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 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.     

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.     

Oxygen permeability and mechanical properties of films from hydrolyzed whey protein

The effects of whey protein hydrolysis on film oxygen permeability (OP) and mechanical properties at several glycerol-plasticizer levels were studied. Both 5.5% and 10% degree of hydrolysis (DH) whey protein isolate (WPI) had significant effect (p 0.05) occurred for film OP between unhydrolyzed WPI, 5.5% DH WPI, and 10% DH WPI films at the same glycerol content. Hydrolyzed WPI films of mechanical properties similar to those of WPI films had better oxygen barrier. Therefore, use of hydrolyzed WPI allowed achievement of desired film flexibility with less glycerol and with smaller increase in OP.     

Lipid oxidation in corn oil-in-water emulsions stabilized by casein,whey protein isolate,and soy protein isolate

Proteins can be used to produce cationic oil-in-water emulsion droplets at pH 3.0 that have high oxidative stability. This research investigated differences in the physical properties and oxidative stability of corn oil-in-water emulsions stabilized by casein, whey protein isolate (WPI), or soy protein isolate (SPI) at pH 3.0. Emulsions were prepared with 5% corn oil and 0.2-1.5% protein. Physically stable, monomodal emulsions were prepared with 1.5% casein, 1.0 or 1.5% SPI, and > or =0.5% WPI. The oxidative stability of the different protein-stabilized emulsions was in the order of casein > WPI > SPI as determined by monitoring both lipid hydroperoxide and headspace hexanal formation. The degree of positive charge on the protein-stabilized emulsion droplets was not the only factor involved in the inhibition of lipid oxidation because the charge of the emulsion droplets (WPI > casein > or = SPI) did not parallel oxidative stability. Other potential reasons for differences in oxidative stability of the protein-stabilized emulsions include differences in interfacial film thickness, protein chelating properties, and differences in free radical scavenging amino acids. This research shows that differences can be seen in the oxidative stability of protein-stabilized emulsions; however, further research is needed to determine the mechanisms for these differences.     

Principal component similarity analysis of Raman spectra to study the effects of pH,heating, and kappa-carrageenan on whey protein structure

Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.     

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.     

Adsorption of whey protein isolate at the oil-water interface as a function of processing conditions: a rheokinetic study

In this paper we present surface dynamic properties (interfacial tension and surface dilational properties) of a whey protein isolate with a high content of beta-lactoglobulin (WPI) adsorbed on the oil-water interface as a function of adsorption time. The experiments were performed at constant temperature (20 degrees C), pH (5), and ionic strength (0.05 M). The surface rheological parameters and the interfacial tension were measured as a function of WPI concentration (ranging from 1 x 10(-)(1) to 1 x 10(-)(5)% w/w) and different processing factors (effect of convection and heat treatment). We found that the interfacial pressure, pi, and surface dilational modulus, E, increase and the phase angle, phi, decreases with time, theta, which should be associated with WPI adsorption. These phenomena have been related to diffusion of the protein toward the interface (at short adsorption time) and to the protein unfolding and/or protein-protein interactions (at long-term adsorption) as a function of protein concentration in solution and processing conditions.     

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.     

Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate

The effects of heat-induced denaturation and subsequent aggregation of whey protein isolate (WPI) solutions on the rate of enzymatic hydrolysis was investigated. Both heated (60 °C, 15 min; 65 °C, 5 and 15 min; 70 °C, 5 and 15 min, 75 °C, 5 and 15 min; 80 °C, 10 min) and unheated WPI solutions (100 g L(-1) protein) were incubated with a commercial proteolytic enzyme preparation, Corolase PP, until they reached a target degree of hydrolysis (DH) of 5%. WPI solutions on heating were characterized by large aggregate formation, higher viscosity, and surface hydrophobicity and hydrolyzed more rapidly (P < 0.001) than the unheated. The whey proteins exhibited differences in their susceptibility to hydrolysis. Both viscosity and surface hydrophobicity along with insolubility declined as hydrolysis progressed. However, microstructural changes observed by light and confocal laser scanning microscopy (CLSM) provided insights to suggest that aggregate size and porosity may be complementary to denaturation in promoting faster enzymatic hydrolysis. This could be clearly observed in the course of aggregate disintegration, gel network breakdown, and improved solution clarification.     

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.     

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.     

Aging of whey protein films and the effect on mechanical and barrier properties

This work focuses on the aging of whey protein isolate (WPI) films plasticized with glycerol (G) and sorbitol (S). The films were cast from heated aqueous solutions at pH 7 and dried at 23 degrees C and 50% relative humidity (RH) for 16 h. They were stored in a climate room (23 degrees C, 50% RH) for 120 days, and the film properties were measured at regular intervals. The moisture content (MC) of the WPI/G films decreased from 22% (2 days) to 15% (45 days) and was thereafter constant at 15% (up to 120 days). This affected the mechanical properties and caused an increased stress at break (from 2.7 to 8.3 MPa), a decreased strain at break (from 33 to 4%), and an increased glass transition temperature (T(g)) (from -56 to -45 degrees C). The barrier properties were, however, unaffected, with constant water vapor permeability and a uniform film thickness. The MC of the WPI/S films was constant at approximately 9%, which gave no change in film properties.     

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.     

Drying temperature effect on water vapor permeability and mechanical properties of whey protein-lipid emulsion films

The water vapor permeability (WVP) and mechanical properties of whey protein isolate (WPI) and WPI-lipid emulsion films dried at different conditions were investigated. As drying temperature increased, WVPs decreased significantly. Significantly lower WVP was observed for emulsion films compared to WPI films. WPI-Beeswax (BW) and WPI-anhydrous milkfat fraction emulsion films dried at 80 degrees C and 40% RH gave the lowest WVP compared to 25 degrees C, 40% RH and 40 degrees C, 40% RH. A large drop in WVP of WPI-BW emulsion films was observed at 20% BW content. The decrease in WVP for emulsion films as drying temperature increased could be due to change in the lipid crystalline morphology and/or lipid distribution within the matrix. Mechanical properties of WPI and WPI-lipid emulsion films, on the other hand, were not modified by drying conditions.     

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   

高压处理对牛乳清蛋白体外消化性的影响

为了评价牛乳清蛋白(WPI)加压凝胶的消化性,使用未加压WPI及加压(200～600MPa)WPI凝胶进行了体外模拟胃蛋白酶-胰蛋白酶消化率的测定,并用十二烷基磺酸钠-聚丙烯酰胺(SDS-PAGE)电泳的方法分析了不同压力对WPI构成蛋白消化性的影响。结果表明,经过胃蛋白酶和胰蛋白酶的消化后,加压WPI凝胶消化率显著高于未加压WPI,分别为72.59%和61.02%;400MPa以上加压处理后,加压WPI凝胶消化率未见有明显的变化(p<0.05)。尽管未加压WPI中β-乳球蛋白不易被胃蛋白酶消化,400MPa加压WPI凝胶中的β-乳球蛋白则能被胃蛋白酶部分消化,而600MPa加压后β-乳球蛋白几乎被完全消化。此外,在胃蛋白酶消化阶段,有3500～6500u范围的多肽产物生成。     

Preparation and characterization of beta-carotene nanodispersions prepared by solvent displacement technique

This work demonstrated the preparation of protein-stabilized beta-carotene nanodispersions using the solvent displacement technique. The emulsifying performance of sodium caseinate (SC), whey protein concentrate (WPC), whey protein isolate (WPI), and a whey protein hydrolysate (WPH, 18% degree of hydrolysis) was compared in terms of particle size and zeta-potential of the nanodispersions. SC-stabilized nanodispersions exhibited a bimodal particle size distribution: large particles (stabilized by casein micelles) with a mean particle size of 171 nm and small particles (stabilized by casein submicelles) of 13 nm. This was confirmed with transmission electron microscopy analysis. Most of the beta-carotene precipitated (87.6%) was stabilized in the small particles. On the other hand, the nanodispersions stabilized by the whey proteins were polydispersed with larger mean particle sizes. The mean particle size of WPC and WPI was 1730 and 201 nm, respectively. The SC-stabilized nanodispersion was expected to be more stable as indicated by its higher absolute zeta-potential value (-31 mV) compared to that of WPC (-15 mV) and WPI (-16 mV). Partially hydrolyzed whey protein possessed improved emulsifying properties as shown by WPH-stabilized samples. It was interesting to note that increasing the SC concentration from 0.05 to 0.5 wt % increased the particle size of beta-carotene stabilized by casein micelles, while the reverse was true for those stabilized by SC submicelles. Microfluidization at 100 MPa of SC solution dissociated the casein micelles, resulting in a decrease in mean particle size of the casein micelle-stabilized particles when the SC solution was used to prepare nanodispersions. The results from this work showed that protein-stabilized beta-carotene nanodispersions could be prepared using the solvent displacement technique.     

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.     

Lipid particle size effect on water vapor permeability and mechanical properties of whey protein/beeswax emulsion films

Lipid particle size effects on water vapor permeability (WVP) and mechanical properties of whey protein isolate (WPI)/beeswax (BW) emulsion films were investigated. Emulsion films containing 20 and 60% BW (dry basis) and mean lipid particle sizes ranging from 0.5 to 2.0 microm were prepared. BW particle size effects on WVP and mechanical properties were observed only in films containing 60% BW. WVP of these films decreased as lipid particle size decreased. As drying temperature increased, film WVPs decreased significantly. Meanwhile, tensile strength and elongation increased as BW particle size decreased. However, for 20% BW emulsion films, properties were not affected by lipid particle size. Results suggest that increased protein-lipid interactions at the BW particle interfaces, as particle size decreased and resulting interfacial area increased, result in stronger films with lower WVPs. Observing this effect depends on a large lipid content within the protein matrix. At low lipid content, the effect of interactions at the protein-lipid interfaces is not observed, due to the presence of large protein-matrix regions of the film without lipid, which are not influenced by protein-lipid interactions.