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.     

The influence of temperature on shortening and rigor onset in beef muscle

At sufficient ATP concentration and temperatures below about 15°C, pre-rigor beef muscles (neck muscles) contract; this phenomenon is known as cold shortening. There is also a contracture at higher temperatures occurring just before rigor onset which is called rigor shortening. While rigor shortening starts in neck muscles at pH around 6·3–6·0 and at about 2 μMol ATP/g muscle, cold shortening can begin at pH around 7·0 and the full ATP concentration (4 μMol ATP/g) in the muscle. Shortening can take place as long as there is no irreversible formation of the actomyosin complex in the muscle, i.e. before rigor onset occurs, which can be measured by intermittent loading of the muscle. The degree of extensibility which follows starts to decrease at the moment of rigor onset. This irreversible loss of extensibility at temperatures between the freezing point (?1°C) and physiological temperatures (38°C) starts at various pH values and ATP concentrations in the muscle. At 38°C the rigor onset occurs at pH 6·25 and about 2 μMol ATP/g muscle, dropping at 15°C to pH 5·75 and 1 μMol ATP/g muscle. At 0°C, as at all temperatures below 10°C, the loss of extensibility at medium loads (about 250 g/cm²) begins shortly after cold shortening. This loss of extensibility is reversible by increasing the load or raising the temperature. The irreversible loss, or rigor onset, however, occurs at 0°C with pH of 6·1–6·2 and 1·8–2·0 μMol ATP/g muscle. Thus, the onset of rigor is influenced by more than one factor. Temperature, pH and ATP concentration each play a rôle.Maximum loss of extensibility or completion of rigor is reached between 10°C and 38°C at pH 5·5–5·6 and less than 0·5 μMol ATP/g muscle. At 0°C the completion of rigor takes place at pH 6·0, but still at 0·5 μMol ATP/g muscle. The latter fact shows that the completion of rigor is solely dependent on the ATP concentration in the muscle; nevertheless, the pH of rigor completion is higher in the extreme cold shortening range. This is apparently due to a different pH/ATP relationship in muscles at low temperatures.The results are discussed in terms of changes in the concentration of Ca²⁺ ions and ATP.The results are of particular interest for the handling of hot-boned meat; that is, for both the cooling of pre-rigor muscle and the use of hot-boned meat for processing.     

Acceleration of Physicochemical Change in Carp Muscle by Washing in Either Chilled or Heated Water

“Arai” (washing) is a traditional cooking method of fish preparation in Japan. Carp muscle slices were washed in water (49°C for 15 sec, or 0°C for 10 min), and examined for physical and biochemical changes. At 49°C, rigor-mortis was accelerated, resulting in a marked decrease of muscle slice thickness. ATP degradation proceeded faster at 49°C than at 0°C. Arai treatment at 49°C increased myofibrillar Mg²⁺ - AT-Pase activity 8-9 times in the absence of Ca²⁺, but did not affect activity in the presence of Ca²⁺ as much, giving only a 1.2 times increase.     

Effect of Storage Temperature on Rigor-Mortis and ATP Degradation in Plaice Paralichthys olivaceus Muscle

Live specimens of the plaice Paralichthys olivaceus were spiked at the brain, stored at various temperatures ranging from 0° to 20°C and examined for changes in rigor tension and ATP degradation in the muscle. The ATP degradation rate was clearly slower at 5–15°C than at WC, resulting in retardation of rigor- mortis onset at the former temperatures. Lactic acid accumulation in the muscle correlated well with the decrease of ATP. The muscle showed an ATP concentration of 3 μmol/g and “rigor index” (full rigor = 100%) at 30% when lactic acid increased up to 30 μmol/g at most storage temperatures. The muscle showed full rigor when ATP completely disappeared and lactic acid attained the maximum plateau (40–50 μmol/g).     

Temperature-Dependency of Rigor-Mortis of Fish Muscle: Myofibrillar Mg2+-ATPase Activity and Ca2+ Uptake by Sarcoplasmic Reticulum

Myofibrillar Mg²⁺-ATPase activity of plaice and red sea bream increased with increase in reaction temperature and Ca²⁺ concentration. However, temperature decrease tended to lower Ca²⁺ sensitivity of Mg²⁺-ATPase activity. While it was assumed that spiked fish muscle was relaxed and the myofibrillar Ca²⁺ concentration was low, the progress rate of rigor-mortis correlated well with Mg²⁺-ATPase activity in the presence of a few micromolar Ca²⁺ at 0°C. Correspondingly, it was found that Ca²⁺ uptake by plaice sarcoplasmic reticulum decreased with the decrease in reaction temperature. These results strongly suggest the acceleration of fish rigor-mortis at 0°C is due to the increase of Ca²⁺ concentration in myofibrils during storage at this temperature.     

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.     

Influence of Prerigor Pressurization on Postmortem Beef Muscle Creatine Phosphokinase Activity and Degradation of Creatine Phosphate and Adenosine Triphosphate

Semimembranosus muscle was excised immediately following slaughter and pressurized at 103.5 MN⁻² for 2 min at 35°C. Creatine phosphate (CP), adenosine-5′-triphosphate (ATP) and R-values (a measure of metabolic rate) were determined at 1, 2, 4, and 24 hr postmortem. Creatine phosphokinase activity (CPK) was measured at 1 and 24 hr postmortem. Pressurization accelerated catabolism of both CP and ATP which, in turn, resulted in a higher ATP turnover value than that of the control. R-values were significantly correlated (r=−0.95) with degradation of ATP and indicate onset of rigor mortis in treated muscle immediately after pressurization. CPK activity was significantly higher (P<0.05) in the pressurized muscle than in the control at 1 hr postmortem. However, this difference did not exist at 24 hr postmortem.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

Dissociation of natural actomyosin from kuruma prawn muscle induced by pyrophosphate

Effect of pyrophosphate (PP) on the dissociation and stability of natural actomyosin (NAM) from kuruma prawn muscle was studied in comparison with adenosine 5′-triphosphate (ATP). In the presence of PP up to 5 mM, NAM exhibited lower Mg²⁺-ATPase activity (P < 0.05), while no marked change was observed in NAM treated with ATP at all concentrations tested (0.25–10 mM) (P > 0.05). Ca²⁺-ATPase activity of NAM treated with 5 mM PP decreased markedly when incubated at temperatures greater than 30 °C, suggesting lowered thermal stability of the liberated myosin molecule. Nevertheless, Ca²⁺-ATPase activity of ATP-treated NAM was similar to the control NAM. In the presence of 5–10 mM MgCl₂, NAM treated with 5 mM PP underwent dissociation effectively, as evidenced by a greater decrease in Mg²⁺-ATPase activity as well as an increased band intensity of actin released. Therefore, addition of PP in combination with MgCl₂ was more effective than was ATP in dissociating the actomyosin complex of prawn muscle.     

Carp Sarcoplasmic Reticulum Changes Due to Heat Treatment

Electron microscopy revealed that heat treatment in the range 35–55°C caused no observable change in membrane structures of the sarcoplasmic reticulum (SR). Proteins with molecular weights about 200 and 400 kDa, (probably dimers and tetramers of Ca²⁺-ATPase, respectively) were observed in SDS-PAGE when the light SR was treated at 49 and 55°C respectively. Ca²⁺ leakage from the SR was markedly enhanced with increasing temperatures, especially for the heavy SR. Results suggest that the decrease of Ca²⁺ uptake ability following heat treatment is at least partly caused by the acceleration of Ca²⁺ leakage together with irreversible denaturation of Ca²⁺-AT-Pase protein.     

Effect of lightly salt and sucrose on rigor mortis changes in silver carp (Hypophthalmichthys molitrix) stored at 4°C

To evaluate the effect of salt and sucrose on rigor mortis changes in silver carp during 72 h at 4 °C, physical (texture parameters, water holding capacity (WHC), cooking loss, L* value) and chemical adenosine triphosphate (ATP‐related compounds, K value, pH value) characteristics were studied. Silver carp were subjected to following treatments: left untreated (CK), treated with 1.8% salt (S) and 1.8% salt incorporating 1.8% sucrose (S+S). Curing treatments predominantly increased IMP content, WHC and texture parameters, retarded cooking loss and discoloration compared with untreated samples. The highest IMP concentration measured in S and S+S all occurred at 4 h, which was prior to that of CK (48 h). K value of S+S did not follow a linear relationship with time. The results indicated that the best eating times of CK, S and S+S were 2, 4 and 4 h at 4 °C, respectively, and curing treatments improved the quality of silver carp during postmortem process.     

Ca2+-Induced Gelation of Pre-heated Whey Protein Isolate

Addition of CaCl₂ to pre-heated whey protein isolate (WPI) suspensions caused an increase in turbidity when pre-heating temperatures were ≥ 64°C. Pre-heating to ≥ 70°C was required for gelation. WPI suspensions which contained CaCl₂ became turbid at 45°C and formed thermally induced gels at 66°C. Thermally and Ca²⁺-induced gels showed significant time/temperature effects but the penetration force values in the Ca²⁺-induced gels were always lower. However, Ca²⁺-induced gels were higher in shear stress at fracture. The Ca²⁺-induced gels had a fine-stranded protein matrix that was more transparent than the thermally induced gels, which showed a particulate microstructure.     

Development of Muscle Thermal Rigorometer and Characterization of Heat-induced Muscle Shortening of Tilapia

A muscle thermal rigorometer was constructed, allowing muscle shortenings induced by dynamic heating or isothermal aging to be monitored. Operating isothermally like a traditional rigorometer at 10 °C, postmortem dorsal muscle shortening (S_10°C) developing from 0% to 10% of its initial length in corresponding to RI_fiber along fiber‐direction developing from 0% to 100% within 16 h was monitored for freshwater tilapia. Monitoring meat cooking in the dynamic heating mode, heat‐induced shortenings could be observed for all muscle samples possessing different degrees of rigor induced by 10°C aging. The heat‐induced shortening (S_dynamic) plus its 10°C aging shortening (S_10°C) for each sample was the same, S_overall= S_10°C+ S_dynamic= 10%. Their heat‐induced shortening peak temperatures (Ts) from 30°C to 48°C were inversely correlated with the sample RI_fiber from 0% to near 100%. These findings together with an additional calcium/adenosine triphosphate (ATP) model studies showed that the ATPase related myofibrillar contractile system was responsible for these low‐temperature cooking shortenings, which along with Ts values could thus be adopted as new rigor indices.     

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     

Molecular Properties of Post-Mortem Muscle.

SUMMARY— Post-mortem changes in nucleoside triphosphatase activity of bovine myosin B have been studied by using several different modifiers with either 5 mM ATP or 5 mM ITP as substrate at ionic strengths (r/2) of 0.09, 0.19, or 0.52. Enzymic activity was determined by measuring the release of inorganic phosphate. There was very little difference in enzymic activity between myosin B isolated from prerigor, rigor (24 hr post-mortem) or post-rigor (312 hr post-mortem) muscle stored at either 2° or 16°C except that the specific activity of myosin B prepared from muscle stored for 12–24, hr post-mortem was higher than activity of myosin B prepared immediately after death. This increase cannot be explained in terms of rigor shortening, but suggests that a change in myosin conformation or in the nature of the actin-myosin interaction occurs in post-mortem muscle. If an actin-myosin interaction occurs during rigor mortis and if this association remains unchanged during extraction of myosin B, then the very low Mg⁺⁺-modified myosin B enzymic activities obtained at Γ/2 = 0.19 and 0.52 indicate that this interaction is not irreversible. Extraction in the absence of ATP produced a myosin B whose ATPase activity was markedly inhibited by trace amounts of Mg⁺⁺. This may be due to the absence of a-actinin in these myosin B preparations. No consistent differences in activation energies were found either at Γ/2 = 0.19 or 0.52 among the NTPase reactions of myosin B samples prepared from muscle after various times of post-mortem storage.     

Production of Cyclodextrins by Simultaneous Actions of Two CGTases from Three Strains of Bacillus

A moderate thermophile, which produces a large amount of extracellular and thermostable cyclomaltodextrin glucanotransferase, was isolated from soil samples at hot spring areas in Japan. The isolate (A-147), that grew at 30–60°C with an optimum at 45–50°C, was identified as a strain of Bacillus coagulans. The enzyme, which was most active at pH 6.5 and 60°C, was stabilized by 2–3 mM Ca²⁺, and degraded about 40% (as dry basis) of 13% (w/w) potato starch solution into cyclodextrins composed of α- and β-cyclodextrins dominantly at 74°C and pH 6.5 in the presence of 3 mM Ca²⁺. The ratio of α-, β- and γ-cyclodextrins was almost constant (2:2.6:1) during all stages of the reaction. By simultaneous actions of two enzymes from the bacterium and other 2 kinds of Bacillus, starch hydrolysates containing various amounts and rations of cyclodextrins were produced from starch. Ein mäßiges Thermophiles, das eine große Menge an extracellulärer und thermostabiler Glucanotransferase produziert, wurde aus Bodenproben in Heißquellengebieten von Japan isoliert. Das Isolat (A-147), das bei 30–60°C mit einem Optimum von 45–50°C wuchs, wurde als ein Stamm von Bacillus coagulans identifiziert. Das bei pH 6,5 und 60°C aktivste Enzym wurde durch 2–3 mM Ca²⁺ stabilisiert und baute etwa 40% (TS) einer 30%igen (w/w) Kartoffelstärkelösung zu Cyclodextrin ab, bestehend aus α- und β-Cyclodextrin, vorwiegend bei 74°C und pH 6,5 in Gegenwart von 3 mM Ca²⁺. Das Verhältnis von α-, β- und γ-Cyclodextrinen war nahezu konstant (2:2,6:1) während sämtlicher Reaktionsschritte. Durch gleichzeitige Einwirkung von zwei Enzymen aus dem Bakterium und zwei anderen Arten von Bacteria wurden Stärkehydrolysate hergestellt, die verschiedene Mengen und Verhältnisse an Cyclodextrinen enthielten.     

Influence of Postmortem Changes in Bovine Muscle on the Water-Holding Capacity of Beef. Postmortem Storage of Muscle at 20°C

Rigor mortis occurs in bovine neck muscles as soon as pH 5.9 and an ATP level of about 1 μMol/g are reached. At 20°C muscle contraction does not occur before the onset of rigor. Postmortem changes in water-holding capacity (WHC) were followed by measuring the cooking loss of unsalted and salted (2% NaCl) muscle homogenates prepared after storage of the intact muscle tissue at 20°C for different periods postmortem. At tissue pH above 5.9 addition of salt causes a strong increase of WHC of muscle homogenates. There is a small decrease of WHC of both unsalted and salted homogenates during the prerigor phase which is apparently caused by the postmortem fall of pH. Rigor mortis does not influence the WHC of unsalted muscle homogenates but causes a strong decrease of WHC of salted homogenates. The reason for this difference is discussed. No more than one-third of the total postmortem decrease of WHC and protein solubility in salted muscle homogenates was attributed to the fall of pH, so at least two-thirds were due to the development of rigor.     

鲢鱼新鲜度对鱼糜凝胶品质的影响

通过对4 ℃条件下鲢鱼死后10 h感官特性、pH值和僵直指数的变化过程以及鲢鱼死后不同时间鱼肉的挥发性盐基氮（total volatile basic nitrogen,TVB-N）值和Ca2＋-ATP酶活性变化的检测,研究鲢鱼肉新鲜度对鱼糜凝胶品质的影响。结果表明：鲢鱼在死后2～3 h开始进入僵直期,鱼体僵直指数升高,pH值下降;随着鲢鱼死后时间的延长,鱼肉的TVB-N值显著升高,Ca2＋-ATP酶活性显著降低。采用死后不同时间的鲢鱼肉制作的鱼糜凝胶,其凝胶强度、保水性和白度均有显著差异,其中以新鲜鲢鱼为原料制作的鱼糜凝胶的凝胶强度最高,而鱼糜凝胶的保水性和白度在鱼肉的僵直期最差。     

A Comparative Biochemical Characterization of Microbial Transglutaminases: Commercial vs. a Newly Isolated Enzyme from <Emphasis Type="Italic">Streptomyces</Emphasis> Sp.

A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples was characterized in crude and purified forms. The aim of this work is to provide relevant information about a new transglutaminase and to compare its characteristics with the well-known commercial transglutaminase from Ajinomoto Co. Inc. (Activa® TG-BP). The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0–6.5 pH range and at 35–40°C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. The fact that this second maximum of activity does not appear on the purified form of the enzyme may suggest the presence of an isoenzyme on the crude extract. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45°C. The decline in activity of the commercial transglutaminase above 45°C and pH 8.0 was more gradual. The activities of all the MTG samples were independent of Ca⁺² concentration, but they were elevated in the presence of K⁺, Ba²⁺, and Co²⁺ and inhibited by Cu²⁺ and Hg²⁺, which suggests the presence of a thiol group in the MTG’s active site. The purified enzyme presented a K _m of 6.37 mM and a V _max of 1.7 U/mL, while the crude enzyme demonstrated a K _m of 6.52 mM and a V _max of 1.35 U/mL.     

The function of endogenous cathepsin in quality deterioration of grass carp (Ctenopharyngodon idella) fillets stored in chilling conditions

Changes of textural properties and cathepsin activity of grass carp fillets during storage in superchilling (?1.5 ± 0.2 °C) and ice (0.2 ± 0.1 °C) was investigated, and the function of cathepsin in quality deterioration of grass carp fillets was discussed. Results showed that shear force of superchilled and ice‐stored fillets decreased by 50% and 55% after 6 days of storage, respectively. The cathepsin activies in different fractions changed significantly during storage at both conditions. Cathepsin B, B+L activities in sarcoplasma, myofibrillar and heavy mitochondrial fraction significantly increased during the early 3 days postmortem, accompanied by remarkable reduction of corresponding activity in lysosome. Cathepsin D activity in sarcoplasma and myofibrillar significantly increased during 6 days of storage with corresponding decrease in lysosome and heavy mitochondria. Correlation analysis showed that changes of shear force of grass carp fillets were significantly correlated with integration of cathepsin B, D and B+L throughout storage time (r² > 0.94). As derived from the first‐order exponential decay model, the enzyme efficiencies (k_a) of cathepsin B and B+L were more than twofold higher than those of cathepsin D, suggesting the major role of cathepsin B and B+L in textural deterioration of grass carp fillets in chilling storage.