Postmortem Biochemistry of Meat and Fish Herbert 0. Hultin Denartment of Food Science and Nutrition. Marine Foods Laboratory, University of Massachusetts Marine Station, Over the centuries m e of the most highlv prized foods i n the human diet has been the muscle tissue of a variety of animals. Although I shall he using the terms "muscie" and "meat" interchangeably, there are some differences in common usage. Muscle tissue is usually used to indicate the organ in the living animal which is responsible for locomotion. After the death of the animal this tissue goes through changes which will be described below. After these changes occur, the muscle tissue is referred to as meat. Meat also implies a product which includes some fat and hone. Furthermore, it is common to reserve the term "meat" for the converted muscle tissue of mammals, while the muscle tissue of other animals is referred to by species name, such as chicken, duck, cod, flounder or more general terms such as poultry or fish. Besides its esthetic appeal, meat is important because of its nutritive value. Although water accounts for the bulk of muscle (about 70-80% by weight) a high percentage of lean muscle tissue is protein (1&23% by weight) and this protein has a high proportion of the essential amino acids. The next major component (by weight) is lipid. Lipid is the most variable comoonent in meat. Its content in commercial cuts depends in large part on the amount of adipose (fat) tissue associated with the meat. In some species of fish, e.g., those termed "fatty fish" like mackerel and herring, the amount of fat in the tissue can vary markedly, e.g., from 5 to 23% (1).On the other hand, with some lean fish, the total lipid content is less than 1%.The relative com~ositionof fatty acids in various species varies considerably. Generally, beef tissue is high in its content of saturated fatty acids whereas fish has a high content of highly polyunsaturated fatty acids. Pork and poultry are intermediate (2). Muscle tissue also is an excellent source of vitamins of the B complex and is a good source of iron. Structural and chemical features of muscle tissue will he discussed below with emphasis on the chemical and physical changes which occur and how these changes affect eating quality.
. .
h e a w coverine" of connective tissues. the enimvsium. Other connective tissues penetrate the interior of the muscle from this eoimvsium. seoaratine eroum of fibers into hundles. ~ h e s e - c o n ~ e c ttidsues i v ~ maki upathe perimysium. From the ~erimvsiumextend finer sheaths of connective tissue surrounding vach musclr fiber; this is termed the endomysiutn. The arranrement of musrle wlls in fish is quite dittrrent and is related'to the different mechanics o f locomotion. The structural arrangement of muscle tissue from a typical bony fish is shown in Figure 2. The segments, shaped like the letter W, are called myotomes and have two backward and one forward flexure. Myotomes intercept the midplane of the fish at a sharp angle. The fish myotomes are one cell deep, and the individual muscle fibers or cells are roughly perpendicular to the surface of the myotome. Myotomes are connected to each other by myosepta or myocommata, the connective tissue. Cellular Structure Although the gross arrangement of cells varies in different muscles, the muscle cells o h o s t animals are very similar. A typical muscle cell or fiher is shown in Figure 3. The surface
BLOOD VESSELS
PERIMYSIUM
Muscle Structure Gross Structure A tvoical arraneement of muscle com~onentsfor mammals and l;i;ds is shuwi in Figurc I. The muscle reils or fibers are arranred in ii parallel fashion to form hundles. Groups of these bundyes make up the muscle. Around the whole muscle is a
Figure 1. Cmss-section of muscle showing relationship of muscle fibers and muscle bundles and illustrating the location of epimysiurn, perirnysium, and endornysium. Location of blood vessels is also indicated. (Reprinted from Relerence (3)by courtesy of J. J. Lippincon Company.)
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Number 4
April 1984
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of the muscle cell is the sarcolemma which is comprised of an outer network of collagen fibrils, an amorphous layer, and the inner cell memhrane ( 6 ) .Invaginations of this cell membrane form the transverse (T) system (7-9) and the ends of this T svstem come together inside the cell close to two terminal sacs z t h e sarcop~aBmicreticulum. The latter is a memhrane that generally runs parallel to the main axis of the cell. The meeting point of the T system and sarcoplasmic reticulum is called the triadic joint and occurs at different locations in different muscles. The T system brings the cell memhrane to the interior of the muscle cell and allows the muscle cell to r e s ~ o n d as a unit, i.e., the inside of the cell can react concurrently with the outside. Mitochondria serve as the prime energy transducers for the musrle cell, and they are d t e n located nvar the Z-lme or near the cell membrane. I.ysosomt.s are the digestive organ of the cell and contain large quantitivs of hydrol\tic en7ymes. It has nie~nhrane, been suggestrd t hat on hredkup uf the lys~~wmal 1)roteost.i are released which play an important role in the tenderization process in muscle. These proteases are termed "cathespins." All of the cellular structures are surrounded by the sarcoplasm, a semi-fluid material which contains soluble components such as enzymes, metabolic intermediates, and myoglobin, which is responsible for meat color. Another type of cell associated with muscle tissue is the fat cell which is a oart of the connective tissue. Fat cells are derived from undifferentiated mesenchyme cells, often developing around the blood vessels in muscle. As a fat cell develops, i t accumulates droplets of fat. These droplets grow by coalescing and eventually only a single large drop of fat exists. Around this fat droplet is the cytoplasm; all the subcellular oreanelles are located in this thin cvto~lasmiclayer. Fat cells a; generally located in the perimysial spaceLor suhcutaneously but not in the endomysial area. There may, however, be tiny fat droplets distributed in the muscle fiher itself. The amount of fat that an animal accumulates depends on age, nutrition, exercise, and other, physiological, factors. Initially fat accumulates under the skin and only later does it accumulate around the muscle. The latter accumulation leads to "marbling" of meat. Since this is the last fat to he deposited, a large quantity of feed is required to obtain this marbling. Myofibrillar Structure The principle components of the musrle cell in terms of auantitv are the myoiibrils. The characteristic striated avpearance of skeletai muscle is due to the repetitive arrangement of proteins in the myofihril (Figure 4). Viewed in polarized light, one set of hands is anisotropic or birefringent, and the hands are termed the A-hands. The lighter bands are isotopic and are called the I-bands. At the center of each Ihand is a dark line called the Z-line or Z-disc. At the center of each A-hand is a light area which is called the H-zone. In the center of the H-zone there is a darker M-line. The distance from one Z-disc to the next is termed the "sarcomere" and is the basic contractile unit of the fibril. In the myofibril the A-hand is composed of thick filaments while the I-hand contains the thin filaments. The latter project outward from the Z-disc in both directions and overlao the thick filaments of the A-band in certain areas. The light'zone in the center of the thick filaments (H-zone) is that part where the thin filaments do not overlap the thick filaments. The extent of contraction of the muscle determines the size of the various hands and zones since the thick and thin filaments slide oast each other during contraction. The l e n ~ t hof the A-badd remains constant during contraction while tbe I-hand and the H-zone shorten. The thin filaments are connected to Z-disc material; the latter probably serves as an anchor during the contractile process. Each thin filament in the I-band is linked through 290
Journal of Chemical Education
the Z-disc to the four closest thin filaments in the adjacent sarcomere. The M-line located in the center of the thick filaments is where myosin head pieces are not present. The M-line prohably serves to keep the filaments in correct alignment. The cells of striated muscle from other vertebrates as well as invertebrates have much the same structure as discussed above for mammalian muscle. There may he some differences, however, in the amount of sarcoplasm, the arrangement of the myofibrils, the relation of mitochondria and nuclei to other components in the cell, the location of the triadic joint, and the arrangement of the sarcoplasmic reticulum. In fish muscle cells, the inner myofibrils have the normal arrangement parallel to the long axis of the muscle fiher (11) while the myofihrils at the periphery are perpendicular to the surface of the cell. In a few muscles such as those of scallop adductor muscle and squid muscle, the striations are oblique rather than being cross-wise (12).
Figure 2.Peltem of myotoms in musculature of typical bony fish. Detailedlateral views of a single rnyatome are also given. (Reprinted Com Reference (4) by courtesy of John Wiley & Sons.)
Figure 3. Muscle fiber cutaway showing outer membrane and T system (invagination of outer membrane). which runs horizontally and meets wilh two terminal Sacs of iongaudi~lsarmplarmic reticulum. The meeting point is called the b i d . Repeating cross-striations are also shown. (Reprinted fmm Reference (3by courtesy of Marcel Dekker.)
Muscle Proteins
For convenience, the proteins found in muscle can be broadly categorized as contractile, soluble, or insoluble. The soluble protehs are those which can he easily extracted from the muscle in water or dilute salt solution. The contractile protein can he soluhilized by solutions of relatively high ionic strength hut not in solutions of lower ionic strength. The insoluble proteins are not soluhle in salt solutions of either high or low ionic strength. Soluble Proteins
Figure 4. Schematic structure of the myofibrii in longitudinal section indicating the basic contractile unit. the sarcomere. The l-band consists of lhin filaments. In the darer area of me A oand. Mere are overlappmg m~ckand mm tdamsmr The I ghter area of the H-zone is wnere only thtcr 11 smenls are present M M I ne 19a oulge ,n te center at each tn ck fllament Theresa pseuda-rl-zone. a bare region on each side of the M-line. (Reprimed t o m Reference (3by courtesy of Marcel Dekker.)
Heavy merom 051 n
w o polypeptide choins in W-helical supercoil F2
Figure 5. Schematic representation of myosin. Globular head with light chains and elongated tail sectionare indicstedas well as pointsof enrymicfragmentation. (Reprinted t o m Reference (3by courtesy of Marcel Dekker.)
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The soluhle nroteins in the muscle cell have been desienated by various names such as myogen or simply the sarcoplasmic extract. Soluble rote ins make un anvwhere from 20-30% of the total proteins of the muscle Ee11. They consist mostly of enzymes, particularly the glycolytic enzymes. The oxygenstorage component of the cell, myoglobin, is also found in this fraction. The sarcoplasm has a high viscosity probably due to the high concentration of these soluble proteins which may he as much as 20-30s by weieht. Low-molecular-weight components such as sugar derivat&es and nucleotides are &o found in the soluble fraction, as are salts. h4yofibrillarlContractile Proteins
The myofihrillar or contractile proteins are those which participate directly in the process of muscle contraction by which the chemical energy of ATP is converted to mechanical energy. These are the proteins which constitute the sarcomere. The major contractile protein is myosin which accounts for some 50 to 60% of the total myofihrillar proteins and is found in the thick filaments. I t is a molecule with a high axial ratio and a molecular weight of approximately 470,000 daltons. Myosin contains two polypeptide chains that are identical; each has a high degree of a-helical structure. The two chains are supercoiled, i.e., wound around each other as illustrated in Figure 5. At one end of the molecule are two globular heads which are responsible for its enzymic (ATPase) activity; these heads interact with actin. Two "lieht chains" are associated with each globular head section; i.e, there are four light chains with each myosin molecule. These light chains are com~osed of two different chemical classes whicb are named on the basis of the way that they can he removed from the myosin molecule. The light chains range in size from 16,000to 25,000 daltons. Each myosin globular head has one of each of the different chains associated with i t (14). Enzymes such as trypsin may cleave the myosin near the head region producing two protein fractions. One of these is termed "light meromyosin," and the other, containing the. glohular head, is termed "heavy meromyosin." The heavy meromyosin retains its ATPase activity and its ability to combine with actin. The globular section of heaw meromvosin, termed "suhfragneni 1," is attached to the straight of the myosin molecule with rather flexible linkages and can assume awide variety of positions in relation to the rod. Light meromyosin associates readily and is responsible for the backbone structure of the thick filament. The rod section of heavy meromyosin (subfragment 2) is free to swing out from the surface to allow the globular heads to make contact with the thin filaments. Some 400 molecules of myosin are contained in each thick filament. These molecules interact in a specific orientation, joining head to tail in two directions as illustrated in Figure
Figure 6. Arrangement of myosin in mick filament. Stralaht - .Dortion indicates tail region: laggered portion, the globular head region. Myosin molecules have revened polarity on either side of the center. (Reprinted from Reference (15) by courtesy of Academic Press. inc.)
~i~~~ 7. ~ o u b l ehelical anangement at actin indicating tropomyosin in the pave of the actin double helix with periadic location of boponin complex. Reference (16) by Periodicity of latter is approximateiy 400 A. (Reprinted courtesy of Cambridge University Press.)
6. This polarity of the arrangement of the myosin molecules in the thick filaments allows contraction to occur. The second most abundant protein of the myofibril is actin which some 15-30% of the total orotein and is lo. ----~ renresents cated in the thin filament. Monomeric actin is a globular protein with a molecular weight of 43,000 to 48,000 daltons. In muscle tissue it most likely exists as the so-called fibrous actin (F-actiu), which has a double helical structure as illustrated in Figure 7. Each actin monomer in F-actin binds to one globular head of myosin. There is polarity in the way in which the actin interacts with the myosin. F-actin filaments on opoosite sides of the Z-disc point in opvosite directions. This h o w s the thick filaments in adjacent sarcomeres to move towards the Z-disc. In uitro, actin and myosin can interact to form a complex called actomvosin. Actomvosin has, like myosin, ATPase activity, but the activity has significantly different characteristics. ATP and ADP can serve as plasticizing agents and prevent the interaction of actin and myosin. Tropomyosin, like actin, is a two-stranded, coiled coil. I t has a molecular weight of 65,000 to 70,000 daltons and has a very hizh a-helical content (over 90%).The two subunits that comprise it are different. I t has a tendency to aggregate end to end, and lies in the groove of the supercoiled actin thin filaments (Fie. , " 7). , Troponin is closely associated with tropomyosin. There are three snecific subunits desienated trooonin-C (17,000-18.000 dalkni), troponin-l (20.00ii-24,000dsltons), andtwponin-T ~:i7.00~1-1U.000dnltons).'I'ro~onin-C binds calcium, trooonin-I inhibits the enzymic act&ity of actomyosin, and t r k o n in-T is the major site for binding troponin t o tropomyosin. Troponin and tropomyosin each comprise about 5% of the contractile proteing. Another protein found in the thick filament is "C-protein!' This protein is a single polypeptide chain of 140,000daltons. I t has been suggested that it may he wrapped around the thick filament surface to protect the filament from physical or chemical destruction. Crentine kinase, an enzyme involved in ATP rewneration and which can he prepared as a sduhle enzyme, h& turned out surprisingly to be a&ructural protein of the M-line. a-Actinip is located in the Z-disc, has a molecular weight of 180,000 daltons, and is composed of two polypeptide chains of similar size. Z-nin has a molecular weight of some 300,000 to 400,000 daltons and is also part of the Z-disc. More recentlv it has been observed that there are other sets of filaments in the muscle cell which may play an important role both physiologically and in the use of this tissue as food. The so-called gap filaments run longitudinally along the myofibril. A schematic drawing of their possible location is shown in Figure 8. The term "gap filament" derives from the fact that when muscle is stretched and the I-bands are pulled away from the A-hands, these filaments are observed in the "ean" between the thin and thick filaments. The protein of tgese filaments has been termed "connectin" or "titin." I t comorises about 5% of the total contractile protein. ~esmin (also called skeletio) is a protein which forms filaments of 10 nm and connects adjacent Z-discs horizontally. Desmin also appears to be part of the Z-disc and serves to keep the myofibrils in register.
M
2
M
Figure 8. Arrangement of gap filaments (dotted line) in a stretched sarcomere. serve as to two thick in sarcomeres, (Adapted from Reference (17)).
~.~ ~
~
~
~
~
292
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~~~
Journal of Chemical Education
~
Figure 9. Diagramatic @)and schematic (b) representations of how molecules are aligned in native collagen fibril. (Reprint* from Reference (20)by cowtesy of A. J. Bailey.)
Collagenllnsoluble Proteins . Some proteins of the muscle cell are soluble in neither low nor high ionir strength salt solutions. These include some of the contractilt: prowins such as conncrtin and desmin, portions uf membrane systrms, and the connertive tissue proteins. Connective tissue represents the major portion quantitatively and is comprised chiefly of the protein collagen. Collken m ~ o & n tin muscle tissue used for f w d because ~ , is. i ~ it contributes to the textural properties of the tissue. In addition. oartiallv denatured cdlaeen is ralled relatin and is a usefuihgrediek in many food products d u e t o its high viscositv and its abilitv to form eels a t low temperature. It bas beenuestimated that collagenkakes up one third or more of the protein in mammals ( I S ) . The proportion of collagen in fish protein is lower since fish do not need as extensive support as do land animals. The collagen molecule is along cylindrical protein about 280,000 A in length and 1 6 1 5 A in diameter. It has three ~ lv~ i d e wound around each other in . o ". . e ~ tchains suprahelical fashion. When the individual v v ~.e ~ t i dof e scollagen are not con.o l.. nected by covalent crosslinking they are teimed a components. When two of them are joined, it is called a P component, and when all three are joined the product is designated a y comvonent. Solubilitv of collazen is a function of the intermol&dar crosslinking, decreasing as the crosslinking increases. Collagen exists in several polymorphic forms. The most common is known as Type I collagen, and it has two polypeptide chains which are identical (called a-l(1)) and a third chain, a-2, with a different amino acid sequence. Each chain has a molecular weight of approximately 100,000 daltons giving a total weight of 300,000 daltons for the whole collagen molecule (19). Type I11 collagen is also found in muscle and is made up of three identical a chains designated a-l(II1).This type of collagen has intramolecular disulfide bonds in the end portion of the molecule. Type IV molecules are more complex ~~
and consist of polypeptide chains of dissimilar sizes. Type V collagen probably represents more than a single type. Type I collagen appears in the epimysium of muscle, Type I and I11 in the perimysium, and Types 111, IV, and V in the endomysium (19).Type 111is of particular importance in determining the textural properties of muscle. Collagen molecules are linked end to end and adjacently to make the collagen fibril (Fig. 9). The cross striations in collagen have a periodicity of about 640 to 700 A. The fibrils may he arranged in parallel fashion to give great strength as is found in tendons, or they may he highly branched and disordered as found in skin. Glycine represents nearly one third of the total residues, and this amino acid is distributed uniformly at every third position throughout most of the molecule. The presencc of glycine and its small size means that the helical structure of is different from the tvoical the collaeen ~-~~ -~ .. n-helix. beine more closely packed. At both ends of each collagen molecile are several amino residues, about 14 a t the N-terminus and about 10 at the C-terminus, which are termed the "telopeptides" and do not have the helical structure and repetitive occurrence of glycine of the rest of the molecule. Collagen contains a large content of hydroxyproline (up to lo%), and this forms the basis of the determination of collagen in food products. In the oolvoentides which make UD collagen. >. . .. . two imvortant oxidations take place. These are the conversions of lysine to hydroxylysine and proline to hydroxyproline. The hydroxy-
~~-
.
'iCHOH
lysine, as well as lysine, can be further oxidized to a-aminoadipic-A-semialdehyde. Enzymes catalyzing these oxidations require molecular oxygen, ry-ketoglutarate, Fez+, and a reducing substance such as ascorhate. It appears that the role of ascorbic acid in wound-healing comes about through its role in the synthesis of collagen. The covalent crosslinks involved in the beta and gamma components of collagen as well as the intermolecular crosslinks among collagen molecules form spontaneously by the condensation of aldehyde groups in reactions such as aldol condensations, or Schiff base formation, where the aldehyde reacts with an amino group. When hydroxylysine reacts with hydroxylysinealdehyde,the product of the reaction may undergo an "Amadori-type" rearrangement, forming a keto structure, hydroxylysino-5-keto-norleucine. Some of these reactions are presented in Figure 10. As an animal increases in age, the crosslinks of collagen are converted from a reducible to a more stable, non-reducible form. The exact nature of these mature "non-reducible crosslinks" is not known but is thought to involve several functional groups in an extensive network of proteins. These intermolecular crosslinks are confined to the telopeptide portion of one chain involving a lysine-aldehyde and a hydroxylysine in the helical region of an adjacent chain. Since the crosslinks in collagen increase with increasing age, this may explain why meat from older animals is tougher than that from younger animals even though muscle tissue from younger
(CH,),
( Hz)z
I I
CHOH
I
NH, hydroxylysine
+
H e 0
I CHOH I
ciH2 NH
-
I I C=O I
CH,
Sehiff
base
F
( H2)2
-NH-CHw hydroxylysino-5-ketonorleucine
hydroxylysine aldehyde
c.
-NH--CH--CO--
I I CHOH I
(CH,),
F"' NHI
hydroxylysine
+ Hc==O
I
Z-a-amino-adipic-6semi-aldehydes
a$-unsaturated aldol
a-amino-adipic4semi-aldehyde
Schiff base dehydrohydroxylysinenorleucine
Figwe 10. Formation of crosslinks in collagen by side chain groups. (a) Schiff-base formationfollowed by Amadori rearrangement. (b) Aldoi condensation followed by dehydration. (c)Schiff-base formation. Lyrine reacts in a manner analogous to hydroxylysine.
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April 1984
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animals contains more collagen. This situation is, however, not ohserved fish.-Althoueh older~ fish tend to have more ~ . in ~ ~" ~ collagen, the collagen has fewer crosslinks. It appears that the collagen of fish muscle is turned over, or renewed, yearly. ~
Muscle Contradlon Some background in the normal functioning and interactions of contractile proteins in the living muscle cell is necessary to understand the changes which occur in muscle after the death of the animal and which contribute to the eating quality of the muscle tissue. Muscle is stimulated by an electrical impulse received from the nerves. This causes depolarization of the muscle cell membrane. The transverse tubular system of the muscle is simply an extension of the cell membrane (7-9) and carries this stimulus (depolarization) rapidly to the interior of the muscle cell so that the whole cell can resoond as a unit. The transverse membrane system adjoins the bulbous end proiections of the sarco~lasmicreticulum near the mvofibrils. bepolarization of t i e transverse tuhular system-in some manner causes a release of calcium from the terminal sacs of the sarcopl:~smicreti~ulum121).The calrium that is rrleaied modifies the ronformation ui the trooonin comolex which in some way activates the contractile apparatus. A suggestion for how this miaht occur is the followina (14). Trooonin-I is strongly houndio both actin and troponin-C. when the calcium that has been released interacts with tropouin-C, the tropinin complrx undergoes a structural rhnnge which causes tro~onin-lto bind more tightly to troponin-C while at the same time weakening the interaction with actiu. This weakening of the interaction of tropinin-I with actin causes it to move which allows the tropom~osinto move further into the groove of the actin superhelix. The movement of a tropomyosin molecule uncovers seven actin hinding sites, allowing interaction with the subfragment 1portion of myosin. The tropomyosin is in a position to interfere with the binding of. MgATP a t the inhibitory site of the myosin. This overcomes the inhibition and allows ATP to he hydrolyzed by the actomyosin complex. The hydrolysis of this high-energy com~~~~~~~~
~
pound provides the energy for the changes which take place in the contractile nroteiu and which lead to contraction. In other words, contrktion is regulated by the amount of free calcium ions released into the sarconlasm la~uroximatelv 1 . NM of Ca2+activates the system). The actual contraction of the muscle takes olace bv a nrocess which allows the thin and thick filamenis to slide past each other (Fie. 11).The exact mechanism bv which the energy released &ring ATP hydrolysis is con;erted into mechanical energy of muscle contraction is not fully understood a t the molecular level. Presumably the globular head portion of the myosin interacts with actin and then pulls the actin filament parallel to the fiber axis. Since interaction of the filaments occurs throuah the crosshridaes, tension must he developed and transmitted by means ofthe crosshridges. An important factor which probably allows this to occur is the flexible nature of the rod-like portion (subfragment 2) of the heavy meromyosin section of myosin. I t allows both the attachment of the globular head to the thin filament and also allows the globular portion of the head to change its position in relation to the thin and thick filaments. The maximal amount of contraction varies with species but generally represents 20-50% of the rest length of the sarcomere. Muscle relaxes when the Ca2+ is removed from the sarcoolasm. This can take olace bv enerev-suu~ortedtransnort or by sequestering of the calcium by tie sa~oplasmicreti'culum. When the calcium ion concentration becomes low enough, i.e., less than 0.5 pM, the ATP can no longer be hydrolyzed, and ATP then functions as a olasticizina arent, causina the actin and myosin to separate a i d allowing themto s ~ i d ~ p aeach st other to their rest length. A.
Blochemical Events Postmortem: Role in Quality Chanoes Occurrino Soon After Death The major event which occurs after an animal is slaughtered is the cessation of circulation, the means bv which oxygen is transpor~t:d10 muscle t i s s u e . ~ h i is s rapidly fdowedby develorment of anaerobic conditionj within the musrle which brings about certain biochemical changes which significantly affect its properties as a food. In general, the biochemical control systems of most cells are such as to maintain a high content of ATP in the cell. In living muscle, most of the ATP under tnost circumitances is obtnintd f r o ~ nrespiring mitochondria that utilize molecular oxygen. aft^ the circulation stops following death of the animal, oxygen is no longer available, and muscle cells can no longer produce ATP by means hiehlv" efficient. aerobic resoiration. Initially - - ~ of~ the ~ ~ muscle cells can generate ATP from storeiof creatine phosphate and ADP. Another reaction immediately available to the cell is that catalyzed by the enzyme, adenylate kinase, which converts two molecules of ADP to one molecule of ATP and one molecule of AMP. These energy resources are not extensive, however, and may be depleted within the first several minutes postmortem. The maior anaerobic source of ATP after the death of the animal is glycolysis which converts either glycogen or glucose through a series of phosphorylated sugar derivatives to lactate with the concomitant production of ATP. In the absence of circulation of blood to the muscle, however, the extent of this glycolytic activity is limited. Generally either of two factors causes it to cease. The most common is a drop in pH to a level that inhibits the glycolytic enzymes. ~ u r i hydrolysis n ~ of ATP, hydrogen ion is produced and the tissue becomes more acidic. As the ATP is broken down, more of it is synthesized by the glycolytic enzymes which are under cellular controls to maintain a high ATP level. The end product of glycolysis in the muscle cell is lactate, and the amount of lactate produced corresponds in a rough fashion to the amount of ATP that has been synthesized and hydrolyzed. Thus, the amount of lactate builds up and is a useful measure of the amount of acid that has been produced by the hydrolysis of ATP.
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Frg~re11 Sareomere conlraclm %ate> na!cal ng thach (dark) 11 aments and tn n tloght) I laments altachea to 2-d scs B may be taken as representat 4e of asarcomerefromrsst ng muscle A represent9 rhc mrcomere homa stretched muscle, while C and Dare representations of sarcameres from muscle contracted to different extents. The jaggered lines of the thin filaments in D indicate portions of thin filaments overlapping one another. 294
Journal of Chemical Education
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In some cases with some species of animals, alvcolrsis may stop because the m w l e tissue is depleted of its&bstiate, e.;, glycogen. This very seldom happens in certain species like the cow where even vigorous exercise prior to slaughter is insufficient to lower the glycogen reserves sufficiently to deplete substrate postmortem. With other animals, e.g., the pig, i t is not uncommon for the glycogen of the muscle tissue to he depleted under relativel; moderate stress. When the muscle tissue is depleted of substrate forglycolysis or an unfavorable pH is reached, the production of ATP ceases. The enzymes in the muscle tissue that can break down ATP, however,continue to function so that there is a gradual loss of ATP with time. The ATP goes through a series of degradative reactions producing ADP, AMP, inosine monophosphate, inosine, and hypoxanthine. Both ATP and ADP are nlasticizers of thin and thick filaments. When they are they prevent the interaction of the thin and thick filaments. When they are depleted, the thin and thick filaments interact. When this happens the muscle loses its natural extensibility. This state is called rigor mortis (literally, the stiffness of death). Accompanying rigor mortis there is often some contraction (see below). The time that it takes to reach rigor mortis depends on a large number of factors including the snecies of animal, the particular muscle under consideratio;, the way it is handled after slaughter, the temperature at which it is held, etc. Prior to rieor mortis muscle is generally tender when it iscooked. After ;igor mortis muscle becomes tough. As oreviouslv. the attemnt of the muscle cells ~ - -mentioned - - - - ~ to maintain a high ATP le;h after deaih brings about acidification of the muscle. Both the rate and extent of the pH drop are important variables in the food quality of the muscle. The final pH reached by the muscle tissue is termed the ultimate pH, and it is dependent in part on the species, although it can v a n with bandline conditions. Beef aenerallv has an ultimate p ~ i the n range of5.5 to 5.7, while chcken is around 6.2, and fish mav be as hieh as 6.6 to 6.7. The ultimate nH is an importantrical sti&ulation works in the same way as the normal nerve impulse in the living animal causing strong contractions of the muscle. This causes amore raoid develo~mentof rieor mortis with a more rapid decline in pH and improved color and tenderness in some muscles. The improvement in texture may come from the fact that the muscles that are stimulated undergo supercontraction in certain areas and stretched sarcomeres in other areas, both of which can contribute to improved tenderness. Other suggested causes for the improvement brought about by electrical stimulation are: (1) stimulation occurs before the temnerature of the carcass is low enough for cold-shortening to occur, and (2) the rapid glycolvsis which causes the pH to fall rapidlv releases Droteases from the lysosomes whi;,h modiiy the structural components of the muscle. It is oossihle that all three of these contribute to improved tenderness.
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PERCENTAGE OF SHORTENING Figure 13.Relationshipbetween shatenirg of periga beef mrscktard Jhearing force required f w the waked muscle. (Reprinted from Reference (24) by courtesy of MacMillan Journals. Ltd.).
A o w i b l e explanation for the occurrence of a minimum in the shortening versus temperature curve is that the process of shortening declines with decreasing temperature, as is typical of alichemical, as well as enzymic, reactions. Below 10-14"C, however, another process becomes rate-limiting. This is probably the loss of the ability of the muscle system to control the calcium level in the sarcoplasm. The increase of Ca2+ in the soluble phase of the cell more than offsets the tendency for shortening to decrease with decreasing temperature. A phenomenon related to cold-shortening is "thaw-rigor." If muscle is frozen before it enters the state of rieor mortis. i t will contract vigorously on thawing, sometimes & as much'as 70%of its original rest length with the accompanying release of a large amount of exudate. It is believed that this is caused bv destruction of mitochondria and sarco~lasmicreticulum hy ice crystal formation during the freeling proress.'l'his a1lows the releaw of CaL' into the sarcodasm which trinrers -- the shortening. Advantaee is taken of these properties of muscle by "hotboning." ~ k classical e way to &eat a carcass after slaughter has been to cool it in a refrigerated room. The meat may then be removed from the carrass Into whatever cuts are required. This meration is wasteful in that rnerrv is rewired to cool parts oythe animal which do not have tobe coofed and which are later going to he trimmed off and used for purposes other than food, e.g., excessive fat and bone. The process of hotboning involves waiting a short period of time for the muscle to losesome of its heatand then to carry out the removal of the muscle tissue from the carcass in the temperature range of 1620°C. At this point the amount of shortening is minimal. After rigor, the temperature of the meat may be lowered. The bone and excess fat does not have to be cooled in this process. The temperature used allows only minimal contraction, and a t the same time is low enough to prevent excessive denaturation of the muscle proteins. The process of rigor may be speeded up by electrical stimulation. Benjamin Franklin observed in 1749 that stimulation of turkevs " bv " electricitv . imoroved their tenderness (25). . . In electrical stimulation the carcass is suhjected to an electric current of 500 to 600 V and 2-6 A applied in 16-20 pulses of ~~~
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Long-term Changes Occurring in Muscle Tissue Stored at Refrigerated Temperatures When muscle tissue goes into rigor, it is at its toughest state. With storage time this toughness decreases, and this phenomenon has been termed "resolution of rigor." It is clear that the resolution of rigor is not a simple reversion of the rigor process. There is no general agreement as to what is responsible for the increase in tenderness on storage although it is clear that very significant changes are taking place that may be related to tenderization. Some workers have reported chanees in actin-mvosin interaction with time oostrieor (26. 27), while other wo;kers have not been able to bhserie thesd chanees (28). The amount of tro~omvosinand t r o ~ o n i nhas been;ep&ted to decrease with time; chis is taken a: evidence that there are structural alterations in the mvofihrillar proteins (29). The disintemation of the Z-disc amears to have an im.. " portant role in the process of postrigor tenderization (30,31) in both poultry (32) and in beef (33). There is good evidence that the Z-disc is destroyed by the action of proteolytic enzymes. In particular a neutral protease which is activated by Ca2+ has been isolated from several species of animals, and this protease has been shown to cause disintegration of the Z-disc (34). a-Actinin is released by this process from the Z-disc. This calcium-activated factor (CAF) is active against troponin, tropomyosin, and C-protein but does not hydrolyze myosin, actin, connectin, or a-actinin (35). Some work has suggested that there is a "Z-disc actin" which is susceptible by CAF (36). When CAF hydrolyzes tropouiu-T, to hydrolysis . a snecies of molecular weight " of 30.000 daltons is oroduced. The amount of this component is related to the tenderness of heef (371. An endoeenous orotein inhibitor of CAF has been " shown to exist in skeletal muscle and may help regulate the activity of this enzyme (38). At concentrations known to exist in muscle, Ca2+ has been shown to weaken the Z-disc in nostrieor muscle (39.40). This weakening, which is evidencedby fr&mentation of the myofibrils. does not release a-actinin and so a . ~.o a r e n t l v does not involve CAF. The cathespius are a group of proteases which are associated with the lysosomes. Like most lysosomal enzymes they have acid pH optima. These enzymes may contribute to the tenderization of muscle tissue, particularly if the pH drops to a low value at a relatively high temperature. The high temperature causes the lysosomal membranes to break down releasing these enzvmes which can act on the muscle proteins at the'relati\.ely low pH. In ~ d d i t i mit, has heen demonstrated that some of the cathe~sinswill attack myofibrillar proteins at pH values which may be attained in stored meat (26, 41 ). Connectin, the protein that forms the gap filament between Z-discs, has also been shown to decrease with time postmortem. The concentration of calcium has an effect on the loss of ~
a-Actinin is the most heat-lahile of the muscle proteins and becomes denatured and insoluble a t approximately 50°C. The heavy and light chains of myosin are denatured around 55"C, and actin is denatured in the range of 7MO0C. Tropomyosin and troponin are the most heat-stahle of the muscle proteins and a temperature of approximately 8O0C is required to insoluhilize them (47). If one looks at the major structural features of the contractile elements in the muscle cell, it can he observed that the filaments in the A-band, i.e., thick filaments, denature ouite ranidlv. usuallv at 55°C or below. The filaments of t6e Lbands denature somewhere between 60°C and 70% I t has recentlv been demonstrated that the eao filaments start to denature a t 60°C; however, unlike tLe A and I filaments. thev are not comoletelv destroved even hv severe heating ( I 7). 1 h a s been suggested that they are responsible for the mvofihrillar contribution to texture in cooked meats. Perimysial collagen exists in the form of a net which is slack and crimped in relaxed muscle but becomes taut during stretching or contraction. Heating can also cause this net to contract and produce tension. e his contraction generally occurs in beef in the range of 60-70°C. With more prolonged cooking there is a tendency for the collagen to break down, and if the cooking is done in the presence of water, such as with a pot roast, the collagen may he denatured and converted to unfolded gelatin. This latter does not contribute significantly to the textural properties of a piece of meat. After the collagen network has been destroyed, the gap filaments still remain intact. and thev"mav.he the orinci~alcomoonent res~onsible for the textural properties of meat cooked to convert collagen to eelatin. Whv denaturation of the connectin in t h e g a p filaments doesnot lead to breakup of the filaments is nit known. In summary, as meat is cooked most of the myofibrillar oroteins are denatured, affecting structure and texture. At this point, rhe textural properties a;e primarily dependent on the collagen network and. ~ossihlv,on the rap filnmenrs. It is this influence of collagen that explains why cut of meat that is tough due to its collagen content (nature and amount) remains tough when cooked rare, e.g., broiled. On prolonged cooking, the collagen breaks down and is converted to gelatin in the presence of water. This conversion explains why wet cooking (e.g., braising or stewing) is better for the less tender cuts of meat. The textural characteristics of extensivelv cooked meat are based on those components of the muscle cell that are relatively unaffected by beat. In addition to the gap filaments, it may he that the desmin filaments (which connect the Z-discs in a horizontal olanel as well as other elements are involved. h,'eat that has been overcwked and allowed to dehydrate (for example. broiled to excess). could become tough s i m ~ l vfrom the diiving off of the waterand the coagulation, aggregation, and dehvdration of masses of oroteins. As mentioned earlier, the above n~nsiderationsdo nnt apply well t u fish since fish ~ d l a a e niadrstroved at such n low tem. perature during cooking, and there are no extensive connections longitudinally of fish muscle cells (the horizontal adhesion of fish muscle cells would be ruptured by the destruction of the endomysial collagen). Significant changes, nevertheless, do occur in fish muscle on heating. The flesh hecomes opaque and firm. This is undoubtedly due to denaturation of the proteins, especially the contractile proteins. The lower the pH of the fish muscle the greater will be the firmness developed. Although the emphasis here on the effects of cooking on meat quality has dealt principally with texture, it should he noted that there are other very important quality changes brought about by the heating process. Heating denatures the meat pigment and predisposes it to oxidation. Upon oxidation, the iron in the heme group converts from the ferrous to the ferric state, producing metmyoglobin. It is the brown color of
the denatured metmyoglohin that, in large part, is the cause of the browning of meat during cooking. At the same time that the pigment oxidizes, the high temperature causes the fat to melt leading to l i ~ i doxidation and also induces reactions and sugars (as well as some of the between amino breakdown products of lipid oxidation) corres~ondingto the \Iaillanl reaclion. hes sere actions are very important in the dwt.lt,prnent d m e a r flavor. Heactiuns involr,inc the s1111'11rcontaining amino acids are especially important in the development of meat flavor. Artificial meat flavors are produced by the heating of the proper sugars and amino acids. Final Remarks Workers in many different scientific disciplines have contributed to our knowledge " of the relationshin of the chemical and physical changes in muscle tissue during processing and storage to eating quality of meat and fish. This work has elucidated many problems in the processing of muscle tissue and has led to the high quality products that can be produced today. Progress in this area will continue, and before long the fields of molecular bioloev and eenetic eneineerinr undoubtedly will be called u&n to make their contribution to the area, leading to ever-better food for mankind through basic knowledge of chemistry. Literature Cited 111 Leu. S-S.,Jhausri.S. N., Ksrskoltsidis. P. A,, and Consfantinides,S. M., J. F o d S c i . , 46,1635(1981). (2) Hultin, H. 0.. in ' T o d Chemintry." 2nd ed.. (Editor Fenmema. 0.).Marcellkkker, NewYork, (in press). . (3) Ham, A. W.,"Histalopy."6th ed., Lippincott, Philadelphia, 1 9 6 9 , ~ 563. (4) La&. K. F.,I3ardach.J. E..Miller, R. R.,snd Paasino, 0. R.M.,"Iehthyology." 2nd ed..John Wiley& Sons.New York, 1917.p. 74. (5) Purler, K. R., and Franrini-Armstrong. C., Sei. Amer., 212 [a], 72 (1965). (6) KO,,O,T., ~ s k u m sF.. , Homms, M., end Fukuda, S.,Biochim. Biophya. Acto.88.155
a
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\.*,-,.
1351 Msruvama. K.. Kimura. M.. Kimura, S., Ohashi. K., Suzuji, K.. and Katunums.
N.,
11981). Hattori, A.,andTakshashi. K., J. Rimhem.. 85.47 11979). Hattori,A.,andTakahashi. K.. J. Hiorhem.. 92,381 119821. NC&,T.. lsogai, K., Hayashi,H., sndKstanuma, N..J. Hiach~m.,90,371119811. Takahashi,K..sndSaito,H., J. Hiorhsm.85, 1539(1979). Laakkonen,E..Sherbnn,J, W.,and Wel1ington.G. H., J. Foad. Sci.,35.181(19701. Knqgel. W. G..andFieid, R.A.. J FmdSci.36.1114 (19711. Herring, H. K.,Casrenr. R.G..and Brirkey, E. J.. J. FoodSei.32.534 (1967). Price, J. F.,and Schueigerl, B. %"The Science olMeat and Meat Pmduetn," 2nded.. Fnod and Nutrition Press. Westport, CT, 1978. Cheng.C.-%and Parrish. F.C.. J Fnod Sci.44.22 11979).
