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ADVANCES IN FOOD RESEARCH, VOL. 26

THEORIES OF PROTEIN DENATURATION DURING FROZEN STORAGE OF FISH FLESH

SOLIMAN Y. K. SHENOUDA*

National Murine Fisheries Service Gloucester Laborarory, NOAA, United States Department of' Commerce

Gloucesrer, Massachusetts

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Tests Used to Detect Protein Denaturation in Frozen Fish . . . . . , . . , . . , . . ,

A. Tests for Protein Solubility or Extractability . . . . . . . . . . . . . . . . . . . . . . . B . Tissue Properties and Objective Parameters C. Microscopic Examination . . . . . . . . . . . . . . D. Changes in the Reaction Velocity of Enzymatic Acti E. Tests on Extracted Proteins.. . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Measurements of Low-Molecular-Weight Degradation Products . . . . . . .

111. Factors Causing Protein Denaturation during Frozen Storage of Fish A. Moisture as a Factor in the Denaturation of Fish Protein . . . . . . . . . . . . . B . Factors Related to Fish Lipids . . . . . , . . . . . . C. Enzymatic Activity of TMAOase as Related to Protein Denaturation

during Frozen Storage . . . . . . . . . . . . . . . . . . . D. Interaction between Factors: Indirect Effect o

during Frozen Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Research Needs. . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 277 271 218 279 219 280 280 28 1 28 1 290

298

304 306 301

I. INTRODUCTION

Deterioration in the quality of frozen fish product has long been noted; studies to understand and to prevent the loss of quality during frozen storage were begun as far back as five decades ago. The changes that take place during frozen storage of fish are of great commercial importance, for they determine the storage life of

*Present address: General Foods Corporation, Technical Center, Tarrytown. New York, 10591,

215 Copynghr 0 1980 by Academic Press, Inc.

All rights of reproduction In any form reserved. ISBN 0-12-016126-4

276 SOLIMAN Y. K. SHENOUDA

the frozen seafood. Deterioration in texture, flavor, and color is the most serious problem, particularly when poor freezing practices are used or when the quality of the fish is low.

The effect of various freezing conditions on quality has received considerable attention from fish technologists. The results of their work have furthered our understanding of the problems and have contributed to the development of better frozen seafood. Numerous studies have revealed important criteria such as:

1 . The advantages of rapid freezing of the catch without unnecessary delay-

2. The superior quality produced by a fast freezing rate. 3. The advantages and disadvantages of different freezing methods (shelf

freezer, blast freezer, refrigerated seawater, fluidized bed, direct contact with freezing media-that is, immersion freezing in Freon or liquid nitrogen).

4. The deterioration in quality due to fluctuations in storage temperature and relative humidity.

5. The effectiveness of prefreezing treatments for enzyme inactivation or the addition of antioxidants or cryoprotective agents.

6 . The importance of packaging as related to moisture vapor and oxygen permeability.

that is, freezing at sea.

Unfortunately, deterioration in quality, and especially in texture, as a consequence of long storage periods remains an unsolved problem for many fishery products. Deterioration in flavor (such as off-flavor, rancidity, bitterness, or an undesirable fishy taste) is believed to be due to the formation of low-molecular- weight compounds from lipid oxidation or protein degradation. Undesirable changes in color and appearance (such as loss in intensity of the colored tissues, loss of surface glossiness, development of freezer bums and surface dehydration, drip, or muscle opacity or chalky appearance) are thought to be due to irreversible changes that occur in muscle proteins or protein-bound pigments, or to changes in certain pigmented proteins-for example, myoglobin and oxymyo- globin into metmyoglobin, such as is found in frozen tuna meat (Matumoto and Matsuda, 1967). Finally, undesirable textural changes are a major consideration in judging the quality of frozen seafood. Changes in fish texture (extra firmness, toughness, springiness, sponginess, stringiness, dryness, rubbery texture, lack of succulence, loss of water-holding properties, or loss of juiciness) are recognized as being due to protein denaturation during frozen storage, particularly the myofibrillar proteins (Dyer, 195 1).

In summary, freezing and frozen storage are believed to furnish favorable conditions for the irreversible denaturation of fish muscle proteins.

PROTEIN DENATURATION IN FROZEN FISH 277

II. TESTS USED TO DETECT PROTEIN DENATURATION IN FROZEN FISH

To quantify the undesirable deteriorative characteristics mentioned, scientists have tried to correlate them with various analytical parameters, mostly associated with the phenomena of protein denaturation. Table I lists the most common parameters used to detect or monitor changes or deterioration occurring in fish or fish proteins during frozen storage.

A. TESTS FOR PROTEIN SOLUBILITY OR EXTRACTABILITY

The most popular tests used to study the changes that occur in fish protein during frozen storage are related to the loss in solubility or the loss in extractability of total fish protein, or of a particular group of proteins (myofibrillar, sarcoplasmic, or the actomyosin group), or even of particular protein species, such as myosin, tropomyosin, or actin. The extracting conditions usually em- ployed in these tests are not standardized. For example, the extracting solution may vary in the type of salts used, the ionic strength, the concentration of divalent cations, the incorporation of detergents such as sodium dodecyl sulfate (SDS), the pH, or the buffering capacity. Variations also exist in the ratio of muscle to solution, and in the duration and speed of blending. All these variables are rarely duplicated in the literature, in spite of their importance in determining the type and degree of solubilizing of various proteins.

It is generally considered that the water-extracted fractions represent the sarcoplasmic proteins, and the higher-ionic-strength extractions (0.3- 1 .O p ) represent the myofibrillar proteins. Myosin and actomyosin are considered to be the prominent components of the latter; they are easily separated from the extract by preferential precipitation from the solution, either by ultracentrifugation, or by lowering the ionic strength to 0.3 for actomyosin precipitation and to less than this value for myosin precipitation. Actin, on the other hand, is considered to be the prominent component of the divalent cation precipitate of the water extract from the acetone-fish powder, Pure protein preparations are rarely used in solubility tests because of the complexity of the purification steps. However, since the data obtained from extractability tests are used primarily in comparative analysis, they serve the purpose of assessing the changes in (crude) proteins that take place during frozen storage. Numerous studies in this area show a clear relationship between the decrease in protein extractability and the increase in toughness of fish fillets. When compared with myofibrillar proteins, sarcoplasmic proteins seem to be more stable, and their solubility remains unchanged ex- cept after a long storage time. Within the myofibrillar group, myosin is by far the most sensitive protein to denaturation, whereas actin (Connell, 1960b) shows a


TABLE I PARAMETERS USED TO DETECT OR MONITOR CHANGES OR DETERIORATION IN

FISH PROTEINS DURING FROZEN STORAGE

1.

2.

3 .

4.

5 .

6 .

7.

Extractability of fish proteins Total extractable proteins Protein groups: myofibrillar, sarcoplasmic, actomyosin Protein species: myosin, actin, tropomyosin, etc. Protein solubility in Aqueous buffers Detergents Proteolytic enzymes Tissues and texture Drip-thaw Water-holding properties Objective textural measurements: shear, deformation, tensile, and compressibility forces Ultrastructure features Light microscopy, scanning electron microscopy, transmission electron microscopy Extracted proteins Viscosity, molecular weight, specific volume Functional groups: available lysine, reactable SH Spectrometric analysis: UV, ORD, NMR, JR, x-ray patterns Mobility and fractionation under external forces:

Ultracentrifuge sedimentation pattern Electrophoretic pattern Isoelectric focusing pattern Chromatographic separation (ion exchange, molecular sieves, adsorption systems)

Enzymatic activity ATPase, aldolase, TMAOase, malic enzyme, glycerophosphate dehydrogenase Formation of low-molecular-weight degradation products Lipid hydrolysis: FFA Lipid oxidation: ketones, aldehydes, peroxides, free radicals, TBA TMAO hydrolysis: FrHO, DMA

very small change. Tropomyosin is considered the most stable myofibrillar protein in fish during frozen storage (Matsumoto, 1980).

B. TISSUE PROPERTIES AND OBJECTIVE PARAMETERS

This group of indices is distinctive in that intact fish tissues are used in the testing. Measuring the volume of drip-thaw and determining the changes in the water-holding properties of fillets are among the simple tests used to reflect the decrease in the capacity of fish muscle proteins to reabsorb the water of melted ice crystals during thawing. This decrease is attributed to the surface dehydration of protein or, to a lesser extent, to physical damage in the cells or cell membranes.

In addition to sensory evaluations, which are the major determinations of


quality, objective textural measurements are now recognized as powerful tools in studying the changes in rheological properties of tissues during frozen storage. Instruments have been designed to simulate the mouth-feel characteristics-for example, the cutting effect, mastication, hardness, chewiness, and cohesiveness. The relationship between various physicomechanical values (such as compressibility, deformation, tensile, rupture, and shear values) and the subjectively de- termined textural status of tissues has been established. As a result, textural changes in tissues during frozen storage can be quantitatively monitored with greater accuracy.

C. MICROSCOPIC EXAMINATION

Visual examination of the ultrastructural arrangements of fish muscles under a light microscope, a transmission electron microscope (TEM), or a scanning electron microscope (SEM) has proved useful in detecting disturbances or damage to the macro- or microstructures of tissues or cells during frozen storage. The fixing processes of tissues or tissue sections, which are necessary in such techniques, should not create artifacts by altering the ultrastructural images or by masking the microchanges we are looking for.

D. CHANGES IN THE REACTION VELOCITY OF ENZYMATIC ACTIVITY

Biochemical parameters such as changes (decreases) in the enzymatic activity of fish muscles, or in the susceptibility of fish proteins to the effects of various proteolytic enzymes, are sensitive indicators for monitoring protein denaturation as a consequence of conformational changes that may occur during frozen storage.

Few endogenous enzymes reflect a correlation between freeze damage and storage time, among them adenosine triphosphatase (ATPase), aldolase, malic enzyme, and glycerophosphate dehydrogenase. ATPase hydrolyzes ATP into ADP and inorganic phosphate. Its enzymatic activity is found in two different types of protein: myofibrillar ATPase, and sarcoplasmic ATPase. ConneLl (1960a) showed that there was .a loss in myofibrillar ATPase activity in cod muscles during frozen storage, which was dependent on the storage temperature. Although there was no detectable change in its activity in fish stored at -24"C, a noticable decrease was observed at higher storage temperatures (-22" or -14°C). Matsumoto (1980) reported that the decrease in ATPase activity of myosin was faster than actomyosin isolated from the same fish. Moreover, the ATPase activity showed faster decline, due to freezing, in the heavy meromyosin (HMM) than in intact myosin. Aldolase, a member of the Embden- Meyerhoff glycolytic group, which catalyzes hexose-diphosphates into two triose phosphates, showed a continuous decline in activity in cod and haddock stored at

280 SOLIMAN Y . K . SHENOUDA

- 14°C and was completely diminished after 60 weeks (Connell, 1966). Malic enzyme and glycerophosphate dehydrogenase are also temperature-sensitive enzymes (Gould, 1965; Gould and Peters, 197 1). Their activity declined noticeably in cod and pollack stored at -7"C, but both enzymes were quite stable at -24°C. Another enzyme that has attracted the attention of researchers is trimethylamine oxidase (TMAOase), an enzyme that, at freezing temperatures, hydrolyzes trimethylamine oxide (TMAO) into dimethylamine (DMA) and formaldehyde. Formaldehyde has been condemned for its detrimental effect on the storage quality of certain groups of fish.

E. TESTS ON EXTRACTED PROTEINS

Changes in the physical and chemical properties of extracted fish proteins give more in-depth information on the changes that have occurred at the molecular level during frozen storage. Simple tests, such as those that determine emulsify- ing capacity, or viscosity, or gel-forming properties, show the general condition of proteins. Other tests monitor changes in the more susceptible functional groups of proteins, such as free sulfhydryl groups, reactable SH groups, and available €-amino groups on the lysine residue. These tests can reveal the existence of protein cross-linking, or they can predict deformation and explain aggregation phenomena. A slight decrease in available lysine in frozen-stored fishery products has been frequently reported in the literature. The decrease in reactive SH groups in fish myosin was finally confirmed by Buttkus (1970), who explained that the presence of the other 32 stable SH groups on the myosin molecule masks the slight decrease that occurs in the few reactable SH groups near the meromyosin heads. One should note that such changes, particularly in lysine, are considered nutritionally insignificant, since lysine is not a limiting factor in fish (Poulter and Lawrie, 1977).

The mobility of protein groups subjected to external forces, as indicated by ultracentrifuge sedimentation patterns or isoelectric focusing or electrophoretic patterns, would reflect the changes that occur in the shape, size, charge, and weight (aggregation or dissociation) of the protein molecules.

Spectrometric absorption methods such as optical rotary dispersion (ORD), circular dichromism (CD), x-ray analysis, ultraviolet (UV), infrared (IR), and nuclear magnetic resonance (NMR) are other powerful techniques that explore the changes in the three-dimensional structure of proteins.

F. MEASUREMENTS OF LO W-MOLECULAR-WEIGHT DEGRADATION PRODUCTS

These tests give an indirect indication of certain reactions that ultimately lead to protein denaturation, often accompanied by off-flavors. For example, free

PROTEIN DENATURATION IN FROZEN FISH 28 1

fatty acids are formed as a result of lipid hydrolysis. Aldehydes and ketones, whether measured directly or estimated by other simple tests such as thiobar- bituric acid (TBA) or peroxide value, are good indications of the oxidative rancidity of lipids. The enzymatic breakdown of trimethylamine oxide in certain fish species into formaldehyde and dimethylamine has been used to evaluate the quality of the frozen stored seafood products.

In conclusion, many tests and methods are available to monitor alterations in fish proteins during frozen storage. Some of these tests are conducted on whole or intact fish flesh, whereas others are done on total or limited fish protein groups. Still other tests are applied to model systems utilizing purified protein preparations. These tests differ in simplicity, accuracy, and the amount of information they reveal. Consequently, they vary in their ability to suggest hypotheses or to confirm theories.

Ill. FACTORS CAUSING PROTEIN DENATURATION DURING FROZEN STORAGE OF FISH

Many factors have been reported to cause protein denaturation during frozen storage of fishery products. These factors can be grouped into three categories: ( a ) factors related to changes in fish moisture; ( 6 ) factors related to changes in fish lipids; and ( c ) factors related to the activity of a specific enzyme (TMAOase).

A. MOISTURE AS A FACTOR IN THE DENATURATION OF FISH PROTEIN

Changes in the moisture phase during freezing or during frozen storage of fish create an environment that is conducive to protein denaturation. The effects of these changes can be classified according to three major patterns: ( a ) damage due to formation and accretion of ice crystals; ( 6 ) damage due to dehydration; and ( c ) damage due to an increase in salt concentration.

1. Ice Crystal Formation and Accretion

The concept of damage during freezing as a result of the formation of ice crystals was well recognized in early studies. It has been shown that freezing, particularly at a slow rate, causes the formation of inter- and intracellular ice crystals, which leads to breakage in the cells, rupturing of membranes, and disordering of the ultrastructure of the cells and tissues. Love (1968) summed up the factors that influence the size and location of ice crystals found in frozen fish

282 SOLIMAN Y. K . SHENOUDA

tissue: the physiological status of the fish, the freezing rate, the storage time, and temperature fluctuations. In the prerigor state, the cell fluid is tightly bound to the intracellular proteins, which limit its diffusibility from inside to outside the cell. This causes the dry appearance seen in unfrozen fish tissues. It was found that, when this type of tissue (prerigor) is frozen, ice crystals are formed, mainly intracellularly, regardless of the speed of freezing. On the other hand, with the onset of rigor mortis, leading to muscle contraction, some of the cellular fluids are set free to diffuse into the extracellular spacing, giving a moist or watery appearance to the unfrozen tissues. Consequently, when this type of tissue is frozen, inter- and intracellular ice crystals are formed, depending in this case on the rate of freezing. At a slow freezing rate, the exterior of the cell cools more rapidly than the interior parts, and with the continuous dropping of temperature, the supercooled extracellular fluid reaches a critical temperature at which point water separates from the solute, forming ice crystals outside the cells. At the same time, this slow freezing rate induces a high salt concentration in the extracellular fluid, which in turn draws out moisture from inside the cell fluids by osmosis. The formation of ice crystals proceeds along the extracellular channels in the tissues. In contrast, a fast freezing rate presumably does not allow the migration of water into the extracellular spacing, and consequently smaller ice crystals, usually spear-like and separated by proteins, form within the cell, building a discontinuous ice crystal column (Love, 1968).

During storage, the small ice crystals have a tendency to melt and aggregate to the larger ones. Kent (1973, using "complex dielectric permittivity," the elec- tromagnetic technique that is capable of differential measurements of frozen (ice) and unfrozen (bound and liquid) water, showed that, during frozen storage of fish fillets at constant temperature, there is a gradual accretion of ice in the frozen system at the expense of the unfrozen water fraction.

It has also been shown that fluctuation in the storage temperature, even if the freezing temperature is quite low, accelerates the growth in the size of the ice crystals formed. With a slight rise in the storage temperature (still below freezing), the small ice crystals presumably melt faster than the larger ones, and when the temperature drops down again, the melted ice refreezes around the large (nuclei) ice crystals, forming larger and larger crystals. Obviously, the effect of temperature fluctuation on ice crystal accretion is more prominent in the critical freezing zone of -0.8" to -5.O"C (Dyer and Dingle, 1961).

Another physical phenomenon of water is that at temperatures below +4"C there is a reversal in its specific gravity; that is, decreasing the temperature below +4"C increases the volume of a specific weight of water. Therefore, storage at temperatures below freezing results in a continuous pressure from the ice crystals being exerted upon the ultrastructure, causing a disrupture of the orientation and organization of the microorganelles. Such changes were revealed through electron microscopic studies by Jarenback and Liljemark (1975a). These authors


studied the cross sections of fibers from unfrozen and frozen-stored fish tissues. On measuring the center-to-center distance along the hexagonal array between the thick filaments in the A-band of the sarcomere, where the thick and thin filaments overlap (illustrated in Fig. l ) , they found a significant decrease in the distance between the contractile units, but only after long frozen-storage periods. For example, the average distance (Table 11) between filament centers in fresh cod muscles was found to be 47.4 nm, which decreased to 46.5 nm upon freezing. But the effect of ice pressure on the compactness of the fibrils was apparent in fish stored for 2-3 years, giving an average interfilament distance of 42.5 nm. Moreover, the electron micrographs showed a collapse and deformation of the sarcoplasmic reticulum in the frozen-stored specimen. It has been postulated that a reduction of the distance between filaments favors the formation of cross-bridges between them and stiffens the fibers. Traditional tissue preparation for electron microscopy work, such as dehydration, fixation in glutaraldehyde or osmium tetroxide, causes shrinkage of embedded specimens. This artifact introduced by the preparation technique can overshadow the effect of the pressure-exerted by ice crystals-on the reduction of interfilament spacing during frozen storage. Thus, Jarenback and Liljemark recommended the use of freeze etching technique for accurate measurements of distances between myofilaments.

r-- s" +sarcomere-- . +=*=*

2-line A 1 t s a *

,thick f i l a m e n t s

cross section at "S-S" FIG. 1 . Schematic representation of the rnyofibrils. Top: Longitudinal arrangement of thick and thin filaments in the sarcomere. Bottom left: Diagram showing arrangement of thin filaments, which have a double helical structure, and thick filaments in which myosin globular heads protrude for cross-bridging with the thin filaments. Bottom right: Cross section in the sarcomere at which thick and thin filaments overlap. This also shows the hexagonal array of the thick filaments. d is the center-to-center distance of the thick filaments. used in Table 11.


TABLE Il DENSITY OF FILAMENTS, MEAN OF INTERFILAMENT SPACING, AND MINIMUM

INTERFTLAMENT SPACING IN TRANSVERSE FRACTURE OF COD MUSCLE"

Filament Interfilament Minimum Muscle and treatment packing spacing interfilament

(cod muscle) density (nm) spacing (nm) ~

Unfrozen 161 41.4 43.0 Frozen at -40°C for 1 week 166 46.5 42.1 Frozen-stored at -20°C for 3-3.5 years 209 42.5 36.0

"After Jarenback and Liljemark (1975a). The data show a significant decrease in the distance between the thick filaments in the sarcomere with freezing and frozen storage.

2. Dehydration

Proteins are amphiphilic molecules. They are built up from hydrophobic as well as charged or polar amino acids, which vary in their number, ratio, and sequence. As an example, water-soluble proteins are generally constructed from 25-30% hydrophobic amino acids, 45-50% ionic or uncharged hydrophilic amino acids, and the rest of amino acids have relatively little preference for being in an aqueous or a hydrophobic environment (Tanford, 1973). The conformation of most proteins usually follows a general pattern in which a substantial fraction of the hydrophobic side chains are buried inside the molecule. In most native proteins, however, some hydrophobic groups remain exposed at the molecule surface or in crevices. It has been postulated (Lewin, 1974) that water molecules also adhere to those exposed hydrophobic group side chains in a highly organized water barrier and mediate the hydrophobic-hydrophilic linkages between molecules.

Moreover, the stability of the three-dimensional structure of protein molecules is highly dependent on a network of hydrogen bonds, many of which are mediated through water molecules.

Thus, dehydration of protein molecules through freezing-that is, migration of hydration water molecules to form ice crystals-would result in a disruption of the hydrogen bonding system as well as the exposure of surface regions (hydrophobic or hydrophilic) of the protein molecules, and consequently would leave these regions unprotected and vulnerable.

Hydrophobic-hydrophobic and hydrophilic-hydrophilic interactions could then take place, either within the same protein molecule, causing deconformation of the three-dimensional structure, or between adjacent protein molecules, inducing protein-protein interactions and consequently aggregation.

The onset of the dehydration effect in protein molecules at freezing temperatures is not clearly demonstrated in the literature. However, model system studies by Suzuki (197 1) at nonfreezing temperatures showed that the maximum dena-


turation rate of myofibrillar proteins during dehydration occurred when the dehydration exceeded a critical point that coincided with an abrupt change in the line plotting the residual moisture against half the value of the NMR spectra of dehydrated sea bass muscle, which is calculated as 20-28% moisture.

3 . Increase in Solute Concentration

An increase in solute concentration is also the basis for one of the earliest theories of protein denaturation during frozen storage. During the freezing of fish muscle, part of the water will freeze out, but a considerable portion may remain unfrozen, even at temperatures well below the freezing point. This fluidity is presumably due to energetically or entropically preferred states-that is, the influence of polar forces from sites on the protein matrix and the influence from ions in solution (Kent, 1975). Several analytical techniques, such as calorimetry, thermodynamics, nuclear magnetic resonance, or electrical conductivity (dc conductivity or complex dielectric permittivity), are capable of detecting the existence of unfrozen water quantitatively at freezing temperatures. Sussman and Chin (1966), using NMR spectroscopy (Fig. 2 ) , showed that the percentage (by weight) of liquid water in cod or flounder tissue decreased

8 0

50

4 0 .

3 0-

20-

10-

0 C O D FLOUNDER

/+ 0 -10 -20 -30 - 4 0 -70

TEMPERATURE ( 'C I

FIG. 2 . Percentage by weight of liquid water in cod and flounder muscles (calculated from NMR pea!-area ratio) as a function of temperature. After Sussman and Chin 1966; copyright 1966 by the American Association for the Advancement of Science.


rapidly, almost in an exponential pattern, as a function of temperature, and the liquid water phase persisted down to about -70°C.

The amount of liquid water remaining unfrozen is stated to be temperature- and time-dependent, The liquid water phase is shown to decrease very rapidly at temperatures between 0” and -1O”C, and more slowly at lower temperatures (Dyer and Dingle, 1961). This rapid freezing rate of tissue water followed by slow freezing may be interpreted in terms of the “free” and “bound” water phases. Charm and Moody (1966) were able to show that all the unfrozen “free” liquids in haddock fillets disappeared at temperatures between 0” and 10°F (-17.7” to -23”C), and what was left unfrozen at temperatues below these levels was in “bound” form.

The effect of frozen storage time on liquid solidification is not as drastic as the effect of temperature. A slight, gradual decrease is seen as the liquid water fractions form into ice crystals, or enlarge the existing ice crystals, in frozen fish muscle (Kent, 1975).

Accordingly, at common freezing temperatures (- 10” to -20°C) more than 90% of the moisture will freeze out, leading to roughly a tenfold increase in the concentration of soluble solutes.

An increase in salt concentration is known to affect cell permeability and protein properties. A drastic increase in cell permeability to solutes was observed when fish muscles were soaked in concentrated brine at 0°C (Duerr and Dyer, 1952) and also when frozen fish muscles were immersed in brine solutions at temperatures above freezing (Deng , 1977).

Theoretically, the effect of salt concentration on protein denaturation, aggregation, or dissociation could be based on the effect of salts on the secondary forces (ionic, van der Waals, hydrogen, and hydrophobic forces), which help to stabilize the tertiary and quaternary configuration of protein macromolecules. For example, ionic bonding is basically possible between appropriately charged groups within the protein molecules or between different molecules or sub- molecules of proteins, lipids, carbohydrates, nucleotides, etc. In addition, groups with prominent or inducible dipoles (that is, -OH, -CO) would also be expected to interact with ionic groups. The stability of ionic binding, as well as all other secondary forces, is dependent on the dielectric constant, the pH, and the ionic strength of the media. Thus, increasing the salt ions presumably will cause competition with the existing electrostatic bonds and the breakdown of some of them. It will also disturb the other secondary forces; and, although the net result cannot be precisely predicted, it will be a mixture of dissociational, aggregational, and conformational changes. The critical salt concentration-the concentration at which the maximum rate of protein change and damage occurs-differs according to the type of salts and the type of tissues involved. As cited by Connell(1964), however, it is calculated to be around 10% NaCl or the equivalent.


a. Effect of Salt Concentration on Specific Fish Myofibrillar Proteins. Un- fortunately, the identification of all the myofibrillar proteins that build up the sarcomeres of fish tissues is not yet complete, and the presence or absence of certain protein species discovered in mammal tissues has not been confirmed in fish tissue. Table 111 lists the proteins found in mammalian sarcomeres, as compiled by Lowey (1972). One would expect the existence of the same (or counterpart) proteins in fish tissues similar to those found in mammals. A survey of the literature showed that studies on fish myofibrillar proteins as related to freeze-denaturation are centered mainly on the two major proteins-myosin and actin.

Myosin molecules are the building units of the thick filaments in the sarcomeres. Myosin possesses ATPase enzymatic activity, which transfers the chemical energy of ATP into the contractions of the muscles. The myosin molecule is a large molecule, composed of two heavy polypeptide chains existing in a supercoil helical conformation. At the end of the molecule, both of the polypeptide chains are folded into a globular structure. The molecular weight of this part of the myosin molecule is estimated at 470,000. In addition, some (light) peptide chains are bound to the globular head with secondary forces. In most animals, these light chains are estimated to be three or four in number, with an average molecular weight of 20,000. In the case of carp myosin, however, only a single light chain is found (Tsuchiya and Matsumoto, 1975). It is believed that the characteristics and properties of myosin are strongly related to the SH groups it possesses, particularly those located near the globular head on the light

( i ) Myosin.

TABLE 111 TYPES AND PROPERTIES OF CONTRACTILE PROTEINS FOUND IN MAMMALIAN MUSCLES"

Subunit Localization Total protein Sedimentation Viscosity Molecular molecular a-Helix in myofibril (%) Protein coefficient (mVgm) weight weight (%)

Thick filament 55 Myosin 6.4

Thick filament 2 C-Protein 4.6 Thin filament 25 G-Actin 3.3 Thin filament 5 Tropomyosin 2.6 Thin filament 5 Troponin 4.0

Z-line Trace a-Actinin 6.2 M-line Trace M-Protein* 5.1

5.4

210

14 4

34 4

9

4.5 -

470,000

140,000 42,000 w 0 0 0 80,000

180,000 165,000 88,000

200,000 20,000

140,000 42,000 32,000 37,000 24,000 21,000 90,000

165,000 43 ,000

57

10 26 90 35

60 0

26

"Reproduced from S. Lowey, 1972. *Modified according to Trinick and Lowey, I977


chains. Myosin properties such as extractability, sensitivity to proteolytic diges- tion, and ATPase activity vary according to its source. Differences are found in myosin extracted from different animals or even from different parts of the same animal (Chung Wu, 1969).

Myosin is considered the most sensitive myofibrillar protein with respect to freeze denaturation studied so far. Its loss in extractability during frozen storage reaches 80% of that of the total cod myosin. In frozen trout, the aggregation process of myosin reaches its maximum near the eutectic point of the myosin- KCI-water solution, which is estimated to be near - 11°C (Buttkus, 1970).

Model system studies of myosin preparations show that, at high ionic strength, myosin molecules experience a rapid, reversible monomer-dimer equilibrium (Godfry and Harrington, 1970). With an extended period of exposure to a concentrated solution, the myosin molecules dissociate into subunits: a heavy core, and light components (Dreizen and Gershman, 1970). Furthermore, the light chains undergo irreversible aggregation during prolonged salt treatment, especially in the absence of thiol proteciton. The heavy chain core also forms insoluble aggregates, accompanied by conformation changes. Figure 3 diagram- matically illustrates the reactions that take place in myosin in high salt concentrations.

(ii) Actin. Actin is the second most abundant protein in the contractile units. It forms the backbone of the thin filaments, where the globular actin beads (G-actin) are arranged in a double-stranded right-handed helix, with tropomyosin and troponin in the two grooves of the coiled ribbon of actin. Actin monomers (G-actin) are relatively small, having a molecular weight close to 46,000 and a diameter of 55 A.

Actin is relatively stable during frozen storage, compared with myosin. Con- nell (1960b), working with cod, showed that after storage at - 14°C for up to 30 weeks, when over 80% of the myosin was rendered insoluble, only a small change in actin solubility was found.

Relatively little work has been done on fish actin. Moreover, it has recently been recognized that most of the actin preparations from fish are probably ser-

MYOSIN Dissociation Aggregation

FIG. 3. Illustration showing the effect of storage time at high ionic strength (p) on myosin molecules. Increased ionic strength caused reversible dissociation of myosin into heavy cores and light polypeptide chains, but with extended storage at high ionic strength, irreversible aggregation between these subunits takes place.


iously contaminated with other proteins. Troponin, actinin, and tropomyosin were found to be tightly bound to the crude actin preparation, and additional purification (chromatographic) steps are required to obtain pure actin (Ebashi and Maruyama, 1965; Drabikowski et al., 1968; Shenouda and Pigott, 1975a).

Model system studies using pure fish actin (Shenouda and Pigott, 1975b) showed that increasing the ionic strength of the buffer solution caused a gradual polymerization of the soluble actin (G-actin), which finally precipitated as an insoluble gel. This polymerization process was accompanied by a significant increase in the actin’s ability to bind polar and neutral lipids, forming insoluble lipoprotein complexes. When the ionic strength was increased over 1.0 p , a steeper increase in the binding of neutral lipids to actin was observed (Fig. 4), indicating that a higher concentration of salt (KCl in this case) induces changes in the actin molecules pertaining to a more hydrophobic nature. Later studies (Shenouda and Pigott, 1976) showed that this hydrophobic interaction between neutral lipids and actin in fish is stronger than the forces that bind the polar (charged) lipids to the actin.

b. Effect of Divalent Cations Logically, the increase in solute concentration, as a result of freezing, will include an increase in the concentration of certain cations, such as calcium and magnesium. The increase in concentration of these divalent cations has been shown to cause the contraction of muscle actomyosin associated with ATP splitting (Briskey and Fukazawa, 1971). In model system studies, Ca2+ and Mg2+ initiated polymerization of G-actin into an uncontrollable length of fibrous actin (F-actin). Further studies with fish actin by Shenouda and Pigott (1972b, 1977) showed that polymerization of actin in the presence of excess divalent cations gives a different binding pattern with lipids than that given in the absence of such cations (Table IV). Their results showed that the presence

$100 .- a .- - u 5 80 0 n

60 c c

40 2

FIG. 4. Effect of increasing

7 .*- - - - - - -- -.

c- neutral Lipids * polar Lipids

- i

0.1 0.5 10 2.0 3.0

Ionic Strength

ionic strength on the formation of fish lipid-actin complex. A linear increase in the lipid (polar or neutral) bound to actin was notice4with increasing KCI concentration up to 0.3 p. A further increase in KCI concentration beyond 1 .O p caused a noticeable increase in bound neutral lipids, indicating the effect of high salt concentration in inducing changes in the actin molecules, causing them to be more hydrophobic. After Shenouda and Pigott, 1975b.


TABLE IV EFFECT OF PRESENCE AND ABSENCE OF DIVALENT CATIONS ON

LIPID-ACTIN INTERACTION n , b

Incubation temperature Lipid bound to actin Treatment of fish actin ("C) ('70)

Actin + neutral fish lipids Actin + polar fish lipids Actin + neutral fish lipids Actin + polar fish lipids Actin + neutral lipids + Ca'+ Actin + neutrallipids + Mg'+ Actin + polar lipids + Ca'+ Actin + polar lipids + Mg'+

4

4 Room temperature Room temperature

4 4 4 4

33 48 46 57 93 89 39 33

" After Shenouda ( 1974). bFour hundred micrograms of '*C-labeled fish lipids were incubated with 7 mg of actin, and the

mixture was fractionated over sucrose gradient centrifugation. In the absence of the divalent cations, the actin interacts with polar lipids more than neutral lipids do, particularly at higher incubation temperatures, indicating the role of hydrophilic interaction. In the presence of divalent cations, there was a remarkable increase in neutral lipid-actin interaction, indicating an increase in the hydrophobicity of actin molecules.

of excess Ca2+ or Mg2+ induced the exposure of more hydrophobic regions on the F-actin molecules, which stimulates the binding of neutral lipids and depresses the hydrophilic interaction between polar lipid and actin. In other words, the increase in Ca2+ or Mg2+ cations changes the nature of the actin, and the molecules are more water-repellent or less soluble in aqueous media. These findings show that the role of the fish actin-lipid-metal system is different from the role designated for divalent cations in other protein systems, such as wheat gluten (Fullington, 1969) or cell membrane (Braun and Radin, 1969), in which calcium or magnesium ions act as a bridge between charged groups on the protein and lipid molecules and manifest the formation of polar lipid-protein complexes.

Finally, one should note that these model system studies on fish actin clearly demonstrate the detrimental effect of increased salt concentration, including divalent ions, on the solubility of actin. But, since actin is a relatively stable protein compared with the rest of the myofibrillar proteins, one would expect that the increased salt concentration, due to freezing, would play an important role in the denaturation of these less-stable proteins.

B. FACTORS RELATED TO FISH LIPIDS

The effect of fish lipids on protein during frozen storage varies according to the state of the lipids. Intact (unhydrolyzed, not oxidized) lipids differ in their


action from their hydrolyzed products; also, the free fatty acids will have a different influence from the oxidized lipids. In this section the role of each of these lipid forms on fish proteins during frozen storage will be discussed sepa- rately.

I. Intact Fish Lipids

The term “intact,” as used here, refers to lipids that have not been subjected to partial or total hydrolysis or oxidation.

The role of intact lipids on the stability or instability of fish myofibrillar proteins is still unclear. Although there is some evidence that their presence, particularly in biological systems, is essential and plays a protective role for the proteins involved, other evidence, particularly from model system studies, indicates that they can have a detrimental effect on proteins by forming lipoprotein complexes, which denature the protein properties or make them more liable to denaturants.

Early frozen-storage studies (Dyer, 1951; Simidu and Simidu, 1957) on various fish species showed an apparent relationship between their stability and their fat content. Dyer and Dingle (1961) found that lean fish such as lizard fish, cod, and haddock, with a fat content of less than 1%. were less stable and showed a fairly rapid decrease in protein (actomyosin) extractability when compared with fatty fish species ( 3 - 10% lipids) such as yellowtail, halibut, and rosefish. These findings led to the hypothesis, originated by Dyer, that the presence of such moderate levels of lipids may protect the fish proteins or increase their resistance to denaturation during frozen storage. This protective effect was credited to the neutral lipid fractions such as triglycerides. Their presence is presumed to diminish or counteract the detrimental effect of the free fatty acids (FFA). It is assumed that neutral lipid droplets will dissolve the FFA and neutralize their hydrophobic effect on protein; or it is possible that, by dissolving the FFA, neutral lipids will dilute their action or compete with them for the binding sites on the protein.

Love and Elerian (1965), in a study on the cryoprotective effect of glycerol on frozen cod muscles, postulated that the added glycerol might indirectly protect the protein by lowering the concentration of cell salts formed in the frozen material. As a corollary to this hypothesis, one should not exclude the possibility that the protective effect of lipids in fatty fish could also be due to a similar effect in which the lipid dilutes or hinders the increased concentration of solute formed during freezing.

Other evidence of the protective effect of lipids-in this case phospholipids--on proteins is derived from studies on the coexistence of actomyosin and lecithin in fish tissues (Ikeda and Taguchi 1967, 1968; Taguchi and Ikeda 1968a,b). These studies showed that the solubility, stability, and ATPase

a . The Protective Effect of Intact Lipids.


activity of tuna actomyosin are proportionally affected by increasing amounts of lecithin, in reduced form. They also showed that delipidation of actomyosin by phospholipase-C (which breaks down lecithin) caused a decrease or total loss of actomyosin ATPase, which was completely restored with the re-addition of lecithin.

Lipids have also been shown to have a synergistic effect in protecting frozen- stored proteins. In their studies on cryoprotective additives, Akiba et al. (1967) found that the presence of lipids such as lecithin or plant oil enhances the effect of other protein-protective additives, such as polyphosphates or sugars, in pre- serving the quality of frozen minced fish. The synergistic, cryoprotective role of these added lipids was observed in the decreasing amount of drip and in the improvement of some of the textural properties of frozen minced fish compared with frozen mince that had not been treated with a combination of lipid and phosphates (or sugars).

b. The Detrimental Effect of Intact Lipids, During frozen storage of whole or minced fish tissues, the pressure exerted by the ice crystals that have formed on the cellular structure and the breakdown in membranes or deformation in other microorganelles can lead to disorientation or liberation of lipid and protein components from their natural compartments and open the way to new forms of contact between lipids and proteins. Since these lipid and protein moieties are derived from different locations in the cell, they would be dissimilar to natural lipoprotein complexes, and their interaction would probably form unconven- tional lipid-protein complexes, usually absent in Nature, which affect the textural quality of the muscle tissue. This hypothesis was extrapolated from the results of model system studies by Shenouda and Pigott (1974-1977), in which lipids and proteins extracted from the same fish were incubated together. A lipid-protein interaction took place, and insoluble lipoprotein complexes were formed.

In their work, fish actin (G-form) was incubated with fish polar or neutral lipids, at refrigerated temperatures. The mixtures were then separated (centrifug- ally) over sucrose gradient, resulting in the formation of an insoluble lipoprotein fraction. When other mixtures of actin-tropomyosin preparations were separated from the incubated lipid electrophoretically, over SDS-polyacrylamide gels (Fig. 3, a major portion of the actin and all the accompanying tropomyosin stayed on top of the gels (that is, did not pass through), indicating the existence of high- molecular-weight, insoluble lipoprotein aggregates, which were not dissociable by sodium dodecyl sulfate.

Fish myosin, which behaved differently from actin, did not form complexes with added lipids after incubation. However, when a myosin preparation was stored at high ionic strength before the addition of lipid, a significant amount of lipid-protein complexes was detected (Shenouda and Pigott, 1974). As discussed


FIG. 5 . Electrophorograms showing the formation of high-molecular -weight aggregates of fish proteins that did not dissociate with urea and SDS and remained at the top of the gels when an actin-tropomyosin preparation was incubated (at room temperature) with fish lipids, particularly polar lipids. The same pattern was obtained, to a lesser extent, when the preparation was incubated at refrigerated temperatures. (A) SDS-polyacrylamide gels of fish actin and tropomyosin preparations (actin is the top band). (N) and (P) are neutral and polar fish lipids, respectively. (A+N) and (A+P) are neutral and polar lipids incubated with fish protein preparation before electrophoresis, respectively. After Shenouda and Pigott, 1977.

earlier, a buildup of high salt concentration in frozen fish tissues can be formed from the partial freezing of tissue water at temperatures above -70°C. Again, the valid deduction that freezing causes an increase in the divalent cations, indicates not only an increase in lipid-protein interaction but also an increase in the hydrophobicity of the proteins (Table IV).

Another indirect detrimental action of fish lipid is seen in the possibility that it might enhance other denaturant effects. That is, when denaturation of fish protein is triggered by other factors such as heat, ionic strength, or foam formation, the degree of denaturation may be intensified in the presence of fish lipids. This was shown qualitatively (Shenouda, 1974) in studies of the effect of heat in the absence and in the presence of fish lipid on the aggregation of actin and tropomyosin. The presence of lipid, particularly the polar fraction, significantly enhanced the effect of heat on the aggregation and insolubilization of both actin and tropomyosin. Nevertheless, whether the presence of lipid intensifies the

294 SOLIMAN Y, K, SHENOUDA

effect of other factors responsible for the denaturation of fish proteins during frozen storage is a question that still needs a more specific answer.

The forces of interaction between intact fish lipid and proteins (actin and myosin, so far) indicate the contribution of both electrostatic and hydrophobic forces. Electron paramagnetic resonance (EPR) studies (Shenouda and Pigott, 1976), as well as evidence of the resistance of the lipoprotein aggregates to dissolve or dissociate in urea and SDS, suggest the existence of stronger hydrophobic participation.

2 . Lipid Hydrolysis: Free Fatty Acid Interaction

As the correlation between the toughness of frozen-stored fish and the decrease in protein extractability has been established, a similar correlation between the decreased protein extractability and the accumulation of FFA in the frozen-stored tissue has been observed. As a result, the detrimental effect of FFA on the textural quality of frozen-stored fish has been repeatedly documented. Free fatty acids are derived from enzymatic or nonenzymatic hydrolysis of lipids, particularly the phospholipids, which are located primarily in the cell membrane. Both lipase and phospholipase enzymes have shown significant activity in producing FFA ip various fish species stored at - 12" or - 14°C (Olley et al., 1962).

The accumulation of FFA was found (Dyer and Dingle, 1961) to increase with prolonged storage time and at elevated frozen storage temperatures. The maximum rate of lipid hydrolysis in many fish species (cod, sole, halibut, and many gadoid species) was found at temperatures just below freezing-at - 4°C (Lovern and Olley, 1962). Also, the rate of lipid hydrolysis in fish was shown to be faster in dark muscles than in white tissues (Olley et af., 1962).

During frozen storage of fish, an initial rapid rate of FFA formation was noticed, followed by a much slower rate. The decrease in protein extractability followed more or less the same pattern of FFA accumulation. Anderson and Ravesi ( 1970b) reported that a decrease in protein extractability occurred more rapidly in the first 8-10 weeks than in subsequent storage periods. Also, the decrease in extractability was slower in muscle stored at - 18°C than in muscle stored at - 12°C. The same authors (1969) further explored the fact that storage in ice (before freezing) stimulates fat hydrolysis; they found a pronounced decrease in protein extractability from ice-stored frozen fish, which indicates that FFA-protein interaction may begin before freezing and cause the frozen fish to deteriorate at a much faster rate.

Jarenback and Liljemark (1975b) were concerned about lipid oxidation. By protecting their model system (myofibrils and linoleic acid) with antioxidants (propylgallate) and a nitrogen atmosphere, they were able to prove that the loss in protein extractability from the myofibrils in this case could be ascribed to the FFA effect rather than to oxidative products.

King et al. (1962) studied the factors that influence FFA-myofibrillar protein


interaction. They showed that the rate of interaction depended on the type and degree of unsaturation of the fatty acids, their concentrations, and the incubation (storage) time. Polyunsaturated fatty acids insolubilized more fish myofibrillar proteins than did less unsaturated ones, and shorter fatty acids were more powerful than high-molecular-weight FFA.

Free fatty acids are believed to attack primarily the myofibrillar proteins. Their binding to sarcoplasmic proteins is not excluded but apparently is less effective in insolubilizing them. Actomyosin was considered by many researchers to be the prime target and to be largely unextractable in the presence of FFA. Childs (1974) reported that tropomyosin was also highly insolubilized, owing to the effect of hydrolyzed oil.

The effect of FFA on the myofibrillar structure as revealed by electron microscopy (Jarenback and Liljemark, 1975b) showed that low levels of FFA induced aggregation of the extracted proteins, but the fibrils retained a great deal of their original shape. Also, at low levels of FFA concentration, there was no apparent micellar formation, indicating an even spread of FFA over the entire myofibrillar structure. At higher levels of FFA, however, micelles were found adhering to the actomyosin filaments. And when the myofibrils were treated first with high levels of FFA (over 10 pmoles of linoleic acid per gram of muscle) and then extracted with a high-ionic-strength buffer, most of the thick filaments disappeared (dissolved) from the residual (unextractable) myofibrils, while the extracted proteins showed fewer actomyosin filaments but were still rich in myosin, indicating that myosin extraction is not seriously affected during the treatment.

The mechanism of FFA-myofibrillar protein interaction has not yet been explained sufficiently. It is generally perceived that covalent bonding is not a major participant and that the interaction mechanism occurs primarily through secondary forces+lectrostatic, van der Waals, hydrogen, and hydrophobic forces. The dependence on pH (Hanson and Olley, 1965) suggests the participation of ionic and hydrogen bonding. The low sensitivity of the FFA-protein linkages to salt solution (Anderson and Ravesi, 1970a) and their sensitivity to sodium dodecyl sulfate (Connell, 1965) are evidence of the involvement of hydrophobic forces in the formation of aggregates. However, covalent bonding between the double bonds of the hydrocarbon chain pf the fatty acids and the sulfhydryl groups of the protein, as suggested by Robenson (1966), could possibly be in the route of the protein-FFA interaction mechanism during frozen storage.

In conclusion, the apparent results of the presence of FFA, as postulated by Sikorski et al. (1976), is that the FFA attach themselves hydrophobically or hydrophilically to the appropriate site on protein surfaces. Consequently, they may create more hydrophobic regions in place of polar or charged groups and surround the protein surface with a more hydrophobic microenvironment. Thus, the end result is a decrease in protein solubility in aqueous buffers, or further intermolecular linkages extensive enough to decrease extractability.


3. Lipid Oxidation

The oxidation of lipids in frozen fish greatly shortens the shelf-life of many species, especially fatty fish, because of adverse consumer reaction to rancidity. In addition to the problem of rancidity, oxidized lipids interact with proteins, causing undesirable changes in the nutritional and functional properties of the proteins. It has been demonstrated that during frozen storage the product of lipid oxidation renders the fish tissue proteins into harder, more elastic, insoiuble complexes (Takama et al., 1972; Takama, 1974a). Loss of specific amino acids such as cysteine, lysine, histidine, and methionine, as well as damage to other pigmented proteins such as cytochrome c and hemoglobin, also have been reported (Roubal and Tappel, 1966). Browning is also attributed to the formation of certain oxidized lipid-protein complexes. For example, fluorescent compounds have been isolated from the oxidative reaction of linoleate and myosin in frozen Coho salmon. These compounds were shown to contain phosphorous and C=N functional groups (Braddock and Dugan, 1973).

Fish lipids are known for their susceptibility to oxidative rancidity, particularly during frozen storage. They generally contain a high proportion of unsaturated fatty acids with four, five, and even six double bonds. For example, lipids from Atlantic mackerel (Ackman and Eaton, 197 1) contain about 70% unsaturated fatty acids, of which 30% are polyunsaturated. At temperatures of -18" to - 26"C, the polyunsaturated fatty acids oxidize much faster than the monoethylenic acids (Ke et al., 1976), yielding various oxidative products including propanal, pentanal, malonaldehyde, and hexanal.

In a study of jack mackerel stored at -25"C, Shono and Toyomizu (1973) found that phospholipids underwent faster hydrolysis and oxidation than did neutral lipids, but the oxidation of FFA was the most severe, particularly of those of G2:6, indicating the importance of enzymatic hydrolysis of lipids in increasing the rate of their oxidation.

The rate of lipid oxidation in food systems has been recognized as a definite function of moisture content (Labuza, 1974). As the water activity decreases-in this case as a result of freezing-the rate of lipid oxidation increases. This increase is attributed to the effect of an increased concentration of metal catalysts in food systems (Labuza et al . , 1970). Oxidative rancidity reactions are also accelerated in the presence of natural pro-oxidants such as blood hemoglobin; their presence in certain tissues or their distribution, as in minced fish products, increases the potential for oxidation and accelerates protein denaturation during frozen storage.

The mechanism of the reaction between oxidized lipids and proteins has not been fully elucidated. Varma ( 1967) has proposed two mechanisms in proteinaceous food systems. The first mechanism works through unstable free radical inter- mediates of lipid peroxidation, which can abstract hydrogen from labile side- chain groups-such as SH-and cause two types of polymerization: Water-


soluble polymers in which lipid is not incorporated, and water- insoluble polymers which are formed by cross-linking of proteins through an additional reaction involving lipid peroxy radicals. The second mechanism works through stable oxidation products such as carbonyl compounds-for example, malonaldehyde, propanal, and hexanal-which react covalently with side-chain groups of protein, primarily histidine, methionine, cysteine, and lysine. More attention has been given recently to the free radical route as a cause of protein damage. It is believed that the free radicals are transferred to proteins, forming protein-free radicals (Karel et al., 1975; Schaich and Karel, 1975), which in turn could initiate various reactions such as cross-linking with other proteins or lipids, forming protein-protein and protein-lipid aggregates.

Thus, the free radical mechanism could explain the high capacity of oxidized lipid to interact with fish proteins. An experiment conducted by Jarenback and Liljemark (1975b) indicates that the oxidized form of linoleic acid (hydroperoxide linoleic acid) was far more reactive and effective in insolubilizing fish proteins than was the free fatty acid itself. It required only one-tenth the concentration of hydroperoxide linoleic acid (compared with linoleic acid on a molar basis) to completely insolubilize the same amount of fish protein in solution.

Differences were also noted between oxidized lipids and FFA in the type of protein aggregates they induce and in their mode of action. Model system studies of fish actomyosin (Takama 1974b) showed that the aliphatic aldehydes-for example, propanal-react with the sulfhydryl groups of actomyosin (at -20°C) to form inter- and intramolecular mercaptal during the initial stage of frozen storage, whereas short-chain FFA-for example, caproic acid-are bound to actomyosin through secondary forces causing an imbalance in the net charge on the actomyosin. Moreover, electron microscopic examination of fish fibers revealed that the oxidized lipids (hydroperoxide linoleic acid) have a direct effect on myosin itself. After extraction of the myofibrils, which were incubated with the hydroperoxides, with 0.5 ionic strength buffers, the remaining sarcomeres retained the A-bands (which are composed primarily of myosin). In other words, the myosin of the thick filaments was not extractable after its contact with peroxidized lipid. In contrast, free fatty acids did not affect the extractability of myosin and the thick filaments as much they affected total actomyosin extractability.

Thus, it is believed that the oxidized products of lipids bind (attack) specific, susceptible functional groups on proteins, among them cysteine-SH, the €-amino groups of lysine, the N-terminal amino groups of aspartic acids, tyrosine, methionine, and arginine (Kuusi et al., 1975; Buttkus, 1967). Consequently, these interactions increase the hydrophobicity of the filament proteins (depriving them of the charged or polar groups), and the proteins will be less water-soluble. Also a stronger hydrophobic interaction may occur between individual filaments, entangling them into aggregates.


C. ENZYMATIC ACTIVITY OF TMAOASE AS RELATED TO PROTEIN DENATURATION DURING FROZEN STORAGE

It has been observed that some fish species, even those with similar chemical composition, are less stable than others during frozen storage. In particular, such species undergo rapid textural deterioration, resulting in toughness and sponginess, accompanied by a noticeable loss in protein extractability. There have also been a number of reports on the presence of formaldehyde (FrHO) and dimethylamine (DMA) in those species that are characterized by their poor keeping qualities. But not until fairly recently has the fate of trimethylamine oxide (TMAO), a component that occurs in marine fish and shellfish, been carefully studied and its degradation in fish muscles into the secondary amine DMA and FrHO in a 1: 1 molar ratio elucidated (Amano and Yamada, 1965). Soon after, a correlation between the formation of FrHO and a deterioration in texture in fish species, particularly the gadoid family, was confirmed. However, it was the work of Amano and Yamada that first showed that FrHO and DMA are produced from TMAO through the existence of a specific enzyme. Subsequent investiga- tions by other workers confirmed the fact that the enzyme, TMAOase, exists in a limited group of marine animals and is capable of activating, in vivo and in vitro, the reaction

TMAO DMA + FrHO

at freezing temperatures.

1. Distribution of TMAO and TMAOase

Trimethylamine oxide is a compound naturally present in many marine animals. Its physiological role is believed to be similar to the function of urea or uric acid in land mammals; it is excreted to maintain nitrogen balance. Surveys of the distribution of TMAO in various marine animals showed that its presence and the amount vary widely. Generally, TMAO is either extremely scant or entirely absent in freshwater fish. In marine species, the elasmobranchs (cartilage fish) contain a higher amount of TMAO than the teleosts (bony fish) (Konosu et al . , 1974; Suyama and Suzuki, 1975). Among the teleosts, the gadoid family (cod, pollack, haddock, whiting, hake, and cusk) contain the highest amounts of TMAO, whereas the flatfish (plaice, flounder, sole, sand dab, etc.) have the lowest amounts. Mollusks, such as squid and octopus, resemble the elasmobranchs in having a high level of TMAO, whereas crustaceans, such as shrimp and crab, contain moderate amounts of TMAO (an average of 50 mg N per 100 gm of muscle). On the other hand, the bivalves (clams, oysters, etc.) and echinoderms (starfish, sea urchins) contain very small amounts of TMAO.


The distribution of the enzyme (TMAOase), which is usually detected by measuring its end products (DMA and FrHO), has been surveyed by many workers (Castell et al., 1970, 1971, 1973: Babbit et al., 1972; Harada, 1975; Hiltz et al., 1974, 1976; Tomioka et al., 1974). Their results showed that the enzyme exists only in a limited group of marine animals, and its activity varies widely among species, types of tissue, and storage temperatures. Besides fish, a few members of the invertebrates (a few species of squid, bivalves, and gas- tropodes) show some capacity to form FrHO and DMA (Harada, 1975). On the other hand, crustaceans and other commercial invertebrates, such as scallops, lobsters, and shrimp, apparently lack this enzyme (Castell et al., 1970). The highest activity of the enzyme was reported in fish species belonging to the gadoid family, in which the formation of DMA and FrHO was greatest in species that had the largest amount of dark lateral muscle in the fillets. Red hake showed the highest activity, and haddock showed the least. In contrast to the gadoids, many other commercial fish species contained no enzyme, among them the flatfish group (halibut, flounder, etc.), ocean perch, rockfish, and many others.

The activity of the enzyme was not confined to the muscle tissues; in fact, the highest activity was found in some organs of the viscera (Amano and Yamada, 1965) such as the pyloric caeca, bile bladder, liver, spleen, and kidney. Dark muscles formed DMA and FrHO, whereas white muscles did not, even if they possessed high contents of the precursor TMAO (Tokunaga, 1974; Castell et al . , 1971).

Consequently, processing operations used in the fish industry that cause distribution of the enzyme throughout other tissues that lack it would result in rapid deterioration in the textural quality of the frozen-stored products. An example of such a process is the use of bone-meat separators for producing mince, in which the white flesh is mixed with the dark. The stability of frozen minced meat made from fish species that form DMA and FrHO is exceeding low, and deterioration takes place twice as fast as it does in intact fillets of the same species (Babbitt et al., 1972; Hiltz et al., 1976; Sorensen, 1975), particularly if the mincing has been done without effective cleaning and removal of blood and intestinal organs. Also, frozen flesh that has been mechanically separated from the fish frames (backbone skeletons remaining after removal of fillets from beheaded and gutted fish, usually including the kidney) shows a rapid textural deterioration as well as a rapid loss in protein extractability accompanied by a higher rate of DMA and FrHO formation (Jarenback, 1975; Dingle and Hines, 1975). These findings are attributed to the high activity of TMAOase, which presumably is derived from the kidney. Furthermore, when minced flesh from flounder, plaice, or Atlantic mackerel, which are known to lack TMAOase but do contain TMAO, and are considered to be relatively stable as mince (Fig. 6), are mixed in a 4: 1 proportion with minced hake (a gadoid fish), there is a noticeable and rapid deterioration in the mixed mince equal to the deterioration rate of minced hake alone (Dingle and


HAKE at -1O'C

0 10 2 0 30 40 5 0

DAYS OF STORAGE

FLATFISH 8 MIXTURE at -1O'C

B

EPN (mix.)

-i-.-L

0 10 2 0 30 40 5 0

DAYS OF STORAGE

FIG. 6 . The decrease in extractable protein nitrogen and formation of formaldehyde and DMA during frozen storage of minced fish at - 10°C. (A) Typical pattern of minced hake (gadoid family), in which a drastic loss in protein extractability accompanied by a noticeable accumulation of FrHO and DMA was observed after a relatively short storage time. (B) The stability of minced flatfish as indicated by the slight decrease in protein extractability and no formation of DMA or FrHO. How- ever, a mixture of4: l flatfish-hake showed a poor storage stability pattern similar to that of minced hake alone. After Dingle er a[., 1977.

Hines, 1975; Dingle et al. , 1977). This is attributed to the ease of diffusibility of either the substrate (TMAO) or the enzyme (TMAOase) in the mixed mince and consequently the rapid formation of FrHO.

The influence of temperature on TMAOase activity during frozen storage was studied by Tokunaga (1974), who found that the highest accumulation of DMA in minced pollack stored at various temperatures (from -5" to -40°C) occurred at temperatures near - 10°C.


2 . Formaldehyde: Effect and Mode of Action

It is widely accepted that the deterioration in quality and texture of frozen- stored fish due to the enzymatic breakdown of TMAO is basically attributed to the formation of FrHO rather than DMA. Although FrHO has a harmful effect on humans and its use in food has been banned in many countries, it is generally believed that the amounts accumulated during the frozen storage of minced fish are unlikely to cause a health hazard. In fact, it has been observed that the development of toughness and sponge-like textures in fish meat due to FrHO renders the product unacceptable by the time the FrHO has reached a very low concentration: 0.5 mM per 100 gm of flesh (Dingle et a l . , 1977). In any case, cooking would probably eliminate any free FrHO either by volatilization or by binding it to the proteins.

The effect of the formation and accumulation of FrHO in fish tissues was observed in relation to both textural qualities and protein properties. Sensory evaluations showed that tissues containing FrHO became tougher and their ability to hold water increased, but they lacked the juicy and moist mouth-feel desired, and in advanced stages their texture became rubbery, with a structure resembling that of an open sponge. Instrumental analyses of the texture-that is, changes in deformation forces and changes in deformation works evolved during two consecutive deformations-confirmed the deterioration in organoleptic mouth-feel, and indicated clearly that the changes that took place in the firmness and cohesiveness of the fish tissues were due to the action of the FrHO. The presence of FrHO also caused a noticeable decrease in the extractability of total proteins, particularly the myofibrillar group; the tropomyosin and heavy chains of myosin were most affected, with actin and troponin components least affected (Childs, 1973).

The mechanism by which FrHO affects proteins at the molecular level was postulated from the fact that FrHO had the ability to bind covalently to various functional groups in the protein and hence would cause a deformation accompanied by cross-linking between the protein peptide chains via methylene bridges. Various reaction sites in protein molecules, sensitive to FrHO attack, were identified (Walker, 1964); these included amino, amido, guanido, thiol, phenolic, imidazole, and indolyl residues. Using model systems of various amino acids and amino acid analogs, Dewar et al. ( 1 975), employing 13CNMR spectroscopy, were able to detect and confirm the FrHO-derived methylene bridges. They were also able to identify the sites of linkage of various amino acids by FrHO such as the following:

Tyrosine tC,,,)-CH ,-Lysine (NH ,) Lysine (NH,) ‘-CH ,-Lysine (NH >)


R-(NH)-CH2-S-R Tryptophan (N)-(CH,)-Lysine (NH,) Lysine (NH 2) ‘-CH ,-Glutmine (NHY) Tyrosine (C~*,)-CH,,-GIutamine (NH y ,

However, the theory of the intermolecular covalent cross-linking of protein with the methylene group derived from aldehyde has been challenged by Connell (1965, 1975), who was able, using SDS, to almost completely extract the protein from cod that had been frozen-stored until its texture deteriorated badly and it became unacceptably tough because of formation of FrHO. The extracted proteins were reported to have approximately the same average molecular weight as unfrozen fish proteins. Since SDS is known for its ability to break down and disrupt the secondary forces of interactions, Connell came to the conclusion that FrHO could irreversibly or covalently bind to protein but that proteins probably cross-link through noncovalent binding. However, this did not exclude the prob- ability of covalent cross-linking of short peptides or small protein subunits, which would still be soluble in SDS. In a subsequent study (Connell, 1975), when FrHO was added in vitro to fish muscles at temperatures above freezing, an apparent decrease in the ability of SDS to extract the proteins was noted. Con- nell interpreted this as a different protein-FrHO mechanism (that is, a covalent methylene cross-bridging), which does not occur widely in frozen storage of fish.

3 . Properties of the TMAOase

Although TMAOase has not yet been isolated in a highly purified form, the pioneer work of Harada ( 1 975) revealed fundamental information on its nature and properties. In his work, the extracted enzyme was purified only by adsorbant-type clarifying agents (such as calcium acetate, kaolin, or charcoal) and pH or temperature adjustment. His studies showed that the enzyme (crude) is relatively heat-stable; that is, its activity was maintained at temperatures up to 60°C for 5 min, a fact that might be used advantageously in purification steps of the enzyme (Fig. 7). Electrophoretic separation of the enzyme preparation showed more than one zymogen, indicating that TMAOase is probably a complex enzyme composed of subunits. Gel filtration chromatography separated the accompanying low-molecular-weight, heat-stable cofactors, which were further fractionated on an ion exchange column into two major components, one of which was characterized by its high absorbancy spectra at 265 nm, signaling the existence of a nucleic acid type of structure.

Kinetic studies (Tomioka et al., 1974; Harada, 1975) showed that the enzyme was most active at pH 5.0, but most stable at pH 6.2. Its enzymatic activity was


10

8

C .- 4 2

L

a 2

0

Temperature ('C )

FIG. 7. The increase in specific activity of TMAOase preparation (as monitored by formaldehyde formation and decrease of protein concentration of the supernatant) with heating of the crude enzyme preparation at 60°C for 5 minutes. After Harada, 1975.

inhibited with an increased substrate concentration (TMAO), and to a lesser degree with increased formaldehyde concentration. However, DMA had no inhibitory effect.

Evidence obtained in vitro suggested that the enzyme action involves an oxidation-reduction type of reaction in transforming TMAO into DMA and FrHO, and the presence of hydrogen donor coenzyme(s) is essential to its activity. For example, methylene blue, in reduced form, is essential for assaying enzyme activity. Flavin compounds such as riboflavin, flavin mononucleotides (FMN), and flavin adenine dinucleotides (FAD) also activate the enzyme. Other compounds such as nicotinamide adenine dinucleotides (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), ascorbic acid, Fez+, and biotin stimulate the activity of the enzyme only in the presence of methylene blue, whereas Fe3+, Ca2+, EDTA, TMA, and choline inhibited its entire activity. Hence, methylene blue compounds are probably capable of reducing any essential functional groups on the zymoprotein, which in turn acts as a general base (nucleophil) for the reaction to proceed.

From the fact that there was qn equimolar relationship between the amount of decreased TMAO and the FrHO and DMA formed under various reaction conditions, and from nonenzymatic catalysis studies, Harada (1975) suggested the participation of both carboxylic and amino groups in the protein moiety of the enzyme for the rearrangement of TMAO to form the intermediate compound dimethylaminomethylol, which then yields DMA and FrHO. The following con-


version reaction was postulated by Harada as the mechanism of FrHO and DMA formation:

H3Y

CH,-N-0 - I

J

The heat pattern of TMAOase showed maximum activity in in vitro systems at 30"C, with stability up to 60°C. However, Tokunaga (1974) reported that he was able to arrest the enzymatic activity in frozen-stored (- 17°C) pollock by preheating the muscles to 50°C for a hold-up period of 30 min. Nevertheless, La11 et al. (1975) showed that preheating treatments of silver hake fillets or mince to an internal temperature of up to 60°C (without holding time) were not effective in inactivating the enzyme during subsequent frozen storage at - 10°C, but when the internal temperature reached 80°C the preheating treatment was highly effective in arresting the enzyme action.

D. INTERACTION BETWEEN FACTORS: INDIRECT EFFECT ON PROTEIN DENATURATION DURING FROZEN STORAGE

All the factors discussed in the previous sections showed their direct effect or potential on denaturing fish proteins during frozen storage, as illustrated in Fig. 8 by the vertical arrows. However, these factors could also influence protein denaturation indirectly through their effect on each other (the horizontal arrows in Fig. 8). The formation or accumulation of one of these factors could have a positive effect (accelerate, catalyze, or trigger) or a negative effect (inhibit or decrease) on the reaction rate of others.

For instance, in addition to the direct effect of salt concentration (as a consequence of freezing) on protein deformation and denaturation, high salt concentrations usually stimulate the hydrolysis of lipids and accelerate the liberation of FFA .

The dehydration of localized areas or subcellular structures due to migration of water to form ice crystals could cause a substantial decrease in the water activity of these confined places, which in turn would provide an excellent environment for faster lipid oxidation reactions.


FIG. 8. indicating positive or negative effect on each other) fish protein denaturation during frozen storage.

Various factors that affect directly (vertical pathways) or indirectly (horizontal arrows

The rupture of membrane systems by the formation of ice crystals probably liberates the membrane lipids and consequently may increase their chances of reacting with myofibrillar proteins. Also, the liberated lipids could be deprived of their natural protection compounds and thus could be subjected to faster hydrolytic or oxidative reactions. Hydrolysis of lipids into FFA will lead to a faster rate of oxidation than is found for intact lipids. On the other hand, dis- pense in some of the deposited fats might help to dissolve the FFA and thus diminish their detrimental action on the proteins.

TMAO has shown a synergistic effect on the activity of y-tocopherols in the inhibition of lipid oxidation, in which TMAO acts as a decomposer of peroxide (Ishikawa et al., 1978a,b). Thus, the depletion of TMAO, due to the activity of the TMAOase, will indirectly accelerate the autoxidation reaction of lipids.

Formaldehyde was found to accelerate the hydrolytic decomposition of fish lipids (Ostyakova and Kosvina, I975), especially the triglycerides, phospholipids, and sterol esters. The products of lipid oxidation could furnish the necessary reduced media for maximum activity of TMAOase.

Unfortunately, the majority, if not all, of these secondary interactions have not yet been studied, in spite of their potential importance in intensifying or activating the other reactions, and all conclusions regarding their actual influence is mere speculation.


In summary, in order to comprehend the problem of the stability of fishery products in frozen storage, one should consider all the factors mentioned-their direct action, their reaction mechanisms, and their interaction or effect on each other as they relate either directly or indirectly to the protein denaturation processes.

IV. RESEARCH NEEDS

This review on fish protein denaturation during frozen storage has presented some novel routes and mechanisms that have not been completely elucidated or have not yet received adequate consideration. Obviously, basic research pro- grams, utilizing modem biochemical techniques to study fish proteins at the molecular level, will help to reveal their structure, properties, and their mode of denaturation during frozen storage. Also, they will reveal the relationship between different proteins at the onset of denaturation. Little is known of the sensitivity of all fish proteins to denaturation caused by freezing, or to the mechanisms involved, or to the influence of the individual proteins on each other during frozen storage. Another area of research that would increase our insight into the problem deals with the complex interactions of the factors involved during frozen storage and their effect on the mechanisms and rate of denaturation.

There is also a need to collect more comprehensive information on the enzyme TMAOase-its physiological function in certain fish species, its structure, mode of action (that is, whether the enzyme acts directly or indirectly on TMAO), stability, and properties, and its naturally existing activators, as well as the most effective way to inhibit or depress its activity during frozen storage without using severe heat or conditions that would sacrifice the quality of the products.

Concurrently, there is an obvious need to find effective cryoprotective additives that would provide the best overall protection of proteins at freezing temperatures. It is equally important to determine their mode of action in protecting proteins against freeze damage so that more effective and safer additives can be developed. Studies should also be made on the unusual stability of certain proteins found in arctic and antarctic fish; these proteins show resistance to freeze denaturation and are able to function normally in these cold-blooded animals at subzero temperatures.

Research is needed on moisture behavior in frozen fish and shellfish. Finding ways to control the migration of intra- and intercellular moisture during freezing or frozen storage, or to regulate the moisture retention (hydration) of tissue proteins, would bring valuable benefits to the freezing industry.

Conditions that affect fat hydrolysis and factors that control the rate of hydrolysis in fish tissues or that circumvent their oxidation would certainly help to increase the shelf-life of frozen seafood.


Finally, little is known about the role of other muscle constituents, such as the nucleotides, carbohydrates, small peptides, and free amino acids, in the various protein denaturation mechanisms encountered during frozen storage.

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