt (1979), who concluded that MHCs play an important role by binding meat pieces together for manufacturing of restructured products. Samejima et al. (1985) found that the solubility of cardiac myosin was strongly affected by pH, ionic strength, and temperature, with pH and ionic strength also interacting together. Although cardiac myosin was completely soluble and remained in the monomeric form at low ionic strengths (<0.2 Ì KC1) at pH 7.0, at low pH a high ionic strength was needed to keep the myosin in the monomeric form. At low ionic strength and low pH, the cardiac myosin formed aggregated filaments. Like skeletal muscle myosin, the gel strength of cardiac myosin was also influenced by pH, ionic strength, and temperature. Production of maximum strength gels was achieved at pH 5.5 and 0.1 Ì KC1 on heating of cardiac myosin to 60°C. Cardiac myosin formed much stronger gels than was the case for skeletal muscle myosin. The greater gel strength of cardiac myosin is surprising: it would be expected to resemble red muscle myosin but in reality produced a stronger gel than white muscle myosin. Although myosin appears to be the major protein involved in emulsification of fat and in gelation, which stabilizes the emulsion, there is some evidence that actin may also be involved, probably through actomyosin. The action of actin may be through F-actin serving as a cross-link between bound and free myosin (Yasui et al., 1982). Tropomyosin and the troponins may also play a role in gelation of myosin (Yasui et al., 1982) as they are contaminants in crude myosin extracts that have been used in most studies. It is also possible that C-protein and titin may be involved in emulsification and gelation as they are also usually extracted with myosin and actin and are frequent contaminants. The work with MHC, however, would indicate that it is the major myofibrillar component participating in gelation. In the case of emulsification, the evidence is not so clear. Some of the other myofibrillar proteins could be contributors to this desirable property. It is well known that prerigor meat has a greater fat emulsifying capacity than postrigor meat. This concept is supported by the fact that gel strengths decrease on aging of myosin at ionic concentrations

VI .

of less than 0.3 Ì KC1. These phenomena support the well-established fact that prerigor muscle has superior properties in meat emulsions. More details on the rationale for prerigor meat having superior properties in processed meats are discussed in an extensive review by Asghar et al. (1985).

Mea t Flavo r

433

A number of researchers have summarized the information available on meat flavor (Herz and Chang, 1970; Chang and Peterson, 1977; Wasserman, 1979; MacLeod and Seyyedain-Ardebili, 1981), which indicates that heterocyclic compounds containing oxygen, nitrogen, and sulfur are major contributors to the characteristic meaty flavor. There is also evidence that the sulfur compounds, including H S , may be important in development of the basic flavor that occurs during the heating of meat (Minor et al., 1965; Mussinan and Katz, 1973). Some of these substances may be produced by the Maillard or browning reaction in which the carbonyl groups of reducing sugars present in the meat react with the free amino groups of the proteins to produce desirable flavor constituents during cooking (Hsieh et al., 1980a,b; MacLeod and Seyyedain-Ardebili, 1981). A number of model systems have been developed for studying meat flavor or producing synthetic meatlike complexes (Batzer et al., 1960, 1962; Hsieh et al., 1980a; Schroedter and Wolm, 1980). Most of these have consisted of a reducing sugar, usually glucose, a source of amino acids, and flavor enhancers, such as inosine monophosphate (IMP). On heating these compounds together, they produce meatlike flavors and aromas, which may be generated by the Maillard reaction. The sulfurcontaining amino acids appear to play a key role in production of the heterocylic compounds that make most important contributions to the meaty aromas and/or flavors of meat (Pearson et al., 1983). 2

VI . M E A T F L A V O R Conversion of muscle to meat results in production of a highly prized protein food that is desirable not only because of its nutritional contributions but also because of its pleasing flavor. Because of its fat content, however, meat is also susceptible to oxidation and the subsequent development of undesirable flavors. The desirable flavors that develop in meat postmortem are briefly reviewed in this section. DESIRABL E MEA T FLAVO R Probably the most important single attribute that makes meat desirable to the human species is the delicious flavor of cooked meat, which is not only distinctive but extremely difficult, if not impossible, to duplicate. During the conversion of muscle to meat, the flavor components become more intense and distinctive. There is also evidence that meat flavor takes on characteristics that add to its desirability during the aging process, although some population groups may object to the aged flavor of meat. 1. Origi n of Meat y Flavo r Raw meat has a serumlike or bloodlike bland flavor, which on heating is altered to produce compounds that impart a full rich flavor. This flavor becomes richer and deeper as the meat passes into rigor. A number of researchers (Crocker, 1948; Bouthilet, 1951a,b; Kramlich and Pearson, 1958) have demonstrated that the components contributing to the meaty flavor are localized in the watersoluble fraction. It has been shown that the characteristic flavor is essentially the same for meat from all species (Hornstein and Crowe, 1960; Hornstein etal, 1963).

2. Characteristi c Specie s Flavor s Each species produces meat with a typical flavor that is characteristic of the particular species, that is, beef, lamb, and pork all have different aromas and flavors. These characteristic species-specific flavors and aromas are localized in the lipid fraction (Minor et al., 1965; Hornstein et al., 1963; Wasserman and Talley, 1968). Taste panel studies by Wasserman and Talley (1968) demonstrated that beef and lamb, but not pork and veal, were identified significantly less often when using ground lean roasts in comparison to normal fat-containing ground roasts. These studies clearly demonstrated that the components contributing to the speciesspecific flavor of meat resides in the lipid fraction.

434

13. Postmortem Conversion of Muscle to Meat

The aromas appear to be more important than taste in contributing to the flavor of the various species, although there appears to be some variability for different species.

VII . SUMMAR Y Postmortem changes involved in the conversion of muscle to meat are dependent on the concentrations of glycogen and of the high-energy phosphate compounds (ATP, ADP, and CP) and their metabolites at the time of death. After death, muscle undergoes both physical and chemical changes that account for its conversion to meat. The physical changes result in alteration of the tissues from a flaccid, highly extensible state into a relatively inextensible and rather rigid state known as rigor mortis, which appears to be associated with the interaction of actin and myosin to form actomyosin. The chemical changes occurring in postmortem muscle result in breakdown of glycogen and highenergy phosphates ultimately to produce lactic acid, which causes a fall in pH. The pH declines from neutral (pH 7.0) or above until it reaches about pH 5.4 in normal meat. The extent and rate of pH fall can influence the physical properties of the meat produced, being associated with such conditions as dark cutting beef and PS Å and DFD pork, which are discussed from the standpoint of factors that are responsible for their development and consequences for the properties of the meat produced. Methods for preventing these undesirable conditions are also reviewed. Several other conditions developing in postmortem meat are also discussed, including cold shortening and the related cold-induced toughening and thaw rigor, all of which occur in postrigor meat. Aging of meat and its influence on meat tenderness and its probable mechanism of development is also discussed. Among those factors considered are the indigenous muscle proteinases, which are present not only as muscle alkaline proteases, neutral proteases, and acid proteases but also as lysosomal enzymes and cathepsins. It was proposed that a number of these native muscle enzymes are probably involved in meat tenderization. Finally,

the role of meat aging and the nature of the flavor compounds responsible for meat flavor were discussed and reviewed in light of their contributions. Water binding, fat binding, and emulsification of fat were also discussed from the viewpoint of the effects of prerigor muscle and postrigor meat on these properties. The myofibrillar proteins, especially myosin and actin, appear to play a major role in these properties.

LITERATUR E CITE D Asghar, Á., and Yeates, Í . Ô. M. (1978). The mechanism for promotion of tenderness in meat during the postmortem aging process. CRC Crit. Rev. Food Sci. Nutr. 10, 115. Asghar, Á., Morita, J., Samejima, K., and Yasui, T. (1984). Biochemical and functional characteristics of myosin from red and white muscles of chickens as influenced by nutritional stress. Agric. Biol. Chem. 48, 2217. Asghar, Á., Samejima, K., and Yasui, T. (1985). Functionality of muscle proteins in gelation mechanisms of structured meat products. CRC Crit. Rev. Food Sci. Nutr. 22 , 59. Atkinson, J. L., and Follett, M. J. (1973). Biochemical studies on the discoloration of fresh meat. J. Food Technol. 8, 51. Balls, A. K. (1960). Catheptic enzymes in muscle. Proc. Res. Conf., Am. Meat Inst. Found., Univ. Chicago, Chicago 12, 73. Bandack-Yuri, S., and Rose, D. (1961). Proteases of chicken breast muscle. Food Technol. 15, 186. Bate-Smith, E. C. (1948). The physiology and chemistry of rigor mortis, with special reference to the aging of beef. Adv. Food Res. 1, 1. Bate-Smith, E. C , and Bendall, J. R. (1947). Rigor mortis and adenosine triphosphate. J. Physiol. (London) 106, 177. Bate-Smith, E. C , and Bendall, J. R. (1949). Factors determining the time course of rigor mortis. J. Physiol. (London) 110, 47. Batzer, O. F., Santoro, A. F., Landmann, W. Á., and Schweigert, B. S. (1960). Meat flavor chemistry. Precursors of beef flavor. J. Agric. Food Chem. 8, 498. Batzer, O. F., Santoro, A. F., and Landmann, W. A. (1962). Identification of some beef flavor precursors. J. Agric. Food Chem. 10, 94. Beecher, G. R., Briskey, E. J., and Hoekstra, W. G. (1965a). A comparison of glycolysis and associated

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