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.     

Mild protein oxidation enhanced hydration and myofibril swelling capacity of fresh ground pork muscle packaged in high oxygen atmosphere

Abstract: This study investigated the influence of an oxygen‐enriched modified atmosphere packaging (HiOx: 80% O₂/20% CO₂) in comparison with air‐permeable polyvinylchloride (PVC) wrapping and partial vacuum (VP: 60%) packaging on the ability of myofibrils to imbibe water during retail display of fresh ground pork at 2 to 4 °C. Both HiOx and PVC muscles after 4 d showed maximum myofibril swelling and A‐band dissolution when isolated myofibrils were subjected to a graded series of salt solutions (0.2→0.4 M NaCl with 10‐mM sodium pyrophosphate, pH 6.2), while VP samples exhibited no remarkable change. Protein carbonyl content increased substantially from day 0 to 4 in all muscle samples. For HiOx, muscle hydration capacity increased linearly (P < 0.05) during storage up to 14 d, corresponding to protein carbonyl production. No significant changes in hydration properties were noted in VP muscle samples, which also maintained lower levels of protein oxidation, during storage up to 21 d. These results indicated that packaging in modified atmosphere with high levels of oxygen could result in increased protein oxidation but enhanced hydration capacity of fresh meat. Practical Application: Packaging of fresh meat, including ground pork, under an oxygen‐enriched atmosphere condition is widely used in the industry to preserve red color of meat. Results from the present study indicate that high oxygen packaging has a discordant effect on fresh ground pork: it enhances hydration but decreases water‐binding, and this seems to be caused by increased swelling of muscle fibers due to mild protein oxidation. Hence, it is advisable to employ a water‐binding agent in this type of packaging system so as to retain the moisture in stored fresh pork products while maintaining the color stability.     

Lysosomal Enzyme Effects on the Postmortem Changes in Tilapia (Tilapia nilotica X T. aurea) Muscle Myofibrils

To investigate the effects of lysosomal proteases on myofibril fragmentation, tilapia (Tilapia nilotica X T. aurea) muscle myofibrils were incubated with isolated lysosomal fraction containing 12 units of cathepsin D/mL (G-II) and pure cathepsin D (12 units/mL G-III) at pH 5.5, 6.0, and 6.5 for 3 days at 4°C. Among samples incubated at pH 5.5, the degree of myofibril fragmentation (DMF) of G-III was highest, while, at pH 6.0 and 6.5, that of G-II was highest. At pH 6.5, the higher DMF of G-III than that of G-I suggested that cathepsin D still had proteolytic activity on myofibrils. Difference in the decrease of protein content between G-III and G-I, and between G-II and G-I indicated that proteolysis caused by cathepsin D was highest at pH 5.5, while that caused by lysosomal enzymes was highest at pH 6.5. This suggested the participation of lysosomal enzymes in the fragmentation of myofibrils.     

Thermal Gelation of Brown Trout Myofibrils: Effect of Muscle Type, Heating Rate and Protein Concentration

The thermal gelation properties of myofibril solutions (KCl 0.6M; pH 6.0) from reared brown trout white and red muscles were analyzed by thermal scanning rheometry. With a heating rate of 1°C/min, red muscle myofibrils exhibited a lower gelation capacity than white muscle myofibrils at low temperatures. No difference was observed above 60°C where solid gels were formed from the two myofibril types. Increasing protein concentration or reducing heating rate increased the values of the rheological parameters at 80°C for the two muscle type myofibrils. With a low heating rate (0.25°C/min), white muscle myofibrils formed stronger gels whatever the temperature.     

Oxidation‐Initiated Myosin Subfragment Cross‐Linking and Structural Instability Differences between White and Red Muscle Fiber Types

Both white and red muscles are commonly used in meat processing, and protein cross‐linking, which may be affected by oxidants, is a key factor affecting the product quality. In this study, myofibrillar proteins (MPs) extracted from postrigor chicken Pectoralis major (PM, predominantly white) and Gastrocnemius (GN, predominantly red) muscles were subjected to a ?OH‐oxidizing system (10 μM FeCl₃, 0.1 mM ascorbic acid, with 0, 5, 10, or 20 mM H₂O₂) at pH 6.2, 4 °C for 18 h. The solubility of nonoxidized (control) PM MPs (63%) was higher than that of control GN MPs (41%). After oxidation with ?OH generated at 5 mM H₂O₂, protein solubility decreased by 46% and 21% for PM and GN, respectively, due to aggregation. Chemical and electrophoretic analyses indicated H₂O₂‐dose‐dependent losses of sulfhydryls and the concomitant formation of disulfides which were more pronounced in PM protein samples. Oxidation favored cross‐linking of myosin rod or tail in PM MPs compared to an equal susceptibility of myosin subfragment‐1 (s‐1) and rod to ?OH in GN MPs. Both Ca‐ and K‐ATPase activities in GN myosin were more sensitive to ?OH than their PM counterparts, indicating a less stable s‐1 region of GN myosin to oxidation. The uncoiling of rods from PM myosin was more rapid than that in GN myosin during heating. Oxidation induced cross‐linking via disulfide bonds hindered the unfolding of rod, particularly in PM myosin. These data revealed the molecular events that underscore the necessity of meat processing and formulation control based on muscle fiber types.     

Nitrite-cured color and phosphate-mediated water binding of pork muscle proteins as affected by calcium in the curing solution

Calcium is a mineral naturally present in water and may be included into meat products during processing thereby influencing meat quality. Phosphates improve myofibril swelling and meat water-holding capacity (WHC) but can be sensitive to calcium precipitation. In this study, pork shoulder meat was used to investigate the impact of calcium at 0, 250, and 500 ppm and phosphate type [sodium pyrophosphate (PP), tripolyphosphate (TPP), and hexametaphopshate (HMP)] at 10 mM on nitrite-cured protein extract color at various pH levels (5.5, 6.0, and 6.5) and crude myofibril WHC at pH 6.0. Neither calcium nor phosphates present in the curing brines significantly affected the cured color. Increasing the pH tended to promote the formation of metmyoglobin instead of nitrosylmyoglobin. The ability of PP to enhance myofibril WHC was hampered (P < 0.05) by increasing the calcium concentration due to PP precipitation. Calcium also decreased the solubility of TPP but did not influence its enhancement of WHC. On the other hand, HMP was more tolerant of calcium but the soluble Ca-HMP complex was less effective than free HMP to promote water binding by myofibrils. The depressed muscle fiber swelling responding to added calcium as evidenced by phase contrast microscopy substantiated, to a certain extent, the deleterious effect of calcium, suggesting that hardness of curing water can significantly affect the quality of cured meat products. PRACTICAL APPLICATION: Although not affecting nitrite-cured color, calcium hampers the efficacy of phosphates to promote water binding by muscle proteins, underscoring the importance of water quality for brine-enhanced meat products.     

CHARACTERISTICS OF SARCOPLASMIC PROTEINS AND THEIR INTERACTION WITH MYOFIBRILLAR PROTEINS

The protein solubility and molecular‐weight distribution of freeze‐dried sarcoplasmic proteins (SPs) from rockfish treated under low and high pH as well as various NaCl concentrations were elucidated. The solubility of SPs was significantly suppressed at an acidic pH (2.0–4.0) and in the presence of high salt concentration (0.5 M NaCl). The least amount of protein was lost when SPs were treated at pH 2.0 or 3.0 followed by precipitation at pH 5.5. The interaction of SPs with Alaska pollock surimi (myofibrillar proteins) was also investigated. The addition of SPs appeared to delay the thermal denaturation of myosin and actin. The SPs positively contributed to the gelation of myofibrillar proteins as judged by breaking force.     

Origin of variable extraction of myosin from myofibrils treated with salt and pyrophosphate

When isolated myofibrils are treated with solutions containing NaCl and pyrophosphate to imitate conditions in meat being cured, considerable dijfirences are observed between myofibrils in the concentration of NaCl required for extraction of the myosin-containing A-band. The diferences in extent of extraction at an intermediate concentration of NaCl are between bundles of myofibrils rather than within them, indicating that the variation derives from differences between muscle fibres. The extraction behaviour is determined by the myosin isoenzyme content of the muscle fibre. Thus, the proportion of myofibrils extracted at an intermediate concentration of NaCl in preparations from various bovine and rabbit muscles reflects the documented proportion of fast-contracting fibres in each muscle. Rabbit M plantaris myofibrils from fast white fibres are extracted in a low concentration of NaCl, whereas those myofibrils containing slow myosin are extracted only in a high Concentration of NaCl. Myofibrils probably from fast red fibres require an intermediate concentration of NaCl for extraction.     

Protein Extraction From Chicken Myofibrils Irrigated with Various Polyphosphate and NaCl Solutions

Physical changes in chicken gastrocnemius myofibrils incubated in 0.1 to 1.0 M NaCl solutions with or without 10 mM ortho-(P), pyro-(PP), tripoly-(TPP) or hexameta- (HMP) phosphate at pH 6.0 were examined by phase-contrast microscopy, electrophoresis, and solubility. PP and TPP performed similarly in promoting protein extraction, P had no apparent effect, and HMP exhibited an intermediate effect. PP, TPP, and HMP treatments markedly improved protein solubility in 0.3 and 0.4 M NaCl through the release of myosin, but the phosphate effect diminished in ≥ 0.6 M NaCl. Overall, phosphates influenced the ultrastructure of myofibrils and extraction of their constituents in the order: PP ∼ TPP > HMP > P ∼ nonphosphate control.     

Myosins from red and white bovine muscles: Differences measured by turbidimetric,calorimetric and rheological techniques

The aim of this study was to elucidate the functional performance of the most abundant protein component in meat, ie myosin, which is recognised as important for binding in meat products. As several genetic variants of skeletal myosin exist, myosins from two bovine muscles of different fibre type composition, M masseter and M cutaneus trunci were compared with respect to filament forming properties and denaturation characteristics. The principal methods used were turbidimetric measurements, which were used to monitor filament formation, calorimetry and rheology. The myosin systems were examined at two different salt levels (0.2 and 0.6 M NaCl) and at pH 5.5–7.0. The method of preparing myosin suspensions/solutions was also examined. Differences in the filament-forming process for the two myosins were detected. Measurements of turbidity revealed that at conditions of low pH and low ionic strength white myosin had a higher ultimate turbidity compared with red myosin. Early in the transition from low to high turbidity, red myosin had a higher turbidity than white myosin corresponding to reduced solubility. The turbidity increased with time of storing the myosin suspensions/solutions. This change was attributed to formation of filaments and further association of filaments. White myosin had a smaller apparent enthalpy of denaturation than red myosin. The calorimetric measurements recorded in 0.2 M NaCl suggested that the head and the rod of white myosin were less stable than the corresponding parts of red myosin. However, exceptions to this rule were found at pH 6.0. In 0.6 M NaCl the identification of the transitions for red myosin was more difficult. The method of preparing myosin suspensions affected calorimetric and rheological measurements. In 0.6 M NaCl and pH 6.0 calorimetric thermograms of both myosins were affected by the preparation method. At pH 5.5 this change was interpreted as caused by denaturation promoted by the dilution/rapid titration technique compared with dialysing the systems to pH 5.5 from pH 7. Differences in the filaments formed might, however, also contribute to the variations seen in the calorimetric ther-mograms. The gelling properties of white myosin were most sensitive to the preparation method used. Systems prepared by dialysis gave stronger heat-induced gels than those prepared by ‘dilution’. White myosin always produced stronger gels than red myosin independent of the preparation technique. The rheological properties (at 80°C) of red myosin were less affected by the preparation method than were those of white myosin. At lower temperatures, however, there was more variation in the shapes of the rheological thermograms (? versus temperature) for red myosin than in the corresponding thermograms of white myosin.     

Protein Extractability and Thermally Induced Gelation Properties of Myofibrils Isolated from Pre- and Postrigor Chicken Muscles

Purified chicken myofibrils were suspended in 0.6M NaCl at various pH values to study gelation properties of the myofibrils. Postrigor breast myofibrils showed a greater protein extractability and gel strength than prerigor breast myofibrils, but the reverse was found for leg myofibrils. Salt-soluble protein was least extractable at pH 5.50 for both breast and leg myofibrils. The pH for optimum gelation, indicated by increased penetration force, was 6.00 for breast and 5.50 for leg myofibrils. Heating at 1°C/min from 20 to 70°C produced stronger breast but weaker leg myofibril gels than isothermal heating at 70°C for 20 min. Muscle rigor state showed a greater effect on protein extractability and gel strength for breast myofibrils than for leg myofibrils.     

Changes in Protein Solubility and Gelation Properties of Chicken Myofibrils during Storage

The effect of storage on protein solubility and heat-induced gelation properties of chicken hen breast and leg myofibrils was investigated. Myofibrils suspended in 0.6M NaCl, pH 6.0, showed increasing protein solubility, viscosity, gel strength and water holding capacity with storage at 4°C. However, the effect of storage was most dramatic only during the initial 10 hr for all of the parameters studied. The relative distribution of the proteins comprising the salt soluble protein (SSP) extract changed during storage. Although storage had little effect on breast SSP, it was detrimental to leg SSP gelation. Breast myofibril suspensions, for all storage times, contained a greater amount of SSP and had better gelation properties than leg myofibril suspensions.     

Functionality of native and denatured cashew nut kernel protein isolates at isoelectric pH as a function of salt concentration

The reduced solubility of proteins near the isoelectric pH limits their use in food formulations whose pH lies in the range 5.0–6.0 because of poor functionality. In the present study, the effect of salt on the functionality of native and denatured cashew nut kernel protein isolates at the isoelectric pH was investigated. Both isolates showed improvement in their functional properties, but the improvement was greater for the denatured protein isolate. The solubilities of denatured and native protein isolates at the isoelectric pH increased from 26.4 g l^?1 and 64 g l^?1, respectively, without salt to maxima of 363 and 308 g l^?1, respectively, at 0.75 M salt concentration. The water binding capacity of the isolates increased with increase in NaCl concentration from 1.70 ml g^?1 to 1.77, 1.82, 1.92 and 2.2 ml g^?1 for denatured protein isolate and from 1.45 ml g^?1 to 1.65, 1.69, 1.82 and 1.97 ml g^?1 for native protein isolate at 0.25, 0.50, 0.75 and 1.0 M salt concentrations, respectively. When the properties of the isolates in 0.75 M NaCl solutions were compared with those in salt‐free water there were 15% and 116% increases in emulsifying capacity, 40‐fold and 45‐fold increases in emulsifying activity and 4.6‐fold and 40‐fold increases in emulsion stability for native and denatured protein isolates, respectively, whilst the corresponding foaming capacities increased from 4 to 5.5 and 0 to 8.9 ml g^?1 protein. Statistically, no difference in the foaming capacity of either of the isolates was observed above 0.5 M NaCl. The foam stability also exhibited similar behaviour. Copyright © 2004 Society of Chemical Industry     

Effect of β‐cyclodextrin on pasting properties of wheat starch

Changes of viscosity characteristics of genetically diverse hexaploid wheat starches during pasting in water, 1% NaCl solution, or at pH 4 or pH 10 were studied using a Rapid Visco‐Analyzer. Peak viscosity (PV( hot paste viscosity (HPV) and cool paste viscosity (CPV) of all the wheat starches was little affected in pH 4 and pH 10 treatments. In 1% NaCl, all starches showed substantial increases in all three parameters relative to pasting in water. The use of 1% β‐cyclodextrin (β‐CD) (cycloheptaamylose) solution increased PV of high‐swelling starches, but generally slightly decreased that of low swelling starches in all treatment conditions. HPV was always reduced by addition of 1% β‐CD, but CPV was increased in most treatments for Anza and Yecora Rojo (low swelling( but decreased for Klasic (high swelling). Bacterial α‐amylase was added to starch or flour. The effect of β‐CD was shown to be independent of α‐amylase inhibition in wheat starch, but β‐CD strongly inhibited α‐amylase in wheat flour.     

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.     

Heat Gelation Properties and Protein Extractability of Beef Myofibrils

At a heating rate of 1^oC/min suspensions (pH 6.0) of isolated beef myofibrils were found to start forming gels at 43-56^oC, as detected by dynamic rheological measurements. The increase in gel storage modulus levelled off at temperatures > 65^oC. At medium to high (0.3-0.6M) concentrations of sodium chloride, addition of pyrophosphate (plus magnesium chloride) had the following effects: (1) both the protein extractability of non-heated myofibrils and the storage moduli of heat-induced gels were markedly increased; (2) the apparent activation energy for gel formation was decreased. Increasing concentrations of sodium chloride, up to 0.5-0.6M, increased the protein concentration of the liquid phase of the gels.     

A COMPARISON OF THE RHEOLOGICAL CHARACTERISTICS OF DIFFERENT FRACTIONS OF CHICKEN MYOFIBRILLAR PROTEINS1

ABSTRACT Suspensions of myofibrils and salt-soluble (SSP) or insoluble (SIP) proteins of chicken breast muscle in 0.6 M NaCl at pH 6.0 were heated to induce gels. Dynamic oscillating measurements showed multiple transitions in the shear storage modulus and loss modulus for all three protein fractions in the temperature range of 40–65C. However, changes in these viscoelasticities were most pronounced for SSP and least appreciable for SIP. Gel penetration test also revealed a descending order of SSP < myofibrils < SIP in gel strength. The three fractions of myofibrillar proteins appeared to follow a similar gelation mechanism but vary in the density of the gel networks.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER (PSEUDOSCIAENA CROCEA)

A myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker (Pseudosciaena crocea) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S‐200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio‐Sil SEC‐125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N‐terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α‐tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α‐tropomyosin‐binding proteinase.     

Thermally Induced Interactions and Gelation of Combined Myofibrillar Protein from White and Red Broiler Muscles

The gelling properties of broiler myofibrillar protein were studied by determining protein-protein interactions during heating. Breast and leg salt-soluble protein (SSP) showed 1–3 transitions in protein-protein interactions within pH 5.5–6.5. The maximum transition temperatures of leg SSP decreased when leg SSP was mixed with breast SSP. The combined breast/leg myofibrils formed stronger gels than leg myofibrils alone at pH ≥ 6.0, and stronger gels than breast myofibrils alone at pH < 6.0. The results suggest that interactions existed between breast and leg myofibrillar proteins, and the transitions in these interactions were useful for predicting gel strength of the combined breast/ leg myofibrils.     

Chemical Modification and Functional Properties of Acylated Beef Heart Myofibrillar Proteins

Beef heart myofibrils were acylated with 0.1, 0.3, 0.6, 0.8, 1.0, 1.5, 2.0, 3.0, and 5.0 mmoles anhydride/g protein, at pH 8.0–8.5 and 2–3°C, with acetic anhydride (AA), succinic anhydride (SA), cis,cis,cis,cis-tetrahydrofuran-2,3,4,5-tetracarboxylic dianhydride (FA), and 1,2,4-benzenetricarboxylic anhydride (BA). The anhydride reacted with e-amino groups of lysine, sulfhydryl groups, and hydroxyl groups of tyrosine, serine, and threonine. Chemically modified beef heart myofibriliar proteins were superior to native heart myofibriliar proteins in solubility, emulsifying capacity, emulsion activity, and emulsion stability in a low salt solution of 0.1M NaCl, 0.05M potassium phosphate at pH 7.4 and 6.0. Protein modified with 0.5 mmole anhydride/g protein in 0.2M NaCl had a solubility greater than unmodified proteins in 0.6M NaCl at pH 7.4. Chemical modification also altered the pH-solubility profile. The chemically modified beef heart myofibrillar proteins exhibited an emulsifying capacity at pH 6.0 and 7.4 that was greater than that of the native proteins at pH 7.4. The recommended extent of acylation for modifying beef heart myofibrils on a gram protein basis is 0.6 mmole AA, 1.5 moles SA, 0.6 mmole FA, and 0.6 mmole BA.