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.     

Interactions of whey proteins during heat treatment of oil-in-water emulsions formed with whey protein isolate and hydroxylated lecithin

The interactions of proteins during the heat treatment of whey-protein-isolate (WPI)-based oil-in-water emulsions with and without added hydroxylated lecithin were studied by examining the changes in droplet size distribution and the quantity and type of adsorbed and unadsorbed proteins. Heat treatment at 90 degrees C of WPI emulsions resulted in an increase in total adsorbed protein; unadsorbed beta-lactoglobulin (beta-lg) was the main protein interacting with the adsorbed proteins during the first 10 min of heating, but after this time, unadsorbed alpha-lactalbumin (alpha-la) also associated with the adsorbed protein. In emulsions containing hydroxylated lecithin, the increase in total adsorbed protein during heat treatment was much lower and the unadsorbed beta-lg did not appear to interact with the adsorbed proteins during heating. However, the behavior of alpha-la during heat treatment of these emulsions was similar to that observed in the emulsions containing no hydroxylated lecithin. In the presence of NaCl, the particle size of the emulsion droplets and the quantities of adsorbed protein increased markedly during heating. Emulsions containing hydroxylated lecithin were less sensitive to the addition of NaCl. These results suggest that the binding of hydroxylated lecithin to unfolded monomers or intermediate products of beta-lg reduces the extent of heat-induced aggregation of beta-lg and consequently decreases the interactions between unadsorbed beta-lg and adsorbed protein. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated whey protein and hydroxylated lecithin solutions.     

Heat-induced soy-whey proteins interactions: formation of soluble and insoluble protein complexes

The aggregation behavior during heating of a solution containing soy protein and whey protein isolate (WPI) was studied using rheology, confocal microscopy, gel filtration, and electrophoresis. Soy/WPI mixtures formed gels at 6% total protein concentration with a high elastic modulus (G') and no apparent phase separation. The ratio of soy to WPI was fundamental in determining the type of network formed. Systems containing a high soy to WPI ratio (>70% soy protein) showed a different evolution of the elastic modulus during heat treatment, with two apparent stages of network development. Whey proteins formed disulfide bridges with soy proteins during heating, and at low ratios of soy/WPI, the aggregates seemed to be predominantly formed by 7S, the basic subunits of 11S and beta-lactoglobulin. Size exclusion chromatography indicated the presence of high molecular weight soluble complexes in mixtures containing high soy/WPI ratios. Results presented are the first evidence of interactions between soy proteins and whey proteins and show the potential for the creation of a new group of functional ingredients.     

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.     

Molecular structure, physicochemical characterization, and in vitro degradation of barley protein films

Barley protein films were prepared by thermopressing using glycerol as a plasticizer. The combined effects of heating temperature and amount of plasticizer interacted to determine protein conformation and, subsequently, the properties of the film matrix. The film barrier and mechanical properties were systematically investigated using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), SDS-PAGE, and protein solubility tests. These experiments demonstrated that heat treatment induced barley protein unfolding and then protein aggregation and the formation of covalent disulfide bonds to enhance film strength. Increasing the amount of plasticizer reduced protein denaturation and limited protein interactions, resulting in significantly improved film flexibility at the cost of reduced film moisture barrier property and tensile strength. In vitro degradation experiments demonstrated that barley films were resistant in gastric conditions, yet can still be completely degraded by intestinal enzymes, and they possess low cytotoxicity to Caco-2 cells. The prepared barley films have potential for development as delivery systems for gastric-sensitive bioactive compounds to the intestine for release.     

Glucose slows down the heat-induced aggregation of β-lactoglobulin at neutral pH

The behavior of β-lactoglobulin (β-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that β-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.     

Influence of pH and ionic strength on heat-induced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions

The influence of pH and ionic strength on gel formation and gel properties of soy protein isolate (SPI) in relation to denaturation and protein aggregation/precipitation was studied. Denaturation proved to be a prerequisite for gel formation under all conditions of pH and ionic strength studied. Gels exhibited a low stiffness at pH >6 and a high stiffness at pH <6. This might be caused by variations in the association/dissociation behavior of the soy proteins on heating as a function of pH, as indicated by the different protein compositions of the dissolved protein after heating. At pH 3-5 all protein seems to participate in the network, whereas at pH >5 less protein and especially fewer acidic polypeptides take part in the network, coinciding with less stiff gels. At pH 7.6, extensive rearrangements in the network structure took place during prolonged heating, whereas at pH 3.8 rearrangements did not occur.     

不同热处理大豆分离蛋白凝胶冻藏特性

为探究冻藏过程中不同加热温度处理大豆分离蛋白(soybean isolate protein,SPI)凝胶特性变化及评估不同热处理对SPI凝胶冻藏特性的影响。该文以65、90和135℃3个不同温度处理所得SPI为研究对象(分别记为65SPI、90SPI和USPI),采用离心法、质构分析法、可溶蛋白含量测定和电泳等方法对其冻藏过程中的凝胶持水性、凝胶硬度、凝胶弹性、可溶蛋白含量及亚基组成和凝胶作用力进行了分析研究。结果表明:随冻藏时间延长,不同温度处理SPI凝胶持水性、凝胶弹性和凝胶可溶蛋白含量呈下降趋势,而凝胶硬度呈增大趋势。凝胶持水性、弹性的下降和凝胶硬度的升高标志着凝胶品质的劣变。不同温度处理对SPI凝胶的冻前凝胶特性和冻藏特性有较大影响,65和90℃的温度处理降低了冻前SPI凝胶的持水性,增强了冻前SPI凝胶硬度,有更多的β和B亚基参与了凝胶形成,冻藏前后的亚基组成没有变化;超高温瞬时加热(ultra high temperature,UHT)处理则降低了冻前SPI凝胶硬度,冻藏过程中可溶蛋白含量大幅下降且可溶蛋白中β和B亚基含量下降。3种温度处理SPI的凝胶劣变程度均高于未处理SPI。加热处理会造成SPI发生部分或完全变性,变性后疏水基团的暴露会加快蛋白凝胶形成过程中聚集速率,进而增大粗糙凝胶结构形成的几率,而粗糙凝胶网络在冻藏过程中其劣变程度更甚于未加热SPI。由此可知,加热处理尽管在一定程度上增大了凝胶硬度,但会加速其凝胶品质冻藏劣变。     

Behavior of protein in the presence of calcium during heating of whey protein concentrate solutions

The effect of added CaCl(2) on heat-induced changes in whey protein (WP) solutions prepared from whey protein isolate (WP1), acid whey protein concentrate (WP2), and cheese whey protein concentrate (WP3) was investigated. The loss of native-like, proteins, aggregation, and gel firmness of WP were maximum at certain levels of added CaCl(2). These levels were different for different WP products. The effect of added CaCl(2) on these changes appeared to be related to the initial calcium concentrations of these solutions. The higher the calcium content of the product, the less available sites for added CaCl(2) to bind. It was considered that addition of CaCl(2) changed the types of protein interactions that formed the protein aggregates during heating. Added calcium caused dramatic decreases in fracture stress of WP gels due to the formation of large protein aggregates.     

Heat-induced destabilization of oil-in-water emulsions formed from hydrolyzed whey protein.

The emulsifying ability, heat stability, and coalescence stability of oil-in-water emulsions prepared with whey protein of varied degrees of hydrolysis (DH), and at varied protein contents, was studied. Whey protein hydrolysates (WPH) with a DH of 4% and 10% had poorer emulsifying ability than non-hydrolyzed whey protein concentrate (WPC), but were more heat stable. Increasing DH between 10 and 27% improved emulsifying ability and further improved the heat stability of the emulsion droplets. Increasing DH from 27 to 35% led to a big decrease in both emulsifying ability and heat stability. The quiescent coalescence stability of WPH emulsions was relatively good up to a DH of 27%. Above DH 27% emulsions become highly unstable. It appears that two mechanisms of instability are at work here. At low DH heat-induced denaturation and aggregation occur. In the DH range of 4-20% heat stability increases as protein globular structure is disrupted. At a DH greater than 27% we see a change from a hydrolysis-induced increase in heat-stability to coalescence instability, with a resultant large increase in emulsion breakdown during heating.     

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.     

Effects of ionic strength and sulfate upon thermal aggregation of grape chitinases and thaumatin-like proteins in a model system

Consumers expect white wines to be clear. During the storage of wines, grape proteins can aggregate to form haze. These proteins, particularly chitinases and thaumatin-like proteins (TL-proteins), need to be removed, and this is done through adsorption by bentonite, an effective but inefficient wine-processing step. Alternative processes are sought, but, for them to be successful, an in-depth understanding of the causes of protein hazing is required. This study investigated the role played by ionic strength (I) and sulfate toward the aggregation of TL-proteins and chitinases upon heating. Purified proteins were dissolved in model wine and analyzed by dynamic light scattering (DLS). The effect of I on protein aggregation was investigated within the range from 2 to 500 mM/L. For chitinases, aggregation occurred during heating with I values of 100 and 500 mM/L, depending on the isoform. This aggregation immediately led to the formation of large particles (3 μm, visible haze after cooling). TL-protein aggregation was observed only with I of 500 mM/L; it mainly developed during cooling and led to the formation of finite aggregates (400 nm) that remained invisible. With sulfate in the medium chitinases formed visible haze immediately when heat was applied, whereas TL-proteins aggregated during cooling but not into particles large enough to be visible to the naked eye. The data show that the aggregation mechanisms of TL-proteins and chitinases are different and are influenced by the ionic strength and ionic content of the model wine. Under the conditions used in this study, chitinases were more prone to precipitate and form haze than TL-proteins.     

Heat-induced gelation of pea legumin: comparison with soybean glycinin

Gel network formation of pea legumin (8.4% on a protein basis, pH 7.6) was monitored via dynamic rheological measurements. Gelation was performed in the absence and presence of the thiol-blocking reagent N-ethylmaleimide, at different rates of heating and cooling. Overall, it was shown that pea legumin gel formation was not effected by changes in the heating rate, and the two differently heated samples were unaffected by the addition of 20 mM NEM, which indicated that disulfide bonds were not essential within the network strands of these legumin gels. However, slowly cooling the legumin samples caused disulfide bonds to become involved within the network; this was observed by a large increase in gel strength that was then substantially reduced when repeating the sample in the presence of NEM. These experiments were repeated with soybean glycinin in order to determine whether a common model for gel formation of legumin-like proteins could be built, based upon molecular reasoning. The two proteins were affected in the same way by changes in the conditions used, but when applying a procedure of reheating and recooling the gel networks responded differently. Pea legumin gel networks were susceptible to rearrangements that caused the gels to become stronger after reheating/recooling, yet glycinin gel networks were not. It was concluded that the same physical and chemical forces drove the processes of denaturation, aggregation, and network formation. Each process can therefore be readily targeted for modification based upon molecular reasoning. Pea legumin and soybean glycinin gel networks had structurally different building blocks, however. A model of gelation aimed at texture control therefore requires additional information.     

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.     

Antioxidant properties and metal chelating activity of glucose-lysine heated mixtures: relationships with mineral absorption across Caco-2 cell monolayers

Model Maillard reaction products were generated by heating glucose-lysine mixtures (GL) at 150 degrees C for different times (15, 30, 60, and 90 min). Samples were characterized by free lysine, browning, and UV-visible spectra and assessed for antioxidant properties, metal chelating ability, and effects on mineral absorption across Caco-2 monolayers. It was found that the capacity to retard lipid peroxidation in a model linoleic acid emulsion system increased with heating time up to 60 min and then leveled off, whereas the scavenging activity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals increased in early periods of the reaction (15 and 30 min of heating) and decreased thereafter. The iron binding affinity of the different samples was not correlated with antioxidant properties, and iron transport in Caco-2 cells was unchanged between samples. On the contrary, copper chelating activity showed significant correlation with free radical scavenging activity and with copper absorption across intestinal cells. It can be concluded that severe heat treatment of GL mixtures maintained the ability to reduce lipid peroxidation but decreased the free radical scavenging activity. Moreover, antiradical activity, copper chelation ability, and positive effects on copper absorption were correlated and associated to compounds formed at early stages of the Maillard reaction.     

Effect of pH on the thermal denaturation of whey proteins in milk

The effect of pH on thermal denaturation of four main whey protein fractions in skim milk was examined by gel permeation FPLC. On heating skim milk at 80 degrees C for 0.5-20.0 min over the pH range 5.2-8.8, the extent of denaturation, based on loss of solubility at pH 4.6, increased with heating time and was usually in the order immunoglobulins > serum albumin/lactoferrin > beta-lactoglobulin > alpha-lactalbumin. Rates of denaturation of the immunoglobulins and the serum albumin/lactoferrin fraction were highest at the lower end of this pH range, whereas those of beta-lactoglobulin and alpha-lactalbumin increased over most of the pH range. The effects of pH, addition of Ca, and reduction of disulfide bonds on the rates of the unfolding and aggregation stages of denaturation are discussed.     

Inhibiting effects of egg white dry-heated at 120 degrees C on heat aggregation and coagulation of egg white and characteristics of dry-heated egg white.

Dialyzed and freeze-dried egg white (FDEW) was dry-heated at 120 degrees C for up to 6 h. The inhibiting effects of the dry-heated egg white (DHEW) on the heat aggregation and coagulation of egg white (as 10% FDEW solution) and characteristics of the DHEW were examined. From the changes in turbidities and soluble protein contents of supernatant in various mixtures of 10% FDEW and DHEW solutions induced by heating (60 degrees C, 5 min), it was found that the inhibiting capacity increased with increases in the dry-heating time (DHT). The FDEW proteins were denatured with a mild conformational change (not secondary but tertiary structure) with the increase in DHT and aggregated partially. However, the more transparent solutions of DHEW containing soluble aggregates according to DHT were also obtained after heating. The transparency according to DHT came to be scarcely affected by the NaCl concentration and the dilution with diluents containing SDS, urea, and 2-mercaptoethanol. These findings suggest that the heat aggregations and coagulations of ovotransferrin and lysozyme in the FDEW were inhibited by their bindings with the soluble aggregates in DHEW.     

Influence of heating on oil-in-water emulsions prepared with soybean soluble polysaccharide

The effect of heating on the physicochemical properties of emulsions prepared with soybean soluble polysaccharide (SSPS) was investigated. The emulsions were stable after heating at 90 degrees C for up to 30 min. Heating at different pH values or in the presence of CaCl2 (<10 mM) did not affect the stability; however, at higher concentrations of calcium ions, the emulsion particle size increased. Two fractions, a high molecular weight (HMF) and a low molecular weight (LMF) fraction, were separated from the crude SSPS preparation by gel fitration. Emulsions prepared with SSPS/HMF (MW = 310-420 kDa) showed little change in size with heating, while the protein impurities of the SSPS/LMF fraction formed aggregates by heating at pH 7. Analysis of the heat-induced aggregation of the two fractions of SSPS suggested that the changes in SSPS functionality with heating can be attributed to the protein impurities (LMF) present in the SSPS.     

Adsorption and dilatational rheology of heat-treated soy protein at the oil-water interface: relationship to structural properties

We evaluated the influence of heat treatment on interfacial properties (adsorption at the oil-water interface and dilatational rheology of interfacial layers) of soy protein isolate. The related structural properties of protein affecting these interfacial behaviors, including protein unfolding and aggregation, surface hydrophobicity, and the state of sulfhydryl group, were also investigated. The structural and interfacial properties of soy protein depended strongly on heating temperature (90 and 120 °C). Heat treatment at 90 °C induced an increase in surface hydrophobicity due to partial unfolding of protein, accompanied by the formation of aggregates linked by disulfide bond, and lower surface pressure at long-term adsorption and similar dynamic interfacial rheology were observed as compared to native protein. Contrastingly, heat treatment at 120 °C led to a higher surface activity of the protein and rapid development of intermolecular interactions in the adsorbed layer, as evidenced by a faster increase of surface pressure and dilatational modulus. The interfacial behaviors of this heated protein may be mainly associated with more flexible conformation and high free sulfhydryl group, even if some exposed hydrophobic groups are involved in the formation of aggregates. These results would be useful to better understand the structure dependence of protein interfacial behaviors and to expand utilization of heat-treated protein in the formulation and production of emulsions.     

Rheological properties and characterization of polymerized whey protein isolates.

Whey protein polymers were formed by heating whey protein isolate solutions at 80 degrees C. Flow behaviors of whey protein polymers produced from different protein concentrations and heating times were comparable to various flow behaviors of hydrocolloids. Polymer formation was found to be a two-phase process. The initial protein concentration was a significant factor that determines the size and/or shape of the primary polymer in the first phase as shown by intrinsic viscosity. Heating time was a factor in determining the aggregation in the second phase as shown by apparent viscosity. Intrinsic viscosity of whey protein polymers was as high as 141.7 +/- 7.30 mL/g, compared to 5.04 +/- 0.20 mL/g for native whey proteins. The intrinsic viscosity and gel electrophoresis data suggested that disulfide bonds played an important role in whey polymer formation.