Thermal Denaturation of Hake (Merluccius hubbsi) Myofibrillar Proteins. A Differential Scanning Calorimetric and Electrophoretic Study

Denaturation of hake (Merluccius hubbsi) proteins was studied with DSC by monitoring T_max of transitions and denaturation enthalpies. Whole muscle free of connective tissue showed two transitions (T_max 46.5°C and 75.3°C), and ΔH of 4.27 cal/g. The exudative sarcoplasmic fraction showed three transitions (T_max 45.2°C, 59.0°C and 75.5°C) and ΔH of 3.92 cal/g. The sarcoplasmic proteins from whole hake muscle contributed to both denaturation peaks. Muscle depleted of sarcoplasmic proteins by chloride extraction showed a higher thermal sensitivity and a diminished denaturation enthalpy on the second transition. This suggested an additional effect of chloride upon actin in addition to sarcoplasmic protein extraction. pH had an effect upon the native conformation of thick filament proteins, specifically myosin.     

Effects of carrageenan on thermal stability of proteins from chicken thigh and breast muscles

Thermal stability of ground chicken meat and myofibrillar proteins mixed with κ-, ι-, and λ-carrageenan (CGN) at different NaCl concentrations was investigated with differential scanning calorimetry. Three transitions, characteristic of myosin head (63.3, 62.2°C), sarcoplasmic proteins/myosin tail (67.7, 68.6°C), and actin (78.3, 81.4°C), were observed for nontreated thigh and breast muscles, respectively. The influence of CGNs on the thermal transitions was dependent on salt concentrations. κ-CGN with 2.5% NaCl decreased (P<0.05) transition temperature (T_max) of thigh myosin head and actin, while all three CGNs with or without 2.5% salt decreased (P<0.05) T_max for breast actin. Total enthalpy of denaturation decreased slightly in the presence of κ-CGN for thigh muscles only. The gum effects on myofibril isolates were variable and were salt-dependent. The results suggested molecular interactions between the gums and meat proteins, but the response of the specific proteins to gums appeared to be muscle type-dependent.     

Effect of Sodium Chloride and Phosphates on the Thermal Properties of Chicken Meat Proteins

Maximum thermal transition (T_max) and denaturation enthalpy (ΔH) of water-washed myofibrils and finely cut chicken breast muscle, treated with 1–4% NaCl and/or 0.25–1% of either pyrophosphate (PP) or tripolyphosphate (TPP), were monitored by differential scanning calorimetry. Increasing the concentration of NaCl destabilized the heat resistance of the proteins in water-washed myofibrils and in meat specimens. Actin showed the greatest reduction of T_max, a 16°C decline in the presence of 4% NaCl. In a meat system, the addition of 4% NaCl resulted in one T_max instead of five transitions, as seen in untreated meat. The presence of PP and TPP, especially in concentrations of 0.25 and 0.50%, enhanced the thermal stability of myosin. Changes in denaturation temperatures of proteins were accompanied by corresponding changes in AH.     

Differential scanning calorimetric studies of muscle and its constituent proteins

Rabbit muscle and its constituent proteins were investigated by differential scanning calorimetry (d.s.c.). Post-rigor muscle yielded a complex thermogram comprising at least three endothermic transitions with T_max values of 60, 67 and 80°C. Comparison with the purified proteins or fractions indicated that these transitions corresponded to denaturation of myosin, sarcoplasmic proteins and actin respectively. In addition to these endothermic transitions, pre-rigor muscle produced a single large exotherm of T_max 54°C. The evidence suggested that this transition was closely linked with the process of contraction.     

Carrageenan Effects on Thermal Stability of Meat Proteins

Meat myofibrillar protein (MP) gels and ground pork were used as model systems for studying thermal transition temperatures. Addition of up 2% of κ, ι, or λ carrageenan (CGN) to MP caused a very slight change in the thermal denaturation of the meat proteins. Three transition temperatures were found in ground pork samples, which were characteristic of myosin (59.4°C), sarcoplasmic proteins (67.8°C), and actin (82.4°C). Mixtures of high ionic strength had lower thermal transition peaks. CGN did not cause major shifts in transition temperatures, suggesting that molecular interactions between CGN and meat proteins did not occur.     

Relationship Between Thermal Denaturation of Porcine Muscle Proteins and Water-holding Capacity

ABSTRACT: Differential scanning calorimetry was used to investigate denaturation characteristics of pork muscle proteins from carriers and noncarriers of the RN-gene. Pork from RN-carriers deviated from noncarriers in maximum denaturation temperatures and denaturation enthalpy, with proteins of RN-carriers being the most heat-labile. Correlation studies on the results showed that water-holding capacity was significantly correlated to changes in enthalpy of the population mainly representing myosin tails and sarcoplasmic proteins (p < 0.001). Finally, the influence of ultimate pH and preheating on thermal characteristics of porcine muscle proteins was studied. Myosin tails and sarcoplasmic proteins were most sensitive to pH changes, while myosin heads were most sensitive to preheating simulating stress-induced temperature increases.     

Thermal Denaturation and Aggregation of Proteins in Minced Fish as Studied by a Thermomechanical Method

A thermomechanical method of heating and mixing, was developed to study directly protein denaturation and aggregation in minced fish. It is based on simultaneous and continuous measurement of torque and temperature in a meat sample mixed while being heated. The torque and temperature were continuously recorded. From curves thus obtained, thermal denaturation temperatures of minced samples from 6 marine fish species were determined. The curves showed 4-6 peaks at 32-38°C, 44-46°C, 52-56°C, and 62-75°C, presumably corresponding to denaturation of myosin, actomyosin, sarcoplasmic proteins, and actin, respectively. The temperature range of peak 1 was 4-5°C higher in fatty fish (herring, mackerel) than in lean fish (cod, blue whiting).     

A DSC study on the effects of various maltodextrins and sucrose on protein changes in frozen‐stored minced blue whiting muscle

A DSC heat denaturation study on the effects of various maltodextrins and sucrose on protein changes in minced blue whiting muscle during frozen storage at −10 and −20 °C was carried out. All maltodextrins slowed down the decreases in the denaturation enthalpies (ΔH_d) ascribed to myosin and actin, making evident a noticeable effectiveness against protein denaturation, especially at −20 °C. Sucrose was as effective as maltodextrins at −20 °C, but was the least effective treatment at −10 °C. Significant correlations between both ΔH_ds and either protein solubility or formaldehyde production were found at each storage temperature. A low protein sensitivity to the small amounts of formaldehyde produced during the first weeks of storage and errors associated with the determination of enthalpies led to poorer correlations at −20 °C. Maximum denaturation temperatures (T_max) correlated with protein solubility only at −20 °C. No clear relationship between either T_max and the effectiveness of cryostabilisation was found, as T_max also depends on the effectiveness of the treatments against the thermal denaturation of proteins. © 2001 Society of Chemical Industry     

Thermal denaturation of proteins in Post rigor muscle tissue as studied by differential scanning calorimetry

Post rigor bovine M. semimembranosus was analysed by differential scanning calorimetry (d.s.c.). After extractive removal of sarcoplasmic proteins, subsequent pH adjustment and manual connective tissue removal, d.s.c. yielded reproducible thermograms which permitted investigation of the individual major myofibrillar proteins in various pH and salt environments without prior isolation. The positions of two major peaks, interpreted as myosin transitions, proved to be strongly pH dependent. At pH 5.4, the peak maxima occurred at 58 and 65°C, respectively, at a heating rate of 10°C min-¹. Above pH 6.5 their order of denaturation was reversed. In the pH range 5.4–6.5 the peak ascribed to actin had its maximum near 80°C in intact muscle. Above this pH range it was displaced to lower temperature. The thermal stability of actin was studied after treatment of the muscle tissue with different salt solutions. At equal ionic strengths (μ = 0.15) at pH 5.5, calcium chloride and sodium chloride caused 6.5°C and 4°C displacement to lower temperature, respectively. The thermograms of bovine semimembranosus muscle were compared to those of two red and two white muscle types (bovine cardiac and rabbit soleus muscles, chicken breast and rabbit semimembranosus muscles, respectively) at two pH levels. Greater myosin differences were found between red and white muscles than between muscles from different animal species. All muscles gave similar actin transitions, with exception of the heart muscle where the actin peak appeared at 3 °C lower temperature. The necessity of a strict pH control in order to obtain reproducible muscle thermograms is demonstrated.     

Extractability and thermal stability of frozen hake (Merluccius merluccius) fillets stored at −10 and −30 °C

The relationship between thermal stability changes and functionality loss was monitored in hake muscle fillets stored for 40 weeks at ?10 and ?30 °C. The evolution of changes in apparent viscosity, dimethylamine formation and extractability of muscle proteins in NaCl, sodium dodecyl sulphate (SDS) or SDS plus mercaptoethanol showed drastic differences as a function of temperature. At the higher storage temperature, both myosin heavy chain and collagen were the most severely unextracted in salt and SDS solutions, with actin becoming unextractable at the end of storage. Differential scanning calorimetry showed differences with storage time and temperature in both onset temperature and thermal denaturation enthalpy, mostly affecting the myosin transitions. Some protein denaturation occurred with little or no functionality loss. A considerably high fraction of hake muscle proteins remained in the native‐like condition even at the higher frozen storage temperature. In these conditions both apparent viscosity and myosin and actin extractability in NaCl were very low. © 2002 Society of Chemical Industry     

Changes in firmness and thermal behavior of ice-stored muscle of jumbo squid (<Emphasis Type="Italic">Dosidicus gigas</Emphasis>)

Changes in firmness, muscle total protease activity, and the thermal behavior of jumbo squid (Dosidicus gigas) were measured throughout 15 days of ice-storage. A significant decrease (p<0.05) in the shear force of raw mantle muscle was observed after 7-days ice-storage. The highest total protease activity detected was 1.24 U/g mantle. The thermograms obtained at day zero showed four transition states. The first three transition states were endothermic and correspond to myosin (50 °C), sarcoplasmic proteins (69 °C), and actin (79 °C). The fourth transition state was exothermic at 107 °C, and was probably associated with protein aggregation. The thermal behavior of the muscle showed a decreasing trend in temperature and enthalpy of transition for myosin, sarcoplasmic proteins, and actin with storage time. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a change in the myofibrillar protein pattern, which with the shear force, and differential scanning calorimetry data, suggests a partial denaturation of that protein fraction during ice-storage.     

Thermal Transitions of Salt-soluble Proteins from Pre- and Postrigor Chicken Muscles

Salt-soluble protein (SSP) was extracted from pre- and postrigor chicken muscles at various pH values, and protein thermal denaturation was studied using several techniques. Heating at 1°C/min from 20 to 70°C induced a three- to fourfold increase in breast and leg hydrophobicity. Differential scanning calorimetry of breast and leg SSP showed a major transition occurring within the range 55 to 64°C, with the value dependent on rigor state and pH. Protein-protein association, as measured by turbidity change upon heating, underwent two transitions for leg SSP and two or three for breast SSP. The specific transition temperature and rate were dependent on pH, muscle type and rigor state. However, muscle type and pH had a greater effect than muscle rigor state on SSP denaturation.     

Influence of Sodium, Potassium and Magnesium Chloride on Thermal Properties of Beef Muscle

Sodium, potassium and magnesium chloride, at 1.25, 2.50 and 5.00%, were studied using differential scanning calorimetry. Increasing NaCl resulted in decrease in myosin transition temperature (T₁, first transition) and enthalpy (H₁); both showed significant (P<0.05) linear response. The sarcoplasmic proteins and collagen denaturation temperature (T₂, second transition) increased significantly over the control when 1.25% NaCl was added, but were the same as control when 2.5% and 5.0% were used. Actin denaturation temperature (T₃, third transition) tended to increase when NaCl was added, but enthalpy decreased. Potassium chloride showed similar effects to NaCl on T₁, T₂, H₁ and H₃. Increasing magnesium chloride had much more pronounced effect on decreasing second enthalpy compared to the monovalent salts. At 5% MgCl₂ T₁ increased significantly.     

A differential scanning calorimetric study of the stability of egg white to heat denaturation.

The heat denaturation of egg white and its component proteins was studied by differential scanning calorimetry. At a heating rate of 10 °C/min, egg white at pH 7 shows two major endotherms, at 65 °C and 84 °C, produced by the denaturation of conalbumin and ovalbumin, respectively. The conalbumin endotherm is increased to 70 °C by raising the pH to 9.0, or 77 °C by addition of aluminium at neutral pH. Addition of sucrose stabilises all the proteins; at 10%sucrose, all endotherms are shifted 2 °C to higher temperatures. Within experimental error, the enthalpy of denaturation of egg white equals the sum of the enthalpies of denaturation of its component proteins, and is independent of pH over the pH range 7–9.     

Scanning Calorimetric Behavior of Tilapia Myosin and Actin due to Processing of Muscle and Protein Purification

The thermal behavior of tilapia muscle proteins was investigated by differential scanning calorimetry at various stages in the processing of surimi and during purification of myosin and actin. A shift in the thermal transition of actin to lower temperature was observed and the enthalpies of denaturation for both actin and myosin decreased with further processing. Salt addition also induced shifts in denaturation transitions to lower temperatures and decreased enthalpies of denaturation.     

Thermal properties of Phaseolus angularis (red bean) globulin

The thermal properties of Phaseolus angularis (red bean) globulin were studied by differential scanning calorimetry under various medium conditions. Red bean globulin (RGB) was fractionated by ion-exchange chromatography into a major fraction, with SDS–PAGE pattern corresponding to the 7S vicilin, and two minor fractions, probably representing residual vicilin and the 11S legumin, respectively. The thermogram of RBG showed a major endothermic peak at 86.4°C and a minor transition at 92.2°C. Vicilin exhibited two endothermic peaks (87.7 and 94.1°C), while legumin showed one transition at 89.5°C. The progressive increase in denaturation temperature (T_d) with increase in salt concentration, suggests a more compact conformation for RBG with higher thermal stability. Decreases in enthalpy and T_d were observed under the influence of highly acidic and alkaline pHs, chaotropic salts, and protein perturbants such as sodium dodecyl sulfate, urea and ethylene glycol, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have little effect on the thermal properties of RBG since the major protein component, vicilin, is devoid of disulfide bonds.     

Influence of gender and spawning on thermal stability and proteolytic degradation of proteins in Australian red claw crayfish (Cherax quadricarinatus) muscle stored at 2 °C

Thermal stability and proteolytic degradation of male (M), nonspawning female (F) and spawning female (SF) red claw crayfish (Cherax quadricarinatus) muscle proteins during refrigerated storage (2 °C) were investigated. The thermal transition temperatures (T_max) of myosin and actin remained relatively constant during storage, but their enthalpies of denaturation (ΔH) increased, especially in SF samples. SF muscle proteins were more heat‐stable (greater T_max and ΔH values, P < 0.05) than M and F muscle proteins. Protein degradation occurred in all muscle groups, more rapidly in M and F muscles than in SF muscle. The diminishments of a 69‐kDa component and troponin‐I and the appearance of a 55‐kDa polypeptide represented the most salient proteolytic changes. The results suggested that the spawning status was a more significant factor than gender in affecting the quality of red claw muscle proteins and their changes during refrigerated storage.     

Preliminary studies to determine the chaperoning properties of bovine casein and crystallin proteins at reducing beef muscle protein aggregation during heating

The ability of small heat shock proteins (sHSP) at preventing the aggregation and precipitation of unfolded and misfolded proteins because of changes in pH and temperature is widely recognised. The performance of sHSP from bovine lens extract at protecting sarcoplasmic proteins from heat induced denaturation and aggregation was compared with other chaperones including bovine serum albumin, α_s‐casein, β‐casein and a synthetic peptide based upon αA‐crystallin (AAC). Beef sarcoplasmic proteins were heated in the presence or absence of exogenous chaperone and the solubility, surface hydrophobicity and enzymatic activities of the sarcoplasmic proteins was determined. Lens extract prevented the aggregation of sarcoplasmic proteins, maintaining solubility and clarity up to 65 °C relative to 60 °C for β‐casein. By contrast, α_s‐ and β‐casein proteins protected the activity of endogenous enzymes at temperatures between 37 °C and 52 °C, unlike lens sHSP. Our findings support the addition of casein proteins as potential thermal stabilisers of meat proteins in food systems.     

Low-temperature transitions in cod and tuna determined by differential scanning calorimetry

Differential scanning calorimetry measurements have revealed different thermal transitions in cod and tuna samples. Transition temperatures detected at −11°C, −15°C and −21°C were highly dependent on the annealing temperature. In tuna muscle an additional transition was observed at −72°C. This transition appeared differently than the thermal events observed at higher temperatures, as it spanned a broad temperature interval of 25°C. The transition was comparable to low-temperature glass transitions reported in protein-rich systems. No transition at this low temperature was detected in cod samples. The transitions observed at higher temperatures (−11°C to −21°C) may possibly stem from a glassy matrix containing muscle proteins. However, the presence of a glass transition at −11°C was in disagreement with the low storage stability at −18°C during practical time scales. It was proposed that freezing of cod could be associated with more than one glass transition, with a glass transition at a temperature lower than −11°C being too small to be detectable with instrument, yet governing important deterioration processes. In order to optimize frozen storage conditions, the relationship between deterioration processes important for preservation of quality and glass transition temperatures still needs to be established.     

Low‐field nuclear magnetic resonance analysis of the effects of heating temperature and time on braised beef

We investigated the characteristics of water mobility and distribution in Chinese braised beef after treatment at different temperatures for different times using low‐field nuclear magnetic resonance (LF‐NMR). The beef was heated at 45, 55, 65, 75, 85 or 95 °C for 30, 60, 90 and 120 min. Results showed that T₂ changed significantly with heating temperature. T₂₁ and A₂₁ decreased significantly with increasing temperature below 65 °C, with a steady phase from 75 to 95 °C, which agreed with cooking loss. Inversely, T₂₂ had no changes below 65 °C and changed apparently from 75 to 95 °C. The change in T₂₁ below 65 °C may be related to proteins denaturation and shrinkage and, above 65 °C, T₂₂ possibly induced by the dissolution of connective tissue. The characteristics of braised beef at 65 °C were different from those at other temperatures in T₂ distributions. The findings could provide a theoretical basis for the processing of Chinese braised beef.