CHANGES O C C U R R I N G 0Vi ) lWG B I C M # O R T l S A N D SUBSEQUENT RIPENING OF HUSCLE TISSUES

ERNEST J. B R I S K E Y '

U N I V E R S I T Y OF W I SCONSIN -"--"-----"---"-----"-"----"----"".---"--------"----

In presenting this report especial acknowledgment should be given the major contributions 8nd reviews of the tlistinguished scient is ts : Dr. E. C. Ba te-mth; Dr. J. R e Bendall and Drr R. A. Iamie, ImTemper- ature Research Station, Uhiversity of Cambridge and Department of Scien- t i f i c and Industrial Research; Dr. B. B. Marsh 08 the *at Industry Re- search Ins t i tu te of New Zealand; D r r A. Szent-Gyorgyi formerly of Budapest and more recently a t Marine Biological Zaboratory, Massachusetts; Dr. F, E. Deatherage, Ohio State University; Drr Feiner Hamu, German Meat Research Inst i tute; Dr. Jorgen Udvigsen of the Danish Royal College of Agriculture; and Dr. Eugene Wierbicki, formerly of Ohio State University, as well as many others. These names, I am sum, are familiar to a l l of you.

Muscle Stiffening :

Post-mortem t issue changes are very coqplex. The most obvious of these changes is the s t i f fening of the xtnasculature o r r igor mortis, quently t h i s process is conf'used with the "sett ing of the fat," however, it should be regarded as a manifestation of muscular contraction, extenaion and chemical degradation.

Fre-

Theories of Rigor Mortis:

Before considering the fbdatnentals of the physical and chemical changes, it would perhaps be best t o look at 8 few of the former concepts as w e l l as 8ome of the las t ing theories of r igor mortis.

1, In 1864, &e suggested that the s t i f fening was due t o the spontaneous coagulation of the wscle plasma by a process akin t o the coagulation of the blood.

2. This theory received modification in 1862 by Schipiloff who suggested that the precipitation of the proteins was due t o the l a c t i c acid produced after death.

3, This acidi ty theory was f'urther substantiated i n 1919 by von F k t h , who regarded pH, specifically, as the actual cause of r igor mortis .

4, In 1926 Hoet and Elsrks postulated a t h i r d change suggesting tha t both stiffeniw and acid production were related t o the loss of glycogen and/or of creatine phosphate.

later investigation by Engeulardt (1939) led t o the c lass ica l discovery of the ATPase ac t iv i ty of the s tmc tu ra l protein

5.

109.

myosin. loss of ATP from the muscle a f t e r death which was most closely related t o the onset of st iffening. Also during the early for t ies , Szent-Gyt;rgyi and h i s colleagues were making great strides in their studies of the s tmcture of muscle and the mechanism of contraction and rigor. He considered myosin A (Szent-G$rgyi, 194.5), actin, ATP, K, Ca and Mg ions as es- sen t i a l components of the m s c l e system. In resting muscle myosin A was considered t o be present as a stable complex with a par t icular coqlement of K, Cay Mg and A!LT which is un- combined with actin. The ac t in is i n the fibrous form. He theorized that a post mortem stimulus dislodges part of the combined K, and also part of the combined ATP. potassium (by diffusion) and ATP (by enzymatic breakdown) are removed from miyosin A, as occurs when the muscle dies, ac t in combines with myosin A t o form acto-1Pyosiny which, i n the absence of ATP is extended and confers on dead muscle the r ig id i ty character is t ic of rigor mortis.

In 1943, E r d b demonstrated that it was actually the

If, then, both

6. In 1948, Bate-Smith put for th s t i l l another theory based on the interf i lamentary reactions during rigor. Essentially, his reasoning is as follows: solutione, although uniform i n properties, are not necessarily composed of homogeneous molecules. r igor mortis involves the association of these filaments by weak cross linkages. Thirdly, the cross linkages, which account a l so for the decreased extensibi l i ty of the muscle, must be formed as a resu l t of total removal of ATP.

Firs t , par t ic les present i n m s i n

Secondly, the process of

Stages of Rigor Mortis:

The mrkers, Bate-Smith and Bendall (1949) have also described four stages of r igor morkis. (1) a 'delay period' during which the modulus of e l a s t i c i t y e i the r does not change at a l l o r increases very slightly, and (2) a phase i n which it in- creases rapidly t o i t s maximum which may be 10 t o 40 times greater than the i n i t i a l value. This second phase w i l l be referred t o as the 'rapid phase'. Considerable shortening sonetimes occurs during the rapid phase of rigor, but this shortening is not a necessary concomitant of stiffening. dependent however upon temperature and pH, and according t o Bendall (1951) and Mrsh (1954) is most marked a t high tern ratures (above 90° F. and i n exhausted animals (pH values above 6 .5 ) . & most obvious physical change occurring during the onset of r igor mortis is an increase i n the modulus of e l a s t i c i ty , which i n rabbit psoas muscle was fmnd by these workers t o rise from 500 t o values of about 10,000. Marsh (1954) found that i n beef the modulus rises from a mean i n i t i a l value of 1100 t o a mean f ina l value of 20,000 t o 30,000. Likewise, a shortening i n length accompanied the change i n e l a s t i c i ty . A lengthening of up t o 4% also occasionally preceded the on- s e t of rigor. In a l l cases, whether shortening accompanied the onset of rigor mortis o r not, rigor was resolved within a few hours of i t s onset, the mscle losing the firm, hard appearance of the r i g o r s t a t e . mechanisms involved he= axe quite obscun?.

In these types there were two d i s t inc t phases:

It is

The

Chemical Changes LurLng Rigor :

Now l e t us consider the effect of several chemical constituents on the changes during r igor mortis.

In 1951 Bendall showed that i n rabbit muscle the creative phos- phate level is high immediately post-mortem, but decreases rapidly there- after and i s Educed t o less than 20s of i t s i n i t i a l level at about pH 6.60 betore there is any appreciable loss of ATP. The disappearance of ATP, once started, proceeds at a steady rate u n t i l l e s s than 305 of the resting amount remains. 25% level. It has been suggested that the rate of turnover of ATP, there- fore, determines the rate of glycolysis. breakdown of ATP is exactly balanced by i ts resynthesis from the glycolytic cycle. The rapid phase of r igor occurs when the reserve of glycogen i n the muscle is almost exhausted, resynthesis being unable t o keep pace with the breakdown.

The rate subsequently decreases and is very slow below the

During the delay period, the

In essence, the main chemical changes i n the muscle a f t e r death a m the production of l ac t i c acid by anaerobic glycolysis, the breakdown of creatine phosphate and the resynthesis and the breakdown of ATP. glycolysis and the breakdown of creatine phosphate are mechanisms f o r t h e resynthesis of ATP from ADP. thesized f o r every molecule of l ac t i c acid formed.

Both

One and one half molecules of ATP are resyn-

Slide I -- The first slide taken f r o m par t of Lawrie's work, 1953, I think demonstrates veiy clearly the relationships of the constituents discussed. phate, a slow and then rapid decrease i n adenosine triphoephate, accompanied by a parallel drop i n pH. low there is a rapid decrease i n extensibil i ty.

Immediately a f t e r death there is a rapid drop i n creatine phos-

After about $ hours when all three values are

This balance between breakdown and resynthesis can be maintained only as long as a store of creatine phosphate lasts. according t o Bendall (1951), Iawrie (1953), and Bate-Smith (1956), the dephosphorylation increasingly exceeds rephosphorylation so that the actual ATP level itself begins t o fall , whether glycolytic resynthesis is proceeding o r not. i n amounts equivalent t o t h e amounts of ATP t h a t disappear. suggested the following chain of reactions as shown on Slide 11.

Beyond t h i s point,

A t this stage, ammonia and inosine monophosphate appear Bate-Smith has

111.

Slide I

0.4

0.1

0

Time (min. in nitrogen a t 37' C . ) '7 - 0 I ATP;/\-& CP; 0-0, pH; 8-e extens ib i l i ty

(R. A. Uwrie, J. Physiol. 1953 (121), 275-288)

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Slide I1

ATP - 9 ADP +. P (mPase)

__1___7) ATP 4 AMP (qyokinase)

AMP 3 IMP + % (deaminase)

Ammonia is l iberated from the deamination of AMP t o inosine mono- In phosphate as ske le ta l muscle passes in to r igor mortis, (Webster, 1953).

fac t , i n a recent publication by Dvorak on "Breakdown of Adenine Nucleotides i n Beef Muscle Post-Mortem" the author stated that as ATP i s a c r i t e r ion f o r beginning of r igor mortis i n muscle it may a lso be said that the production of ammonia from nucleotides terminates at the beginning of r igor mortis.

Physical Changes During Rigor Mortis:

As indicated at the beginning of t h i s report, physical changes occur simultaneously with changes in the chemical constituents. One of the observable changes is the change i n muscle appearance.

Marsh (1953) reported three types of whale mat, "dry," "wet" and rubbery." Through several experiments he has shown that these three types of m s c l e are manifestations of different phases of the onset and resolution of r igor mortis. meat i s meat which has passed throu& r igor and the "rubbery" meat is meat i n the stage of rigor. Marsh, f i r ther found tha t as l a c t i c acid was formed it was accompanied by a sizeable decrease i n f luid retention and a relat ive- l y rapid t rans i t ion t o a state of wetness. reported by Ingram (1955) t o occur i n horse muscle during r igor mortis.

H

The 'Id,, meat has not passed through rigor; the "wet"

The same character is t ics were

Shortening and Extensibil i ty:

O t h e r physical phenomena are changes i n shortening and extensi- b i l i t y . A relationship exists (Marsh, 1953) between shortening and extensi- b i l i t y which indicates that fewer cross-bond l inks are present with increased shortening. This suggests that individual bonds become available during the onset of r igor f o r e i t h e r s h o r t e n i q o r an increase i n resistance t o exten- sion which gives fu r the r support t o the cross-linked s t ructure suggested by Bate-Smith. A continued study reported by Marsh i n 1957 held still a further explanation of this shortening phenomenon. He suggested tha t at 50$ shortening the e l a s t i c l i m i t of a containing membrane i s reached and fur ther shortening is possible only a f t e r ruptures of the sarcolemma occur.

pH Changes During Rigor Mortis:

The change i n the pH of the musculature during r igor mortis is of multiple importance as previous speakers have indicated.

Initial pH - It has been shown by Bate-Smith, Bendall, Iawrie and Marsh that the i n i t i a l and ultimate pH values of the muscle are c r i t i c a l i n determining the time course of rigor, and therefore, the fac tors which

113 . predetermine these pH values are of the greatest importance, t o t h e factors determining the i n i t i a l pH has been provided by the Universi- t y of Cambridge workers through the use of myanesin. Through the use of t h i s material it was possible t o study animals paralyzed and fully Elaxed and provided what has been termed as a standard muscle. several of t h e i r experiments with t h i s drug suggested that the i n i t i a l pH is determined mainly by the severity of the death struggle.

The main clue

The resu l t s of

U1timat.e pH - The two factors of greatest importance i n determin- ing the ultimate pH appear t o be the leve l of feeding and degree of fatigue before death. This has been demonstrated in rabbits by Bate-Smith and Bendall, beef by Lawrie and Howard, swine by Iudvigsen, Mackintosh, J. Wismer-Pedersen and the Wisconsin workers. was not correlated with the ultimate pH.

In all cases the i n i t i a l pH

These factors have been mentioned in this report since the con- d i t ion of the animals musculature is highly related t o the chemical and physical changes during rigor mortis.

It has been reported by several workers (Eate-Smith, Lawrie, Ludvigsen and the Wisconsin wrkers ) that muscles contain a variable &mount of glycogen. Arring normal anaerobic glycolysis, glycogen is converted t o l a c t i c acid or possibly other acid components which measurably reduce pH. The severity of t h i s reduction, however, depends on the buffering power of the muscle. Bate-Smith has shown tha t the buffering power of rabbit muscle is about 50 m. eq./100 g. of muscle per pH unit. This means that the pro- duction of 15 l a c t i c acid will usually cause a s h i f t of about 1.8 pH units. As has been stated previously, it has been clear ly demonstrated by the Canibridge workers (Bate-Smith, Bendall, Uwrie and Marsh) t ha t the onset of r igor in rabbit muscle is dependent, not on pH, but on the ATP content; nevertheless, a t any given ultimste pH, the ATP content a t commencement of r igor is approximately constant, and, in noma1 rigor, acid production and ATP decomposition run paral le l .

Slide I11 -- The next slide was taken f r o m a table published by Marsh (1954). "Rigor Mortis i n Beef Muscle". This s l ide also shows the close relationship between a decrease i n pH and Xl!P content. A s t h e pH drops from 6.60 t o about 5.40 there is a concomitant decrease i n ATP from about 20 t o approximately 3.

114.

Slide I11

The Relationship Between pH and ATP content

pH range ATP-P as $I of T.S.P. (man * standard deviation)

above 6.60 20.5 f1 .5

6.59 - 6.40 19 9 2.5

6.39 - 6.20 16 2 2.5

6.19 - 6.00 5.99 - 5.68

13 2.5

+ 5.79 - 5.60 5 - 2.5

5.59 - 5.40 3 - 1.5 9

Part of Table I11 (B. B. Marsh, J. Sci. Food and Agr. 5, 1954)

Thus a knowledge of the pH a t m y time allows an estimate of the progress of t h e onset of r igor , decrease i n the water-binding properties of the muscles. reported, however, that only one-third of the drop in hydration during post-mortem ciianging is explained by the drop i n pH. two-thirds of the loss of hydration is due t o the loss of ATP and t h a t this decrease i n hydration i s proportional t o the decrease i n ATP. breakdown of ATP i t s bound cations are released because AMP and IMP have a much lower a b i l i t y t o form complexes. corporated in to the protein structure causing a t igh te r network of a lower hydration. contain l e s s bound magnesium than those of the r igor o r post-rigor muscle.

With t h i s drop i n pH there is a concomitant Hcumn (1959) has

Hamm suggests that

By the

The cations thus released can be in-

In fac t , the proteins of beef muscle immediately after slaughter

Tissue Death - It has been suggested tha t the accumulation of l a c t i c acid proceeds with increased velocity when a par t icu lar pH i s reached. Apparently synchronizing with t h i s phase (Rowan, 1940) a f a l l occurs i n e l e c t r i c a l resistance and reactance. According t o Hemingway and Collins (1932), these changes are due t o the v i r t u a l elimination of membrane re- sistance and capacitance. It is at t h i s stage that the muscle presumably loses i t s abi l i ty t o contract when stimulated, and the free difYusion of ions through the previously impemable membranes resu l t s i n a rapid equalization of pH throughout the t i s sue with possible exceptions, however, i n pork muscle. of the muscle. ishing ra te u n t i l e i t h e r glycogen i s exhausted o r a second fixed point i n pH is reached at which the glycolytic enzyme s y s t e m i s inhibited.

According to Bate-Smith t h i s is the true point of "death" From t h i s point onward, glycolysis continues at a dimin-

Time - - The time f o r the completion of the onset of rigor will, of course, vary widely according t o several fac tors previously mentioned such

115 e

as cooler efficiency, i n i t i a l pH, temperature and ultimate pH. these works pertain t o rabbit, beef and horse muscle. a post-mortem si tuat ion i n pork involving an abnormally low pH. According t o Iawrie, t h i s might be due t o au abnormally high rate of breakdown of ATP. Inrdvigsen has found that during post-mortem chi l l ing the t issue becomes ex- tremely abnormal and appears t o be loosely bound. The Wisconsin workers have observed that it is the lowest pH which the t issue obtains rather than i t s ultimate pH which dictates color and wateriness.

Most of We do, however, have

Oxidation-Reduction - The last point which I wish t o cover on physical change is the change in the oxidation-reduction potent ia l reported by Bsrnes and Ingram (1955). f a l l i n Eh from positive t o negative values OCCUPG during the first stages of rigor, during the phase of creatine phosphate disappearance.

They have shown very clear ly that the main

Subsequent Ripening of Muscle Tissues:

Most of the reported work of muscle ripening per ta ins primarily t o I w i l l hasten t o add that the present projects underway by Wilson and beef.

Schweigert of the American Meat Ins t i t u t e on "High Temperature Aging" and "Enzymes Concerned with Aging" as well as chemical aspects of tenderization by Deatherage w i l l undoubtedly mark major contributions t o t h i s area of knowledge. moderate degree f o r the past 30 years, Moran and Smith (1929) put forward the view that the connective tissue was the major element contributing t o tenderness and therefore was the component most l i k e l y t o be subject t o change during ripening. The theory of t o t a l connective t i s sue was ref'uted by Wilson and B r a y and temporarily lended fu r the r support t o Steiner's (1939) interpretation that as ripening proceeds the increase i n tenderness i s due exclusively t o an ef fec t upon the muscle fibers.

The area of ripening snd tenderization has been studied t o a

Among the proteins of the f ibers are a considerable number which

Those remaining ac- function as enzymes, but most of these w i l l have been deprived of the i r natural substrates by changes occurring during rigor. t i ve are concerned with the breakdown of protein o r autolysis. enzyme i n t h i s regard is s t i l l thought t o be cathepsin, although current re- search may present a new outlook i n this regard. followed by an increase i n nonprotein nitrogen and an increase i n sulfhydryl groups. proteins. naturation as such should cause meat t o become more tender, since it w i l l be more drastically coagulated when cooked.

The primary

This autolysis could be

These changes may be interpreted as 8 sign of denaturation of the According t o Bates-Smith, there is no apparent reason why de-

In general, the ac t iv i ty of cathepsin increases with a decrease of pH (Bradley, 1938) but several workers (Smorodintsev and Nikolaeva, 1936, 1942) found that in ripening muscle the catheptic ac t iv i ty actually decreased 40-45$ i n the first 24 hours post-mortem and 20$ fur ther during the next 5 days storage at 32-40° F.

Prudent (1947) and Winegarden (1952) reported tha t there are no post-mortem changes i n connective t issue. Wierbicki, e t al., (1954) also s ta ted that connective t i s sue does not appear t o contribute t o increases i n tenderness on post mortem aging inasmuch as t o t a l alkali insoluble protein

116

does not challge. It was also shown by Ramsbottom and Strandine (1949) that beef taken immediately at slaughter and before the onset of r igor was more tender than beef during r igor mortis. The Ohio workers (Wierbicki, I&znkle, Cahi l l and Deatherage) have reported that the changes in muscle plasma are d i r ec t ly associated with tenderness. creases i n tenderness with post mort;em age may be related t o the redis- t r ibu t ion of ions within the muscle, thus causing increased hydration and tenderness. I n 1956 through the use of improved techniques it was shown by these workers that the toughening of meat associated with the onset of r igor mortis is due t o the formation of actomyosin; however t h e actomyosin is not dissociated in to ac t in and myosin on post mortem aging and thus i s not re- sponsible f o r post mortem tenderization. - e t ,', a1 (1956) that post morten tenderization may be related t o changes i n the protein systems of muscle in such a way as t o cause increased hydration. A similar study by Arnold, Wierbicki and Deatherage held as a basic premise t h a t proteins and ions i n f'unctioning muscle are apparently in a high s t a t e of organization and that af ter the onset of r igor mortis it is possible that there is a random diffusion and redistribution of ions. t ha t the ionic s h i f t s might be related t o the degree of hydration of the muscle protein and thereby related t o tenderness, since it is known tha t the degree of hydration is related t o tenderness.

They reported i n 1954 tha t the in-

It was a lso suggested by Wierbicki,

It was thought

Results of the experiment showed that f o r each carcass, without exception, sodium and calcium were released by the meat proteins and potassium was absorbed during post-mortem aging. t o t a l cationic sh i f t was a movement of ions in to the meat proteins. This was due t o the large amount of potassium ions taken up i n re la t ion t o the amount of sodium and calcium released. The r e su l t of t h i s movement of ions was that the tusc le proteins became more posi t ively charged. calcium was a lso thought t o be responsible t o some extent f o r the increase of hydration of the actomyosin complex. increased charge on the meat proteins allowing grea te r hydration and improved tenderness.

In SUmmElry:

For the most part the

The release of

The en t i r e e f f e c t resulted i n an

1.

2.

3.

4.

The most obvious physical change occurring during the onset of r igor mortis o r s t i f fen ing is an increase i n the modulus of elasticity accompanied by a shortening of the f ibers .

There are actual ly two phases of rigor: during which the modulus of e l a s t i c i t y changes only slightly and (b) a "rapid phwe" in which it increases rapidly t o i t s maximum which may be 10-40 time6 greater than the i n i t i a l value.

(a) a "delay period"

The creatine phosphate l eve l is high immediately post-mortem, but decreases rapidly thereaf te r and is reduced t o less than 205 of i t s i n i t i a l l eve l before there is any appreciable loss O f ATP.

The disappearance of ATP, once s tar ted, proceeds at a steady rate u n t i l less than 308 of the rest ing amount remains.

117

5.

6.

7.

8 .

9.

10.

1.

2.

3.

4.

During the "delay period'' the breakdown of ATP is exactly balanced by its resynthesis from the glycolyt;ic cycle and creatine phosphate. reserve of glycogen i n the muscle is almost exhausted,

The rapid phase of r igor occurs when the

During glycolysis, l a c t i c acid is produced presumably i n amounts regulated by the i n i t i a l glycogen concentration. Ammonia i s a l so l iberated f romthe deamination of AMP t o IMP as skeletal muscle passes in to r igor mortis. conferred on dead muscle is actual ly due t o actomyosin which i n the absence of ATP is extended and fim,

The r ig id i ty

The i n i t i a l pH of the musculature i s apparently determined mainly by the severi ty of the death s tmggle, while the ultimate pH is a ref lect ion of the l eve l of feeding and de- gree of fa t igue before death.

The severi ty of pH reduction is governed somewhat by the buffering pover of the muscle and i n turn regulates t o some extent the change i n hydration o r water-binding. suggested recently, however, that only one-third of the drop in hydration i s explained by the change i n pH whereas two- th i rds of the lo s s of hydration is due t o the loss of ATP. Simultaneous with this change is a f a l l in e l e c t r i c a l re- sistance and reactance which is seemingly due t o the elimina- t i o n of membrane resistance.

It has been

The primary enzyme concerned with the breakdown of protein is st i l l thought t o be cathepsin.

The latest information on ripening supports the contention that during post-mortem aging there is a redis t r ibut ion of ions within the muscle which causes increased hydration and consequently increased tenderness,

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MR, PEARSON: Thank you, Ernie. I see our time is up. Our Chair- man has informed me we can hold forth a while longer, so we are going ahead with our panel discussion at t h i s time. Originally I asked Dr, J. L. Hall, t o handle the Panel discussion, but due t o his health, he was unable t o at- tend t h i s year, and he asked I take o v e r t h i s par t icu lar part; of the pro- gram. We have four d i f fe ren t people on our panel this afternoon, Joe Kastelic, University of I l l i no i s ; our f r iend h a t h e m e , you have heard once; Hans Lillevfk, Michigan State University; Dr. Lil levik has been in- terested i n many problems i n proteins during the past winter, and he has had much t o do with the handling of t h i s subject. Am.e~Lcan Ins t i t u t e Foundation. four o r f ive minutes t o point out sone applications they can see from our di6CUSSiOn tNs afternoon. 1'11 cal l on Joe first.

Then, we have D. M. Doty, I am going t o ask each of these men t o take