Physicochemical and enzymatic changes of cod muscle proteins subjected to different freeze–thaw cycles

The effects of freeze–thaw cycles on the physicochemical and enzymatic changes of cod muscle proteins were investigated. The activities of α‐glucosidase and β‐N‐acetyl‐glucosaminidase increased as the the number of freeze–thaw cycles increased. A loss of Ca²⁺‐ATPase and Mg²⁺‐Ca²⁺‐ATPase activities was observed, while Mg²⁺‐EGTA‐ATPase activity increased, especially after one cycle of freezing–thawing, with a concomitant decrease in Ca²⁺ sensitivity. The surface sulphhydryl group content decreased with increasing freeze–thaw cycles, while no changes in total sulphhydryl group content were found. The surface hydrophobicity of actomyosin did not change significantly with the number of freeze–thaw cycles. The loss of protein solubility increased with increasing freeze–thaw cycles. However, no cross‐linked proteins induced by formaldehyde were found. The results revealed that freeze–thaw cycles directly affected the physicochemical and enzymatic properties of cod muscle proteins. © 2000 Society of Chemical Industry     

Effects of brown algal phlorotannins and ascorbic acid on the physiochemical properties of minced fish (Pagrosomus major) during freeze–thaw cycles

The quality loss in fish during freeze–thaw cycles is considered one of the major issues caused mainly by temperature fluctuations during cold storage. The present work is aimed to illustrate the effects of brown algal phlorotannins (BAP) and ascorbic acid (AA) on physiochemical properties of minced snapper muscle through different freeze–thaw cycles. Both AA and BAP could retard lipid and protein oxidation, respectively, and synergistically. The Ca²⁺‐ATPase activity can be protected with the addition of antioxidants. The addition of 0.1% (w/w) AP showed 22.6% higher activity as compared with other groups especially during three freeze–thaw cycles. Cooking loss was efficiently inhibited and 0.1% AA + 0.3% BAP group showed 25.5% lower than control. Antioxidant is also helpful to maintain gel‐forming ability of minced snapper and 0.1% AA + 0.1% BAP group showed the best. These results revealed that both AA and BAP could prevent minced fish by inhibiting the protein denaturation during freeze–thaw cycles.     

Gel‐forming properties of surimi produced from bigeye snapper,Priacanthus tayenus and P macracanthus,stored in ice

The influence of iced storage of two species of bigeye snapper, Priacanthus tayenus and P macracanthus, on the gel‐forming ability of the resulting surimi was investigated. Upon iced storage, whole fish underwent deterioration faster than beheaded/eviscerated fish. Total volatile base and trimethylamine contents of whole fish were higher than those of beheaded/eviscerated fish, particularly after 9 days of storage (P < 0.05). P macracanthus muscle was more susceptible to proteolytic degradation than P tayenus muscle. Ca²⁺‐ATPase activity decreased as the storage time increased (P < 0.05), indicating the denaturation of myosin. A marked decrease in Ca²⁺‐ATPase activity was found in whole fish kept for more than 6 days in ice (P < 0.05). Breaking force and deformation of surimi gels from both species decreased, with a concomitant decrease in whiteness, as the storage time increased (P < 0.05). Beheading and evisceration of fish retarded the deterioration. However, the gel‐forming ability of surimi produced from both species decreased continuously throughout iced storage (P < 0.05), probably owing to the denaturation and degradation of myofibrillar proteins. © 2002 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.     

Ca2+ Affects Physicochemical and Conformational Changes of Threadfin Bream Myosin and Actin in a Setting Model

The effect of Ca²⁺ on physicochemical and conformational changes of threadfin bream (TB) myosin and actin during setting at 25 and 40°C was investigated. Ca²⁺ ion at 10 to 100 mM induced the unfolding of myosin and actin as evident by an increase of surface hydrophobicity (S_o ANS) at 40 °C. Total SH groups also decreased with an increased Ca²⁺ concentration, suggesting that Ca²⁺ promoted the formation of disulfide bonds during setting at 40 °C. Both hydrophobic interactions and disulfide linkages were involved in formation of myosin aggregates at 40 °C and were enhanced by addition of 10 to 100 mM Ca²⁺. Myosin Ca‐ATPase activity decreased when Ca²⁺ was greater than 50 mM, indicating conformational changes of myosin head. Circular dichroism spectra demonstrated that Ca²⁺ reduced the α‐helical content of myosin and actin incubated at either 25 or 40 °C. Ca²⁺ induced conformational changes of TB myosin and actin incubated at 40 °C to a greater extent than at 25 °C.     

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.     

Thermal stability of myofibrillar protein from Indian major carps

The characteristics and stability of natural actomyosin (NAM) from rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were investigated. The total extractable actomyosin (AM) was higher (7.60 mg ml^?1) in the case of rohu compared with that from catla and mrigal (5 mg ml^?1). Although the specific AM ATPase activity was similar (0.43–0.5 µmol P min^?1 mg P^?1) among the three species, the total ATPase activity was lower in mrigal (25 µmol g^?1 meat) compared with the other species (37 µmol g^?1 meat). The inactivation rate constants (k_d) of AM Ca ATPase activity showed differences in the stabilities of actomyosin among these fish, the actomyosin from catla being least stable. The NAM from these species was stable up to 20 °C at pH 7.0. Catla AM became unstable at 30 °C, while rohu and mrigal AM could withstand up to 45 °C. The thermal denaturation with respect to solubility, turbidity, ATPase activity, sulphhydryl group and surface hydrophobicity showed noticeable changes at around these temperatures. Copyright © 2004 Society of Chemical Industry     

Comparative study on physicochemical changes of muscle proteins from some tropical fish during frozen storage

Physicochemical changes of muscle from croaker, lizardfish, threadfin bream and bigeye snapper during storage at −18 °C were investigated for up to 24 weeks. Ca²⁺–ATPase activity decreased, whereas Mg²⁺–EGTA–ATPase activity increased throughout the storage. However, no marked changes in Mg²⁺–Ca²⁺–ATPase and Mg²⁺–ATPase activities were observed. Among all species tested, lizardfish muscle was most susceptible to those changes. Disulfide bond formation with the concomitant decrease in sulfhydryl group was found in all species. However, croaker and lizardfish contained higher disulfide bonds as storage time increased, compared to other species. Surface hydrophobicity increased in all species with the sharp reduction observed in lizardfish after 12 weeks. For all species, α-glucosidase and β-N-acetyl-glucosaminidase activities increased in association with the increased expressible drip. Therefore, extended frozen storage caused the denaturation of protein as well as the cell disruption in all species, but the degree of changes was dependent upon species.     

Physicochemical characterization of muscle proteins from different regions of mackerel (Rastrelliger kanagurta)

Physicochemical characteristics of proteins extracted from muscles excised from four distinct regions of mackerel were examined. Three regions were identified based on their fin position; namely anterior muscle, median muscle, posterior muscle and red muscles from beneath the lateral line. The biochemical characteristics like reactive sulphydryl groups, surface hydrophobicity, Ca²⁺ ATPase activity, turbidity, proximate composition and the functional characteristics such as viscosity, emulsion activity index (EAI) and emulsion stability (ES) were studied in the samples, from different regions. Sarcoplasmic protein (SPP) solubility was found to be higher in red muscles compared to that of white muscles, whereas myofibrillar protein (MFP) solubility was higher in white muscles particularly in the posterior portion. The MFP from posterior white muscles showed better viscosity, surface hydrophobicity and Ca²⁺ ATPase activity, which in turn contributed to the higher EAI and ES of this muscle portion. Variations in composition, electrophoretic pattern of proteins and selected functional properties (EAI) were noticed between red and white muscles, which may be of significance from the utilization point of the fish.     

Application of Antifreeze Protein for Food Preservation: Effect of Type III Antifreeze Protein for Preservation of Gel-forming of Frozen and Chilled Actomyosin

Type III antifreeze protein (AFP) remarkably preserved Ca²⁺ ATPase activity of actomyosin during frozen and chilled storage. Under frozen conditions, AFP helped to retain the Ca²⁺ ATPase activity of actomyosin much higher than that of conventional cryoprotectants (sucrose‐sorbitol mixture). The Ca²⁺ ATPase activity increased with increasing AFP concentration and reached a maximum at 50g/L AFP. After 3‐d chilled storage, the Ca²⁺ ATPase activity of control and sucrose‐sorbitol samples had lost 80% and 50%, respectively, while the AFP samples remained unchanged. A Type III AFP mechanism based on freezing temperature depression (more unfrozen water) and inhibition of ice recrystallization that protects against the freezing of muscle proteins in chilled or frozen conditions is proposed.     

Effect of frozen storage on thermal stability of sarcoplasmic protein and myofibrillar protein from common carp (Cyprinus carpio) muscle

Thermal stability of sarcoplasmic protein and myofibrillar protein extracted from fresh and frozen common carp was comparatively studied. Total sulphydryl content (SH) in sarcoplasmic protein solution from 5‐month frozen carp decreased by 19.43% compared with fresh sample. The SDS‐PAGE patterns showed that all the bands of sarcoplasmic protein from frozen‐stored samples were almost invisible at 80 °C. Myofibrillar protein from fresh sample exhibited lower turbidity and surface hydrophobicity and higher Ca²⁺‐ATPase activity and SH content than frozen‐stored sample when heated from 20 to 80 °C. The Ca²⁺‐ATPase activity from fresh (M0), 2 (M2)‐ and 5 (M5)‐month frozen‐stored carp was completely lost at 48, 46 and 46 °C, respectively. When heated to 80 °C, the SH content of myofibrillar solutions in M0, M2 and M5 decreased by 26%, 60% and 70%, respectively. Sarcoplasmic and myofibrillar proteins from frozen carp were more susceptible to aggregate during heating treatment.     

Physicochemical Changes in Pacific Whiting Muscle Proteins during Iced Storage

No changes in actomyosin Ca²⁺-, Mg²⁺-, or Mg²⁺-Ca²⁺-ATPase activities were observed during iced storage of Pacific whiting fillets, but Mg²⁺-EGTA-ATPase increased with a loss of Ca²⁺-sensitivity. Surface hydrophobicity of actomyosin increased substantially within 2 days, but not total sulfhydryl (SH) content. During longer storage, the SH content decreased gradually, but surface hydrophobicity remained constant. Autolytic degradation products increased in fish muscle with storage time. Myosin heavy chain (MHC) was degraded by 45% within 8 days, but no noticeable difference was observed in actin. Results indicated that autolysis may be the main cause of physicochemical changes in Pacific whiting muscle proteins during iced storage.     

The Cryoprotective Effect of Different Konjac Glucomannan (KGM) Hydrolysates on the Glass Carp (Ctenopharyngodon idella) Myofibrillar During Frozen Storage

The physicochemical properties, Ca²⁺–ATPase activity, and surface hydrophobicity of grass carp myobrillar with different cryoprotectants (0.5 % irradiated degraded KGM, 0.5 % β-glucanase KGM hydrolysate, 8 % sucrose-sorbitol (1:1 w/w)) were investigated. The molecular weight analysis showed that the irradiated degraded KGM and β-glucanase KGM hydrolysate were 1.209?×?10⁴ and 2.093?×?10⁴ Da, respectively. The solubility, Ca²⁺–ATPase activity, and total and reactive sulfhydryl (SH) content of treatments containing KGM hydrolysates were higher than other treatments during frozen storage. Addition of KGM hydrolysates could effectively prevent the increase of surface hydrophobicity. The gel strength and whiteness of surimi showed that the addition of KGM hydrolysates in this experiment resulted in surimi with comparable quality.     

Comparison of the Cryoprotective Effects of Trehalose,Alginate, and Its Oligosaccharides on Peeled Shrimp (Litopenaeus Vannamei) During Frozen Storage

The cryoprotective effects of trehalose, alginate, and its oligosaccharides on peeled shrimp (Litopenaeus vannamei) during frozen storage was investigated by monitoring thawing loss, color, texture, myofibrillar protein content, Ca²⁺‐ATPase activity, and performing microscopic structural analysis. Data revealed significant (p < 0.05) inhibitory effects on thawing loss and textural variables (springiness and chewiness) in trehalose‐, alginate oligosaccharides‐, and sodium pyrophosphate‐treated shrimp compared with the control and alginate‐treated batches. L* values revealed that these saccharides had a positive effect on color stability during frozen storage. In addition, the results of chemical analyses showed that trehalose and alginate oligosaccharide treatments effectively maintained an increased myofibrillar protein content and Ca²⁺‐ATPase activity in frozen shrimp. In addition, hematoxylin & eosin staining and SDS‐PAGE confirmed that these cryoprotective saccharides slowed the degradation of muscle proteins and the damage to muscle tissue structures. Overall, the application of trehalose and alginate oligosaccharides to peeled frozen shrimp might maintain better quality and extend the commercialization of these refrigerated products.     

Actomyosin Stabilization to Freeze-Thaw and Heat Denaturation by Lactate Salts

Stabilization of fish actomyosin (AM) was investigated with respect to freeze- and heat-induced denaturation as affected by concentration of sodium lactate (SL), lithium lactate and sucrose. Recovered Ca²⁺ ATPase activity was used as a measure of denaturation. Optimum cryoprotection was gained with 6% (w/v) SL. However, SL of similar quality from another source destablized AM at low concentration and was less effective at higher concentrations. Lithium lactate was not an effective cryoprotectant. For heat stabilization, SL was two times more effective than sucrose with an optimum concentration of 25%. Sodium lactate appears to have good potential as a cxyoprotectant and stabilizer of AM.     

SARCOPLASMIC RETICULUM Ca2+-ATPase ACTIVITY IN COD (GADUS MORHUA) MUSCLE MEASURED IN CRUDE HOMOGENATES

An assay for sarcoplasmic reticulum Ca²⁺‐ATPase in an unfractionated homogenate from cod(Gadus morhua) muscle has been established. Specificity of the assay was demonstrated by the Ca²⁺ dependence of the enzyme and the effect of a specific inhibitor, thapsigargin. Stimulation and inhibition of enzyme activity was observed at low and high Ca²⁺ concentration, respectively. Half the maximal activity was obtained at 0.2 μM free calcium and no activity could be detected above 20 mM free Ca²⁺. Thapsigargin inhibited 92% of enzyme activity. Sarcoplasmic reticulum Ca²⁺‐ATPase showed maximal activity around pH 7 and 25C. A nonlinear Arrhenius plot was found with pronounced changes in the slope in the temperature interval 6–15C. The activation energies obtained from the approximately linear parts above 15C and below 6C were 25 kJ mot^?1 and 172 kJ mol^?1, respectively.     

Role of formaldehyde in formation of natural actomyosin aggregates in hake during frozen storage

During frozen storage of certain lean species of fish, formaldehyde (FA) is formed, giving rise to changes in texture related to the formation of aggregates of myofibrillar proteins. In order to study these aggregates a model system was prepared with natural actomyosin (NAM) (5 mg/ml) and increasing concentrations of formaldehyde. The system was stored frozen at-20° C for 2 months during which solubility in 0.6 M NaCl, Ca²⁺ATPase activity,cis-parinaric acid (CPA) hydrophobicity, SH groups and sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) were measured. — FA caused an immediate loss of Ca²⁺ATPase activity and a decline in soluble protein and CPA hydrophobicity, an effect that was enhanced when the samples were frozen. The electrophoretic profiles of the proteins that remained soluble showed that in both fresh and frozen samples, when FA reacts with NAM the first protein to be insolubilised is myosin, followed by actin, then the troponins and myosin light chains and lastly tropomyosin, depending on the amount of FA and the reaction time. Aggregates of high molecular mass were found at early stages, probably as a result of covalent binding of myosin molecules. When the amount of FA or the frozen storage time was increased, these aggregates became insoluble, forming high-molecular-mass structures and hence were not found in the soluble fraction.

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Die Bedeutung von Formaldehyd bei der Bildung natürlicher Actomyosin-Aggregate im Seehecht während der Lagerung in gefrorenem Zustand

Zusammenfassung Während der Lagerung gewisser Magerfische in gefrorenem Zustand bildet sich Formaldehyd (FA), was die Bildung von Aggregaten myofibrilärer Proteine verursacht. Diese Aggregate wurden im Modell mit Actomyosin (NAM) (5 mg/mL) und wachsenden Formaldehydkonzentrationen untersucht. Die Muster wurden zwei Monate in gefrorenem Zustand bei -20 °C gelagert. Während dieses Zeitraums wurden die Löslichkeit in 0,6 M NaCl, die Ca²⁺ATPase-Aktivität, die cis-Parinarsäure-(CPA)-Hydrophobie und SH-Gruppen gemessen sowie SDS-Polyacrylamid-Gel Elektrophoresen (PAGE) durchgeführt. — FA verursachte einen sofortigen Verlust von Ca²⁺ATPase-Aktivität sowie einen Rückgang löslicher Proteine und in der CPA-Hydrophobie. Dieser Effekt verstärkte sich bei eingefrorenen Mustern. Die elektrophoretischen Profile derjenigen Proteine, die löslich blieben, zeigten sowohl in frischen als auch in gefrorenen Mustern, daß das erste Protein, das bei einer Reaktion von FA mit NAM gelöst wird, Myosin ist, darauf folgen Actin, die Troponine und leichten Myosinketten und schließlich Tropomyosin, was wiederum von der FA-Menge und Reaktionszeit abhängt. In den Anfangsstadien wurden hochmolekulare Aggregate gefunden, die wahrscheinlich das Ergebnis gleichwertiger Bindung von Myosinmolekülen sind. Bei Erhöhung der FA-Menge oder Verlängerung der Lagerzeit bildeten sich hochmolekulare Strukturen, die im löslichen Teil nicht gefunden werden konnten.

     

冻融次数对镜鲤鱼肌原纤维蛋白功能和结构特性变化的影响

本实验通过测定肌原纤维蛋白溶解性、乳化性、δ-电势、巯基含量、自由氨基酸含量、二聚酪氨酸含量、表面疏水性和α-螺旋含量的变化,探究反复冻融过程引起镜鲤鱼肌原纤维蛋白功能特性和结构特性的变化。结果表明：当冻融次数增加到5 次时,肌原纤维蛋白的溶解度、乳化活性、乳化稳定性分别显著下降至62.85%、20.67 m2/g、34.83%（P＜0.05）;在冻融过程中随着冻融次数的增加,蛋白质的δ-电势、表面疏水性和二聚酪氨酸含量不断增加,而巯基、自由氨基酸和α-螺旋含量不断下降。蛋白质的这些变化表明,冷冻-解冻循环破坏了镜鲤鱼肌原纤维蛋白的完整结构,降低了蛋白质的功能特性。     

Rats fed linseed oil in microemulsion forms enriches the cardiac sarcoplasmic reticulum lipids with docosahexaenoic acid and lower calcium transport

The effect of feeding rats with whey protein or lipoid microemulsion of n-3 rich linseed oil (LSO), and n-6 rich sunflower oil (SNO) compared to native or microemulsion forms of each oil for 60 days on fatty acid composition, Ca²⁺Mg²⁺ ATPase, Ca²⁺ uptake and membrane fluidity of rat cardiac sarcoplasmic reticulum (SR) were assessed. Rats consuming LSO in lipoid microemulsion showed higher levels of ALA (18:3, n-3) conversion to DHA (22:6, n-3) when compared to rats given LSO in native form. Compared to all other dietary treatments, the alterations in the n-6/n-3 PUFA composition of SR of rats given LSO in lipoid microemulsion effected the greatest changes in Ca²⁺Mg²⁺ ATPase activity and Ca²⁺ uptake which effectively reduced Ca²⁺ transport. Therefore, the LSO in lipoid microemulsion confers similar heart healthy fatty acid compositional and calcium transport changes and potentially anti-arrhythmic effects similar to fish oils.     

Gelling Properties of Fish/Pork Mince Mixtures

The gel properties of silver carp/pork mince mixtures were investigated as well as the protein structural changes and interactions during gelling using rheology, SEM, and FT‐Raman spectroscopy. The breaking force values for gels containing 0% to 40% pork was significantly lower (P < 0.05) compared with gels containing 50% to 100% pork. Gels containing 70% to 100% pork had significantly higher (P < 0.05) breaking force values compared with gels containing 50% to 60% pork. Deformation values were more mixed. Dynamic rheological data suggested that mixing fish and pork at 3:7 could strengthen the gel network. The addition of 40% pork or above, significantly decreased (P < 0.05) the water retention of the gels compared with the 100% fish gels. The dimensional ordering of gels was also reduced by addition of pork. The reduced ordering was one of the reasons for the low water retention for fish/pork mixed gels. Raman spectral analysis confirmed that mixing fish and pork in 7:3 and 3:7 ratios could promote hydrophobic interactions such as bringing tyrosine residues into the intermolecular interface. The interactions in the 3:7 fish/pork mixed gels were favorable for forming a stronger gel. However, the interactions in the 7:3 fish/pork mixed gels were adverse. The water retention of gels was related to both molecular interactions and secondary structures of protein as well as the microstructure of the gels.