[Advances in Food Research] Advances in Food Research Volume 26 Volume 26 || Theories of Protein Denaturation During Frozen Storage of Fish Flesh

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<ul><li><p>ADVANCES IN FOOD RESEARCH, VOL. 26 </p><p>THEORIES OF PROTEIN DENATURATION DURING FROZEN STORAGE OF FISH FLESH </p><p>SOLIMAN Y. K. SHENOUDA* </p><p>National Murine Fisheries Service Gloucester Laborarory, NOAA, United States Department of' Commerce </p><p>Gloucesrer, Massachusetts </p><p>1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Tests Used to Detect Protein Denaturation in Frozen Fish . . . . . , . . , . . , . . , </p><p>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 . . . . . . . </p><p>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 </p><p>during Frozen Storage . . . . . . . . . . . . . . . . . . . D. Interaction between Factors: Indirect Effect o </p><p>during Frozen Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Research Needs. . . . . . . . . . . . . . </p><p>References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . </p><p>215 277 271 218 279 219 280 280 28 1 28 1 290 </p><p>298 </p><p>304 306 301 </p><p>I. INTRODUCTION </p><p>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 </p><p>*Present address: General Foods Corporation, Technical Center, Tarrytown. New York, 10591, </p><p>215 Copynghr 0 1980 by Academic Press, Inc. </p><p>All rights of reproduction In any form reserved. ISBN 0-12-016126-4 </p></li><li><p>276 SOLIMAN Y. K. SHENOUDA </p><p>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. </p><p>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: </p><p>1 . The advantages of rapid freezing of the catch without unnecessary delay- </p><p>2. The superior quality produced by a fast freezing rate. 3. The advantages and disadvantages of different freezing methods (shelf </p><p>freezer, blast freezer, refrigerated seawater, fluidized bed, direct contact with freezing media-that is, immersion freezing in Freon or liquid nitrogen). </p><p>4. The deterioration in quality due to fluctuations in storage temperature and relative humidity. </p><p>5. The effectiveness of prefreezing treatments for enzyme inactivation or the addition of antioxidants or cryoprotective agents. </p><p>6 . The importance of packaging as related to moisture vapor and oxygen per- meability. </p><p>that is, freezing at sea. </p><p>Unfortunately, deterioration in quality, and especially in texture, as a conse- quence 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 irreversi- ble 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). </p><p>In summary, freezing and frozen storage are believed to furnish favorable conditions for the irreversible denaturation of fish muscle proteins. </p></li><li><p>PROTEIN DENATURATION IN FROZEN FISH 277 </p><p>II. TESTS USED TO DETECT PROTEIN DENATURATION IN FROZEN FISH </p><p>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. </p><p>A. TESTS FOR PROTEIN SOLUBILITY OR EXTRACTABILITY </p><p>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 ex- tractability 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. </p><p>It is generally considered that the water-extracted fractions represent the sar- coplasmic proteins, and the higher-ionic-strength extractions (0.3- 1 .O p ) repre- sent 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 solu- bility 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, sarcoplas- mic 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 </p></li><li><p>278 SOLIMAN Y. K. SHENOUDA </p><p>TABLE I PARAMETERS USED TO DETECT OR MONITOR CHANGES OR DETERIORATION IN </p><p>FISH PROTEINS DURING FROZEN STORAGE </p><p>1. </p><p>2. </p><p>3 . </p><p>4. </p><p>5 . </p><p>6 . </p><p>7. </p><p>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: </p><p>Ultracentrifuge sedimentation pattern Electrophoretic pattern Isoelectric focusing pattern Chromatographic separation (ion exchange, molecular sieves, adsorption systems) </p><p>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 </p><p>very small change. Tropomyosin is considered the most stable myofibrillar pro- tein in fish during frozen storage (Matsumoto, 1980). </p><p>B. TISSUE PROPERTIES AND OBJECTIVE PARAMETERS </p><p>This group of indices is distinctive in that intact fish tissues are used in the test- ing. 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 de- hydration of protein or, to a lesser extent, to physical damage in the cells or cell membranes. </p><p>In addition to sensory evaluations, which are the major determinations of </p></li><li><p>PROTEIN DENATURATION IN FROZEN FISH 279 </p><p>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 compressi- bility, 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. </p><p>C. MICROSCOPIC EXAMINATION </p><p>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 dam- age 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 tech- niques, should not create artifacts by altering the ultrastructural images or by masking the microchanges we are looking for. </p><p>D. CHANGES IN THE REACTION VELOCITY OF ENZYMATIC ACTIVITY </p><p>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 stor- age. </p><p>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 -14C). 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 mero- myosin (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 </p></li><li><p>280 SOLIMAN Y . K . SHENOUDA </p><p>- 14C and was completely diminished after 60 weeks (Connell, 1966). Malic enzyme and glycerophosphate dehydrogenase are also temperature-sensitive en- zymes (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 -24C. 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. </p><p>E. TESTS ON EXTRACTED PROTEINS </p><p>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 exis- tence 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). </p><p>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. </p><p>Spectrometric absorption methods such as optical rotary dispersion (ORD), circular dichromism (CD), x-ray analysis, ultravio...</p></li></ul>

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