CHANGES IN MYOFIBRILLAR PROTEINS AND LIPIDS OF SQUID (ILLEX ARGENTINUS) DURING FROZEN STORAGE

The biochemical properties of actomyosin (AM) and lipid composition of the mantle from frozen‐stored whole squid were investigated. Irrespective of the sex of specimens, during the first months of storage, there was a trend of decreased protein solubility, reduced viscosity and enzymatic activities of AM. A significant decrease (P < 0.05) in the relative percentage of the myosin heavy chain and a significant increase (P < 0.05) in those of paramyosin and the 155‐kDa component were also observed. After freezing, phospholipids (PL), sterols (ST) and triacylglycerols (TAG) represented 38.6, 29.1 and 21.2%, respectively, of total lipids (TL) from the mantle of male squid. Free fatty acids (FFA) plus diacylglycerols constituted only 11% of TL. TL extract from the mantle of female squid had a higher percentage of PL and had lower ST. In the frozen‐stored male and female squid, TAG were hydrolyzed earlier than PL. At zero time of storage, the relative percentages of saturated and unsaturated fatty acid in TL extracts from the mantle of male and female squid were 39.1, 59.2 and 38.7, 61.1, respectively. Irrespective of sex, the saturated FA fraction significantly (P < 0.05) decreased and the unsaturated one significantly (P < 0.05) increased after 8 months of frozen storage.     

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.     

Biochemical Properties of Actomyosin of Cold Stored Striated Adductor Muscles of Aulacomya ater ater (Molina)

Expressible juice showed highest increase within the second day of storage. Actomyosin was partially purified from stored muscles. Both reduced viscosity and Mg²⁺-ATPase activity of actomyosin decreased about 44% in the first day. These changes were due neither to actomyosin dissociation nor fragmentation of major proteins of the complex. Relative percentage of myosin decreased, and actin increased in actomyosin after the second day. These changes are related to a decrease in the myosin heavy chain and could explain the slow decrease in reduced viscosity and Mg²⁺-ATPase of this protein after the second day.     

Surface hydrophobicity and functional properties of myofibrillar proteins of mantle from frozen-stored squid (Illex argentinus) caught either jigging machine or trawling

The surface hydrophobicity and functional properties of actomyosin from the mantle of frozen squid caught either by jigging machines (AME1) or by trawl (AME2) were investigated. Two components of 155 and 55 kDa were present in the gels at zero time of storage. Degradation of the myosin heavy chain and increase in the 155 kDa component occur earlier in AME2. Irrespective of the catch method used, no significant (P>0.05) changes in protein solubility were observed. The reduced viscosity of both AME1 and AME2 decreased up to months 3 and 5 of frozen storage, respectively. At the beginning of storage, the superficial hydrophobicity of AME2 was 30% higher than that of AME1. So 1-anilino-8-naphthalene sulfonic acid of AME2 significantly increased during 3-5 months of storage period and that of AME1 at the end of storage. The emulsion activity index (EAI) of AME2 significantly (P<0.05) increased during the first month and decreased after 3 months of storage. EAI of AME1 decreased at month 3 and remained unchanged thereafter. Emulsion stability (ES) of AME2 showed a behavior that was similar to its IAE and that of AME1 remained unchanged.     

Freezing and frozen storage of actomyosin from different species

A comparative study was made of the influence of freezing (–24°C) and frozen storage (–12°C) on the functional properties (viscosity, solubility) and physico-chemical characteristics (aliphatic and aromatic hydrophobicity, ATPase activity) of actomyosin (AM) from myosystems (chicken and hake) of differing freezing and frozen stability. The difference in functional behaviour between chicken and hake AM as a consequence of freezing and frozen storage suggests that, for hake AM, denaturation and aggregation occur essentially through direct aggregation of AM molecules to produce AM aggregates, whereas in chicken proteins, AM first dissociates into myosin and actin to produce myosin and myosin-actin aggregates.     

Effect of frozen storage of hake,sardine and mixed minces on natural actomyosin extracted in salt solutions

Natural actomyosin extracted in salt solutions from mixtures of hake and sardine minces (3:1; 1:1 and 1:3 w/w) stored frozen for up to 1 year differed in the amount extracted and in the characteristics of the extracts. In the mixed minces the amount of natural actomyosin extracted decreased during frozen storage at a higher rate than that theoretically corresponding to the amount of hake in the mixes. With increasing storage time and proportion of sardine a lower percentage of myosin heavy chain and actin was observed by electrophoresis. An increased size of aggregates was also observed by electrophoresis and transmission electron microscopy. The stability of emulsions was enhanced when aggregates appeared in the extracts. The decrease in the amount of natural actomyosin extracted does not explain the changes observed in the texture of the minces during frozen storage. This may indicate that the size of the aggregates unextractable in salt solutions, independently of the type of bonds that bind the proteins in the aggregates, plays an important role in the textural changes observed. Copyright © 2003 Society of Chemical Industry     

Biochemical and physicochemical properties of actomyosin from pre- and post-spawned flounder (Paralichtys patagonicus) stored on ice

The biochemical and physicochemical properties of actomyosin from pre- and post-spawned flounder stored on ice were studied. Irrespective of the gonadal condition of the fish, a gradual decrease in reduced viscosity, Mg²⁺ATPase and Mg²⁺Ca²⁺ATPase activities of actomyosin was observed. A similar decrease in the Mg²⁺ EGTA ATPase activity of actomyosin from post-spawned flounder was also observed. The decrease in enzymatic activities was accompanied by an increase in surface hydrophobicity of the protein. No signs of proteolysis of the major components of the actomyosin complex were detected. The relative percentage of myosin decreased and that of actin increased in actomyosin from pre- and post-spawned flounder stored on ice. These changes occur earlier in actomyosin from prespawned flounder. The results of this paper indicate that actomyosin from flounder denatured during fish storage on ice and suggest that this denaturation is due to structural and conformational changes in myosin which led to aggregation of this protein.     

HEAT-INDUCED GELATION OF MYOSIN IN THE PRESENCE OF ACTIN

ABSTRACT The rabbit muscle contractile proteins, myosin, actin and reconstituted actomyosin were mixed in 0.1–1.0 M KCl, 20 mM buffers, pH 5.0–8.0, and were tested quantitatively for thermally induced gelation properties by measuring the rigidity (shear modulus) of the system at 20–70°. Scanning electronmicroscopy (SEM) was also used to study the structure of the gels formed by gelation of myosin in the presence of F-actin. Under the standard condition, i.e. at 0.6 M KCl, pH 6.0 and 65°, decrease of the myosin/actin mole ratio to about 1.5–2.0 in the reconstituted acto-myosin system resulted in substantial augmentation of the rigidity of the gel formed. Further decreases in the myosin ratio relative to F-actin reduced the rigidity value of the gel to close to the level of myosin alone. Gel-formability of the reconstituted actomyosin was maximal at pH 5.5–6.0 and between 0.5 and 0.8 M KCl and decreased considerably at other pH values and KCl concentrations. The SEM studies revealed progressive changes in three dimensional ordering as actin concentration in the actomyosin varied. These were in concordance with the results of gel strength.     

Biochemical and Physicochemical Properties of Actomyosin from Frozen Pre- and Post-spawned Hake

Protein solubility, reduced and intrinsic viscosity, and enzymatic activities of actomyosin showed that frozen fillets from pre-spawned hake, stored at — 20°C, deteriorated faster than those from post-spawned hake. The post-spawned actomyosin showed an 18% increase in reduced viscosity, a larger hydrophilic surface, and an increase (P<0.001) in Mg²⁺-, Mg²⁺-EGTA- and Ca²⁺-ATPase activities. Protein solubilities of 85.5 52.5 ± and 33 ± 2.0% were obtained at the end of storage for post and pre-spawned fillets, respectively. Enzymatic activities of post-spawned fillets persisted up to 60 days. No changes were detected in characteristic polypeptide bands of actomyosin or in the myosin/actin ratio related to either gonadal condition during storage.     

Cryoprotective additives and cryostabilisation effects on muscle fillets of the freshwater teleost fish Rohu carp (Labeo rohita) during prolonged frozen storage

The effects of various cryoprotective additives separately and in combination were studied on the myofibrillar protein integrity, biochemical enzyme activity levels and muscle ultrastructure in the freshwater teleost fish Rohu carp (Labeo rohita). Fish muscle samples were divided into eight groups and immersed in different mixtures of cryoprotective additives (S1–S8), then frozen at ? 20 or ? 30 °C for 24 months. Electrophoretic studies revealed early (within 6 months) alteration of the myofibrillar proteins myosin light chain, α‐actinin and tropomyosin. Reduction of the storage temperature from ? 20 to ? 30 °C slowed down the degradative processes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that fish muscle treated with cryoprotective mixture S8 (40 g L^?1 sorbitol/3 g L^?1 sodium tripolyphosphate/4 g L^?1 sodium alginate) showed minimal post mortem changes in myofibrillar proteins. Ultrastructural results also revealed post mortem damage to the muscle, seen earliest (within 6 months) in the sample frozen‐stored without additives (S2), as compared with the normal, unfrozen muscle (S1). The influence of cryoprotectants alone and in combination on fish muscle structural proteins, myosin and actin filaments (A and I bands), during prolonged frozen storage was investigated. After 12 months, samples frozen‐stored with various cryoprotective additives (S2‐S7), except S8, showed signs of myofibrillar disintegration. Beyond that time the degradative processes started showing up in all samples, with minimal muscle ultrastructural damage in sample S8. Again, reducing the storage temperature from ? 20 to ? 30 °C slowed down the degradative processes. Ultrastructural results correlated well with levels of biochemical enzymes (Ca²⁺ myofibrillar ATPase and succinic dehydrogenase) during frozen storage. This is the first report of the cryoprotective effects of these additives on this popular edible fish species. Of the various combinations of additives tested, cryoprotective mixture S8 was found to preserve the muscle structure longest under frozen storage conditions. However, even this mixture was only effective for 18 months at ? 30 °C. Beyond that time the myofibrillar degradative processes were apparent with correlative electrophoretic, biochemical and ultrastructural studies. Copyright © 2006 Society of Chemical Industry     

Comparison of gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle proteins during iced and frozen storage

Gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle behave differently during storage, whether in ice or deep frozen. Rapid changes have been observed in the texture of hake muscle during frozen storage, while gilthead sea bream has proved to be more stable. In order to ascertain the role of muscle proteins in the changes observed during storage, parameters related to protein functionality and the properties of extracted natural actomyosin (NAM) were studied initially and during storage in ice or at ?20 °C. Initially, the parameters related to functionality had higher values in hake muscle and extracted NAM than in gilthead sea bream. At the end of iced storage (22 days), less myosin heavy chain (MHC) and actin were extracted from hake, but there was practically no change in gilthead sea bream. This decrease was not accompanied by lower Ca²⁺‐ATPase activity. Freezing produced no drastic changes, with lower values for gilthead sea bream. However, this species was more stable after 1 year, except for the Ca²⁺‐ATPase activity of NAM. This suggests that the changes that hake proteins underwent during storage particularly affected properties related to aggregation, whereas in gilthead sea bream the changes hardly affected the formation of soluble or insoluble aggregates but did affect the active sites of myosin. © 2002 Society of Chemical Industry     

冷藏条件下中国对虾肌肉蛋白质的生化特性

以肌肉基本营养成分、蛋白质组成、肌动球蛋白盐溶性、巯基含量、Ca2+-ATPase活性、疏水性及SDS-PAGE分析等作为测定指标,考察中国对虾在冷藏时肌肉蛋白质的生化特性变化规律。结果表明,中国对虾肌肉是典型的高蛋白[(21.75±0.12)%]、低脂肪[(1.21±0.03)%]型食物;随着贮藏时间的延长,肌原纤维蛋白和肌基质蛋白的含量减少,肌浆蛋白含量先增加后减少,碱溶性蛋白含量逐渐增加;肌动球蛋白的盐溶性先上升后下降;巯基(－SH)数贮藏前期下降缓慢,第6天后显著下降;Ca2+-ATPase活性显著下降,且与贮藏时间呈显著线性相关(R0℃= －0.991,R4℃= －0.998),但0℃和4℃两组之间差异不显著。疏水性随贮藏时间延长显著增加,并呈显著线性关系(R0℃= 0.976,R4℃= 0.995),且0℃和4℃两组之间差别显著。SDS-PAGE结果显示,肌动蛋白、肌球蛋白重链、原肌球蛋白和分子质量为67kD的蛋白均发生了明显的降解。综合各指标的变化情况,表明随着冷藏时间的延长,蛋白质变性加剧,总体变性程度为0℃时小于4℃。Ca2+-ATPase活性和疏水性与贮藏时间呈显著线性关系,可以作为蛋白质变性程度的指示指标。     

Biochemical Variations in Salt Soluble Fractions of Ultrasonicated Actomyosin Isolated from Broiler Breast-Muscle

Solubility and quality of actomyosin play crucial roles during storage and processing of a particular muscle type. We report here the effect of low intensity (20 kHz) ultrasonication on the solubility and some other biochemical properties of the actomyosin isolated from chicken breast muscle. Although there was an overall enhancement in the solubility of actomyosin during sonication, the major increase of ~61.55% occurred at 0.2M NaCl after 10–12 min of exposure. The distinctive feature of sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) profiles of this fraction was the presence of free or non-interacting actin in considerable amount. Even at higher salt concentrations (0.4 and 0.5M NaCl), the protein solubility was almost twice as high as that of the corresponding controls. At low salt concentrations (<0.3 M NaCl or KCl) where the protein solubility was high, Ca²⁺-, K⁺(EDTA)- and Mg²⁺-ATPase activities were low. However, a remarkable increase in each of the enzymatic activities occurred at the salt concentrations higher than 0.3 M. More importantly, the high level of Mg²⁺-ATPase strongly indicated that actomyosin was reconstituted. The ultraviolet spectra also supported the reconstitution of actomyosin at high salt concentrations. These data suggested that ultrasonication caused conformational changes which modified properties of myosin and actin affecting the intermolecular relationships within actomyosin complex. The shift in the solubility and functional modifications of sonicated actomyosin provide some basis to explain the processing behavior of sonicated poultry meat. In addition, the low frequency sonication may be suggested as a non-destructive method to compare actomyosin extracted from various sources.     

Properties of actomyosin isolated from cod (Gadus morhua) after various periods of storage in ice

Natural actomyosin was isolated from cod (Gadus morhua L) stored in ice for up to 28 days. The gelling properties, apparent viscosity, Ca^2--ATPase activity and component protein composition by sodium dodecyl sulphate (SDS) electrophoresis were determined for each preparation of natural actomyosin. The apparent viscosity, protease activity, trimethylamine (TMA) content and pH of the fish muscle were also determined. The results showed that the apparent viscosity and Ca^2--ATPase activity tended to decrease slightly during ageing of the fish in ice, whereas some of the gelling properties showed a maximum between 3 and 6 days of storage. However, there was no change in the apparent viscosity of the muscle as a whole even after the fish were considered to be stale according to the TMA values. The ratio of myosin heavy chain to actin in the actomyosin changed with the time of storage of the fish, being highest at 3 days when gelling properties were maximal and decreasing progressively thereafter.     

Untersuchung über Änderungen von Fleischproteinen während der Gefrierlagerung

Investigation of changes in meat proteins during frozen storage. The effect of freezing and frozen storage on proteins was investigated in case of pork L. dorsi muscle. After fresh pork was sliced (1 cm thickness), wrapped and frozen (2 different velocities) samples were taken (control, 48 h-, 2, 4 and 6 month-frozen storage) and changes in proteins investigated by DSC and SDS-PAGE. DSC-thermograms show 4 peaks corresponding to myosin (peak 1) and actin (peak 4). Peak 2 reflects the sarcoplasmic proteins and connective tissue contribution. Peak 3 is not defined. When measuring the enthalpies of the DSC-thermograms, it can be observed that the enthalpy ascribed to myosin decreases during frozen storage, while the enthalpy corresponding to actin is not affected. At the beginning of our experiments SDS-PAGE was supposed to be a proper method for the investigation of protein denaturation. Our results show no significant changes in the electropherograms during the whole period of frozen storage.     

鸭胸肌肉加热过程中肌动球蛋白解离研究

为了解鸭胸肌肉加热过程中肌动球蛋白变化情况,本实验以鸭胸肉为材料,研究了加热温度（45、50、55、60、65、70 ℃）和加热时间（0、1、10、20、30、60 min）对肉中肌动球蛋白解离的影响。采用蛋白质免疫印迹技术测定肌动蛋白的含量。结果表明：在45 ℃加热条件下,肌动球蛋白几乎未发生解离（P＞0.05）;而在50、55 ℃或60 ℃加热条件下,随着加热时间的延长,肌动蛋白含量先增加后降低,但均较对照组显著性增加（P＜0.05）;65 ℃加热60 min,肌动蛋白含量降至对照组水平;70 ℃加热条件下,加热时间为30 min或60 min,检测不到肌动蛋白的存在。因此,加热温度为50～60 ℃,加热时间为10～30 min时能显著促进肌动球蛋白解离。     

Assessment of protein changes in farmed giant catfish (Pangasianodon gigas) muscles during refrigerated storage

Farmed giant catfish (Pangasianodon gigas) muscles (dorsal and ventral sites) were stored in a refrigerator (at 4 °C) for 14 days to determine the effect of refrigerated storage on biochemical and physical changes. The analyses were carried out at 0, 2, 4, 7, 10 and 14 days of storage. At day 14, Ca²⁺ ‐ATPase activity markedly decreased when compared to its value at day 1 (>90%), while a small decrease was observed for surface hydrophobicity and reactive sulfhydryls content. Total volatile basic nitrogen and trichloroacetic‐soluble peptide content gradually increased when the storage period was extended. The myosin heavy chain decreased slightly on SDS‐PAGE for both meat cuts with increased storage time. Expressible drip and cooking loss were highest during the first day of storage and slightly decreased with storage time. Instrumental hardness was significantly higher in the ventral compared to the dorsal muscle, while the toughness was the highest at the second day of storage. The muscle bundles with scanning electron microscopy were less attached, resulting in the observed big gaps over increasing storage time. Results indicated that changes of proteins have detrimental effects on the quality attributes of farmed giant catfish muscles during refrigerated storage, particularly physical and biochemical properties.     

Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus)

The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at ?20 and ?30 °C was studied. Traditional methods including the peroxide value, thiobarbituric acid‐reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of ?20 °C compared with samples stored at ?30 °C. Antioxidants had a significant effect (P < 0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at ?20 than ?30 °C. ATPase activity in the myosin extract of Atlantic mackerel showed a significant decrease (P < 0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6 M NaCl) decreased (P < 0.01) during storage at both ?20 and ?30 °C but was greater at ?20 °C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a significant level (P < 0.01) for up to 1 year at ?20 °C compared with samples stored without antioxidants. This study confirms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty fish. © 2002 Society of Chemical Industry     

CHARACTERISTICS OF MUSCLE FROM TWO SPECIES OF BIGEYE SNAPPER, PRIACANTHUS TAYENUS AND PRIACANTHUS MACRACANTHUS

Composition and some properties of muscle from two species of bigeye snapper, P. tayenus and P. macracanthus, were investigated. Both species had a similar composition with the same myofibrillar protein content. However, muscle proteins from P. tayenus had higher thermal stability than those from P. macracanthus, as indicated by the higher enthalpy for transitions as well as the lower inactivation rate constant (K_D). Upon 15 days of iced storage, natural actomyosin Ca^2*‐ATP ase and Mg²⁺‐Ca²⁺‐ATPase activities decreased, whereas Mg²⁺‐EGTA‐ATPase activity increased, suggesting the denaturation of myosin, actomyosin and troponin/tropomyosin complexes, respectively. Increased surface hydrophobicity and decreased sulfhydryl groups indicated the denaturation possibly occurred via hydrophobic interaction and disulfide formation. Heading and eviscerating offish retarded the denaturation and physicochemical changes of proteins during iced storage. The results indicated that a rapid and proper post harvest handling was of importance to maintain the muscle quality of bigeye snapper.     

NUCLEOTIDE CATABOLISM IN COLD STORED ADDUCTOR MUSCLE OF SCALLOP (ZYGOCHLAMYS PATAGONICA)

The postmortem catabolism of adenosine triphosphate (ATP) in cold stored scallop adductor muscles was examined. The change In the pH of stored muscles was also investigated. The ATP content increased for a short time after death and afterwards decreased up to 24 h of storage. Thereafter, the nucleotide level remained unchanged up to 120 h of storage. The ADP content slightly decreased up to 48 h and after that remained unchanged. The AMP slowly accumulated to around 15% of the total nucleotide concentration when the ATP decreased. Small amounts of IMP were detected in all samples. Conversely, adenosine (Ado) was not detected. Inosine (HxR) slightly increased after 48 h of storage and hypoxanthine (Hx.) significantly increased after 24 h. The 260/250‐absorbance ratio of muscle extracts and the pH of stored muscles fell sharply up to 24 h and then decreased slowly. The Hx contents were positively correlated (P < 0.01) with both the Hx/AMP ratios and the K values.