Heat-induced gelation of chicken Pectoralis major myosin and beta-lactoglobulin

The denaturation, aggregation, and rheological properties of chicken breast muscle myosin, beta-lactoglobulin (beta-LG), and mixed myosin/beta-LG solutions were studied in 0.6 M NaCl, 0.05 mM sodium phosphate buffer, pH 7.0, during heating. The endotherm of a mixture of myosin and beta-LG was identical to that expected if the endotherm of each protein was overlaid on the same axis. The maximum aggregation rate (AR(max)) increased, and the temperature at the AR(max) (T(max)) and initial aggregation temperature (T(o)) decreased as the concentration of both proteins was increased. The aggregation profile of <0.5% myosin was altered by the presence of 0.25% beta-LG. Addition of 0.5-3.0% beta-LG decreased storage moduli of 1% myosin between 55 and 75 degrees C, but increased storage moduli (G') when heated to 90 degrees C and after cooling. beta-LG had no effect on the gel point of > or =1.0% myosin, but enhanced gel strength when heated to 90 degrees C and after cooling. After cooling, the G' of 1% myosin/2%beta-LG gels was about 1.7 times greater than that of gels prepared from 2% myosin/1% beta-LG.     

Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.     

Characterization of beta-conglycinin and glycinin soy protein fractions from four selected soybean genotypes

The beta-conglycinin and glycinin fractions of soy protein were isolated from Macon, Ohio FG1, Enrei, and IL2 genotypes that were grown under the same environmental conditions. The soy protein fractions were evaluated to determine whether chemical composition and gel-forming properties were related. Amino acid analyses suggested that the hydrophobic residues may be the primary cause of differences in soy protein gel characteristics as the storage moduli increased with higher percentages of hydrophobic residues. Reversed-phase high-performance liquid chromatography profiles revealed variations in the composition of each fraction that corresponded to differences observed among the storage moduli. The gel-forming properties may be related to more than just protein content, such as the amount and type of amino acid in the fraction.     

Structure-physicochemical function relationships of soybean glycinin at subunit levels assessed by using mutant lines

Glycinin is a hexameric protein composed of five kinds of subunits. The subunits are classified into two groups, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). We purified four mutant glycinins composed of only group I subunits (group I-glycinin), only group II subunits (group II-glycinin), only A3B4 (A3B4-glycinin), and only A5A4B3 (A5A4B3-glycinin) from mutant soybean lines. The physicochemical properties of these glycinin samples were compared with those of the normal glycinin (11S) composed of five kinds of subunits. The thermal stabilities (as measured by thermal denaturation midpoint temperatures) of 11S, group I-glycinin, and group II-glycinin were similar to each other, although that of A3B4-glycinin was significantly lower than those of the others. The orders of aromatic and aliphatic surface hydrophobicities were the same: A3B4-glycinin > group II-glycinin > A5A4B3-glycinin > 11S > group I-glycinin. The solubility of 11S as a function of pH at mu = 0.5 was governed by that of group I-glycinin and followed this order at acidic pH: 11S = group I-glycinin > A3B4-glycinin > group II-glycinin = A5A4B3-glycinin. The order of emulsifying abilities was A5A4B3-glycinin > group II-glycinin > A3B4-glycinin > 11S > group I-glycinin. This order was consistent with that of the length of their hypervariable regions. Except for this relationship, there was no significant relationship among the other physicochemical properties of the mutant glycinins.     

Heat-induced aggregation of whey proteins: comparison of cheese WPC with acid WPC and relevance of mineral composition

Heat-induced aggregation of whey proteins in solutions made from two commercial whey protein concentrates (WPCs), one derived from mineral acid whey (acid WPC) and the other from cheese whey (cheese WPC), was studied using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), and transmission electron microscopy (TEM). Heat treatment (75 degrees C) of acid WPC solutions (12.0%, w/w, pH 6.9) resulted in formation of relatively small "soluble" aggregates that were predominantly disulfide-linked. By contrast, heat treatment of the cheese WPC solutions (under the same conditions) caused formation of relatively large aggregates, containing high proportions of aggregates linked by noncovalent associations. The rate of aggregation of both beta-lactoglobulin and alpha-lactalbumin at 75 degrees C, measured as the loss of native proteins by PAGE, was higher in the cheese WPC solution than in the acid WPC solution. Cross dialysis of the two WPC solutions resulted in alteration of the mineral composition of each WPC solution and reversing their heat-induced aggregation behavior. The results demonstrated that the mineral composition is very important in controlling the aggregation behavior of WPC products.     

Co-oxidation of beta-carotene catalyzed by soybean and recombinant pea lipoxygenases

A number of products including apocarotenal, epoxycarotenal, apocarotenone, and epoxycarotenone generated by lipoxygenase (LOX) catalyzed co-oxidation of beta-carotene have been tentatively identified through the use of GC/MS and HPLC combined with photodiode array detection. Because of the large number of high molecular weight products detected and their probable chemical structures, a co-oxidation mechanism is proposed that involves random attack along the alkene chain of the carotenoid by a LOX-generated linoleoylperoxyl radical. It is suggested that a direct release from the enzyme of the radical, which initiates the co-oxidation of beta-carotene, is greater for pea LOX-3 than for pea LOX-2 or soybean LOX-1. It is proposed that further products may be formed by free radical propagated reactions and that the formation of 1,10- and 1,14-dicarbonyl compounds may arise by secondary oxidation of the primary products.     

Immunochemical studies on gastric and intestinal digestion of soybean glycinin and beta-conglycinin in vivo.

Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and beta-conglycinin in preruminant calves fed milk replacers containing a mixture of skim milk powder and antigenic heated soybean flour. In experiment 1, duodenal passage of immunoreactive beta-conglycinin lasted for a much longer time after the morning meal than that of glycinin. Western blotting revealed the early abomasal outflow of glycinin subunits that associated nearly intact basic polypeptides to partially degraded acidic polypeptides. Intact beta-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (M(r) = 21000) associated with partially digested acidic glycinin (7000 < M(r) < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. beta-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.     

Characterization of pea vicilin. 2. Consequences of compositional heterogeneity on heat-induced gelation behavior

The gelling characteristics of two vicilin fractions from pea (Pisum sativum L.) were compared over a range of pH and salt conditions after preliminary results showed that despite having equal opportunity to unfold, and expose hydrophobic residues, they had different minimum gelling concentrations (at pH 7.6). Furthermore, at this pH one fraction formed turbid gels and the other formed transparent gels. The fraction that formed transparent gels contained a substantial amount of the 70 kDa alpha-subunits of vicilin, and thus it was hypothesized that the highly charged N-terminal extension region on these 70 kDa subunits hinders gelation of this vicilin fraction at pH 7.6 and I = 0.2 due to repulsion of the net negative charge. The experiments designed to test this hypothesis are presented and discussed in this paper and prove that the hypothesis was true, which offers the possibility to control or modify the gelation behavior of vicilin on the basis of information of its subunit composition.     

Changes in characters of soybean glycinin groups I, IIa, and IIb caused by heating

Soybean glycinin groups I, IIa, and IIb were purified from soybeans composed of only glycinin groups I, IIa, and IIb, respectively. When these protein solutions were heated, the amount of the particulate protein formed in these solutions was greatest in the order of groups IIa, IIb, and I. The protein solubilities decreased upon the addition of magnesium chloride in the order of groups IIa, IIb, and I. It was determined by differential scanning calorimetry analysis that the denaturation temperatures of groups I, IIa, and IIb were 92.8, 96.0, and 97.9 degrees C, and that the enthalpies of their transitions were 24.2, 27.4, and 28.1 J g(-)(1), respectively. The alpha-helix rates of groups I, IIa, and IIb in aqueous solution were analyzed by circular dichroism and were 19, 16, and 15%, respectively. The beta-sheet rates of groups I, IIa, and IIb were 44, 38, and 39%, respectively. In all group proteins, the alpha-helix rates were decreased by heating and the beta-sheet rates were increased. The surface hydrophobicity of these group proteins increased as a result of heating, and those of groups IIa and IIb were larger than that of group I. The surface hydrophobicity of these protein groups increased by heating, and those of groups IIa and IIb were larger than that of group I and beta-conglycinin. Breaking stress of curds prepared from these group proteins containing more than 1 of beta-conglycinin ration showed similar values, but the order of those containing less than 1 in strength was groups I, IIb and IIa. These results suggest that the increase of particulate contents and the curd formation are related to the increase of surface hydrophobicity by heating.     

Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines

Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the high-protein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of beta-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of beta-conglycinin and glycinin, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing beta-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin, beta-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin, beta-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of beta-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.     

Proteomic analysis of high protein soybean (Glycine max) accessions demonstrates the contribution of novel glycinin subunits

Limited biochemical information is available on soybean accessions that have seed protein content greater than 45% of the seed dry weight. SDS-PAGE analysis of seed proteins from nine soybean accessions revealed significantly higher amount of seed storage proteins in these accessions when compared with that of soybean cultivar Williams 82. High-resolution two-dimensional gel electrophoretic analysis of seed proteins revealed significant differences among several seed storage protein components in these accessions. A total of 51 protein spots were identified using peptide mass fingerprinting (MALDI-TOF MS). The contribution of these proteins to the overall protein content of the accessions was quantified using Delta2D image analysis software. Results showed that among the majority of the nine accessions, the largest difference in higher protein quantity was within the seed 11S storage globulins. The high protein trait from PI407788A was successfully transferred to an experimental line, LG99-469, demonstrating that this trait was transferable and robust.     

大豆蛋白限制性酶解模式与产品胶凝性的相关性

为了改善大豆蛋白的胶凝性,对大豆浓缩蛋白、大豆分离蛋白进行限制性酶解处理,并考察相应产品的蛋白酶酶解模式与胶凝性变化的相关性。该研究以蛋白质的水解度为指标,通过中性蛋白酶、胰蛋白酶的酶解作用,水解大豆浓缩蛋白、大豆分离蛋白至蛋白质水解度(DH)为1%、2%,考察酶性质、蛋白质的DH对产品胶凝性影响,并利用SDS-凝胶电泳进行确认。结果表明:大豆浓缩蛋白经中性蛋白酶、胰蛋白酶的酶解后,产品胶凝性均显著下降;大豆分离蛋白经中性蛋白酶的酶解后,产品胶凝性在DH为1%时增加,但在DH为2%时下降;大豆分离蛋白经胰蛋白酶酶解后,胶凝性显著改善。SDS-凝胶电泳确认,蛋白质的酶水解模式和水解度不同是导致产品胶凝性产生不同变化的原因。     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by alcalase: comparison with the plastein reaction and characterization of interactions

Extensive hydrolysis of whey protein isolate by Alcalase 2.4L produces a gel. The objectives of this study were to compare enzyme-induced gelation with the plastein reaction by determining the types of interactions involved in gelation. The average chain length of the peptides did not increase during hydrolysis and reached a plateau after 30 min to be approximately 4 residues, suggesting that the gel was formed by small molecular weight peptides held together by non-covalent interactions. The enzyme-induced gel network was stable over a wide range of pH and ionic strength and, therefore, showed some similarities with the plastein reaction. Disulfide bonds were not involved in the gel network. The gelation seems to be caused by physical aggregation, mainly via hydrophobic interactions with hydrogen bonding and electrostatic interactions playing a minor role.     

Ionic strength and pH-induced changes in the immunoreactivity of purified soybean glycinin and its relation to protein molecular structure

This study examined the immunogenic response of glycinin under varying conditions of pH and ionic strength using enzyme-linked immunosorbent assay. Differential scanning calorimetric (DSC) analysis and Fourier transform infrared spectroscopy (FTIR) were used to investigate the conformational changes induced as a result of these conditions, and the correlation with the changes observed in glycinin immunoreactivity were determined. A highly purified glycinin obtained by isoelectric precipitation followed by native preparative continuous flow electrophoresis was used for these studies. Purity was confirmed by two-dimensional-polyacrylamide gel electrophoresis and mass spectroscopy. DSC and FTIR results suggest that glycinin immunoreactivity is affected by changes in the tertiary and secondary packing of the protein, when flexibility, stability, and accessibility of certain substructures are modified. Aggregation and/or increased compactness of glycinin subcomponents could have potentially prevented epitopes from reacting with the IgG antibodies.     

Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max)

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, catalyzes the conversion of Angiotensin I to the potent vasoconstrictor Angiotensin II and plays an important physiological role in regulating blood pressure. Inhibitors of angiotensin 1-converting enzyme derived from food proteins are utilized for pharmaceuticals and physiologically functional foods. ACE inhibitory properties of different enzymatic hydrolysates of glycinin, the major storage protein of soybean, have been demonstrated. The IC50 value for the different enzyme digests ranges from 4.5 to 35 microg of N2. The Protease P hydrolysate contained the most potent suite of ACE inhibitory peptides. The ACE inhibitory activity of the Protease P hydrolysate after fractionation by RP-HPLC and ion-pair chromatography was ascribed to a single peptide. The peptide was homogeneous as evidenced by MALDI-TOF and identified to be a pentapeptide. The sequence was Val-Leu-Ile-Val-Pro. This peptide was synthesized using solid-phase FMOC chemistry. The IC50 for ACE inhibition was 1.69 +/- 0.17 microM. The synthetic peptide was a potent competitive inhibitor of ACE with a Ki of 4.5 +/- 0.25 x 10(-6) M. This peptide was resistant to digestion by proteases of the gastrointestinal tract. The antihypertensive property of this peptide derived from glycinin might find importance in the development of therapeutic functional foods.     

Effects of Ca2+ and sulfhydryl reductant on the polymerization of soybean glycinin catalyzed by mammalian and microbial transglutaminases

Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.     

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.     

Analysis of downy mildew data on field pea: an empirical comparison of two logistic models

In the years 2002–2005, special trials concerning the level of infection of pea varieties by downy mildew were performed in Poland. In these trials, the large number of varieties were tested in many locations (environments), separately on reach and light soils. Obtained trial data are unique because of the large scale of the performed investigations and also for the fact that all the observations were made by the same observer. In a paper, two methods of statistical analysis of such (ordered) data are compared.

Several models have been proposed for the statistical interpretation of ordinal data. One of the most popular is the cumulative-type fixed logistic model. In the present work, using two field pea data sets, we considered whether adding random effects to the simple logistic model can improve inference. It was investigated whether there is any difference between the decisions concerning varieties resulting from the simple logistic model and the proposed mixed logistic model. The two models were also compared in terms of goodness of fit. According to two applied goodness-of-fit statistics, the mixed model performed better in all the cases. Statistical analysis (what is important for practical agriculture) enabled identification of the most resistant and the most susceptible variety from the analyzed set of cultivars.     

Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation

The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.