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.     

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   

Physical and chemical interactions in cold gelation of food proteins

pH-Induced cold gelation of whey proteins is a two-step process. After protein aggregates have been prepared by heat treatment, gelation is established at ambient temperature by gradually lowering the pH. To demonstrate the importance of electrostatic interactions between aggregates during this latter process, beta-lactoglobulin aggregates with a decreased iso-electric point were prepared via succinylation of primary amino groups. The kinetics of pH-induced gelation was affected significantly, with the pH gelation curves shifting to lower pH after succinylation. With increasing modification, the pH of gelation decreased to about 2.5. In contrast, unmodified aggregates gel around pH 5. Increasing the iso-electric point of beta-lactoglobulin via methylation of carboxylic acid groups resulted in gelation at more alkaline pH values. Comparable results were obtained with whey protein isolate. At low pH disulfide cross-links between modified aggregates were not formed after gelation and the gels displayed both syneresis and spontaneous gel fracture, in this way resembling the morphology of previously characterized thiol-blocked whey protein isolate gels (Alting, et al., J. Agric. Food Chem. 2000, 48, 5001-5007). Our results clearly demonstrate the importance of the net electric charge of the aggregates during pH-induced gelation. In addition, the absence of disulfide bond formation between aggregates during low-pH gelation was demonstrated with the modified aggregates.     

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.     

Effects of pH and the gel state on the mechanical properties, moisture contents, and glass transition temperatures of whey protein films.

The mechanical properties, moisture contents (MC), and glass transition temperature (T(g)) of whey protein isolate (WPI) films were studied at various pH values using sorbitol (S) as a plasticizer. The films were cast from heated aqueous solutions and dried in a climate chamber at 23 degrees C and 50% relative humidity (RH) for 16 h. The critical gel concentrations (c(g)) for the cooled aqueous solutions were found to be 11.7, 12.1, and 11.3% (w/w) WPI for pH 7, 8, and 9, respectively. The cooling rate influenced the c(g), in that a lower amount of WPI was needed for gelation when a slower cooling rate was applied. Both cooling rates used in this study showed a maximum in the c(g) at pH 8. The influence of the polymer network on the film properties was elucidated by varying the concentration of WPI over and under the c(g). Strain at break (epsilon(b)) showed a maximum at the c(g) for all pH values, thus implying that the most favorable structure regarding the ability of the films to stretch is formed at this concentration. Young's modulus (E) and stress at break (sigma(b)) showed a maximum at c(g) for pH 7 and 8. The MC and epsilon(b) increased when pH increased from 7 to 9, whereas T(g) decreased. Hence, T(g) values were -17, -18, and -21 degrees C for pH 7, 8, and 9, respectively. E and sigma(b) decreased and epsilon(b) and thickness increased when the surrounding RH increased. The thickness of the WPI films also increased with the concentration of WPI.     

Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.     

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.     

Light-scattering study of the structure of aggregates and gels formed by heat-denatured whey protein isolate and beta-lactoglobulin at neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.     

Effects of sugars on whey protein isolate gelation

Whey protein isolate (WPI) gels were prepared from solutions containing ribose or lactose at pH values ranging from 6 to 9. The gels with added lactose had no color development, whereas the gels with added ribose were orange/brown. Lactose stabilized the WPI to denaturation, which increased the time and temperature required for gelation, thus decreasing the fracture modulus of the gel compared to the gels with added ribose and the gels with no sugar added. Ribose, however, favored the Maillard reaction and covalent cross-linking of proteins, which increased gel fracture modulus. The decreased pH caused by the Maillard reaction in the gels containing ribose occurred after protein denaturation and gelation, thus having little if any effect on the gelation process.     

Influence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins

The influence of sucrose (0-40 wt %) on the thermal denaturation and functionality of whey protein isolate (WPI) solutions has been studied. The effect of sucrose on the heat denaturation of 0.2 wt % WPI solutions (pH 7.0) was measured using differential scanning calorimetry. Sucrose increased the temperature at which protein denaturation occurred, for example, by 6-8 degrees C for 40 wt % sucrose. The dynamic shear rheology of 10 wt % WPI solutions (pH 7.0, 100 mM NaCl) was monitored as they were heated from 30 to 90 degrees C and then cooled to 30 degrees C. Sucrose increased the gelation temperature and the final rigidity of the cooled gels. The degree of flocculation in 10 wt % oil-in-water emulsions stabilized by 1 wt % WPI (pH 7.0, 100 mM NaCl) was measured using a light scattering technique after they were heated at fixed temperatures from 30 to 90 degrees C for 15 min and then cooled to 30 degrees C. Sucrose increased the temperature at which maximum flocculation was observed and increased the extent of droplet flocculation. These results are interpreted in terms of the influence of sucrose on the thermal unfolding and aggregation of protein molecules.     

Formation of whey protein isolate (WPI)-dextran conjugates in aqueous solutions

The conjugation reaction between whey protein isolate (WPI) and dextran in aqueous solutions via the initial stage of the Maillard reaction was studied. The covalent attachment of dextran to WPI was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with both protein and carbohydrate staining. The formation of WPI-dextran conjugates was monitored by a maximum absorbance peak at approximately 304 nm using difference UV spectroscopy. The impact of various processing conditions on the formation of WPI-dextran conjugates was investigated. The conjugation reaction was promoted by raising the temperature from 40 to 60 degrees C, the WPI concentration from 2.5 to 10%, and the dextran concentration from 10 to 30% and lowering the pH from 8.5 to 6.5. The optimal conjugation conditions chosen from the experiments were 10% WPI-30% dextran and pH 6.5 at 60 degrees C for 24 h. WPI-dextran conjugates were stable under the conditions studied.     

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.     

Rennet-induced aggregation of heated pH-adjusted skim milk

Heated (20-100 °C/0-30 min) skim milks (pH 6.5-7.1) were diluted in buffer (pH 7.0). Rennet was added, and the particle size with time was measured. For all samples, the size initially decreased (lag phase) and then increased (aggregation phase). Milks heated at ≤60 °C had short lag phases and rapid aggregation phases regardless of pH. Milks heated at >60 °C at pH 6.5 had long lag phases and slow aggregation phases. As the pH increased, the lag phase shortened and the aggregation phase accelerated. The aggregation time was correlated with the level of whey protein associated with the casein micelles and with the level of κ-casein dissociated from the micelles. Heated milks formed weak gels when renneted. It is proposed that the milks heated at low pH have whey proteins associated with the casein micelles and that these denatured whey proteins stabilize the micelles to aggregation by rennet and therefore inhibit gelation. In the milks heated at higher pH, the whey proteins associate with κ-casein in the serum and, on rennet treatment, the κ-casein-depleted micelles and the serum-phase whey protein/κ-casein complexes aggregate; however, the denatured whey proteins stabilize the aggregates so that gelation is still inhibited.     

Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched α-lactalbumin and β-lactoglobulin food ingredients

An environmentally friendly protein fractionation process using supercritical carbon dioxide (SCO(2)) as an acid was developed to produce enriched α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) fractions from whey protein isolate solutions containing from 2 to 10% WPI. This study investigated the effects of pH, temperature, WPI concentration, and residence time on the precipitation kinetics and recovery yields of individual whey proteins and the relative enrichment and composition of both protein fractions. At 5.5-34 MPa and 60-65 °C, solubilized SCO(2) decreased solution pH and induced the formation and precipitation of α-LA aggregates. Gel electrophoresis and HPLC of the enriched fractions demonstrated the production of ≥ 60% pure α-LA, and ≥ 70% pure β-LG, under various operating conditions, from WPI containing ～57% β-LG and 21% α-LA. The enriched fractions are ready-to-use food ingredients with neutral pH, untainted by acids and contaminants.     

Modification of the rheological properties of whey protein isolate through the use of an immobilized microbial transglutaminase

A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using <10 mg of the same enzyme, illustrating the capacity of immobilized enzymes to be used more frequently in applications of this nature.     

Heat-induced changes in the ultrasonic properties of whey proteins

The physical aggregation of commercial whey protein isolate (WPI) and purified beta-lactoglobulin was studied by ultrasound spectroscopy. Protein samples were dialyzed to achieve constant ionic strength backgrounds of 0.01 and 0.1 NaCl, and gelation was induced in situ at constant temperatures (from 50 to 75 degrees C) or with a temperature ramp from 20 to 85 degrees C. Changes in the ultrasonic properties were shown in the early stages of heating, at temperatures below those reported for protein denaturation. During heating, the relative ultrasound velocity (defined as the difference between sample velocity and reference velocity) decreased continuously with temperature, indicating a rearrangement of the hydration layer of the protein and an increase in compressibility of the protein shell. At temperatures <50 degrees C the ultrasonic attenuation decreased, and <65 degrees C both velocity and attenuation differentials showed increasing values. A sharp decrease in the relative velocity and an increase in the attenuation at 70 degrees C were indications of "classical" protein denaturation and the formation of a gel network. Values of attenuation were significantly different between samples prepared with 0.01 and 0.1 M NaCl, although no difference was shown in the overall ultrasonic behavior. WPI and beta-lactoglobulin showed similar ultrasonic properties during heating, but some differences were noted in the values of attenuation of WPI solutions, which may relate to a less homogeneous distribution of aggregates caused by the presence of alpha-lactalbumin and other minor proteins in WPI.     

Structural changes imposed on whey proteins by UV irradiation in a continuous UV light reactor

The objective of this study was to investigate the structural changes of whey proteins during exposure in a continuous-flow UV reactor. Varying UV irradiation dosages were obtained by controlling the flow rate and the mixing speed. Whey protein isolate (WPI) solutions at concentrations of 1% and 5% (w/v) were circulated at flow rates ranging from 30 to 800 mL·min(-1), and changes in physicochemical properties of the proteins were investigated. Intrinsic fluorescence spectra and surface hydrophobicity measurements suggested changes in the tertiary structure of the proteins with UV exposure. The UV treatment also increased the concentration of total and accessible thiol groups in 1% WPI solutions, while no change was measured in 5% WPI solutions. Size-exclusion chromatography demonstrated the formation of UV-induced aggregates and oxidation products (N-formylkynurenine and dityrosine) of aromatic amino acids. Furthermore, the UV-induced changes in protein conformation increased the susceptibility of whey proteins to pepsin hydrolysis.     

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.     

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.     

Effect of conductivity control on the separation of whey proteins by bipolar membrane electroacidification

Since the limiting factor of the bipolar membrane electroacidification (BMEA) process at 20% WPI (whey protein isolate) was hypothesized to be the lack of mobile ion inherent to the protein solution at pH 5.0, the aim of the present work is to study the effect of the conductivity control on the precipitation behavior of whey protein. BMEA performances were evaluated by measuring electrodialytic parameters, protein kinetic precipitation, molecular profiles, and isolate chemical composition and purity. The highest protein precipitation with 10% WPI solution was obtained at pH 4.6 and at a conductivity level of 200 microS/cm maintained with many 0.4-mL additions of 1.0 M KCl (200 microS[+]), with a 46% precipitation of the total protein, beta-lg composing the main part of the precipitated protein. With a 20% WPI solution, it was possible to reach pH 4.65 with conductivity control at 350 microS/cm. However, the 27% protein precipitation was still low. The changes in viscosity as pH decreases observed at 20% WPI would decreased the final precipitation rate of beta-lg, since the viscosity of the 20% WPI dispersion was very different.