Formation of new protein structures in heated mixtures of BSA and alpha-lactalbumin

The heat-induced protein-protein interactions of alpha-lactalbumin (alpha-La) and bovine serum albumin (BSA), dispersed in a pH 6.8, 10% whey protein concentrates (WPC) permeate, were followed using alkaline and sodium dodecyl sulfate (SDS) 1D and 2D polyacrylamide gel electrophoresis (PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC). Heated (75 degrees C) 5% BSA solution contained large disulfide-bonded BSA aggregates, although some monomer BSA (SDS-monomeric BSA) could be dissociated from the aggregates by SDS. In contrast, similarly heated alpha-La solutions contained small quantities of several monomeric forms of alpha-La and dimeric alpha-La but no large aggregates. When 10% solutions of 1:1 (w/w) mixtures of alpha-La and BSA were heated, large disulfide-bonded aggregates and SDS-monomeric BSA and alpha-La were present. However, heated 2% mixtures contained more modified alpha-La monomers, alpha-La-dimers, and alpha-La-trimers, fewer large disulfide-bonded aggregates, and less SDS-monomeric alpha-La or BSA. These results suggest that BSA forms disulfide-bonded aggregates that contain available thiol groups that can catalyze the formation of differently structured alpha-La monomers, dimers, higher polymers, and adducts of alpha-La with BSA.     

Dielectric study of heat-denatured ovalbumin in aqueous solution by time domain reflectometry method

The dielectric behavior of native and heat-denatured ovalbumins (OVAs) from three avian species in aqueous solution was examined over a frequency range of 100 kHz to 20 GHz, using the time domain reflectometry (TDR) method. For the native OVA solutions, three kinds of relaxation processes were observed at around 10 MHz, 100 MHz, and 20 GHz, respectively; these could be assigned to the overall rotation of protein molecules, the reorientations of the bound water, and the free water molecules, respectively. For the heat-denatured samples, three relaxation processes were also observed. However, the relaxation process at approximately 100 MHz originated via a different mechanism other than the reorientation of bound water, namely, the micro-Brownian motion of peptide chains of heat-denatured protein. From the observed relaxation process at approximately 100 MHz, the relaxation strength of heat-denatured OVA solution for duck was higher than that of OVA solutions for hen and guinea fowl and showed the pH dependency from pH 7.0 to 8.0 for OVAs obtained from all three species. Furthermore, the results demonstrated that the relaxation strength was closely related to surface hydrophobicity of protein molecules and gel rheological properties. It was suggested that the difference in the surface hydrophobicity of protein influenced the dielectric behavior of water around denatured protein, whereas the dielectric behavior of denatured protein could be an indication of the gel rheological properties. Such studies can aid in the understanding of the different network structures of OVA gels from three avian species.     

Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.     

Limited enzymatic treatment of skim milk using chymosin affects the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates

The effects of heat treatment and limited kappa-casein hydrolysis on the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates were investigated as a possible explanation for the gelation properties of combined rennet and acid gels. Reconstituted skim milk was submitted to combinations of 0-67% hydrolysis of the kappa-casein at 5 degrees C and heat treatment at 90 degrees C for 10 min. The protein composition of the ultracentrifugal fractions was obtained by reverse-phase high-performance liquid chromatography (RP-HPLC). The aggregates contained in each phase were isolated by size-exclusion chromatography and analyzed by RP-HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon heating only, 20-30% of the total kappa-casein dissociated, while 20-30% of the total whey protein attached to the micelles. When heated milk was renneted, little changes were observed in the distribution and composition of the aggregates. Conversely, the heat treatment of partially renneted milk induced the formation of essentially micelle-bound aggregates. The results were discussed in terms of the preferred interaction between hydrophobic para-kappa-casein and denatured whey proteins.     

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.     

Uniaxial compression of thermal gels based on microfluidized blends of WPI and heat-denatured WPI.

The effect of heat-denatured whey protein isolate (dWPI)/whey protein isolate (WPI) ratio (0-0.6), microfluidization pressure (0-1000 bar), and number of passes (1-10) on the uniaxial shear stress at 10% (sigma(10)) and 80% (sigma(80)) relative deformation of dWPI/WPI heat-induced gels (14% total protein, w/w) was studied. No correlation between the average diameter of aggregates and the dWPI/WPI ratio, microfluidization pressure, or number of passes was found. However, increasing the microfluidization pressure or the number of passes resulted in a narrower size distribution of aggregates. Increasing the dWPI/WPI ratio and the number of passes resulted in a decrease and an increase of gel hardness, respectively. The results were interpreted in terms of more random aggregation/gelation of proteins in the presence of aggregates that could result in localized heterogeneities into gels and more dissipation of the deformation energy during compression. The positive effect of the number of passes on the gel hardness was also considered to be due to a more homogeneous aggregation/gelation of proteins in the presence of smaller aggregates.     

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.     

Formation of soluble and micelle-bound protein aggregates in heated milk

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.     

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.     

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 redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate

Cold gelation of whey proteins is a two-step process. First, protein aggregates are prepared by a heat treatment of a solution of native proteins in the absence of salt. Second, after cooling of the solution, gelation is induced by lowering the pH at ambient temperature. To demonstrate the additional formation of disulfide bonds during this second step, gelation of whey protein aggregates with and without a thiol-blocking treatment was studied. Modification of reactive thiols on the surface of the aggregates was carried out after the heat-treatment step. To exclude specific effects of the agent itself, different thiol-blocking agents were used. Dynamic light scattering and SDS-agarose gel electrophoresis were used to show that the size of the aggregates was not changed by this modification. The kinetics of gelation as determined by the development of pH and turbidity within the first 8 h of acidification were not affected by blocking thiol groups. During gelation, formation of large, covalently linked, aggregates occurred only in the case of unblocked WPI aggregates, which demonstrates that additional disulfide bonds were formed. Results of permeability and confocal scanning laser microscope measurements did not reveal any differences in the microstructure of networks prepared from treated or untreated whey protein aggregates. However, gel hardness was decreased 10-fold in gels prepared from blocked aggregates. Mixing different amounts of blocked and unblocked aggregates allowed gel hardness to be controlled. It is proposed that the initial microstructure of the gels is primarily determined by the acid-induced noncovalent interactions. The additional covalent disulfide bonds formed during gelation are involved in stabilizing the network and increase gel strength.     

alpha-Casein improves the gel properties of dried egg white

The effects of addition of alpha-casein (alpha-CN) to dried egg white (DEW) were investigated by measuring transparency, hardness, and water-holding capacity (WHC) of the heat-induced gels. A DEW concentration of 8% (w/w) was required for formation of a self-supporting gel following heating at 80 degrees C for 20 min at pH 7. Solutions of alpha-CN, even up to a protein concentration of 12% (w/w), did not gel under the same conditions. The addition of alpha-CN (0.5-4%) to 8% DEW caused the increase in gel hardness gels, as compared with DEW gels alone at a total amount of protein concentrations, and the mixed gels became transparent with the increase of added alpha-CN concentrations. The 10% mixed protein solutions of alpha-CN (3-6%) and DEW (4-7%) formed transparent gels, although each protein did not gel individually at their protein concentrations. Mixture with 2:8 mixing ratio of alpha-CN to DEW at a total protein concentration of 10% showed synergistic effects in improving DEW gel properties above pH 7 and below 25 mM NaCl. The improvements (hardness, transparency, and WHC) of DEW gel by alpha-CN seem to be caused mainly by the inhibition of alpha-CN against heat coagulation of DEW protein.     

Acid gelation properties of heated skim milk as a result of enzymatically induced changes in the micelle/serum distribution of the whey protein/kappa-casein aggregates

Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.     

Control of heat-induced aggregation of whey proteins using casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P相似文献   

肉蛋白-畜产源非肉蛋白互作的物理化学研究

为了研究典型畜产源非肉蛋白与肌原纤维蛋白(MP)的相互作用,建立了模拟肉制品加工条件下二者等比例用量的溶液及热致凝胶模型,将血浆蛋白(PPP)、鸡蛋白分离蛋白(EPI)和酪蛋白酸钠 (SC)分别与MP 按照1∶1比例混合,以各单一蛋白为对照,利用流变仪、质构仪和低场核磁等仪器测定各蛋白粘度、加热过程的动态粘弹性、凝胶强度和水分子状态等指标。结果表明,PPP和EPI在加热过程中自身可形成凝胶,但与MP相比,储能模量(G')较弱,SC在加热过程中未能形成凝胶。将PPP和EPI分别与MP混合时,流变结果显示,PPP+MP、EPI+MP相互作用指数均大于零,与单独的MP相比,其G'无显著差异;加入PPP未能显著改变MP的凝胶强度,但加入EPI显著提高了MP的凝胶强度(P<0.05);PPP、EPI的加入均能使凝胶保水性显著提高,不易流动水比例增大(P<0.05)。SC会对MP产生一定不利影响,二者相互作用指数小于零,其G'、凝胶强度及凝胶保水性显著降低(P<0.05)。总体而言,PPP、EPI与MP之间在加热后均产生正向相互作用,而SC对MP产生不利影响。本研究结果为凝胶乳化类肉制品中非肉蛋白的应用提供了一定的理论借鉴。     

Acid-induced cold gelation of globular proteins: effects of protein aggregate characteristics and disulfide bonding on rheological properties

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.     

Thermally induced gelation of squid (Illex argentinus) actomyosin. Influence of sexual maturation stage.

The biochemical properties and the characteristics of heat-induced gelation of actomyosin from mature and immature squid were investigated. Both Mg(2+)-ATPase activity and reduced viscosity values of actomyosin showed no significant differences between mature and immature squids. A lower content of myosin heavy chain and a higher content of a 160 kDa component were observed in the SDS-PAGE 10% pattern of actomyosin from immature specimens. Gelation of both actomyosins at 10 mg mL(-)(1) protein concentration was optimal at 80 degrees C and pH 6.0. The highest rigidity was reached at 0.25 M KCl with actomyosin from both mature and immature squids. Irrespective of the heating temperature, ionic strength, and pH condition, the rigidity of mature squid actomyosin gel was greater than that of immature squid. Scanning electron micrographs of gels obtained with actomyosin from mature squids showed a better tridimensional structure than those of immature squids.     

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.