Thermally induced aggregates in mixtures of alpha-lactalbumin with ovalbumins from different avian species

Interactions between alpha-lactalbumin (alpha-La) and ovalbumin (OVA) in mixed systems (1:1 ratios; 2, 4, and 8% w/w total protein, respectively) heated at pH 7 and 80 degrees C for 15 min were studied using sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), gel filtration chromatography (GFC), and competitive enzyme-linked immunosorbent assay (ELISA). Although alpha-La alone did not form aggregates upon heating, it formed large aggregates when heated with OVA. The aggregated molecules eluted at the void volume had a molecular mass >300 kDa. The aggregation process was quantitatively affected by different avian OVAs from five species, possessing different numbers of free sulfhydryl groups. The amount of aggregates (M(w) > 300 kDa) increased in proportion to total protein concentration, and the amount of intermediate components (M(w) < 300 kDa) and monomeric OVA and alpha-La also changed, correlating with total protein concentration during heating. The results also indicated that the aggregates and intermediates, which contained dimeric and trimeric alpha-La, were mainly formed by the intermolecular disulfide bonds. The different interactions observed in several avian OVAs may explain heat-induced gelation of various avian OVAs as well as the enhancement of heat-induced gelation of OVA by alpha-La.     

Heat-induced gels of egg white/ovalbumins from five avian species: thermal aggregation, molecular forces involved, and rheological properties

Product processing (heating, pH change, etc.) usually alters protein structure, improves rheological properties, and gives a unique texture to foods. The thermal aggregation and structural properties of ovalbumins from five avian species were studied at different pH values by polyacrylamide gel electrophoresis (PAGE) and determinations of sulfhydryl group content and surface hydrophobicity. The results showed that sulfhydryl group content changed insignificantly in heat-denatured ovalbumins other than hen ovalbumin (pH-independent), and surface hydrophobicity markedly increased (pH-dependent) after heating, with a significant difference among species. Furthermore, it was demonstrated that the hydrophobic interaction and sulfhydryl-disulfide interchange reaction were necessary in the aggregation and cross-linking of gel networks. Creep tests were also used to characterize the gel network structures of various egg white/ovalbumins upon heating. The viscoelastic behavior of the ovalbumins of all species was dependent on pH values, and changed significantly with the phylogeny of these species. With increases in pH value (7.0-8.5), the heat-induced gels of ovalbumins gradually changed from turbid to translucent, the instantaneous modulus (E(0)) increased slightly and reached a nearly constant value, and the Newtonian modulus (etaN) increased significantly in each sample. The heated egg white from these five avian species also formed highly viscoelastic gels, with a good correlation of viscoelastic behavior between ovalbumin and egg white in corresponding species.     

Relation between solubility and surface hydrophobicity as an indicator of modifications during preparation processes of commercial and laboratory-prepared soy protein isolates

Because water solubility is the main hydration property of proteins, solubility values of commercial and laboratory soy protein isolates, prepared under different conditions, were comparatively analyzed. In contrast, the surface hydrophobicity manifested by proteins is a physicochemical property that determines, to a great extent, the tendency of protein molecules to aggregate and so to lose solubility. On these grounds, the solubility of isolates was analyzed as a function of the surface hydrophobicity of their proteins, and, as a result, three well-defined groups of laboratory isolates were identified: (A) native, (B) partially or totally denatured with high solubility and surface hydrophobicity, and (C) totally denatured with low solubility and surface hydrophobicity. Commercial isolates could not be included in any of these groups; they were grouped as (A') partially native and (C') totally denatured. Solubility values in these two groups were similar to those of group C, but the surface hydrophobicity levels were much lower. The different processes leading to the groups mentioned above are discussed, along with the way the soy proteins are influenced by the specific preparation conditions, namely, protein concentration, chemical or thermal treatments, presence of salts, drying, and phospholipid addition, among others.     

Effect of water content on dielectric relaxation of gelatin in a glassy state

The dielectric properties of gelatin in the glassy state were measured from 100 Hz to 1 MHz over a temperature range of -20 to 60 degrees C. Samples with different water contents were prepared by varying the drying time for desalted gelatin solution; they were confirmed to be in the glassy state from DSC measurements. The dielectric relaxation (the decrease in dielectric constant, epsilon', and the maximum of the dielectric loss, epsilon' ') was observed for each sample; the relaxation time tau was evaluated from the peak of epsilon' '. The activation energy E obtained from an Arrhenius plot of tau decreased with increasing water content. On the basis of the order of magnitude of E, the dielectric relaxation observed was considered to be beta-relaxation reflecting the local motion of molecules. E and tau seem to describe the enhancement effect of water on the mobility of gelatin molecules in the glassy state; tau and E are considered to be suitable parameters for the characterization of the plasticizing effect of water on a glassy material.     

Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties

The effect of nonenzymatic glycation on the structural changes and gelling properties of hen ovalbumin (OVA) through the Maillard reaction was studied. OVA was incubated at the dry state with a rare ketohexose (D-psicose, Psi) and two alimentary sugars (D-fructose, Fru; D-glucose, Glc) at 55 degrees C and 65% relative humidity. To evaluate the modification of OVA by different reducing sugars during the glycation process, the extent of the Maillard reaction, aggregation processes, structural changes, and gelling behaviors were investigated. Reactivity of Psi with the protein amino groups was much lower than that of both Fru and Glc, whereas Psi induced production of browning and fluorescent substances more strongly than the two alimentary sugars did. Furthermore, OVA showed an increased tendency toward multimeric aggregation upon modifying with Psi through covalent bond. The modified OVAs with reducing sugar were similar to nonglycated control sample in Fourier transform infrared (FT-IR) characteristics, but significantly decreased in intensity of tryptophan-related fluorescence. The results indicate that although glycation brought about similar changes in the secondary structure without great disruption of native structure, its influence on the side chains of protein in tertiary structure could be different. Breaking strength of heat-induced glycated OVA gels with Psi was markedly enhanced by the Maillard reaction. These results suggest that Psi had a strong cross-linking activity with OVA; consequently, the glycated OVA with Psi could improve gelling properties under certain controlled conditions.     

Heat-induced aggregation of Phaseolus vulgaris L. Proteins: an electron spin resonance study

The mechanism of heat-induced aggregation of Phaseolus vulgaris L. proteins and of subunit interactions of importance for susceptibility of proteins to proteolysis was studied by electron spin resonance (ESR) spectroscopy. The mobility of a spin label bound to lysine residues was monitored at two different pH-induced (neutral and alkaline) association states of proteins extracted from raw and cooked common bean. The molecular weight of the protein complexes was assessed by size exclusion-high performance liquid chromatography (SE-HPLC) of labeled proteins. Upon alkaline dissociation, both native and denatured protein subunits underwent a reassociation process to form soluble complexes of molecular weight higher than the species originally present at neutral pH. However, unlike native proteins, impaired mobility of the spin label was observed in the aggregates that are formed after dissociation of subunits of denatured proteins, indicating a reduced accessibility of lysine residues. Trapping of lysine residues inside protein aggregates may explain limited digestibility in the small intestine of proteins in cooked legumes.     

Characterization of Dynamic Viscoelastic Behavior of Wheat Flour Doughs at Different Moisture Contents

Three different flours were examined to study the influence of moisture content on the dynamic viscoelastic behavior of wheat flour dough. Doughs with moisture contents varying from 43 to 58% were submitted to dynamic testing using a mechanical spectrometer operating in frequency sweep mode, obtaining information about rheological response in the linear viscoelastic range. To characterize the influence of moisture content on the dynamic viscoelastic behavior of wheat flour dough, some hypotheses regarding the functional role of the water molecules were verified by applying reduction procedures of the rheological curves. By shifting the rheological curves along the vertical axis, it was possible to verify that varying the moisture content of the doughs not only changed dynamic properties but also modified viscoelastic response. By applying a reduction procedure similar to that used to estimate the constants of the Williams, Landel, and Ferry equation, we demonstrated that not only did the viscoelastic response of doughs vary, but that water molecules interfere with the dynamic by which relaxation phenomena take place. Finally, we proved that the rheological behavior of flour dough is similar to that of concentrated polymer solutions, and that it can be characterized by using a double reduction procedure, shifting the rheological curves along the vertical and horizontal axes, and obtaining a master curve that can be considered inherently characteristic of viscoelastic behavior.     

Flavor-protein binding: disulfide interchange reactions between ovalbumin and volatile disulfides

The irreversible binding of selected sulfur-containing flavor compounds to proteins was investigated in aqueous solutions containing ovalbumin and a mixture of disulfides (diethyl, dipropyl, dibutyl, diallyl, and 2-furfuryl methyl) using solid-phase micro-extraction (SPME). In systems which had not been heated, the recovery of disulfides from the headspace above the protein at the native pH (6.7) was similar to that from an aqueous blank. However, significant losses were observed when the pH of the solution was increased to 8.0. When the protein was denatured by heating, much greater losses were observed and some free thiols were produced. In similar heat-denatured systems at pH 2.0, no losses of disulfides were observed. Disulfides containing allyl or furfuryl groups were more reactive than saturated alkyl disulfides. Interchange reactions between protein sulfhydryl groups and the disulfides are believed to be responsible for the loss of the disulfides.     

蜜瓜和西瓜果汁的射频介电特性及其与糖度的关系(简报)

为了解果汁的介电特性与其主要内部品质——糖度之间的关系,该文利用末端开路的同轴探头和阻抗分析仪测量了10 MHz～1.8 GHz的频率范围内不同成熟度的3个品种密瓜汁和4个品种西瓜汁的介电特性,同时测量了反映果汁糖度的可溶性固形物含量和果肉的含水率,分析了介电特性和糖度间的关系。结果显示：密瓜和西瓜果汁的相对介电常数和介质损耗因数皆随频率的增大单调减小,品种间的差异对介电特性有影响;1.8 GHz下密瓜汁的损耗角正切与糖度有较好的线性关系,其决定系数为0.784,但西瓜汁的介电特性与糖度的相关性较差;密瓜和西瓜果汁的糖度与果肉的含水率有很好的线性相关性,其决定系数分别为0.973和0.906。该研究将为基于介电特性的果汁糖度检测仪的开发提供了研究基础。     

A new technique for measuring broadband dielectric spectra of undisturbed soil samples

A new coaxial line cell for the determination of dielectric spectra of undisturbed soil samples was developed based on a 1.625‐inch ‐ 50 Ω coaxial system. Undisturbed soil samples were collected from a soil profile of the Taunus region (Germany) and capillary saturated followed by a step‐by‐step de‐watering in a pressure plate apparatus as well as oven‐drying at 40°C. The resultant water contents of the soil samples varied from saturation to air‐dry. Permittivity measurements were performed within a frequency range from 1 MHz to 10 GHz with a vector network analyser technique. Complex effective relative permittivity or electrical conductivity was obtained by combining quasi‐analytical and numerical inversion algorithms as well as the parameterizing of measured full set S‐parameters simultaneously under consideration of a generalized fractional dielectric relaxation model (GDR). The measurement of standard materials shows that the technique provides reliable dielectric spectra up to a restricted upper frequency of 5 GHz. For the soil samples investigated, the real part of complex effective relative permittivity ?′_r,eff and the real part of complex effective electrical conductivity σ′_eff decreased with increasing matric potential or decreasing water contents. Soil texture and porosity affect the dielectric behaviour at frequencies below 1 GHz. For frequencies above 1 GHz minor texture effects were found. The presence of organic matter decreases ?′_r,eff and σ′_eff. At 1 GHz, the empirical model of Topp et al. (1980) is in close agreement with the experimentally determined real part of the effective permittivity with RMSEs ranging from 1.21 for the basal periglacial slope deposit and 1.29 for bedrock to 3.93 for the upper periglacial slope deposit (Ah). The comparison of the experimental results with a semi‐empirical dielectric mixing model shows that data, especially for the organic‐free soils, tend to be under‐estimated below 1 GHz. The main advantage of the new method compared with conventional impedance and coaxial methods is the preservation of the natural in‐situ structure and properties such as bulk density of the investigated soil samples.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 1. Foaming properties

Foaming properties of native and chemically modified glycinin were evaluated. Effects of ionic strength and glycinin composition and concentration on foam formation and stabilization were studied. Glycinin was modified by means of combined treatments: cold or hot acidic treatments, with or without later disulfide bridges reduction. Modified proteins obtained from glycinin present different degrees of dissociation, deamidation, and as consequence, varied surface hydrophobicity and molecular size. Parameters of forming and stabilizing of foam were correlated with both deamidation and dissociation degrees of modified and native glycinin samples. A positive relationship was observed between surface behavior and foaming properties of different protein species. Results show that dissociation, deamidation, and reduction have produced structural changes on glycinin (increased surface hydrophobicity, increased net charge, decreased molecular size) which enhance the adsorption and anchorage of proteins at the air-water interface and, consequently, improve the foam forming and stabilizing capacities.     

Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase

Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA. Anti-ALP PAb were specific for milk ALP and did not react with ALP from E. coli or bovine and calf intestinal mucosa. Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP. The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate. Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP. When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA. The ELISA differentiated between native and heat denatured ALP. Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.     

Influence of aging and salting on protein secondary structures and water distribution in uncooked and cooked pork. A combined FT-IR microspectroscopy and 1H NMR relaxometry study

Fourier transform infrared (FT-IR) microspectroscopy and low-field (LF) proton NMR transverse relaxation measurements were used to study the changes in protein secondary structure and water distribution as a consequence of aging (1 day and 14 days) followed by salting (3%, 6%, and 9% NaCl) and cooking (65 degrees C). An enhanced water uptake and increased proton NMR relaxation times after salting were observed in aged meat (14 days) compared with nonaged meat (1 day). FT-IR bands revealed that salting induced an increase in native beta-sheet structure while aging triggered an increase in native alpha-helical structure before cooking, which could explain the effects of aging and salting on water distribution and water uptake. Moreover, the decrease in T2 relaxation times and loss of water upon cooking were attributed to an increase in aggregated beta-sheet structures and a simultaneous decrease in native protein structures. Finally, aging increased the cooking loss and subsequently decreased the final yield, which corresponded to a further decrease in T2 relaxation times in aged meat upon cooking. However, salting weakened the effect of aging on the final yield, which is consistent with the increased T2 relaxation times upon salting for aged meat after cooking and the weaker effect of aging on protein secondary structural changes for samples treated with high salt concentration. The present study reveals that changes in water distribution during aging, salting, and cooking are not only due to the accepted causal connection, i.e., proteolytic degradation of myofibrillar structures, change in electrostatic repulsion, and dissolution and denaturation of proteins, but also dynamic changes in specific protein secondary structures.     

Influence of Added Starch on Mixing of Dough Made with Three Wheat Flours Differing in High Molecular Weight Subunit Composition: Rheological Behavior

The effect of mixing time (6 and 20 min) and starch content were studied on doughs prepared with three wheat flours differing in high molecular weight subunit composition. Rheological measurements were performed in dynamic oscillation: frequency and strain sweeps, stress relaxation, and in large deformation viscosity measurements. The flours were diluted with starch to cover flour protein contents of 10–15%. Water was added to keep the starch‐water ratio constant when doughs were prepared with different protein contents. By increasing the starch content of the doughs, the rheological properties approached those of a starch‐water mixture prepared with the same starch‐water ratio as in the dough. The effect of the starch granules was reinforced by prolonged mixing. This may explain the higher values of the storage modulus and relaxation times observed after 20 min of mixing. Qualities related to gluten properties, appeared more clearly in large deformation viscosity measurements.     

Inhibition against heat coagulation of ovotransferrin by ovalbumin dry-heated at 120 degrees C

Ovalbumin (OVA) was aggregated stepwise by dry heating at 120 degrees C with a gradual increase in its heating times (10 min-6 h). The inhibiting effects of DHOVAs (OVAs dry-heated for various times up to 6 h) on the heat coagulation of ovotransferrin (OT) were studied. DHOVAs and OT were solubilized at 5% (w/w) concentration with 10 mM sodium phosphate buffer, pH 7.4. Their solutions were mixed at the volume ratio of 1:1 and reheated at 60 degrees C for 3. 5 min. Some remarkable differences according to dry-heating time were observed: coagulum formations were greatly inhibited in the solutions mixed with DHOVAs treated for more than around 2.0 h, with decreasing turbidity as dry-heating time increased. In addition, the effects of reheating time and temperature, as well as those of pH and ionic strength, were also examined on coagulum formation and turbidity development in connection with dry-heating time. Thus, the inhibiting effects of dry-heated egg white on the heat coagulation of fresh egg white previously described were confirmed on the molecular level of OVA and OT.     

Beer consumption and changes in stability of human serum proteins

The aim of this study was to evaluate the influence of beer consumption (BC) on the functional and structural properties of human serum proteins (HSP). Thirty-eight volunteers (after coronary bypass) were divided into two groups: experimental (EG) and control (CG). Nineteen volunteers of the EG consumed 330 mL per day of beer (about 20 g of alcohol) for 30 consecutive days. The CG volunteers consumed mineral water instead of beer. Blood samples were collected from EG and CG patients before and after the experiment. Albumin (Alb), globulin (Glo), and methanol-precipitable proteins (MPP) from human serum were denatured with 8 M urea. Fluorescence and electrophoresis were employed in order to elucidate urea-induced conformational changes and structural behavior of proteins. The measured fluorescence emission spectra were used to estimate the stability of native and denatured protein fractions before and after BC. It was found that before BC the fractions most stable to urea denaturation were Glo, Alb, and MPP fractions. After BC in most of the beer-consuming patients (EG) some changes in native and denatured protein fractions were detected: a tendency to lower stability and minor structural deviations. These qualitative changes were more profound in MPP than in Alb and Glo. Thus, Glo is more resistible to alcohol influence than Alb, which in turn is more resistible than MPP. No serum protein changes were detected in patients of CG.     

Rheological and nuclear magnetic resonance (NMR) study of the hydration and heating of undeveloped wheat doughs

The undeveloped doughs of two wheat flours differing in technological performance were characterized at the supramolecular level, by fundamental small-deformation oscillatory rheology and shear viscometry, and at the molecular level, by nuclear magnetic resonance (NMR) spectroscopy. For the harder variety, the higher storage moduli indicated lower mobility of the protein/water matrix in the 0.001-100 s range. Conversely, 1H NMR indicated higher molecular mobility in the sub-microsecond range for protein/water, whereas starch was found to be generally more hindered. It is suggested that faster protein/water motions are at the basis of the higher structural rearrangement indicated by tan delta for the harder variety. Rheological effects of heating-cooling reflect mainly starch behavior, whereas 1H NMR spectra and relaxation times give additional information on component mixing and molecular mobility. The heated softer variety dough formed a rigid lattice and, although a similar tendency was seen for the hard variety, all of its components remained more mobile. About 60% of starch crystallizes in both varieties, which may explain their similar rheological behaviors upon cooling.     

Myowater dynamics and protein secondary structural changes as affected by heating rate in three pork qualities: a combined FT-IR microspectroscopic and 1H NMR relaxometry study

The objective of this study was to investigate the influence of heating rate on myowater dynamics and protein secondary structures in three pork qualities by proton NMR T2 relaxation and Fourier transform infrared (FT-IR) microspectroscopy measurements. Two oven temperatures at 100 degrees C and 200 degrees C corresponding to slow and fast heating rates were applied on three pork qualities (DFD, PSE, and normal) to an internal center temperature of 65 degrees C. The fast heating induced a higher cooking loss, particularly for PSE meat. The water proton T21 distribution representing water entrapped within the myofibrillar network was influenced by heating rate and meat quality. Fast heating broadened the T21 distribution and decreased the relaxation times of the T21 peak position for three meat qualities. The changes in T21 relaxation times in meat can be interpreted in terms of chemical and diffusive exchange. FT-IR showed that fast heating caused a higher gain of random structures and aggregated beta-sheets at the expense of native alpha-helixes, and these changes dominate the fast-heating-induced broadening of T21 distribution and reduction in T21 times. Furthermore, of the three meat qualities, PSE meat had the broadest T21 distribution and the lowest T21 times for both heating rates, reflecting that the protein aggregation of PSE caused by heating is more extensive than those of DFD and normal, which is consistent with the IR data. The present study demonstrated that the changes in T2 relaxation times of water protons affected by heating rate and raw meat quality are well related to the protein secondary structural changes as probed by FT-IR microspectroscopy.     

小麦粉面团形成过程水分状态及其比例变化

为揭示小麦粉面团形成过程水分状态和比例、面团结构的变化,以及这种变化与粉质仪和拉伸仪表征的质量特性之间的关系;认识面团形成过程表征筋力强弱的物质基础和变化机理。选用中筋(宁春4号)和强筋(师栾02-1)小麦品种为试验材料,利用低场核磁共振技术测定粉质仪和面过程、拉伸仪醒发拉伸过程不同时间点面团水分状态和比例的变化;利用红外显微成像技术分析面团形成过程不同取样点蛋白质和淀粉的分布及结构变化。结果表明,面粉原料中主要为弱结合水。面粉在粉质仪加水搅拌形成面团后,水分状态和比例发生显著变化,面团中的水可以分为强结合水(T_(21))、弱结合水(T_(22))和自由水(T_(23))。面团搅拌形成过程中,中筋小麦品种宁春4号面团中的强结合水比例显著降低;师栾02-1的强结合水的弛豫时间在和面终点消失,弱结合水的弛豫时间显著延长,而自由水的比例显著增加(P0.05)。强筋小麦粉强结合水的保持时间较长。拉伸过程加盐和不加盐对同一取样点、同一种水分状态之间的水分弛豫时间和比例无显著影响;宁春4号自由水的弛豫时间在加盐和不加盐处理时都显著缩短(P0.05)。湿面筋含量高、筋力较强面团的蛋白质网络结构致密。粉质仪和面过程强结合水和弱结合水弛豫时间和比例的变化,与面筋含量和强度有关。该结论可为面制品加工过程和面工艺选择与优化等方面提供一定的理论参考。     

Contribution of Hydrophobic Soluble Gluten Proteins,Fractionated by Hydrophobic Interaction Chromatography in Highly Acetylated Agarose,to Dough Rheological Properties

Hydrophobic interaction chromatography with highly acetylated agarose in 1‐mL columns was used to fractionate gliadins and acid‐soluble glutenins. Proteins were eluted in two fractions, the first with acetate buffer (pH 3.6) containing 35% propanol, and the second with Tris buffer in 8M urea. The proportion of eluted protein in the second fraction was called the surface hydrophobicity index. The study included 20 wheat samples of different baking qualities. Multiple regression analysis using the general linear model combined with the stepwise technique was used to relate the surface hydrophobicity index of soluble gluten proteins to specific dough rheological characteristics. Surface hydrophobicity index of gliadins and acetic acid soluble glutenins explained part of the variability of swelling index, extensibility, and work of deformation (dough strength) measured with the alveograph, and part of the farinograph water absorption variability, but showed no relationship to dough mixing characteristics. Hydrophobic soluble gluten proteins fractionated by hydrophobic interaction chromatography (HIC) explained a part of the variability of dough rheological properties.