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.     

FACTORS AFFECTING THE SODIUM CHLORIDE EXTRACTABILITY OF MUSCLE PROTEINS FROM CHICKEN BREAST, TROUT WHITE AND LOBSTER TAIL MUSCLES

The amount of protein extracted from chicken breast muscle at low salt (0–50 mM NaCl) increased as the salt concentration of the extracting solutions increased. The addition of 10 mM sodium phosphate buffer pH 7 (P_i) caused a marked increase in protein extractability at all salt concentrations. A particular polypeptide chain of about 150,000 daltons appeared to be particularly sensitive to the extraction conditions. At high salt (0.6M NaCl, 50 mM sodium phosphate buffer pH 7.0) a second extraction still contained significant amounts of protein. The amount of protein extracted was maximized at a 1/20 dilution. On the other hand, the protein extract-ability of trout white muscle, showed a smaller P_i effect and very little dependence on low salt concentration. The protein extractability of lobster flexor muscle showed little change with either increased salt or P_i. For all three muscles extraction over time with either high or low salt remained essentially constant after the first day with the most protein being extracted from lobster muscle and the least from chicken muscle.     

Fractionating Soybean Storage Proteins Using Ca2+ and NaHSO3

ABSTRACT:  Individual soybean storage proteins have been identified as having nutraceutical properties, especially β-conglycinin. Several methods to fractionate soy proteins on industrial scales have been published, but there are no commercial products of fractionated soy proteins. The present study addresses this problem by using calcium salts to achieve glycinin-rich and β-conglycinin-rich fractions in high yields and purities. A well-known 3-step fractionation procedure that uses SO_2, NaCl, and pH adjustments was evaluated with CaCl₂ as a substitute for NaCl. Calcium was effective in precipitating residual glycinin, after precipitating a glycinin-rich fraction, into an intermediate fraction at 5 to 10 mM CaCl₂ and pH 6.4, eliminating the contaminant glycinin from the β-conglycinin-rich fraction. Purities of 100%β-conglycinin with unique subunit compositions were obtained after prior precipitation of the glycinin-rich and intermediate fractions. The use of 5 mM SO₂ in combination with 5 mM CaCl₂ in a 2-step fractionation procedure produced the highest purities in the glycinin-rich (85.2%) and β-conglycinin-rich (80.9%) fractions. The glycinin in the glycinin-rich fraction had a unique acidic (62.6%) to basic (37.4%) subunit distribution. The β-conglycinin-rich fraction was approximately evenly distributed among the β-conglycinin subunits (30.9%, 35.8%, and 33.3%, for α', α, and β subunits, respectively). Solids yields and protein yields, as well as purities and subunit compositions, were highly affected by pH and SO₂ and CaCl₂ concentrations.     

Influence of sodium chloride and glucose on the aggregation behavior of heat-denatured ovalbumin investigated with a multiangle laser light scattering technique

ABSTRACT:  The molecular characteristics of ovalbumin aggregates formed by heating with NaCl and glucose were investigated with a multi-angle laser light scattering system. The presence of NaCl and glucose affected the formation and molecular structure of the aggregates. Specifically, glucose increased the denaturation temperature of ovalbumin due to thermal stabilization of the native state of ovalbumin, regardless of the content of added NaCl. The surface hydrophobicity of the aggregates was increased by the addition of NaCl, which induced the denaturation of ovalbumin at a lower temperature. Aggregates with a larger weight-average molar mass ( M_w ) and root mean square radius ( R_g ) formed from heat-denatured ovalbumin with NaCl and glucose. The presence of NaCl during heat denaturation caused the formation of aggregates with a larger M_w (1.9 × 10⁵ and 3.5 × 10⁶ g/mol for 0 and 10 mM NaCl, respectively) and R_g (14.8 and 80.4 nm for 0 and 10 mM NaCl, respectively). Over a certain amount of NaCl, the addition of more glucose resulted in the formation of more aggregates with greater M_w and R_g values. In sum, the thermostability of ovalbumin was affected primarily by glucose, but the molecular characteristics of the soluble aggregates formed by heat denaturation varied primarily with NaCl content.     

Effect of NaCl addition during diafiltration on the solubility, hydrophobicity, and disulfide bonds of 80% milk protein concentrate powder

We investigated the surface hydrophobicity index based on different fluorescence probes [1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-propionyl-2-(N,N-dimethylamino)-naphthalene (PRODAN)], free sulfhydryl and disulfide bond contents, and particle size of 80% milk protein concentrate (MPC80) powders prepared by adding various amounts of NaCl (0, 50, 100, and 150 mM) during the diafiltration process. The solubility of MPC80 powder was not strictly related to surface hydrophobicity. The MPC80 powder obtained by addition of 150 mM NaCl during diafiltration had the highest solubility but also the highest ANS-based surface hydrophobicity, the lowest PRODAN-based surface hydrophobicity, and the least aggregate formation. Intermolecular disulfide bonds caused by sulfhydryl-disulfide interchange reactions and hydrophobic interactions may be responsible for the lower solubility of the control MPC80 powder. The enhanced solubility of MPC80 powder with addition of NaCl during diafiltration may result from the modified surface hydrophobicity, the reduced intermolecular disulfide bonds, and the associated decrease in mean particle size. Addition of NaCl during the diafiltration process can modify the strength of hydrophobic interactions and sulfhydryl-disulfide interchange reactions and thereby affect protein aggregation and the solubility of MPC powders.     

MEASURING THE WATER HOLDING CAPACITY OF NATURAL ACTOMYOSIN FROM CHICKEN BREAST MUSCLE IN THE PRESENCE OF PYROPHOSPHATE AND DIVALENT CATIONS

The water holding capacity (WHC) of natural actomyosin (NAM) extracted at pH 9.2 in 0.6M KCl was measured in the presence and absence of various combination of sodium pyrophosphate (PP_i), MgCl₂ and CaCl₂ using a modification of the classical centrifugation technique. Samples, in the presence of 0.15M NaCl and 20 mM sodium phosphate buffer pH 6, were spun at 30,900 X G (as measured at the bottom of the centrifuge tube) for 15 min at 2–4C. The results show that between 17 and 20 g water/g protein were bound over a wide range of NAM concentrations. In each case the amount of water held by the experimental sample was equal to or less than the amount held by a control run at the same time: 5 mM PP_i= 100%; 5 mM PP_i+ 5 mM MgCl₂= 58%; 5 mM MgCl₂= 85%; 5 mM PP_i+ 5 mM CaCl₂= 68% and 5 mM CaCl₂= 92% of the control. Thus polyphosphate addition in the presence of divalent cations lowers the WHC of NAM. The absence of the organized structure of muscle in NAM is postulated to be the reason that polyphosphate plus divalent cation reduced WHC in these samples. A series of preliminary experiments were run in order to determine the effect of experimental parameters on WHC.     

Effects of Alkali and Acid Solubilization on Gelation Characteristics of Rockfish Muscle Proteins

ABSTRACT: Solubility of rockfish whole muscle and actomyosin was minimum at pH 5 and gradually increased as the pH was shifted to acidic or alkaline pH. Acidic and alkaline solubilization was followed by isoelectric precipitation induced degradation of myosin heavy chain, resulting in a protein band of about 120 kDa. Both myofibrillar and sarcoplasmic proteins underwent denaturation after acidic and alkaline treatment, exhibiting minimal solubility and absence of endothermic peaks. Acid- and alkali-treated muscle proteins readily aggregated upon heating, showing different dynamic rheological patterns compared with whole muscle and washed mince. Disulfide linkages occurred at a greater extent in gel prepared by alkaline solubilization, resulting in higher breaking force and deformation.     

Effect of pH and Ionic Strength on the Heat Stability of Rapeseed 12S Globulin (Cruciferin) by the ANS Fluorescence Method

The heat stability of rapeseed 12S globulin (cruciferin) was examined using 8-anilinonaphthalene-1-sulphonic acid (ANS) as a fluorescence probe. Heating cruciferin (0·06–0·3 mg ml⁻¹ in 10 mM glycyl–glycyl piperizine buffer, pH 7·0, with 0·1–1·0 M NaCl) for 20 min increased its hydrophobicity as monitored by ANS fluorescence measurements. The mid-point temperature for the heat effect (T_m) increased linearly with increasing solvent pH (T_m (°C)=4·16 pH+41 (μ=0.1)) or sodium chloride concentration (T_m (°C)=14·7 [NaCl]+71 (pH=7·0)). The range of T_m values for cruciferin was 45–96°C. At 20°C cruciferin was unstable at pH<3·0 but relatively stable under alkaline conditions (pH 8–10). Though possessing an oligomeric structure, cruciferin appears to heat denature in accordance with the two-stage deactivation model for simple globular proteins.     

Solubility of amaranth seed proteins in sodium sulphate and sodium chloride: the main factor in quantitative extraction for analysis

Nitrogen was extracted more efficiently from amaranth seed with 0.04 M Na₂SO₄ (5% w/v) than with either 0.09 M or 0.17 M NaCl (5% or 10% w/v), despite both solutions having the same ionic strength (μ= 1). Solubility of saline soluble proteins (albumin ± globulin) was very poor in either water or 1M NaCl, but increased in 0.4M NaCl at alkaline pH between 7 and 10. Globulins were very soluble in 0.4M NaCl at a pH 9. Albumin was the main storage protein. Saline soluble proteins formed very weak gels.     

Solubility, Hydrophobicity and Net Charge of Succinylated Canola Protein Isolate

Effects of succinylation (54% and 84% modification of free amino groups), pH (3.5–11.0) and NaCl concentration (0.0–0.7M) on solubility, hydrophobicity and zeta potential (net charge) of canola protein isolate were examined. Succinylation markedly enhanced protein solubility at alkaline and slightly acidic pH while effect of NaCl depended on pH. Surface hydrophobicity (S₀) decreased as level of succinylation increased. Effect of NaCl varied with pH but in general, S₀ decreased in a curvilinear manner as pH increased. Zeta potential became more electronegative as both succinylation and pH increased, but decreased with addition of NaCl. Hydrophobicity and zeta potential were closely related in a nonlinear inverse manner demonstrating that ionic environment had opposing influences on number of hydrophobic and charged groups on the surface of protein molecules in solution.     

Acid-Aided Protein Recovery from Enzyme-rich Pacific Whiting

ABSTRACT: Relatively higher protein recovery was obtained in acid-aided processing. Solubility was lowest at pH 5 and gradually increased up to pH 11.0. A sharp increase in solubility occurred at alkaline pH between 9.5 and 11.0 and at acidic pH between 3.0 and 1.5. Cathepsin B and L showed higher activities in acid surimi than conventional surimi. Acid-aided surimi did not show Ca- and Mg-ATPase activity and also had lower surface hydrophobicity and sulfurhydryl contents than conventional surimi. Acid processing resulted in low breaking force, possibly due to the activity of retained cathepsin L enzymes. Myosin heavy chain (MHC) and actin were degraded in acid processing and produced major bands right below MHC and actin.     

Cryoprotection Affects Physiochemical Attributes of Rainbow Trout Fillets

ABSTRACT: Stabilization of trout myofibrillar proteins during −20°C, 90-d storage and after soaking fillets in water, 8.0% sucrose/sorbitol, or 1.0% sodium lactate was investigated with or without 0.5% phosphate and with or without 0.05% MgCl₂. Fillets not soaked were the control. Compared with the control, cryoprotectants increased total protein and myofibrillar protein solubility; decreased surface hydrophobicity, total, free, and disulfide sulfhydryl content, and myosin susceptibility to thermal denaturation. There were no differences in total protein solubility and actin susceptibility to thermal denaturation between cryoprotectants and the water treatment. Phosphate minimized frozen storage effects on actin solubility and reduced protein surface hydrophobicity and myosin susceptibility to thermal denaturation, while MgCl₂ increased the negative effects of frozen storage.     

SOLUBILIZATION AND FUNCTIONAL PROPERTIES OF MOTH BEAN (VIGNA ACONITIFOLIA (Jacq.) Marechal AND HORSE GRAM (MACROTYLOMA UNIFLORUM (Lam.) Verdc. PROTEINS

Horse gram and moth bean seeds contained 23.6% and 21.9% protein (N x 6.25), respectively. Both the legumes are rich sources of iron. The iron contents in horse gram and moth beans were 11.0 and 9.6 mg/100 g, respectively. NaCl at 10% (w/v) and Na₂CO₃ at 0.5% (w/v) were found to be effective in extracting 89% and 80% of moth bean and horse gram proteins from defatted flour The minimum solubility of horse gram proteins from defatted flour was at pH 4.0 whereas proteins from moth bean exhibited minimum solubility at pH 4.5. The water and oil absorption, and foaming capacities in case of horse gram and moth bean flours were 2.0 g/g and 2.2 g/g, 23.0% and 2.0 g/g, 1.6 g/g, 27.6%, respectively.     

Physicochemical and functional properties of acidic and basic polypeptides of soy glycinin

The amino acid composition, some selected physicochemical and functional properties of acidic and basic polypeptides of soy glycinin were investigated and compared. Large amount of these polypeptides were obtained by DEAE-Sepharose fast flow column chromatography. Free sulphydryl contents, surface hydrophobicity, solubility and emulsifying activities (at different pH values) were evaluated. Different polypeptides had different patterns of amino acid composition, especially contents of acidic (and basic) and hydrophobic amino acids. The free sulphydryl contents (including total and exposed) and surface hydrophobicity considerably varied with the type of polypeptides. Compared with glycinin, isoelectric point (pI) of individual polypeptides shifted towards a more acidic pH. At a given pH value (e.g. above or below pI), the solubility and emulsifying ability index of these polypeptides were closely related to their relative contents of acidic (and basic) amino acids. The results indicated that glycinin polypeptides with different amino acid character have different physicochemical and functional properties, especially solubility and emulsifying ability.     

pH 值对大豆分离蛋白构象及表面疏水性的影响

采用Lowery法、ANS荧光探针法、圆二色光谱、荧光光谱方法分别对不同pH值大豆分离蛋白溶解度、表面疏水性、蛋白质二级、三级结构进行分析。结果表明：随着pH值的升高,大豆分离蛋白的二级结构发生由β-折叠结构向α-螺旋结构的转变,其Trp残基所处微环境极性增强。大豆分离蛋白表面疏水性与溶解度呈负相关关系,同时大豆分离蛋白表面疏水性也与α-螺旋结构含量呈负相关关系。     

ATPASE ACTIVITY, SURFACE HYDROPHOBICITY, SULFHYDRYL CONTENT AND PROTEIN DEGRADATION IN REFRIGERATED SEABASS MUSCLE IN MODIFIED ATMOSPHERE PACKAGING

The effect of modified atmosphere packaging (80% CO₂, 10% O₂, 10% N₂) on ATPase activity, surface hydrophobicity, sulfhydryl content and degradation of proteins in seabass muscle during storage at 4C was investigated. No changes in Ca²⁺-, Mg²⁺-, Mg²⁺-Ca²⁺-ATPase activities of natural actomyosin (NAM) in seabass slices kept under MAP were observed throughout the storage for up to 21 days (P > 0.05). However, a slightly increased Mg²⁺-EGTA-ATPase was found. For seabass slices stored under air atmosphere, Ca²⁺-ATPase activity decreased, whereas Mg²⁺-EGTA-ATPase activity increased (P < 0.05) with a concomitant loss in Ca²⁺-sensitivity. Lower decreases in total sulfhydryl content but higher increases in surface hydrophobicity were observed in samples stored under MAP, compared to those kept under air atmosphere. No marked autolytic degradation in samples kept under MAP was observed throughout the storage as monitored by no changes in myosin heavy chain, free α-amino acid and trichloroacetic acid soluble peptide. Conversely, a considerable degradation was found in samples kept under air atmosphere, especially after 9 days of storage. Therefore, MAP is a promising means to retard the changes in muscle proteins, especially degradation.     

花生湿热处理对其分离蛋白的结构和功能特性的影响

本文通过还原/非还原电泳、热差示扫描、表面疏水性及等电点测定等手段,表征了花生湿热处理对其分离蛋白结构和功能特性的影响.结果表明:在105℃随着湿热处理时间延长其分离蛋白中伴球蛋白所占比例逐渐减少,球蛋白在加热75min后产生热聚集,此外通过美拉德反应生成的糖蛋白含量逐渐增加,分离蛋白等电点向酸性偏移,表面疏水性先逐渐...     

pH值对大豆11S球蛋白结构和表面疏水性的影响

采用Lowry法、8-苯氨基萘-1-磺酸铵盐（8-anilinonaphthalene-1-sulfonic acid ammonium salt,ANS）荧光探针法研究pH值对大豆11S球蛋白的溶解性和表面疏水性的影响,并利用圆二色光谱和荧光光谱对不同pH值条件下11S球蛋白二级结构和三级结构进行分析,为研究大豆蛋白结构与表面疏水性之间的构效关系提供理论基础。结果表明：除等电点外,大豆11S球蛋白溶解性和表面疏水性呈负相关,并且随着pH值的升高,大豆球蛋白二级结构中发生β-折叠和无规卷曲向α-螺旋的转变,三级结构中色氨酸（Trp）残基微环境极性降低。大豆球蛋白的表面疏水性与α-螺旋结构含量呈负相关。     

THE EFFECT OF pH, POLYPHOSPHATES AND DIFFERENT SALTS ON WATER RETENTION PROPERTIES OF GROUND TROUT MUSCLE

The water binding potential (WBP) and expressible moisture (EM) curves of ground rainbow trout white muscle versus pH are different, particularly in the region from pH 5.0 to 7.0. The effect of different salts such as CaCl₂, Mg Cl₂, NaCl, Nal and Na₂SO₄ on WBP suggests that for the same salts, cations cause a smaller change than anions. On the other hand, the EM measurements suggest that cations have a greater effect on this measurement than anions. In the case of WBP, the effect of tripolyphosphate and pyrophosphate can be attributed solely to their effect on pH. These results indicate the differences between the two methods and suggest that they measure different components of the water retention properties of a complex system, and thus water holding capacity measurements must be interpreted with care.     

Effects of NaCl Concentration on Salting-in and Dilution During Salting-out on Soy Protein Fractionation

ABSTRACT:  Glycinin and β-conglycinin are the main storage proteins in soybeans that can be fractionated by using alkali extraction, SO₂, salting-in with NaCl, salting-out by dilution and pH adjustment to produce a glycinin-rich fraction, a β-conglycinin–rich fraction, and an intermediate fraction, which is a mixture of the two proteins. Two different strategies were employed to optimize the procedure to achieve high efficiency in recovering the β-conglycinin–rich fraction. The first strategy was to optimize salting-in effects of NaCl, and the effects of NaCl concentration on the yields and purities of the protein fractions were investigated. The maximum protein yield of the β-conglycinin–rich fraction was obtained at 500 mM NaCl, but at the expense of purity. The optimum NaCl concentration was 250 mM, at which good protein yield (18.5%) and purity (84.5%) were achieved. At higher NaCl concentrations, the protein yields of the intermediate fractions were significantly lower, and the protein loss in the whey fraction increased. The second strategy was to improve the salting-out step for the β-conglycinin–rich fraction. At 0- and 0.5-fold dilution, the purities and yields of the β-conglycinin–rich fractions were significantly lower than at 1.0- and 2.0-fold dilution. There were no differences in protein yields or purities when using 1.0- or 2.0-fold dilution. According to these results, the recommended NaCl concentration for the salting-in step is 250 mM and the dilution factor for salting-out is 1.0.