Whey Protein Denaturation Research Articles

Kinetics of Cold-Set Diffusion-Limited Aggregations of Denatured Whey Protein Isolate Colloids

The CaCl2-induced cold-set aggregation kinetics of the denatured whey protein isolate (WPI) colloids has been investigated under dilute diffusion-limited cluster aggregation (DLCA) conditions, using small-angle light scattering. In particular, the structure factor, the scattered intensity at zero angle and the average radius of gyration have been measured for the aggregating system as a function of time. It is found that the fractal dimension of the clusters is df= 1.85, in the range typical of clusters aggregated under DLCA conditions. The aggregation kinetics in this transition region can be described by a power law relation in the initial stage of the aggregation, but the exponent of the power law is equal to 0.7, i.e., significantly larger than 1/df= 0.54, which is the typical value of the DLCA kinetics. Since it is found that the average gyration radius of the clusters has reached a value of 80 microm, leading to a cumulative volume fraction of clusters equal to 0.25, it is legitimate to expect that the process is in the region of transition from aggregation to gelation. This confirmed by the fact that, at the later stage of the aggregation, the growth of the average cluster size further accelerates with time and eventually becomes explosive, leading to gelation. The observed aggregation kinetics has been compared with that reported in the literature from DLCA Monte Carlo simulations, and a good agreement has been found with the data corresponding to the transition region from aggregation to gelation. Numerical simulations using the Smoluchowski kinetic model have also been carried out in order to support the experimental findings.

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70–80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para- κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para- κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness ( G ′ ) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.

Effect of heat treatment at alkaline pH on the rennet coagulation properties of skim milk

Reconstituted skim milk was heated at 90 °C for 30 s at pH values ranging from 6.6 to 8.1, stored overnight at 5 °C then renneted at pH values ranging from 6.2 to 6.6. The heat-induced disimprovement of the rennet coagulation properties of the milk (longer rennet coagulation time and lower gel firmness) were partially reduced as heat-treatment pH increased, except at pH 6.6. These properties were related to the increased heat-induced dissociation of micellar κ-casein and subse- quent formation of serum, rather than micelle-bound, heat-induced whey protein/κ-casein aggrega- tes. It was postulated that such distribution of the denatured whey protein and κ-casein slightly reduced the detrimental effects of heating on the enzymatic coagulation, as well as helped earlier destabilisation of the micelles. These results are discussed with respect to the amplitude of the effect of the heat-treatment pH, as well as the side effects of alkalinisation and heat treatment on casein dissociation. whey protein / heat treatment / rennet coagulation / pH

Modulation of hydrophobic interactions in denatured whey proteins by transglutaminase enzyme

The role of enzyme crosslinking in mediating formation of hydrophobic association in chemically denatured whey protein isolates (WPI) is examined. WPI samples denatured with dithiothreitol (DTT) and incubated at 50 °C with and without transglutaminase enzyme show dramatic differences in viscosity, with the viscosity of the sample exposed to enzyme being lower by several orders of magnitude than the sample without enzyme. Upon further exposure of both samples to sodium dodecyl sulfate (SDS) to eliminate hydrophobic interactions, we observe no change in the viscosity of the sample previously treated with enzyme, suggesting this sample to have minimal hydrophobic associations. In contrast, the sample without enzyme shows a dramatic drop in viscosity indicating it to have had substantial hydrophobic associations. A similar trend but to a lesser extent is observed at a higher WPI concentration. These results taken together suggest that the formation of enzyme catalyzed ε-(γ-glutamyl)lysine bonds attenuates hydrophobic interactions through steric hindrance and formation of compact molecules that limits exposure of the hydrophobic moieties.

Determination of the Heat Stability Profiles of Concentrated Milk and Milk Ingredients Using High Resolution Ultrasonic Spectroscopy

In the present work, we used high resolution ultrasonic spectroscopy for the analysis of heat coagulation process in milks. Two skim milks recombined from powder samples differing by their preheating treatments as well as dairy ingredients (phosphocasein, whey protein isolate, and a milk model) were monitored while submitted to a temperature of 120°C. Three stages in the precoagulation and coagulation processes can be distinguished in the ultrasonic velocity and attenuation profiles. The first stage shows a very sharp decrease in ultrasonic velocity and increase in ultrasonic attenuation. This could be attributed to fast denaturation and aggregation of whey proteins and precipitation of calcium phosphate. In the second stage, small changes in ultrasonic velocity are observed, the rate of which depends on the nature and pH of the samples. In the third stage, a sharp decrease in ultrasonic velocity and attenuation is recorded for all samples, which corresponds to the coagulation and formation of the gel network. After coagulation, a very small change in ultrasonic velocity and attenuation was observed. From the ultrasonic measurements, we determined the heat stability vs. pH profiles of the different samples studied. The observed ultrasonic attenuation profiles were also interpreted in terms of changes in the size of casein micelles and coagulation processes.

Physical characteristics of set yoghurt made with altered casein to whey protein ratios and EPS-producing starter cultures at 9 and 14% total solids

This study investigated firmness and syneresis of set yoghurts made at the CN to WP ratios of 4:1, 3:1, 2:1 and 1:1 using non-EPS-, capsular EPS- and ropy EPS-producing starter cultures. The yoghurts were made at 9 and 14% (w/w) total solids. The total solids, total protein, the concentration of lactose and the ratios of CN to WP as well as the protein's profiles (native- polyacrylamide gel electrophoresis) in heated and unheated milk blends were investigated. The level of soluble denatured whey protein aggregates in heated milk was also determined. The concentration of EPS, firmness and level of spontaneous syneresis in set yoghurt were monitored weekly throughout 28 days of storage. The microstructure of the set yoghurt made with milk blends at the CN to WP ratio of 4:1 and using three types of starter cultures was carried out after 1 day of storage. There was no difference in the total solids, total protein and lactose concentration of liquid milk blends, except the CN to WP ratios. There was no difference in whey protein denaturation between milk blends. The level of soluble denatured whey protein aggregates in heated milk blends decreased with reducing CN to WP ratio. The firmness and the level of spontaneous syneresis in set yoghurts decreased as the CN to WP ratios were reduced. The use of EPS-producing starter cultures reduced firmness and syneresis and changed the protein matrix in the microstructure of set yoghurts made at 9% (w/w) total solids compared to the control products. These were not observed in set yoghurts made at 14% (w/w) total solids.

Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration

Microfiltration and ultrafiltration were used to manufacture skim milks with an increased or reduced concentration of whey proteins, while keeping the casein and milk salts concentrations constant. The skim milks were heated on a pilot-scale UHT plant at 80, 90 and 120 degrees C. The heat-induced denaturation and aggregation of beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA) were quantified by polyacrylamide gel electrophoresis. Apparent rate constants and reaction orders were calculated for beta-lg, alpha-la and BSA denaturation. Rates of beta-lg, alpha-la and BSA denaturation increased with increasing whey protein concentration. The rate of alpha-la and BSA denaturation was affected to a greater extent than beta-lg by the change in whey protein concentration. After heating at 120 degrees C for 160 s, the concentration of beta-lg and alpha-la associated with the casein micelles increased as the initial concentration of whey proteins increased.

Effect of preheating and other process parameters on whey protein reactions during skim milk powder manufacture

Skim milk powder was manufactured in a milk powder plant using different preheating temperatures, concentrate heating temperatures and spray drying temperatures. Varying the preheating conditions from 70 °C for 52 s to 120 °C for 52 s had a marked effect on the denaturation of β-lactoglobulin A, β-lactoglobulin B, α-lactalbumin, bovine serum albumin (BSA), and immunoglobulin G. In contrast, varying concentrate heating temperature (65–74 °C) and inlet/outlet air dryer temperature (200/101 °C–160/89 °C) had a minimal effect on whey protein denaturation. Most of the whey protein denaturation and association with the casein micelle occurred in the preheating section of the powder plant. Aggregation of β-lactoglobulin (β-lg) and BSA predominantly involved disulphide bonds. Although, greater than 90% of the β-lg and BSA was denatured after preheating at 120 °C for 52 s, the extent of association with the casein micelle was lower, 50% for β-lg and 75% for BSA.

Whey protein concentrate gels with honey and wheat flour

Structural and functional properties of whey protein concentrate (WPC) gels with different honey and wheat flour contents, prepared at pHs 3.75, 4.2 and 7.0, were analysed. Gel structure was observed by scanning electron microscopy. The apparent transition temperatures for protein denaturation and starch gelatinization were determined by differential scanning calorimetry. Gels were characterised through solubility assays in different extraction solutions and polyacrylamide gel electrophoresis of the soluble protein components. The firmness, elasticity, relaxation time, adhesivity and cohesiveness of gels were determined, and the water-holding capacity and superficial colour of gels were also studied. Results suggest that wheat flour could interact with whey proteins, and produces a decrease in the protein solubility of WPC gels, and in the temperature of whey protein denaturation. The effect of wheat flour on the functional properties of WPC gels was different at acidic than at neutral pH: the presence of wheat flour produced an increase in the relaxation time and in the cohesiveness of gels prepared at pH 3.75, whereas at neutral pH a decrease in both properties was observed. Honey and flour content increased the water-holding capacity and browning of WPC gels.

Kinetics of combined thermal and pressure-induced whey protein denaturation in bovine skim milk

Abstract A detailed kinetic study of combined pressure and temperature treatment of milk was carried out in order to estimate kinetic parameters for the denaturation of whey proteins. Skim milk was treated under isobaric conditions (200–800 MPa) in combination with isothermal conditions (1–70 °C), and denaturation of β -lactoglobulin A and B and α -lactalbumin were analyzed. Kinetic parameters were determined by means of a one step non-linear regression. Denaturation of β -lactoglobulin and α -lactalbumin were found to follow second and 2.5 order kinetics, respectively. Heat- and pressure-induced denaturation of both β -lactoglobulin fractions did not differ significantly and α -lactalbumin was more resistant to denaturation than β -lactoglobulin. The activation volume, Δ V # , for denaturation of β -lactoglobulin decreased from zero to −80 mL mol −1 with increasing temperature. Activation energy, E A , of denaturation of β -lactoglobulin calculated at various pressures exceeded 200 kJ mol −1 at 100 and 200 MPa and decreased at higher pressures, to about 100 kJ mol −1 , indicating that high pressure promotes unfolding of the molecules.

Effects of Native and Denatured Whey Proteins on Plasminogen Activator Activity

The plasmin system native to bovine milk consists of the caseinolytic serine proteinase plasmin; its inactive zymogen, plasminogen; plasminogen activators; and inhibitors. Evidence in the literature indicates that whey proteins may inhibit plasmin activity, but there is very little mention of their effect on plasminogen activators. The objective of this research was to determine the effect of both unheated and heat-denatured β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and BSA on plasminogen activators.Plasminogen activator activity was significantly stimulated by non-heat treated and denatured α-LA as well as by denatured β-LG. The stimulation effect by these whey proteins was kinetically characterized, which showed that all 3 significantly increased the rate of plasminogen activation. The stimulation effect was shown to be independent of any effect of the whey proteins on plasmin activity by testing 2 different substrates, d-Val-Leu-Lys p-nitroanilide (S-2251) and Spectrozyme PL (Spec PL), in a plasmin assay. Results using S-2251 confirmed the inhibitory effect of whey proteins on plasmin observed by several researchers. However, use of SpecPL did not suggest inhibition. Ligand binding studies showed this discrepancy to be due to significant interaction between S-2251 and the whey proteins. Overall, this study indicates that whey protein incorporation into cheese may not hinder plasmin activity and may stimulate plasminogen activation. Furthermore, the results indicate the need for careful consideration of the type of synthetic substrate chosen for model work involving whey proteins and the plasmin system.

The roles of disulphide and non-covalent bonding in the functional properties of heat-induced whey protein gels.

Heat-induced gelation (80 degrees C, 30 min or 85 degrees C, 60 min) of whey protein concentrate (WPC) solutions was studied using transmission electron microscopy (TEM), dynamic rheology and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (150 g/kg, pH 6.9) were prepared by dispersing WPC powder in water (control), 10 g/kg sodium dodecyl sulphate (SDS) solution or 10 mM-dithiothreitol (DTT) solution. The WPC gels containing SDS were more translucent than the control gels, which were slightly more translucent than the gels containing DTT. TEM analyses showed that the SDS-gels had finer aggregate structure (approximately equal to 10 nm) than the control gels (approximately equal to 100 nm), whereas the DTT-gels had a more particulate structure (approximately equal to 200 to 300 nm). Dynamic rheology measurements showed that the control WPC gels had storage modulus (G) values (approximately equal to 13,500 Pa) that were approximately equal to 25 times higher than those of the SDS-gels (approximately equal to 550 Pa) and less than half those of the DTT-gels after cooling. Compression tests showed that the DTT-gels were more rigid and more brittle than the control gels, whereas the SDS-gels were softer and more rubbery than either the control gels or the DTT-gels. PAGE analyses of WPC gel samples revealed that the control WPC solutions heated at 85 degrees C for 10 min contained both disulphide bonds and non-covalent linkages. In both the SDS-solutions and the DTT-solutions, the denatured whey protein molecules were in the form of monomers or small aggregates. It is likely that, on more extended heating, more disulphide linkages were formed in the SDS-gels whereas more hydrophobic aggregates were formed in the DTT-gels. These results demonstrate that the properties of heat-induced WPC gels are strongly influenced by non-covalent bonding. Intermolecular disulphide bonds appeared to give the rubbery nature of heat-induced WPC gels whereas non-covalent bonds their rigidity and brittle texture.

Effect of milk pasteurization temperature and in situ whey protein denaturation on the composition, texture and heat-induced functionality of half-fat Cheddar cheese

Abstract In recent years, many dairy product manufacturers pasteurize milk at temperatures higher than 72°C, so as to reduce further the risk of survival of Mycobacterium avium ssp. paratuberculosis. However, little information is available on the effect of these temperatures on cheese quality. Half-fat Cheddar cheeses (∼15%, w/w, fat) were manufactured from milk pasteurized at 72°C, 77°C, 82°C or 87°C for 26 s, and analyzed over a 360 d ripening period. The mean levels of whey protein denaturation in the pasteurized milks were 2.8%, 8.4%, 20.2% and 34.1% of total whey protein, respectively. Increasing pasteurization temperature significantly increased the levels of moisture and non-expressible serum and decreased the levels of protein, fat, calcium and free oil. Pasteurization temperature did not significantly affect the levels of pH 4.6-soluble N but influenced the degradation profiles of αs1- and β-caseins. Increased pasteurization temperature significantly reduced fracture stress, fracture strain and firmness of cheeses. Raising the pasteurization temperature from 72°C to either 82°C or 87°C significantly reduced the flowability and stretchability of the heated cheese and increased its apparent viscosity; in contrast, increasing pasteurization from 72°C to 77°C did not affect these properties.

Gelation of casein-whey protein mixtures.

Heated milk consists of a mixture of whey protein-coated casein micelles and soluble whey protein aggregates. The acid-induced gelation properties of heated milk are consistently different from those of unheated milk--i.e., a shift in gelation pH, stronger gels, and a different microstructure of the gels. In this study we investigated the role of the different fractions of denatured whey proteins on the acid-induced gelation, the gel hardness, and the microstructure. Both whey protein fractions contribute to the observed shift in gelation pH, although by a different mechanism. Obtaining gels with high gel hardness occurs most effectively when all denatured whey proteins are present as whey protein aggregates. It was observed that disulfide bridge exchange reactions during the acid-induced gelation at ambient temperature play an important role for both whey protein fractions. Additionally, disulfide interactions seem to occur between the aggregates and the casein micelles during the gel state. In this study, we show the development of a new approach for confocal scanning laser microscopy measurements--i.e., separate staining of the proteins in milk. By using this method, we were able to determine that, although whey protein aggregates are not linked to the casein micelles, they nevertheless gel at the same moment. This work adds to a better understanding of the role of denatured whey proteins during acid-induced gelation and could improve the effective use of whey proteins.

The effect of high pressure on nitrogen compounds of milk

The effect of pressurization at different pressures (from 200 to 1000 MPa, at 200 MPa intervals,tconst. = 15 min) and periods of time (from 15 to 35 min, at 10 min intervals,pconst. = 800 MPa) on the changes of proteins and nitrogen compounds of skimmed milk was studied.The pressurization caused an increase in the amount of soluble casein and denaturation ofwhey proteins. The level of nonprotein nitrogen compounds and proteoso-peptone nitrogencompounds increased as a result of the high-pressure treatment. These changes increasedwith an increase in pressure and exposure time. High-pressure treatment considerablyaffected the changes in the conformation of milk proteins, which was reflected in thechanges in the content of proteins sedimenting and an increase in their degree ofhydration.

Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt

Abstract Bifidobacterium breve R070 (BB R070) and Bifidobacterium longum R023 (BL R023) were encapsulated as freeze-dried or fresh cultures in water-insoluble food-grade microcapsules produced by emulsion and/or spray-drying, using milk fat and/or denatured whey proteins as immobilization material. The encapsulation yield differed significantly according to the method and the strain used. Dispersion of fresh cells in heat-treated whey protein suspension followed by spray-drying was the least destructive immobilization technique tested, with a survival rate of 25.7±0.1% and 1.4±0.2% for BB R070 and BL R023, respectively. Viable counts of BB R070 cells entrapped in whey protein microcapsules using this method were significantly higher than those of free cells after 28 days in yoghurt stored at 4°C (+2.6 log cycles), and after sequential exposure to simulated gastric and intestinal juices (+2.7 log cycles). In contrast, no protective effect of encapsulation was observed with BL R023. Immobilization of probiotic cultures in whey protein-based microcapsules can increase cell survival when subjected to extreme conditions, making this approach potentially useful for delivery of viable bacteria to the gastrointestinal tract of humans via dairy fermented products. However, technological properties of the strains, and particularly heat resistance, should be taken into consideration for spray-dry encapsulation of sensitive microorganisms.

Heat treatment of whey proteins in the presence of anionic surfactants

Heat treatment of whey protein at pH=7.5 in the presence of three anionic surfactants (sodium dodecyl sulfate, sodium stearoyl-2 lactylate and diacetyl tartaric acid ester of monoglyceride) was studied. The effect of surfactants on whey protein denaturation level was evaluated by measuring solubility at pH=4.8 and slow reacting thiol groups. Denaturation temperature and enthalpy were determinated by differential scanning calorimetry. The resulting heat-induced whey protein–surfactant complexes were characterized by solubility profile between pH 2 and 6, hydrodynamic diameter and surface activity. During heat treatment, protein solubility at pH 4.8, slow reacting thiol groups and denaturation temperature increased with increasing surfactant/protein ratio and reached maximum values around 80 μmol surfactant per gram protein. Anionic surfactants decreased the size of heat-induced whey protein polymers. Heat-induced complexes showed low solubility between pH 4.8 and 5.4. The pH where minimal solubility occurred shifted to lower values when the surfactant/protein ratio increased. The surface tension profiles before heating showed three transitions associated with surfactant binding stages. After heating, these transitions disappeared as a result of total protein unfolding. The thermal behavior of whey proteins depended on the nature of surfactant, sodium dodecyl sulfate and sodium stearoyl-2 lactylate being the most effective.

Effect of pH on the viscosity of heated reconstituted skim milk

Reconstituted skim milk was adjusted to pH values between 6.5 and 6.7 and heated (90°C) for up to 30 min. The change in the viscosity, as well as the casein micelle size and the level of whey protein association with the micelles, was monitored for each milk sample. The change in the viscosity was dependent on the pH of the milk at heating. At pH 6.5, the viscosity increased markedly during the early stages of heating, and plateaued on prolonged heating. The magnitude of the change in viscosity decreased as the pH of the milk was increased, so that only small changes in viscosity were observed on heating at pH 6.7. The change in viscosity was linearly correlated with the change in particle volume (calculated from the particle size). This indicates that the viscosity changes were related to a change in the volume fraction of the casein micelles as a consequence of heating. The level of denatured whey proteins associating with the micelles was also markedly dependent on the pH at heating. High levels associated at pH 6.5, and the level associated was lower as the pH was increased to pH 6.7. The changes in viscosity and casein micelle volume were correlated with the levels of denatured whey proteins that were associated with the casein micelles, although there was some deviation from linearity at low levels of association (<15%).

Rheological properties of acid gels prepared from heated pH-adjusted skim milk.

Reconstituted skim milk was adjusted to pH values between 6.5 and 7.1 and heated (90 degrees C) for up to 30 min. The skim milk samples were then readjusted to pH 6.7. Acid gels prepared from heated milk had markedly higher G ' values, a reduced gelation time, and an increased gelation pH than those prepared from unheated milk. An increased pH at heating decreased the gelation time, increased the gelation pH, and increased the final G ' of acid set gels prepared from the heated milk samples. There were only small differences in the level of whey protein denaturation in the samples at different pH values, and these differences could not account for the differences in the G ' of the acid gels. The levels of denatured whey protein associated with the casein micelles decreased and the levels of soluble denatured whey proteins increased as the pH at heating was increased. The results indicated that the soluble denatured whey proteins had a greater effect on the final G ' of the acid gels than the denatured whey proteins associated with the casein micelles.

Effects of high pressure treatment on the yield of cheese curd from bovine milk

Cheesemaking properties of milk are affected by high pressure (HP) treatment; this study focussed on HP-induced changes in curd yield. Yield was not influenced by treatment at pressures ≤250 MPa, but at higher pressures, curd yield was increased, by up to 25% after treatment at 600–800 MPa. In parallel with increased curd yield, increased curd moisture content and reduced protein content in the whey were observed after treatment at such pressures. Denaturation of α-lactalbumin or β-lactoglobulin occurred at >400 or >100 MPa, respectively. Curd yield and curd moisture content increased and protein content in the whey decreased with increasing treatment time (0–30 min of treatment at 400 or 600 MPa). Increased curd yield is probably a result of both the incorporation of denatured whey proteins and enhanced moisture retention in the curd. HP-induced increases in curd yield may indicate possible applications of HP treatment of milk for cheese manufacture.