Whey Protein Denaturation Research Articles

Effect of interactions between denatured whey proteins and casein micelles on the formation and rheological properties of acid skim milk gels

The effect of interactions of denatured whey proteins with casein micelles on the rheological properties of acid milk gels was investigated. Gels were made by acidification of skim milk with glucono-δ-lactone at 30°C using reconstituted skim milk powders (SMP; both low- and ultra-low-heat) and fresh skim milk (FSM). The final pH of the gels was ∼4·6. Milks containing associated or ‘bound’ denatured whey proteins (BDWP) with casein micelles were made by resuspending the ultracentrifugal pellet of heated milk in ultrafiltration permeate. Milks containing ‘soluble’ denatured whey protein (SDWP) aggregates were formed by heat treatment of an ultracentrifugal supernatant which was then resuspended with the pellet. Acid gels made from unheated milks had low storage moduli, G′, of <20 Pa. Heating milks at 80°C for 30 min resulted in acid gels with G′ in the range 390–430 Pa. The loss tangent (tan δ) of gels made from heated milk increased after gelation to attain a maximum at pH ∼5·1, but no maximum was observed in gels made from unheated milk. Acid gels made from milks containing BDWP that were made from low-heat SMP, ultra-low-heat SMP and FSM had G′ of about 250, 270 and 310 Pa respectively. Acid gels made from milks containing SDWP that were made from ultra-low-heat SMP or FSM had G′ values in the range 17–30 Pa, but gels made from low-heat SMP had G′ of ∼140 Pa. It was concluded that BDWP were important for the increased G′ of acid gels made from heated milk. Addition of N-ethylmaleimide (NEM) to low-heat reconstituted milk, to block the —SH groups, resulted in a reduction of the G′ of gels formed from heated milk but did not reduce G′ to the value of unheated milk. Addition of 20 mm-NEM to FSM, prior to heat treatment, resulted in gels with a lower G′ value than gels made from reconstituted low-heat SMP. It was suggested that small amounts of denatured whey proteins associated with casein micelles during low-heat SMP manufacture were probably responsible for the higher G′ of gels made from milk containing SDWP and from milk heated in the presence of 20 mm-NEM, compared with gels made from FSM.

Distribution of Iron Between Caseins and Whey Proteins in Acidified Milk

The association of iron with casein and whey proteins was studied by fortifying pasteurized skim milk with 100 mg of iron (Fe3+)/L and adjusting the pH to 6.7, 6.2, 5.8, 5.3, 4.5, and 4.0. Samples were fractionated by ultracentrifugation, and the pellets and serum collected and analyzed for iron, calcium, and phosphorus contents, and for protein profile. At pH 6.7, 6.2, 5.8, and 5.3, some casein, particularly β-casein was not sedimented. More iron was sedimented at pH≤4. However, expressed on a protein basis, the pellets had lower iron contents, probably because denatured whey proteins have lower iron-binding affinities. As pH decreased, the calcium and phosphorus dissociated from casein micelles, with less solubilization of colloidal calcium phosphate occurring in the iron-fortified samples at pH 6.2, 5.8 and 5.3. In yogurt fortified with casein-chelated iron, iron remained bound to casein and was distributed within the micelles. Yogurt fortified with whey protein-chelated iron showed iron being distributed throughout the nonmicellar portion of the yogurt. Fortification with FeCl3resulted in iron being preferentially located within the casein micelles.

The effect of degree of whey protein denaturation and conditions of milk preparation on functional properties of yoghurt (short communication).

Food / NahrungVolume 42, Issue 03-04 p. 250-251 Short Communication The effect of degree of whey protein denaturation and conditions of milk preparation on functional properties of yoghurt Z. Żbikowski, Z. ŻbikowskiSearch for more papers by this authorA. Żbikowska, A. Żbikowska Institute of Food Biotechnology, University of Agriculture and Technology, Institute of Dairy Technology, M. Oczapowskiego 7, PL-10-957 Olsztyn, PolandSearch for more papers by this authorM. Baranowska, M. Baranowska University of Agriculture and Technology, Institute of Dairy Technology, M. Oczapowskiego 7, PL-10-957 Olsztyn, PolandSearch for more papers by this author Z. Żbikowski, Z. ŻbikowskiSearch for more papers by this authorA. Żbikowska, A. Żbikowska Institute of Food Biotechnology, University of Agriculture and Technology, Institute of Dairy Technology, M. Oczapowskiego 7, PL-10-957 Olsztyn, PolandSearch for more papers by this authorM. Baranowska, M. Baranowska University of Agriculture and Technology, Institute of Dairy Technology, M. Oczapowskiego 7, PL-10-957 Olsztyn, PolandSearch for more papers by this author First published: 08 January 1999 https://doi.org/10.1002/(SICI)1521-3803(199808)42:03/04<250::AID-FOOD250>3.0.CO;2-ECitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume42, Issue03-04August 1998Pages 250-251 RelatedInformation

Effect of heat treatment on the rennet coagulation properties of recombined high total solids milk made from milk protein concentrate powder

The effects of heat treatment on the small and large deformation rheological properties of rennet gels made from recombined high total solids milk made from milk protein concentrate were studied. Heat treatment of recombined milk resulted in a slower rate of increase in the storage modulus (G') of rennet induced gels, a reduction in the gelation time and a decrease in the yield force required to fracture gels. The extent of whey protein denaturation (as a result of heat treatment) was related to the decrease in the G' value of gels as well as the yield force.

The Effect of Heating on Physicochemical and Renneting Properties of Milk: A Comparison between Caprine, Ovine and Bovine Milk

Abstract The effect of heating at 75–90°C for 30 s to 10 min on physicochemical characteristics and renneting properties of ewe’s, goat’s and cow’s milk was studied. In the three species, the kinetics of whey protein denaturation led to the same maximum rate of denaturation corresponding to about 80% of total whey protein, whereas diffusible calcium decreased to about 20% of its initial value after the most severe heating. Micelle size increased from 25% in goat’s milk to 75% in ewe’s milk but remained unchanged in cow’s milk. Renneting properties of goat’s and ewe’s milk were much less impaired by severe heating than in cow’s milk. The latter lost its ability to form a gel when heating intensity was higher than 90°C for 1 min, whereas in goat’s and ewe’s milk, coagulation was slowed down but remained always measurable. Curd whey draining capacity was severely reduced by heating, but to a lesser degree in goat’s milk than in cow’s and ewe’s milk.

Effects of High Pressures Combined with Moderate Temperatures on the Rennet Coagulation Properties of Milk

The effects of milk pressurization (100–400 MPa) at different temperatures (25–60°C) on the denaturation of whey proteins, rennet coagulation time, wet cheese yield and enzymatic phase of coagulation have been investigated. The results indicated that there is a synergistic effect between pressure and temperature on the biochemical properties of milk. Pressurization at 40°C delayed coagulation time and impaired gel strength, although protein and water retention in the curd were improved as compared with milk pressurized at 25°C. The use of temperatures higher than 40°C adversely affected milk coagulation. The combined application of high pressures and moderate temperatures was found to increase the extent of β-lactoglobulin denaturation over treatments at 25°C, and pressurization at 50°C induced denaturation of α-lactalbumin. Enhanced whey protein denaturation is supposed to be responsible for hindering the enzymatic hydrolysis of κ-casein, in particular that of the glycosylated forms.

Kinetics of Denaturation and Aggregation of Whey Proteins in Skim Milk Heated in an Ultra-high Temperature (UHT) Pilot Plant

Skim milk was subjected to various temperature–time (70–130°C for 3–1800 s) treatments in a pilot-scale ultra-high temperature (UHT) plant using a direct steam injection (DSI) system. The rates of denaturation and aggregation of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) were determined by quantitative polyacrylamide gel electrophoresis while that of immunoglobulin G (IgG) was determined by affinity chromatography. The order of reaction, the rate constant and the activation energy were determined for the denaturation of β-lg, α-la and IgG using non-linear regression methods. The kinetic parameters obtained for β-lg and α-la showed a marked temperature dependence. Aggregation of β-lg mainly involved the formation of intermolecular disulphide linkages at all temperatures studied, while the aggregation of α-la, particularly below 80°C, appeared to also involve hydrophobic interactions. The kinetic parameters obtained for β-lg aggregation, due to disulphide bond formation, were similar to those observed for β-lg denaturation.

Microstructure, permeability and appearance of acid gels made from heated skim milk

The effects of a range of heat treatments on the microstructure and appearance of acid skim milk gels were investigated using confocal scanning laser microscopy, permeability measurements and photography. Gels were made from reconstituted skim milk heated at 75, 80, 85 and 90 °C for 15 or 30 min, by acidification with glucono-δ-lactone at 30 °C, with a few samples at 40 °C. Heating milks, at temperatures ≤80 °C, resulted in a gel microstructure that appeared to have thinner but more numerous branches, and had a higher ‘apparent interconnectivity’ (in the thin optical section of the x - y plane) of aggregates compared with unheated or mildly heated milks that had tortuous, bent or irregular clusters and strands making up the gel network and much less ‘apparent interConnectivity’ of strands and clusters. There were no major differences in the microstructure of acid milk gels formed from milk heated in the range 80–90 °C. Permeability measurements, which gave information on the size and number of the largest pores in the gel matrix, indicated that heat treatment had little effect on the overall porosity of the gels. It was proposed that the aggregation of denatured whey proteins during the acidification of heated milk altered gel formation and were responsible for the modified microstructure. Heating probably reduced the thickness and altered the orientation of the strands in the network. High treatment of milk also resulted in gels having large visible cracks and a rough surface appearance, but gels made from unheated milk had a smooth, unblemished appearance. It was observed that these structural rearrangements of the network, which were responsible for the large cracks and rough appearance of gels made from heated milk, occurred just after gel formation. It was proposed that a reduction in the shear deformation at fracture of gels made from heated milk may have contributed to the greater susceptibility of these gels to localized fracture

Composition, Gel Properties and Microstructure of Quarg as Affected by Processing Parameters and Milk Quality

Abstract Qarg cheese was manufactured on a small scale from batches of milk which had received different heat treatments (72°C for 15 s or 90°C for 10 min), different levels of added KIO 3 (0 or 0.1 m m ) and different levels of plasmin proteolysis premanufacture (none or 6 mg L -1 plasmin added and incubated at 37°C for 6 h). Plasmin digestion premanufacture affected the microstructure (changed appearance in electron micrographs) and rheology (lower G ′ values) of quarg. Plasmin digestion also increased cheese moisture ( P P β -lactoglobulin and α -lactalbumin levels in whey were decreased by this treatment ( P 3 before high-heat treatment reduced α -lactalbumin denaturation, but gave a cheese of similar composition to that made from high heated milk controlled. Rheological properties of the KIO 3 -treated samples were poor, and microstructures appeared different. Milk quality (plasmin content) and processing conditions used (extent and nature of whey protein denaturation) appear to significantly affect the composition and structure of quarg.

Inhibitory Activity Against Plasmin, Trypsin, and Elastase in Rennet Whey and in Cheese Fortified with Whey Protein

The inhibitory activity against trypsin, elastase, and plasmin was determined in samples of Danbo 45+ that were manufactured from milk pasteurized at 72, 80, and 90°C for 15, 30, and 60s; the corresponding rennet wheys; and Havarti 45+ manufactured from milk concentrated 1.8-fold, 2.7-fold, and 4.6-fold by ultrafiltration. A sensitive colorimetric assay demonstrated that the incorporation of thermally denatured whey proteins into the cheese curd by pasteurization resulted in a decreased proteinase inhibitory activity against trypsin and elastase in Danbo 45+ and against trypsin, elastase, and plasmin in the corresponding rennet wheys. However, incorporation of native whey proteins into Havarti 45+ by ultrafiltration of the cheese milk resulted in an increased inhibitory activity against trypsin and elastase in the cheeses. Cheese manufactured from milk concentrated 1.8-fold, 2.7-fold, or 4.6-fold displayed trypsin inhibitory activity that was 1.8, 2.9, and 5.1 times, respectively, that of the reference cheese. Similarly, the elastase inhibitory activity in the cheeses increased 2.2, 3.2, and 7.8 times. The increased inhibitory activity in cheese fortified with native whey protein likely contributes to the decreased proteolysis and altered ripening characteristics of the resulting cheeses, and, further, the method can be adapted to detection of other inhibitors if sufficiently sensitive substrates are available.

The influence of milk pasteurization temperature and pH at curd milling on the composition, texture and maturation of reduced fat cheddar cheese

Reduced fat milks were pasteurized, for 15 s, at temperatures ranging from 72 to 88°C to give levels of whey protein denaturation varying from ˜ 3 to 35%. The milks were converted into reduced fat cheddar cheese (16–18% fat) in 500 litre cheese vats; the resultant cheese curds were milled at pH values of 5.75 and 5.35. Raising the milk pasteurization temperature resulted in impaired rennet coagulation properties, longer set‐to‐cut times during cheese manufacture, higher cheese moisture and moisture in the non‐fat cheese substance, lower levels of protein and calcium and lower cheese firmness. Increasing the pH at curd milling from 5.35 to 5.75 affected cheese composition and firmness, during ripening, in a manner similar to that of increasing milk pasteurization temperature. Despite their effects on cheese composition and rheology, pasteurization temperature and pH at curd milling had little influence on proteolysis or on the grading scores awarded by commercial graders during ripening over 303 days.

Gel Formation from Industrial Milk Whey Proteins under Hydrostatic Pressure: Effect of Hydrostatic Pressure and Protein Concentration.

The effects of high hydrostatic pressure and protein concentration on the denaturation and gelation of whey protein were investigated. Industrial whey protein isolate (WPI) and whey protein concentrate (WPC) solutions (pH 6.8) at various concentrations were pressurized for 10 min at 30 degrees C under 200-1000 MPa. With the WPI solution, the concentration for affecting the turbidity was 1% and was 6% for the viscosity at 400 MPa, while for inducing gelation, it was 10% at 600 MPa. With the WPC solution, the viscosity changed at a concentration >12%, and gel formation began at >18% at 400 MPa. The hardness and breaking stress of pressure-induced WPI gels increased with increasing concentration of WPI (12-18%) and hydrostatic pressure, the ratings for the 20% WPC gels being one-third those of the 20% WPI gels. The solubility of proteins from the pressure-induced WPI gels decreased with increasing pressure, while that of WPC gel induced at >600 MPa remained constant at approximately 50%. The microstructure of the WPI gels had a porous network form, whereas the WPC gels were irregular particulates. beta-Lactoglobulin, alpha-lactalbumin, and serum albumin preferentially participated in pressure-induced aggregation and gelation through S-S bonding.

Incorporation of Native and Denatured Whey Proteins into Cheese Curd for Manufacture of Reduced Fat, Havarti-type Cheese

Undenatured and heat-denatured whey proteins were incorporated into Havarti-type cheese by manufacturing cheese from ultrafiltered milk that had been heated to 72°C for 17s or 85°C for 17s. Traditional Havarti also was manufactured and used as a control. Cheeses were ripened for 12 wk. High heat treatment resulted in significantly greater percentages of denatured whey proteins in milk. The rate of αs1-CN hydrolysis, as determined by urea-PAGE, was significantly lower in cheese that had been manufactured from UF milk heated to 72°C than in control cheese and cheese made from UF milk heated to 85°C. More whey proteins, mostly denatured, were recovered in cheese manufactured from milk heated to 85°C than in cheeses manufactured from milk heated to 72°C. The overall flavor intensity among cheeses was indistinguishable by the sensory panel; however, control cheeses were more bitter than UF cheeses.

Whey protein fractionation: Isoelectric precipitation of α-lactalbumin under gentle heat treatment

The selective precipitation of alpha-lactalbumin (alpha-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. In these conditions, beta-lactoglobulin remains soluble, whereas bovine serum albumin and immunoglobulins co-precipitate. Knowledge of the mechanism governing the alpha-LA precipitation influences the choice of operating conditions and enables optimization of the fractionation process. alpha-LA is a calcium metallo-protein and its isoelectric precipitation is governed by the protein-calcium complexation equilibrium. Citrate, a sequestrant of calcium, decreases the free calcium concentration and displaces the precipitation phenomenon to a lower temperature range. A study of the effect of citrate on the precipitation phenomena of whey proteins is presented. Whatever the citrate content, precipitation curves for bovine serum albumin (BSA) and alpha-LA intersect at a temperature around 45 degrees C. For a temperature of heat treatment lower than 40 degrees C, a selective enrichment in alpha-LA of the precipitated phase is observed. As addition of citrate leads to high alpha-LA precipitated fractions at a temperature around 35 degrees C, the precipitation step may be performed at this temperature. It results in a reduced heat denaturation of whey proteins and in a higher alpha-LA purity in the precipitated fraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 391-397, 1997.

Whey protein fractionation: Isoelectric precipitation of α‐lactalbumin under gentle heat treatment

The selective precipitation of α-lactalbumin (α-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. In these conditions, β-lactoglobulin remains soluble, whereas bovine serum albumin and immunoglobulins co-precipitate. Knowledge of the mechanism governing the α-LA precipitation influences the choice of operating conditions and enables optimization of the fractionation process. α-LA is a calcium metallo-protein and its isoelectric precipitation is governed by the protein-calcium complexation equilibrium. Citrate, a sequestrant of calcium, decreases the free calcium concentration and displaces the precipitation phenomenon to a lower temperature range. A study of the effect of citrate on the precipitation phenomena of whey proteins is presented. Whatever the citrate content, precipitation curves for bovine serum albumin (BSA) and α-LA intersect at a temperature around 45°C. For a temperature of heat treatment lower than 40°C, a selective enrichment in α-LA of the precipitated phase is observed. As addition of citrate leads to high α-LA precipitated fractions at a temperature around 35°C, the precipitation step may be performed at this temperature. It results in a reduced heat denaturation of whey proteins and in a higher α-LA purity in the precipitated fraction. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 391–397, 1997.

Effect of Protein Concentration on Rates of Thermal Denaturation of Whey Proteins in Milk

The effects of varying the concentrations of total protein, whey protein, and casein on levels of thermal denaturation of four main whey protein fractions in skim milk, namely immunoglobulins, serum albumin/lactoferrin, β-lactoglobulin, and α-lactalbumin, were examined by gel permeation fast protein liquid chromatography. On heating at 80 °C, the rates of denaturation of the individual whey proteins in skim milk increased with total protein concentration and, to a lesser extent, with increasing whey protein concentration. The levels of denaturation of the whey proteins were not affected by increasing micellar casein concentration but decreased markedly when the casein concentration was reduced below that in skim milk. In milks from different cows, rates of denaturation of individual whey proteins were not affected by β-lactoglobulin phenotype but were higher in milk from cows in late lactation. Keywords: Whey protein; denaturation; aggregation

EDTA-induced dissociation of casein micelles and its effect on foaming properties of milk

The effects of addition of EDTA on the dissociation of caseins and foaming properties of milks (100 g solids/l) reconstituted from skim milk powders given a low-heat (72°C for 30 s) or high-heat (85°C for 30 min) treatment during powder manufacture were determined. The EDTA-induced dissociation of caseins was independent of heat treatment but in high-heat milk was accompanied by release of denatured whey proteins. EDTA changed the proportions of individual caseins in the supernatant. EDTA addition improved both foam overrun and foam stability of low- and high-heat milks. The increase in serum protein on addition of EDTA contributed to the improvement in foaming properties of milks by increasing the availability of the proteins for formation of the air–water interface.

Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk

The effect of a range of milk heat treatments on the rheological properties, at small and large deformations, of acid skim milk gels was investigated. Gels were made from reconstituted skim milk heated at 75, 80, 85 and 90°C for 15 or 30 min by acidification with glucono-δ-lactone at 30°C. Gels were also made from skim milk powder (SMP) samples that had been given a range of preheat treatments during powder manufacture. Heating milks at temperatures [ges ]80°C for 15 min increased the storage moduli (G′) compared with unheated milk and produced gels with G′ in the range 300–450 Pa. Acid gels made from high-heat or medium-heat SMP had higher G′ than gels made from low-heat or ultra-low-heat SMP. Cooling gels to low temperatures resulted in an increase in G′. The yield stress of gels slightly decreased with mild heat treatments of milk, and then increased again to a maximum, finally decreasing slightly with very high heat treatments of milk. The strain at yielding decreased markedly with increasing heat treatment of milk, making these gels brittle and easier to fracture. We propose that denatured whey proteins aggregated with casein particles during the acidification of heated milk and were responsible for most of the effects observed in this study.

Assessment of heat-induced denaturation of whey proteins

Milk proteins are significant for their sensitivity to heat so that they can act as suitable indicators for monitoring the heat treatment of milk. In the present work, raw skimmed milk was heated under laboratory conditions to 60°C, 74°C, 85°C and 90°C for 5 min and the extent of denaturation of major whey proteins (α-lactalbumin and β-lactoglobulin) was determined by using polyacrylamide gel electrophoresis (PAGE), CE and indirect competitive enzyme-linked immunosorbent assay (ELISA). ELISA showed higher sensitivity especially for lower temperatures of heat treatment. PAGE enables semiquantitative evaluation of whey protein fractions and is rather time consuming, while CE is a very rapid and quantitative method. The results given for denaturation were also compared to those yielded from theoretical kinetic equations.

Whey Changes during Processing Determined by Near Infrared Spectroscopy

ABSTRACTWe assessed the potential application of near infrared (NIR) spectrophotometry using a remote optic fiber probe for the development of calibrations. We evaluated the heat denaturation of whey proteins, the chemical composition of a whey protein concentrate (WPC) during production, and the degree of hydrolysis (DH) of whey proteins during trypsin hydrolysis. Partial least square (PLS) calibrations were developed for these applications and were related to a variety of products and processing conditions, so as to increase their reliability. NIR has potential applications in real time analysis for whey processing lines.