Physico-chemical properties and rheological behaviour ofPatella caerula paramyosin

Paramyosin is a muscle protein which is characteristic of all invertebrates but which is not present in vertebrate muscles. Given the functional importance of paramyosin, the purpose of this paper was to study the physico-chemical properties, including the amino acid composition and rheological behaviour, of purified paramyosin and to investigate its mode of interaction with myosin. Paramyosin was purified from the limpet (Patella caerula) by an ethanol precipitation step. It was soluble at ionic strengths below 0.05 m NaCl and its maximum solubility at neutral pH occurred at approximately 0.4 M NaCl. At this high ionic strength, the pH dependence of solubility was such that paramyosin passed quickly into solution when pH exceeded pH 5, the transitional pH value. By using an immunological method, it was shown that interactions between paramyosin and myosin occurred, even in the presence of actin. The molecular assembly of both proteins was probably specified by hydrophobic interactions, as well as by interactions enhanced by divalent cations. The changes in the dynamic shear storage modulus (G′) started between 40°C and 50°C, and reached a maximum at about 75°C.     

Microbial transglutaminase-induced structural and rheological changes of cationic and anionic myofibrillar proteins

This study investigated the effects of microbial transglutaminase (TG) on structural changes in porcine myofibrillar protein (MP) under varying pH (2.0–6.0) and two ionic strength conditions (0.1 M versus 0.6 M NaCl). Lowering the pH below the isoelectric point (pI) of myosin induced protein unfolding as revealed by surface hydrophobicity and differential scanning calorimetry. Although the MP solubility at the low ionic strength (0.1 M NaCl) was maximal at pH 3.0, both SDS-PAGE profiles and dynamic rheology indicated TG could not cross-link MP under this condition. Based on the carboxyl group content, the TG-catalyzed deamidation was dominant at a pH lower than the pI of myosin (pH 5.0) while cross-linking occurred at higher pH. Moreover, deamidation had no effect on rheological properties of MP. The results indicate that the TG reaction was governed by the pH of substrate protein, and the reaction intensity was related to the solubility of protein.     

养殖暹罗鳄肉肌原纤维蛋白的理化性质

筛选养殖暹罗鳄肌原纤维蛋白含量最高部位,提取并分析离子强度、pH值和温度对肌原纤维蛋白溶液的溶解性、乳化性和热诱导凝胶特性的影响。结果表明：暹罗鳄尾部肌原纤维蛋白所占比例最高（（7.95±0.12） g/100 g,以湿质量计）,暹罗鳄尾肉中肌原纤维蛋白主要为肌球蛋白重链、副肌球蛋白、肌动蛋白和原肌球蛋白。在低离子强度条件下,肌原纤维蛋白的溶解度和乳化性较低,但有良好的凝胶特性,随着离子强度的升高,肌原纤维蛋白的溶解度和乳化性升高,凝胶特性则呈现下降趋势。随着pH值升高,肌原纤维蛋白溶解度呈现先迅速下降后升高的趋势,乳化性和凝胶特性则呈现持续缓慢下降的趋势,其中溶解度和保水性在pH 5.5达到最低点。随着热变温度升高,其凝胶特性显著增加,保水性先下降后略有升高,在低温（40 ℃）下有较好的保水性,在80 ℃保水性升高至又一峰值随后下降。结论：肌原纤维蛋白在NaCl浓度0.2 mol/L进行调配,并在80 ℃条件下加热处理,暹罗鳄肉类产品将具有较好的质构特性及保水性。     

Acid-induced aggregation of actomyosin from silver carp (Hypophthalmichthys molitrix)

Acid-induced denaturation and aggregation properties of silver carp actomyosin added with d-gluconic acid-δ-lactone (GDL) during incubation at chilled temperature (4 °C) were studied. Actomyosin underwent aggregation with decreasing pH, as evidenced by increased turbidity and decreased protein solubility in 0.6 M NaCl solution. Ca²⁺-ATPase activity decreased continually with increasing incubation time in the presence of GDL, accompanied by an initial increase in surface reactive sulphydryl (SH) content, indicating the conformation changes of actomyosin during acidification. Protein solubility in selected solvents and electrophoresis analysis showed that the major myofibrillar proteins, particularly, myosin heavy chain and actin were involved in the formation of protein aggregates mainly through noncovalent bonds including hydrophobic interactions and hydrogen bonds during acidification. The slight decrease in total SH content during acidification suggested that disulphide bonds were also involved in acid-induced aggregation of silver carp actomyosin to a lesser extent.     

ROLE OF IONIC STRENGTH IN BIOCHEMICAL PROPERTIES OF SOLUBLE FISH PROTEINS ISOLATED FROM CRYOPROTECTED PACIFIC WHITING MINCE

Biochemical characteristics of Pacific whiting muscle proteins extracted at acidic, neutral and alkaline conditions were investigated as affected by various ionic strength levels. The protein solubility at pH 4 declined, as NaCl was added up to 200 mM, due to protein aggregation through hydrophobic interactions. In contrast, at pH 7 and 10, solubility increased as NaCl was added up to 400 mM after which it remained constant. Changes in total SH content and S_owere highly related to the different molecular weight distributions of the soluble proteins. At pH 4, myosin heavy chain (MHC) was soluble as evidenced by the presence of MHC in the soluble fraction, even though degraded molecules were shown at IS 10–100 mM, and became completely insoluble at IS ≥ 150 mM. At pH 10, the density of the MHC band gradually increased as IS increased and the formation of high MW polymers was observed at IS ≥ 150 mM.     

Heat-induced gelation of myosin in a low ionic strength solution containing L-histidine

Binding properties are important for meat products and are substantially derived from the heat-induced gelation of myosin. We have shown that myosin is solubilized in a low ionic strength solution containing l-histidine. To clarify its processing characteristics, we investigated properties and structures of heat-induced gels of myosin solubilized in a low ionic strength solution containing l-histidine. Myosin in a low ionic strength solution formed transparent gels at 40-50 °C, while myosin in a high ionic strength solution formed opaque gels at 60-70 °C. The gel of myosin in a low ionic strength solution with l-histidine showed a fine network consisting of thin strands and its viscosity was lower than that of myosin in a high ionic strength solution at 40-50 °C. The rheological properties of heat-induced gels of myosin at low ionic strength are different from those at high ionic strength. This difference might be caused by structural changes in the rod region of myosin in a low ionic strength solution containing l-histidine.     

Dynamic oscillatory rheological measurement and thermal properties of pea protein extracted by salt method: Effect of pH and NaCl

The effects of two important factors, pH (3.0-10.0) and NaCl (0-2.0 M), on pea protein gelation properties were studied using dynamic oscillatory rheometer and differential scanning calorimeter (DSC). The strongest gel stiffness was achieved at 0.3 M NaCl; higher or lower salt concentrations lead to weakening of the gel. The gelation temperature was also influenced by ionic strength; salt had a stabilization effect which inhibited pea protein denaturation at higher salt concentrations resulting in higher gelling points (p < 0.05). At a NaCl concentration 2.0 M, pea protein gelation was completely suppressed at temperatures ?100 °C. The pH also played an important role in gel formation by pea protein isolates since acid and base cause partial or even total protein denaturation. In this paper the maximum gel stiffness occurred at pH 4.0 in 0.3 M NaCl; higher or lower pH values resulted in reduced gel stiffness (p < 0.05). pH also altered the denaturation temperature of the pea protein; higher pH values resulted in higher denaturation temperatures and higher enthalpies of denaturation (p < 0.05). At pH 3 pea proteins seem like completely denatured by acid as the DSC curve showed a straight line. The gelation temperature (gelling point) peaked at pH ∼6.0 (89.1 °C). Careful adjustment of pH and NaCl concentration would enable the food industry to effectively utilize the salt-extracted pea protein isolate as a gelling agent.     

Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH,temperature and salt

Hydrolyzed wheat gluten (GH, 77–85% protein) was prepared by limited hydrolysis with chymotrypsin at 37 °C for 4 h (degree of hydrolysis=6.4%) and 15 h (degree of hydrolysis=10.3%). The effect of microbial transglutaminase (MTGase) treatment (55 °C for 1 h, or 5 °C for 18 h) on the emulsifying and foaming properties of GH was evaluated under selected food processing conditions (pH 4.0 and 6.5, 0 and 0.6 M NaCl, and temperature 20 and 5 °C). At pH 4.0 and 0 M NaCl the MTGase treatment substantially increased foaming capacity (FC) of GH compared with their respective control GH samples, as a result of enhanced peptide adsorption to the air–water interface, but FC was similar for both control and MTGase-treated GH at pH 6.5. In contrast, foam drainage stability (FS) of MTGase-treated GH decreased at pH 4.0, but increased significantly (P<0.05) at pH 6.5 when compared with their respective control GH samples. The FC and FS were affected by 0.6 M NaCl in a pH-dependent manner. The MTGase treatments increased emulsion activity index up to 15-fold at pH 6.5, while emulsion stability index was influenced by emulsion temperature and ionic strength conditions. The MTGase-induced changes in functional properties of GH were attributed to pH-dependent solubility changes, the amphiphilic nature of gluten peptides and increased electrostatic repulsion resulting from deamidation.     

Study of Ca<Superscript>2+</Superscript>-ATPase Activity and Solubility in the Whole Kuruma Prawn (<Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>) Meat During Heating: Based on the Kinetics Analysis of Myofibril Protein Thermal Denaturation

Protein denaturation is considered to be the main cause of physicochemical changes in prawns during heating. However, no studies have been analyzed the kinetics of protein denaturation and the relationship between the degree of denaturation and chemical changes. Therefore, we investigated the changes in Ca²⁺-ATPase activity, protein solubility, and total sulfhydryl content of whole prawn meat during heating by determining the thermal denaturation kinetics of the proteins. Activation energies (E _a ) for the denaturation of myosin (183.2 kJ/mol) and actin (178.8 kJ/mol) were obtained by non-isothermal differential scanning calorimetry analysis. Using the kinetic parameters, the distribution of protein denaturation was predicted in whole prawns under arbitrary heating conditions. The results revealed an uneven distribution of the protein denaturation in prawns that was dependent on the heating conditions. Ca²⁺-ATPase activity decreased with increasing heating times at 51 or 85 °C and was strongly related to the average degree of protein denaturation. The results of protein solubility analysis suggested that hydrogen bonds, hydrophobic interactions, and ionic bonds changed with protein denaturation. The number of ionic bonds was reduced, while hydrogen content was enhanced at both temperatures. Hydrophobic interactions increased gradually at 51 °C (p?<?0.05). At 85 °C, hydrophobic interactions increased notably at first (p?<?0.05); however, as heating continued, no significant changes were observed (p?>?0.05). Our results indicate that the extent of protein solubility is significantly correlated with the average degree of protein denaturation during the heating process.     

ROLE OF pH IN SOLUBILITY AND CONFORMATIONAL CHANGES OF PACIFIC WHITING MUSCLE PROTEINS

This study was conducted to better understand biochemical changes of fish muscle proteins as affected by novel surimi process, acid- or alkali-aided solubilization. At 10 mM NaCl, between pH 5 and 10, the solubility of Pacific whiting muscle proteins was low but increased dramatically as the pH was shifted to either acidic or alkaline pH. At 600 mM NaCl, the isoelectric point was shifted to the acidic direction by about 2 pH units, resulting in aggregation of proteins at low pH, but improving the solubility of MHC (myosin heavy chain) between pH 6 and 10. ANS surface hydrophobicity (ANS-S_O) showed much greater values than PRODAN surface hydrophobicity (PRODAN-S_O) for samples treated at pH 2 - 4 perhaps due to an enhancement of the electrostatic interactions between the ANS probe and proteins. At very high pH, according to hydrophobicity results, proteins were partially refolded when the ionic strength increased. Under acidic conditions, SDS-PAGE demonstrated the degradation of MHC at 10 mM NaCl. The formation of MHC polymers was observed under alkaline treatment with a concomitant decrease of SH content.     

Effect of Two-Step Heating on Gel Properties of a Paramyosin-Myosin System

The effect of two-step Heating, including low temperature setting, on the gel properties of the paramyosin-myosin system was investigated. At relatively low paramyosin contents, little effect of the two-step heating on gel properties was observed. When the paramyosin became 25 % or more, the indices of gel properties became significantly larger than those for the gels prepared by one-step heating. Paramyosin was more susceptible to the effect of two-step heating than myosin. It was thus concluded that paramyosin was mainly responsible for the effect of setting in invertebrate muscle gels.     

Influence of environmental conditions on thermal stability of recombinant Aspergillus aculeatus pectinmethylesterase

The thermal stability of purified recombinant Aspergillus aculeatus pectinmethylesterase (PME) in different media was studied. The influence of pH, ionic strength and additives (salts and polyols) was evaluated. At pH 5.0 and a high ionic strength (0.50 M), the enzyme showed a high thermostability (inactivation at temperatures 60 °C). Interestingly, an enhancement of its heat stability was observed at pH 7.0 and temperatures above 55 °C, this behaviour was reflected in an atypical evolution of structural changes in the overall conformation of the enzyme, according to FTIR spectroscopy results. Recombinant A. aculeatus PME thermal inactivation at pH 7.0 could be described by a fractional-conversion model. Addition of NaCl increased the thermal stability at pHs 5.0 and 7.0, while addition of CaCl₂ had no influence. With regard to sugars (sucrose, trehalose, glucose and maltose) and polyols (sorbitol, lactitol and glycerol) addition, at the same concentration and pH, the polyols showed a higher protective effect than sugars. Also, the thermostability of recombinant A. aculeatus PME increased with the additive concentration, although the source of OH groups was the main parameter involved.     

Measurement of ionic calcium, pH, and soluble divalent cations in milk at high temperature

Dialysis and ultrafiltration were investigated as methods for measuring pH and ionic calcium and partitioning of divalent cations of milk at high temperatures. It was found that ionic calcium, pH, and total soluble divalent cations decreased as temperature increased between 20 and 80°C in both dialysates and ultrafiltration permeates. Between 90 and 110°C, ionic calcium and pH in dialysates continued to decrease as temperature increased, and the relationship between ionic calcium and temperature was linear. The permeabilities of hydrogen and calcium ions through the dialysis tubing were not changed after the tubing was sterilized for 1 h at 120°C. There were no significant differences in pH and ionic calcium between dialysates from raw milk and those from a range of heat-treated milks. The effects of calcium chloride addition on pH and ionic calcium were measured in milk at 20°C and in dialysates collected at 110°C. Heat coagulation at 110°C occurred with addition of calcium chloride at 5.4 mM, where pH and ionic calcium of the dialysate were 6.00 and 0.43 mM, respectively. Corresponding values at 20°C were pH 6.66 and 2.10 mM.     

Functional properties of whey protein concentrate texturized at acidic pH: Effect of extrusion temperature

Reactive supercritical fluid extrusion (RSCFX) process at acidic condition (pH 3.0) was used to generate texturized whey protein concentrate (TWPC) and the impacts of process temperature on product's physicochemical properties were evaluated. TWPC extruded at 50 and 70 °C formed soft-textured aggregates with high solubility than that extruded at 90 °C that formed protein aggregates with low solubility. Total free sulfhydryl contents and solubility studies in selected buffers indicated that TWPC is primarily stabilized by non-covalent interactions. Proteins texturized at 90 °C showed an increased affinity for 1-anilino-naphthalene-8-sulfonate (ANS) and a decreased affinity for cis-parinaric acid (CPA), indicating changes in protein structure. Water dispersion of TWPC at room temperature showed thickening function with pseudoplastic behavior. Secondary gelation occurred in TWPC obtained at 50 and 70 °C by heating the cold-set gels to 95 °C. TWPC texturized at 90 °C produced cold-set gels with good thermal stability. Compared to control, TWPC formed stable oil-in-water emulsions. Factors such as degree of protein denaturation and the balance of surface hydrophobicity and solubility influenced the heat- and cold-gelation and emulsifying properties of the protein ingredients. TWPC generated by low and high temperature extrusions can thus be utilized for different products requiring targeted physicochemical functionalities.     

Biochemical and conformational changes of myosin purified from Pacific sardine at various pHs

ABSTRACT:  Biochemical and conformational changes of purified sardine myosin were investigated at various pHs. The purity of myosin, as determined by SDS-PAGE, was approximately 94.6%. One major band at 205 kDa, corresponding to myosin heavy chain, and 3 light chains at 31, 24, and 23 kDa were observed on the SDS-PAGE gel. The greatest myosin protein solubility was observed at pH 7 and remained constant up to pH 11. Sardine myosin showed no solubility at pHs 2.5 to 5.0. Three endothermic peaks were obtained for samples prepared at pHs 7 and 10, while no peaks were shown for pH 2 samples, indicating chemical denaturation of myosin occurred before thermal treatment. The greatest Ca²⁺-ATPase activity was observed at pH 7, while no activity was observed between pHs 2 and 5 and at pH 11. Total sulfhydryl content was not measured at pHs 2.5 to 4 while the greatest measure was obtained for samples at pH 5.5. Surface hydrophobicity was not detected from pHs 2.5 to 5.0; thereafter the content remained consistent through pH 11. Storage modulus, indicating the elastic element of myosin gels, was minimally affected at pH 2, indicating myosin was chemically denatured before the temperature sweep treatment. However, at pH 10, the thermal exposure of myosin, as evidenced by dynamic thermograms with deeper valleys at 40 to 60 °C, was noted, indicating myosin was not damaged by adjustment to pH 10 and therefore was still able to undergo thermal gelation.     

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.     

Effect of endogenous acid proteinases on the properties of edible films prepared from Alaska pollack surimi

The existence of endogenous acid proteinases in Alaska pollack surimi and their effect on mechanical properties of surimi films were investigated. The optimum pH of acid proteinases involved in the degradation of myosin heavy chain (MHC) was 3.0, and the optimum temperature was 45 °C. The degradation of MHC was completely inhibited by pepstatin A together with any one of cysteine proteinase inhibitors, suggesting that acid proteinases present in surimi are mainly cathepsin D and cysteine proteinases. The concomitant decrease of surimi film strength with the extent of MHC degradation was observed, but surimi films were formed even when most of MHC was degraded. The main associative forces responsible for the surimi films prepared at pH 3.0 were ionic bonds and hydrophobic interactions.     

Effect of ionic strength and/or pH on Extractability and physico-functional characterization of broad bean (Vicia faba L.) Protein concentrate

Protein extractability studies showed that the protein of broad bean (Vicia faba L.) was extractable at both acidic and basic pH. The percentage of pH-12 extractable protein that was precipitated at pH 4 (isoelectric point) from protein concentrate, dehulled full-fat seed flour and whole seed flour are 62.0%, 61.2% and 71.6%, respectively. Low ionic strength (μ0.4) increased the solubility of the protein in the bean concentrate at acidic pH, while at alkaline pH, increase in ionic strength (0.1–2.0) had an inverse relationship on the concentrate protein solubility. The capacity to form protein-stabilized foam was least (34%) at pH 4 and highest (97%) at pH 12. These were increased to 62% (pH 4) and 139% (pH 12) in medium with ionic strengths of 0.2 and 0.4, respectively. The foam formed was more stable at pH 4 than at the other pHs. Low ionic strength of 0.1 improved water absorption capacity but reduced it at ionic strength of 0.6.     

Isolation and characterization of acid and pepsin-solubilized collagens from the skin of balloon fish (Diodon holocanthus)

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were successfully extracted from the skin of balloon fish (Diodon holocanthus) with yields of 4% and 19.5% respectively (based on dry weight). According to the electrophoretic patterns, both the ASC and PSC consisted of two different α chains (α1 and α2), were characterized to be type I, and contained imino acid of 179 and 175 residues/1000 residues, respectively. The PSC had a lower content of high-molecular weight cross-links than the ASC. The ultraviolet (UV) absorption spectrum of collagens showed that the distinct absorption was between 210 and 230 nm. A maximum solubility in 0.5 M acetic acid was observed at pH 1–5, and a sharp decrease in solubility above 4% (w/v) in both the ASC and PSC was observed in the presence of NaCl. The denaturation temperatures (T_d) of the ASC and PSC measured by viscometry were 29.01 °C and 30.01 °C, respectively. The maximum temperatures (T_max) of the ASC and PSC were 29.64 °C and 30.30 °C, respectively.     

Influence of salt and pH on the solubility and structural characteristics of transglutaminase-treated wheat gluten hydrolysate

Hydrolyzed wheat gluten (GH, 77–85% protein) was prepared by limited chymotrypsin digestion at 37 °C for 4 h (degree of hydrolysis = 6.4%) and 15 h (degree of hydrolysis = 10.3%). Microbial transglutaminase (MTGase) treatment (55 °C for 1 h, or 5 °C for 18 h) effect on the solubility and structural characteristics of GH was examined under selected food processing conditions (pH 4.0–7.0, 0–0.6 M NaCl). The MTGase treatment increased solubility of GH by 3–29-fold (P < 0.05) within pH 4.0–7.0. Addition of 0.6 M NaCl or changing the conditions of MTGase incubation did not significantly alter solubility characteristics of GH. The MTGase treatment decreased surface hydrophobicity, and increased carboxyl groups in GH, suggesting cross-linking and deamidation. Fluorescence and UV spectra attributed the improved GH solubility to MTGase-induced polar environment, and partial masking of some nonpolar aromatic amino acids possibly due to high-molecular-weight polypeptides formed.