Thermal Gelation Properties of Protein Fractions from Pork and Chicken Breast Muscles

Thermal gelation properties of myosin, actin, myofibrils (MF), sarcoplasmic protein (SP) and connective tissue (CTS) from pork semimem-branosus and chicken breast (CB) were evaluated. Gels were prepared at pH 6.0, 2% NaCl and cooked to 70°C at 0.7°C/min. Pork myosin had a higher gel strength and a lower cooking loss than CB myosin at 7% protein. Actin from both species did not form a measurable gel. MF gels from pork and CB had the lowest cooking loss while CTS and SP had the highest cooking loss. Difference in gel strength between muscle and MF+SP+CTS at 7% protein was not consistent with that at 10% protein.     

Characterization of Myosin Subfragment-1 of Summer and Winter Silver Carp (Hypophthalmichthys molitrix) Muscle

Myosin subfragment-1 (S1) was prepared from myofibrils of summer and winter silver carp by chymotryptic digestion in the presence of ethylenediaminetetraacetic acid (EDTA). Two S1 heavy chain isoforms with different molecular sizes of 91 kDa and 95 kDa were detected in the fast skeletal muscle from summer and winter silver carp, respectively. ATPase inactivation assay indicated that winter S1 was about 20-fold unstable comparing to summer S1. Matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry (MALDI-TOF MS) further confirmed that summer and winter myosin S1 heavy chain isoforms were homologous to myosin high-temperature type and myosin low-temperature type S1 heavy chain, respectively. Moreover, both types of myosin S1 heavy chain isoforms were identified at the intermediate stage. The results indicated that myosin was expressed in a season-specific manner; different types of myosin isomer expressed in different seasons, showing different thermostabilities. Practical Application: Silver carp, Hypophthalmichthys molitrix, is one of the most abundant freshwater fish species in China. The structure thermal stability of myosin rod from silver carp was affected by season change. The gel-forming abilities of surimi prepared in different seasons were different. This study investigated the seasonal differences in structure thermal stability of myosin S1 which is vital for gel formation of myosin. The results of this study will aid understanding of the relationship between the structure and function of myosin, and effective production of surimi from freshwater fish species in different seasons.     

Solubilization of Fish Muscle Myosin by Sorbitol

Sugars and their reduced forms decreased the turbidity of myofibril suspension in a concentration dependent relation. The extent of turbidity decrease was compound specific; glucose sorbitol lactitol = lactose=maltitol. Turbidity decreasing effect of sugars correlated well with the number of hydroxyl groups. Sorbitol reduced the NaCl concentration required for solubilization of myofibrils. Moreover, filament formation by myosin was strongly inhibited by sorbitol.     

Effects of Salt and Pyrophosphate on the Physical and Chemical Properties of Beef Muscle

The effects of sodium chloride (NaCl) and pyrophosphate (PP) were examined by treating beef sternomandibularis muscle tissue and isolated beef myofibrils with various concentrations of NaCl, with and without 10 mM PP. Gel electrophoresis showed that higher NaCl concentrations (1.0M > 0.7M > 0.4M) increased the extraction of titin, myosin, and other myofibrillar proteins from beef tissue and that the inclusion of 10 mM PP to NaCl solutions enhanced the extraction of those proteins. Beef tissue water-holding capacity (WHC) was increased by higher NaCl concentrations and the presence of 10 mM PP. Increased myofibrillar/cytoskeletal protein extraction, especially titin, was associated with increased beef myofibril swelling and increased beef muscle WHC.     

Suppression of thermal denaturation of myosin and salt-induced denaturation of actin by sodium citrate in carp (Cyprinus carpio)

The effect of sodium citrate (Na-citrate) on myosin and actin denaturation in myofibrils was investigated. Na-citrate significantly suppressed the thermal inactivation of Ca²⁺-ATPase of carp myosin in a concentration-dependent manner. The effect was greater than that of sorbitol. A similar effect was observed with myofibrils in which myosin is stabilized by F-actin binding. Na-citrate dissolved myofibrils at lower concentration than NaCl. Nevertheless, Na-citrate at 1 M failed to denature F-actin in myofibrils, while 1 M NaCl denatured F-actin almost completely. Na-citrate suppressed the NaCl-induced F-actin denaturation. Sorbitol did not show such protective effect on F-actin denaturation. Moreover, Na-citrate suppressed the freeze denaturation of myofibrils at lower concentration than sorbitol. Thus, Na-citrate was proved to be superior to sorbitol. It was suggested that Na-citrate alone could substitute sorbitol as cryoprotectant in surimi and NaCl as dissolving reagent of myofibril in thermal gel production.     

Suppression of Thermal Denaturation of Myosin Subfragment-1 of Alaska Pollack (Theragra chalcogramma) by Sorbitol and Accelerated Inactivation by Pyrophosphate

Thermal denaturation of myosin subfragment-1 (S-1), prepared from myofibrils (Mf) of Alaska pollack dorsal muscle by chymotryptic digestion, was well suppressed by sorbitol addition in a concentration dependent relationship. ATP and ADP also suppressed the ATPase inactivation significantly at different degrees. On the contrary, pyrophosphate (PPi) accelerated the inactivation by several-fold; half the maximal acceleration occurred at about 30 μn, M PPi concentration. The accelerated inactivation by PPi addition was remarkable at high-salt medium such as 0.5M KC1.     

溶液体系中迷迭香酸与肌球蛋白的相互作用及其对蛋白理化特性的影响

利用荧光光谱、圆二色谱、电泳等技术,研究溶液体系中肌球蛋白与迷迭香酸(rosmarinic acid,RA)的相互作用方式以及不同NaCl浓度条件下RA的添加对肌球蛋白构象和理化特性的影响。结果表明,RA对肌球蛋白的内源荧光具有较强的静态猝灭作用,且主要通过疏水相互作用结合,不存在共价交联。RA可以促进肌球蛋白结构展开,α-螺旋含量降低,更多活性基团暴露,表面疏水性增加。不同NaCl浓度条件下,添加RA会降低Zeta电位的绝对值,导致肌球蛋白的溶解度降低,浊度和粒径增大。低盐浓度下(0.2～0.4 mol/L NaCl),添加RA降低了肌球蛋白的乳化性;中高盐浓度(0.6～1.0 mol/L NaCl)下,RA对肌球蛋白的乳化性影响较小。     

Effect of ageing on the properties of myofibrils of rabbit muscle

The effect of ageing of rabbit muscle at 4° and 15–18° on the extractability and adenosine triphosphatase activity of the myofibrils has been examined. The amount of protein extracted by M-KCL-4 mM sodium glycerophosphate (pH 6.2) and by 0.1 M sodium tetrapyrophosphate-4 mM MgCl₂-10 mM-KH₂PO₄ (pH 7.2) increased as the muscle aged. By using the amount of Ca²⁺ adenosine triphosphatase extracted, i.e. the enzyme associated with myosin, as a measure of the amount of myosin in the actomyosin extracted, it was possible to show that the buffered potassium chloride did not extract all the actomyosin from the myofibrils of pre-rigor muscle. As the muscle aged, more actomyosin was extracted, together with some tropomyosin. Pyrophosphate, however, extracted all the myosin from the pre-rigor muscle, and the increase in the protein extracted from aged muscle was due to actin and tropomyosin in addition to myosin. It is suggested that these changes are caused by a disruption of the Z-band structure during ageing, perhaps due to the hydrolysis of tropomyosin by proteolytic enzymes. The specific Ca²⁺ adenosine triphosphatase activity of the myofibrils was unaltered by ageing but the specific Mg²⁺-activated adenosine triphosphatase, i.e. the enzyme associated with actomyosin, was reduced by about one-third. This latter result may be caused by a change in the mode of linkage of actin to myosin.     

Humectants improve myosin extractability and water activity of raw, cured intermediate moisture meats

Glycerol, propylene glycol and sorbitol were incorporated into salt-based intermediate moisture meats manufactured from porcine M. longissimus thoracis and bovine M. biceps femoris by dry curing and air drying at 4°C. Moisture content and water activity (a(w)) in cured pork were reduced by the addition of propylene glycol and sorbitol. Propylene glycol was more effective than sorbitol in lowering a(w). The extractability of myosin heavy chain, used as an index of alteration of myofibrillar protein, decreased in intermediate moisture porcine meats with the addition of salt and was unaffected by sorbitol. However, use of glycerol and propylene glycol in cured and air-dried pork increased the extractability of myosin heavy chain. Whereas intact myofibrils could not be extracted from salt-cured, air-dried beef, myofibrils could be made from air-dried beef cured in the presence of 10% glycol, 5% propylene glycol and 4% sorbitol. Such myofibrils contracted immediately on addition of Mg(2+)-ATP. In addition, even after storafe for 5 months, including 30 days at 25°C, myosin heavy chain could be extracted from meat cured with this combination of humectants. In comparison with salt curing alone, curing meat with the above three humectants together, plus salt, results in intermediate moisture meats more like fresh meat.     

Myosin heavy chain isoforms influence myofibrillar ATPase activity under simulated postmortem pH, calcium, and temperature conditions

The pH and Ca(2+) sensitivity of myofibrillar ATPase activity plays an integral role in regulating postmortem muscle ATP utilization and likely paces postmortem glycolysis. The objective of this study was to determine the influence of pH and Ca(2+) concentration on the ATPase activity of myofibrils from red semitendinosus (RST) and white semitendinosus (WST) porcine muscles. Myofibrillar ATPase was measured at 39 °C over a pH range 5-7.5 and a [Ca(2+)] range pCa 4-9 (10(-4)-10(-9)M). At maximum Ca(2+)-dependent activation (pCa 4), RST myofibrils had lower (p<0.0001) ATPase activity than WST myofibrils. This maximum activity of myofibrils from both muscle regions was not influenced from pH 7.5 to 6.5, declined between pH 6.5 and 5.75 (Hill coefficient, n(H)=2.7-3.4; pH at half maximum activity, pH(50)=5.97) and was near zero at pH 5.5. At pH 7, pCa-activity relationships showed that RST required less Ca(2+) for half-maximum activation (higher pCa(50); 6.50) than WST myofibrils (pCa(50)=6.35) but had no difference in n(H). At pH 7, both RST and WST myofibrils had maximum Ca(2+)-dependent, actin-activated ATPase activity at pCa ?6 and Ca(2+)-independent myosin ATPase activity at pCa ?6.75. pCa-activity relationships at different pH levels indicated that pCa(50) decreased with pH from pH 6.5 to 6.125 in both RST and WST myofibrils. At pH <5.75, [Ca(2+)] did not influence ATPase activity in RST or WST myofibrils. These data show that myofibrils with predominantly fast MyHC (WST) have a higher actin-activated myosin ATPase activity than myofibrils with primarily slow MyHC isoforms (RST) at Ca(2+) concentrations and pH values characteristic of postmortem muscle.     

Protein Extractability of Turkey Breast and Thigh Muscle with Varying Sodium Chloride Solutions as Affected by Calcium, Magnesium and Zinc Chloride

Ground turkey breast and thigh muscle were extracted with various NaCl solutions with or without added CaCl₂, MgCl₂, or ZnCl₂ (0.05%). Protein solubility was increased by CaCl₂ and decreased by ZnCl₂ in each muscle type. At 4% NaCl, MgCl₂ increased thigh myosin solubility by 30%, compared to the control, whereas CaCl₂ had no effect. At 2% and 4% NaCl, breast myosin was not affected by MgCl₂ or CaCl₂. Myosin was not detected for either muscle type when ZnCl₂ was used. All three salts increased breast actin solubility but only MgCl₂ increased thigh actin solubility. The CaCl₂ resulted in the highest overall protein solubility and MgCl₂ resulted in the highest thigh myosin and actin solubility at 4% NaCl.     

Heat-Induced Gelation of Myofibrillar Proteins and Myosin from Fast- and Slow-Twitch Rabbit Muscles

Heat-induced gelation of myofibrillar proteins and myosin (0.6M; pH 6.0) from rabbit fast- and slow-twitch muscles was analyzed by thermal scanning rheometry. Proteins from slow-twitch muscle exhibited higher thermostability and lower gel strength than those from fast-twitch muscle. Purifying myosin from myofibrillar proteins changed heat-gelation profiles and generally increased gel rigidity at 80°C. However, the effect of some proteins on the gelation of myosin was muscle dependent. Complete elimination of actin decreased the heat-gelling ability of slow myosin and increased that of fast myosin. Also, elimination of C-protein led to a greater increase in rigidity of gels from slow myosin than from fast myosin. The heat-behavior of the different protein fractions was related to the degree and type of aggregation in the gel.     

Curtailing Oxidation‐Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron‐redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl₂ at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross‐linking was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation‐initiated cross‐linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G′) of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation‐induced G′ by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.     

Some observations on protein changes in washed myofibrils of bovine muscle

It was found that the protein and water contents of washed myofibrils obtained from bovine longissimus dorsi muscles vary with the rate of pH change during the first hour post mortem. Neither myosin nor actin is responsible for the variation, which is due to changes in the other myofibrillar components. The amount of remaining myofibrillar proteins (RMP), obtained after the sum of myosin and actin is deducted from the total myofibrillar protein (TMP), shows a minimum at a rate of pH change corresponding to a 1-h pH value of 6·7. Apart from this pH-dependent decrease, a further substantial reduction of the RMP fraction was observed, particularly in myofibrils extracted from electrically stimulated muscles. It is considered that the observed changes result from partial proteolysis taking place in muscle during the early hours post mortem. A possible involvement of connectin as a primary substrate for the action of calcium-activated neutral protease is discussed. On the basis of the observed correlation between Warner-Bratzler peak shear force and TMP, a significant influence of pre-rigor muscle status on meat tenderness is suggested.     

Action of NaCl and polyphosphates in meat processing: Responses of myofibrils to concentrated salt solutions

When meat is treated with salt, some parts of the meat are exposed to very high salt concentrations which then fall during equilibration. To gain a better understanding of the consequences of this treatment, we have observed the swelling and extraction of isolated myofibrils treated with series of salt solutions designed to mimic conditions found in salt-treated meat. Myofibrils swelled most in 1m (5·8%) NaCl and hardly at all in 5m (29%) NaCl. Similar results were obtained when the concentration of NaCl was raised in steps to 5m. The pronounced extraction of the A-band that occurs in 1m NaCl was decreased if higher NaCl concentrations were used instead. Lowering the concentration from higher values to 1m caused some swelling and extraction of the A-band, but not as much as when 1m was used directly. Inclusion of pyrophosphate in the solutions had marked effects. There was little swelling in 1m NaCl and none in 5m. Lowering the NaCl concentration to 1m from higher concentrations gave more swelling than observed when 1m was used directly. Extraction of the A-band was greatest in 1m, and was strongly inhibited in 5m. Myofibrils treated with 5m NaCl lost the ability to respond to the presence of pyrophosphate in 1m NaCl. The behaviour of myofibrils in high concentrations of NaCl alone is similar to that of pieces of meat. However, the persistent swelling of meat when the NaCl concentration is slowly raised to 5m is not found with myofibrils, suggesting that significant water compartments exist outside the myofibrils in such meat.     

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.     

高密度CO2对虾肌球蛋白微观形貌的影响

以凡纳滨对虾肌球蛋白为研究对象,用原子力显微镜观察高密度CO2（dense phase carbon dioxide, DPCD）处理压强、温度和时间对肌球蛋白微观形貌的影响,探讨DPCD处理过程中肌球蛋白的聚集行为特征。结果 表明：在不同的DPCD处理压强、温度和时间条件下,肌球蛋白变性和聚集的程度有显著差异;DPCD诱导肌球蛋白 变性聚集过程中,不仅有热效应引起的蛋白质分子间的相互作用,还有高压下CO2与蛋白质分子间的相互作用。     

Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin

The kinetics of myosin dissociation from actin was investigated and also the impact of salt, MgPPi, and myosin heavy chain isoform on myosin subfragment 1 (S1) dissociation from actin using purified proteins and fluorescence spectroscopy. Both NaCl and MgPPi increased myosin S1 dissociation rate. When salt concentrations increased from 0.1 to 1.0 M, the dissociation rate of S1 from bovine masseter (slow) and cutaneous trunci (fast) muscle increased 38 and 78-fold, respectively. MgPPi had an even greater effect on S1 dissociation from actin. With the addition of MgPPi to the mixture of pyrene actin and S1, the fluorescence increased about 85% within the dead time of the mixing approach. Unlike salt, MgPPi had no apparent difference in its ability to dissociate slow or fast S1 isoforms from actin. The results reveal that salt and MgPPi increase myosin extraction and functionality in meat by weakening the actomyosin interaction and that some of the difference in the functionality of red and white muscle may be related to actomyosin dissociation.     

提取条件对鲨鱼肉盐溶蛋白热诱导凝胶特性的影响

以鲨鱼肉为材料,研究NaCl 浓度、pH 值、静置提取时间及加热时间对鱼肉盐溶蛋白热诱导凝胶保水性和质构特性的影响。结果表明：鲨鱼肉盐溶蛋白凝胶的保水性、硬度、弹性和黏聚性与NaCl 溶液浓度呈正相关;在NaCl 溶液浓度0.8mol/L、pH6.5～7.0、静置提取时间24h、40℃加热40～60min 时形成的凝胶保水性及其质构指标较理想;40℃加热盐溶蛋白,有利于形成凝胶结构,但随着加热时间的延长,盐溶蛋白凝胶硬度增加,保水性降低。     

Autolysis of Squid Mantle Muscle Protein as Affected by Storage Conditions and Inhibitors

Autolysis of squid mantle muscle was investigated under various conditions using muscle homogenate as a model system. Cleavage of myosin into heavy and light meromyosin was prominent during autolysis. Storage conditions such as NaCl concentration and temperature affected not only the rate of autolysis but also the products formed. Conditions for maximal autolysis were: NaCl concentration 0.3M, pH 7 and temperature 40°C. Autolysis was well inhibited by ethylenediaminetetra acetic acid and Na-pyrophosphate at <25°C and by phenyhnethylsulfonyl fluoride and chymotrypsin inhibitor extracted from potato tuber at >35°C suggesting two different types of proteinases were involved.