Conditions limiting the influence of thiol–disulphide interchange reactions on the heat-induced aggregation kinetics of β-lactoglobulin

The effects of calcium and N-ethylmaleimide addition on the heat-induced denaturation/aggregation of β-lactoglobulin at neutral pH were examined. Aggregation of unfolded β-lactoglobulin was found to be rate limiting on heating aqueous protein solutions, while stoichiometric additions of calcium indicated that the unfolding reaction was limiting. Increased binding of calcium reduced the zeta potential of β-lactoglobulin to a minimum of −5 mV, resulting in first-order denaturation/aggregation kinetics at pH 7.0. Thiol–disulphide interchange reactions had a positive effect on the aggregation of unfolded β-lactoglobulin at low protein concentrations and low ionic strength. In the presence of calcium, the absence of thiol–disulphide interchange reactions did not affect the protein aggregation kinetics. Thiol–disulphide interchange reactions had little effect on storage modulus development of heated β-lactoglobulin (3%, w/w) acid gels; however, they enhanced the strain stability. Results showed the importance of non-specific forces in controlling the heat-induced aggregation of unfolded β-lactoglobulin.     

Isolation and characterisation of disulphide peptides from wheat flour

A method has been devised for the isolation and fractionation of disulphide peptides from flour. Oxidised glutathione is only a minor member of a family of disulphide peptides, the rest of which are basic and of molecular weight about 2000. The basic disulphide peptides could not be fractionated into individual components, but they contained some disulphide groups that were very reactive towards glutathione at pH 5.8, which is approximately the pH of dough.     

Disulphide interchange in cereal proteins

A method of measuring the extent of disulphide interchange between a model compound, cystamine, and cereal flour proteins has been applied to wheat, steamed wheat, rye, barley and maize. The results indicate that, if the extent of protein-protein S.S interchange is of the same order as that found in the model reaction, it is several times greater in wheat than the calculated minimum necessary to create a continuous protein network. In the case of rye the extent seems to be sufficient to just comply with this minimum condition. Steamed wheat, barley and maize (none of which can form doughs) fall below the limiting extent of interchange required. It has been assumed that insoluble proteins are precluded from taking part in protein-protein S.S interchange reactions, and evidence from the literature has been cited to support this.     

Disulphide interchange in dough proteins

Appreciable disulphide interchange has been found between wheat proteins and cystamine when the latter was incorporated into doughs. If the extent of the protein-protein reaction is of the same order it has been calculated that interchange of this magnitude would be enough to cross-link the glutenin into a continuous structure. Cysteamine was more reactive than cystamine in the disulphide interchange reaction.     

三聚磷酸钠改性花生蛋白的研究

采用三聚磷酸钠对花生蛋白进行化学改性,探讨了改性中三聚磷酸钠的添加量、反应温度、时间、pH等因素对花生蛋白化学改性的影响。实验结果表明,通过化学改性使花生蛋白的溶解性达到2.01g/mol,乳化度为56.98%,乳化稳定性为55.54%,高于原料花生蛋白的水平。     

Determination of thiol groups and disulphide bonds in wheat flour and dough

A study was made of the conditions for determining all thiol (SH) groups and disulphide (S—S) bonds in wheat flour and dough. Thiol groups were determined by amperometric titration with Ag₂SO₄, the flour or dough which contains proteins and peptides being suspended in a solution of 6 m urea and 10^?4 M sodium dodecyl sulphate (SDS). The potential of the platinum electrode was ?0.24 V vs calomel reference electrode. Disulphide bonds were determined in two ways: (a) Using an excess of Ag₂SO₄ in the presence of 9 M urea; when the reaction was finished, the excess Ag₂SO₄ was removed by adding an exact quantity of glutathione. The glutathione which remained after binding the Ag₂SO₄ excess was determined by amperometric titration with a 5.10^?5 M Ag₂SO₄ solution, (b) Determination after reduction with NaBH₄; the disulphide bonds were reduced to thiol groups and the total amount of thiol groups was determined with Ag₂SO₄. The results indicated that wheat flour contains 5–7 μmol SH-groups, which is about six times the level found by previous workers, and 11-18 μmol disulphide bonds per gram flour. A correlation with baking quality was not observed.     

The reaction of wheat proteins with sulphite. II.—The accessibility of disulphide and thiol groups in flour

The disulphide groups of flour which are accessible for reaction with sulphite, have been measured at different concentrations (0–8 M) of urea. The content of thiol groups has also been determined over the same range of urea concentrations. Results are given for two flours which yield doughs of widely different rheological properties. The accessibility of disulphide groups in the stronger flour showed a greater response to increasing urea concentration, and reached a maximum at a lower urea concentration than did the weaker flour. The results are discussed in relation to current theories of the mechanism of dough formation, involving protein thiol-disulphide interchange reactions.     

Modification of plant proteins by immobilized proteases

A potential application of plant proteins could be a replacement of animal proteins now in use in the food industry on the basis of certain specific functional properties plant proteins have. Modification of the chemical structure of selected plant proteins is needed to replace more expensive animal proteins as food ingredients that have specific functional characteristics. Structure modification may be achieved by physical, chemical, or microbiological methods, or by a combination of these. Immobilized enzyme techniques offer significant advantages for protein modification. Knowledge of the molecular properties of plant proteins is essential to understand the basis of protein functionality, to modify proteins so that they acquire desirable functional properties, and to predict potential applications of modified plant proteins. This paper reviews all the above mentioned aspects of plant protein chemistry and potential utilization.     

Fractional extraction of cereal flour proteins

Wheat (Wichita), rye, barley, oat and maize flours were successively extracted with 0.04 m sodium chloride solution (3 × ), water (3 × ), 70% ethanol (3 ×) and 0.1 N acetic acid. The fractions and residues were analysed for nitrogen and subjected to starch-gel electrophoresis. Saline extractions chiefly removed the albumins and globulins of all five cereals. The prolamins of wheat, rye and barley appeared mostly in the three aqueous extracts but also, particularly, those of very low mobility, occurred in the alcoholic solutions. The acetic acid-soluble fractions of wheat, rye and barley were shown by electrophoresis under reducing conditions to be crosslinked by S.S bonds; those of rye and barley showed some characteristic differences from glutenin. Oats and maize were practically devoid of this fraction. Most of the protein of oats and maize was insoluble. Electrophoresis patterns after reduction were obtained from insoluble fractions, though there were differences among the genera. Both overall protein solubility and the quantity of the acetic acid-soluble fraction were correlated with baking quality. The presence of covalent crosslinks other than S.S in glutelin fractions is suspected.     

Amino acid analyses of cereal flour proteins

Amino acid analyses have been made on flours of wheat, rye, barley, oats and maize. The overall pattern is sufficiently similar (particularly between rye and barley) to indicate a common ancestor, but significant individual differences occur. Wheat protein differs from the other four in its higher capacity for polar and H-bonds and lower content of salt links. Other factors such as S.S interchange potential may be more important than the pattern of intermolecular forces since the latter does not change much from rye to barley in spite of their rheological differences. The factors for conversion of nitrogen to protein are, in the above order, 5.7, 5.8, 5.8, 5.7, 5.9.     

Extraction and fractionation of wheat flour proteins

Extraction of wheat flour with 1.5% sodium dodecyl sulphate (SDS) solution dissolved 65–67% of the total flour nitrogen. The SDS-insoluble proteinaceous material was separated into glycoproteins-I, II and III by ultracentrifugation. Part of the SDS-soluble proteinaceous material was precipitated by addition of ethanol and separated into glycoproteins-IV, glutenins and globulins. The rest of the dissolved proteinaceous material was separated into glycoproteins-V, gliadins, glycoproteins-VI, and albumins by (NH₄)₂SO₄-precipitation and gel filtration chromatography.     

Extraction and fractionation of wheat flour proteins

With the aid of a 1.5% sodium dodecyl sulphate solution (SDS) the proteins of two Dutch wheat flours were separated into an SDS-soluble and an SDS-insoluble fraction. The SDS-soluble fraction was fractionated with the aid of ethanol precipitation and gel filtration chromatography into albumins, globulins, gliadins, glutenins II and III, and glutelins I, II, III and IV. The SDS-insoluble proteinaceous material was separated into glutenins I and glycoproteins. The protein fractions were identified with the aid of SDS-polyacrylamide gel electrophoresis and amino acid analysis. The glutenins I and II consist of A, B and C subunits. Their molecular weights ranged from about 600 000 to 10 million. The glutenins III consist only of B and C subunits and their molecular weights ranged from 300 000 to 600 000. A hypothesis explains how the glutenins III are the precursors for glutenins I and II and how they contribute to gluten's structure. The glutelins I consist of α β and γ subunits and their molecular weights ranged from about 600 000 to several millions. The glutelins II consist also of α β and γ subunits with a molecular weight of about 300 000 to 600 000, while the molecular weights of the glutelins III and IV ranged from 10 000 to 200 000. All glutelins are insoluble in 70 % aqueous ethanol and 5 M urea, but soluble in SDS and 0.1M NaOH. Only the glutelins III and IV are soluble in 0.1M HAc. The globulins consist of a heterogeneous fraction of components, their molecular weights arranged from 98 000 to 10 000. The gliadins form a heterogeneous but homologous group of polypeptides with an average molecular weight of 35 000. The albumins consist of components with molecular weights lower than 15 000.     

Fractionation and characterization of tartary buckwheat flour proteins

Protein fractions (albumin, globulin, prolamin and glutelin) were extracted from defatted tartary buckwheat flour. Albumin was the predominant protein fraction (43.8%) followed by glutelin (14.6%), prolamin (10.5%), and globulin (7.82%). Albumin was relatively rich in histidine, threonine, valine, phenylalanine, isoleucine, leucine and lysine. Globulin had high levels of methionine and lysine. Prolamin was high in histidine, threonine, valine, isoleucine, and leucine. Glutelin was rich in histidine, threonine, valine, isoleucine, and leucine. SDS–PAGE analysis, under non-reductive and reductive conditions, showed that disulfide bonds existed in the four fractions. Non-reduced albumin showed major bands at 64, 57, 41, and 38 kDa, and globulin at 57, 28, 23, 19 and 15 kDa. Reduced albumin and globulin shared two common bands at 41 and 38 kDa. Reduced prolamin showed major bands at 29, 26, 17 and 15 kDa. In vitro pepsin digestibility of the four fractions (from high to low) was: albumin > globulin > prolamin and glutelin.     

Copper modulates the heat-induced sulfhydryl/disulfide interchange reactions of β-Lactoglobulin

This study describes the effect of copper on the heat-denaturation/aggregation of β-Lactoglobulin AB at neutral pH. The kinetics of disappearance of native β-Lactoglobulin under different ionic strength and Cu²⁺/β-Lactoglobulin molar ratio conditions were followed and the type of interactions (covalent or non-covalent) shared between non-native structures during the heating process were examined. On heating, the rate of disappearance of native β-Lactoglobulin was accelerated by increasing the Cu²⁺/β-Lactoglobulin molar ratio. Copper induces oxidation of the free sulfhydryl group of β-Lactoglobulin resulting mainly in the formation of covalent dimers, which were further associated into large non-covalent aggregates under high ionic strength conditions. Characterisation of the β-Lactoglobulin dimers reveals the existence of three different molecular species arising randomly (dimers A–A, A–B and B–B), in which tertiary structure was completely lost. The quantity of added copper constitutes a powerful way to control the heat-denaturation/aggregation process of β-Lactoglobulin in particular regarding the relative proportion of covalent and non-covalent interactions into formed aggregates.     

Isolation and characterisation of wheat flour proteins. III.—Amino-acid composition of Sephadex-gel fractionated wheat flour proteins

Wheat flour proteins were extracted with 0·05M -acetic acid, 0·01M -sodium pyrophosphate buffer, pH 7·0, and 0.05M -acetic acid after pyrophosphate buffer, and were fractionated by Sephadex-gel filtration. Seventeen amino-acids were determined by ion-exchange Chromatography in hydrolysates of 19 protein extracts and fractions. Concentrations of amino-acids with ionisable cationic chains and with hydroxylic groups, and of cystine, glycine, alanine, valine, leucine and methionine were higher, and of dicarboxylic acids, and their amides, and of proline were lower, in the pyrophosphate- than in the acetic acid-extracted proteins. The distribution of amino-acids in Sephadex-gel separated fractions depended on the elution volume of the fraction. It was affected also by the system used in extracting the proteins from the flour and by the presence of salt-dispersible proteins in the acetic acid-extractable proteins.     

Stabilization of milk proteins in acidic conditions by pectic polysaccharides extracted from soy flour

Pectic polysaccharides were extracted from soy flour at either room temperature (SPRT) or 121°C (SPH), and their abilities to stabilize milk proteins in acidic conditions were evaluated. Both SPRT and SPH were found to contain proteinaceous components that were difficult to dissociate from polysaccharide components using size exclusion chromatography, whereas the molar mass of the former was approximately twice that of the latter. Due to the higher molar mass, SPRT was expected to provide stronger steric effects to prevent aggregation between milk proteins in acidic conditions than SPH. Alkaline treatment of SPRT for breaking O-linkages between AA and monosaccharide residues decreased its molar mass by approximately 160 kDa, indicating that they contained naturally occurring conjugates of pectic and proteinaceous moieties. Particle size distributions in simulated acidified milk drink samples containing 0.2% SPRT or SPH showed monomodal distributions with median diameters of around 1.2 μm at pH 4. The presence of large protein aggregates (～5 μm) was detected at 0.2% SPRT and pH 3.2, 0.6 to 0.8% SPRT and pH 4, or 0.2% SPH and pH 3.4. The presence of excess polysaccharide molecules unbound to proteins was detected at 0.2% SPRT and pH 3.2 to 3.4, 0.4 to 0.8% SPRT and pH 4, 0.2% SPH and pH 3.4 to 3.6, and 0.4 to 0.8% SPH and pH 4. The present results suggest that molecular characteristics of pectic polysaccharides vary depending on extraction conditions and hence their functional behavior.     

Determination of carboxyl groups in wheat and barley flour proteins

The method of Hoare and Koshland for the estimation of free carboxyl groups in proteins has been scaled down and applied to wheat proteins. With standard proteins, provided the carboxyl groups were not inaccessible (as in lysozyme), the error was as large as 19%. This level of error is acceptable for estimating by difference amide contents when these are high, as in gluten proteins, because the error is greatly reduced by the molar ratio of side-chain CONH₂:COOH—a ?leverage’? effect. About 95% of the side-chain COOH groups appear to be amidated in the prolamins and about 50% in the albumins. The proportion of Glx + Asx in the amide form seems to decrease slightly as the electrophoretic mobility of a prolamin increases. The errors in calculating the numbers of positive and negative charges that a protein bears at pH 8.9 become serious when superimposed on the net charge, which is the small difference between these numbers. The net charge at pH 8.9, therefore, could not be obtained accurately by calculation. The polarity of the net charge can be determined by electrophoresis using a marker for electroendosmosis.     

Isolation and characterisation of wheat flour proteins. IV.—effects on wheat flour proteins of dough mixing and of oxidising agents

Proteins dispersible in 3M -urea increased from about 65 to 85% during mixing of dough in Farinograph. The increase was positively correlated with length of mixing. Protein dispersibility was highest in doughs mixed with five-fold excess potassium iodate, and was lowest in flours. Intermediate amounts of proteins were extracted from doughs mixed in air, in a nitrogen atmosphere, or in air with a five-fold excess of potassium bromate. Proteins, extracted with 3M -urea from doughs mixed to optimum consistency, were separated by fractionation on Sephadex G-100 columns into at least 6 fractions, constituting 3 distinct peaks. Distribution of proteins in the peaks was measured by Kjeldahl-N, biuret, and absorbance at 280 mμ methods. Mixing in air, but not in a nitrogen atmosphere, increased substantially the amount of proteins with a molecular weight above 150,000 and decreased the amount of low molecular-weight proteins. The effects of mixing bromated doughs were similar to mixing plain doughs in air, but the chemical modifications in bromated doughs were much more pronounced. No such changes were observed in iodate-treated doughs. Similar changes in distribution of protein fractions were observed when the gross protein extracts or Sephadex-fractionated proteins were studied by electrophoresis on starch gel in the presence of 3M -urea.     

Modification of functional properties of egg-white proteins

Egg-white proteins are extensively utilised as food ingredients due to their unique functional properties. Several attempts have been made in order to improve the functional properties of egg-white proteins and to identify the optimal formulations for unique food products. Experimental data proves that controlled denaturation of egg-white proteins can have a beneficial impact on various functional applications in the food industry such as emulsifying ability, heat stability, and gelation. This review describes the effect of heat-induced denaturation on protein structure and functionality. Studies on the impact of Maillard reaction, which aim to elucidate the structure-function relationship of egg-white proteins, are presented. A novel approach which could be the basis for the development of new methods aiming to improve the functional properties of egg-white proteins is also discussed.