Whey Protein Denaturation Research Articles

Reconstituted skim milk: effect of re-dilution after heating concentrated milk at different pH and temperatures

The change in particle size in milk is dependent on the concentration, pH and the temperature of heating. Many of the changes are attributed to a partial aggregation of the casein micelles during heating. κ-Casein dissociation and serum-phase whey protein levels increased with increasing milk concentration at each pH and temperature, whereas αS-casein/β-casein dissociation increased to a maximum at intermediate temperatures before decreasing and then increasing again. Small changes in non-sedimentable calcium and phosphate were observed. Casein micelle solvation decreased with increasing pH and milk concentration at temperatures above 80 °C. This decrease is related to the pH-dependent dissociation of κ-casein from the casein micelles and the extent of denatured whey protein in the serum phase. It is likely that the properties of milk could be manipulated by choosing appropriate milk concentrations pH and/or and heating conditions, even if the milk is subsequently diluted.

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5-90°C for 0.5-30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.

Effects of the thermal denaturation degree of a whey protein isolate on the strength of acid milk gels and the dissociation of κ-casein.

In this study, the effects of the degree of thermal denaturation of whey protein (WP) added to milk on the dissociation of κ-casein from casein micelles were investigated, since they are related to the strength of acid milk gel and its factors. Acid milk gels were prepared by heating thermally denatured WP isolate (WPI) and undenatured milk mixtures and treating them with glucono-δ-lactone as a coagulant. The strength of these gels was negatively correlated with the WPI denaturation degree and strongly positively correlated with the extent of κ-casein dissociation from casein micelles. This behavior was ascribed to the fact that α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) contained in WPI denatured after heating and engaged in disulfide bond formation with each other. With an increase in the degree of denaturation and disulfide bond formation, the bonding between β-lactoglobulin and κ-casein was suppressed to decrease the amount of κ-casein-WPI complexes. When β-Lg forms SS bonds with α-La, the number of highly reactive, free SH groups decreases, which complicates the formation of SS bridges between β-Lg and κ-casein. Thus, the denaturation degree of WPI largely determined the degree of κ-casein dissociation from casein micelles and, consequently, the strength of acid milk gels. Adding WP to milk increases the strength of acid milk gel, and it can be controlled by changing the degree of thermal denaturation of the WP. Furthermore, it was clarified for the first time that the dissociation of κ-casein from casein micelles influences this effect. Further studies are needed to elucidate the structural features of κ-casein-dissociated micelles.

Structural changes and cholesterol-lowering in denatured whey protein isolate: Malic acid combined enzymolysis

Binding of bile salts (BS) and protein has been linked to cholesterol reduction. However, the interaction between the protein structural and binding of BS ability was unclear. The goals of this study were to investigate the relationship among the structure, morphology, and functional properties of hydrolytic peptides of whey protein isolate (WPI) for four groups: control (no treatment), malic acid acidolysis [WPI + MA(DL)], enzymolysis (WPI-E), and enzymolysis combined with MA acidolysis [WPI-E+MA(DL)]. We found that the combination of enzymolysis and acidolysis could significantly improve the bile salt-binding ability of WPI (p < 0.05) by changing the structure and morphology of the protein compared with that in the other groups. An increase in the degree of hydrolysis, reduction in sulfhydryl group content, and exposure of aromatic amino acids indicated that WPI bound to the bile salts through hydrogen bond interactions. Additionally, the increase in surface hydrophobicity and β-sheet, β-turn, and random structure contents according to Fourier transform infrared spectroscopy indicated an exposure of hydrophobic groups. This further confirmed that WPI exhibited higher bile salt-binding ability through hydrophobic interactions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresi (SDS-PAGE), Tricine-SDS-PAGE, scanning electron microscopy, and particle size analysis revealed that the hydrophobic groups exposed to the protein surface were aggregated and formed fragmented polymers with a molecular weight of approximately 3.3 kDa. Moreover, differential scanning calorimetry and rheology revealed that these polymers exhibited higher thermal stability, apparent viscosity, and viscoelasticity, and the viscosity was positively correlated with binding ability. Thus, the relationship between the structure, morphology and functional characteristics of hydrolytic peptides in the study can provide good data for designing innovative new peptides that ameliorate cholesterol and lipid metabolism. And it has good application value for the development of healthy and functional food.

Effect of cheese composition and cheese-making conditions on salt and moisture transfer during brining

Using a model system, based on laboratory scale curd production, the influence of curd composition on salt uptake during brining was examined by varying the composition of milk or the cheese-making conditions. Cheese moisture, minerals and salt concentrations before and after brining were determined. Salt and moisture transfer was mainly driven by the initial curd moisture content. However, the approach used to vary the curd moisture seems to influence salt uptake. The rate of salt uptake was higher when the moisture content was increased by adding denatured whey protein than by decreasing the protein or fat concentration in milk. It was also higher when the moisture content was increased by reducing the cooking time than by reducing the cooking temperature or the pressing strength. These results suggest that salt uptake during brining could be controlled by the approach used to adjust the moisture content of cheese curd.

Assessment of milk protein digestion kinetics: effects of denaturation by heat and protein type used.

Knowledge about how molecular properties of proteins affect their digestion kinetics is crucial to understand protein postprandial plasma amino acid (AA) responses. Previously it was found that a native whey protein isolate (NWPI) and heat denatured whey protein isolate (DWPI) elicit comparable postprandial plasma AA peak concentrations in neonatal piglets, while a protein base ingredient for infant formula (PBI, a β-casein-native whey protein mixture) caused a 39% higher peak AA concentration than NWPI. We hypothesized that both whey protein denaturation by heat as well as changing protein composition by including β-casein, increases the rate of intact protein loss, and that changing the protein composition (by including β-casein), but not whey protein denaturation, yields a faster absorbable product release. Therefore NWPI (91% native), DWPI (91% denatured) and PBI hydrolysis was investigated in a semi-dynamic in vitro digestion model (SIM). NWPI and DWPI hydrolysis were also compared in a dynamic digestion model with dialysis (TIM-1) to exclude potential product inhibition effects that may occur in a closed vessel digestion model as SIM. In both models, the degree of hydrolysis (DH), loss of intact protein, and release of absorbable products (SIM: <0.5 kDa peptides and free AA, TIM-1: bioaccessible AA) were monitored. Additionally, in SIM, intermediate product amounts and their characteristics were determined. DWPI showed considerably faster intact protein loss, but similar DH and absorbable product release kinetics compared with NWPI in both models. Furthermore, more, relatively large, intermediate products were released from DWPI than from NWPI. PBI showed increased intact protein loss, similar DH, and absorbable product release kinetics, but more, relatively small, intermediate products than NWPI. In conclusion, both whey protein denaturation and β-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during in vitro digestion. Our results suggest that intermediate digestion product characteristics are important in relation to postprandial AA responses.

Impact of heat treatment, casein/whey protein ratio and protein concentration on rheological properties of milk protein concentrates used for cheese production

In fresh/hard cheese manufacture, a high casein concentration is desired, whereas, for quark production, a high total protein concentration and a high degree of whey protein denaturation is needed. However, concentration impacts inevitably the concentrate's viscosity—particularly after heat treatment. Our research question was therefore, which mechanisms are responsible for changes in viscosity upon increasing the total protein concentration, varying the casein/whey protein ratio, and heating. In order to answer this question, we investigated the viscosity of unheated and heated milk protein concentrates (3–14% total protein) and three casein/whey protein ratios (85:15, 92:8, 98:2). It turned out that a 37% whey protein depletion was sufficient to 1) inhibit the impact of the casein micelles' repulsive forces on the viscosity; 2) achieve the highest polydispersity and, therefore, particle packing density of all concentrates; and 3) allow up to 95% whey protein denaturation without changing the viscosity of the concentrates after heating.

Microbial inactivation of milk by low intensity direct current electric field: Inactivation kinetics model and milk characterization

Microbial inactivation by pulsed electric field (PEF) has been studied widely although with high operational risk, while few studies on the potential of low intensity electric fields for microbial inactivation have been reported. In this study, the feasibility of inactivating microorganisms in milk by low intensity direct current (DC) electric field was investigated. Then a kinetics model was proposed based on the inactivation curves. Finally, the effect of electric field on the microflora and physicochemical properties of milk was analyzed. Results showed that the bacterial reduction >5 log CFU/mL could be achieved at 50–55°C, 0.3 A–0.6 A, and with 5 min starting intensity of 5 V/cm-9 V/cm. The inactivation kinetics consisted of three stages, therein, the middle stage, main part of the inactivation curve, followed 1st-order reaction kinetics, and the effect of temperature on it was consistent with the Arrhenius Law, which implied that the electric field itself can inactivate bacteria without thermal inactivating effect. The microflora analysis showed that naturally occurring bacteria in the milk contained typical potential pathogenic bacteria (e.g., 56.9% of Acinetobacter spp.) and spoilage bacteria (e.g., 27.5% of Pseudomonas spp.), and the electric field can inactivate them. Moreover, the inactivation chemically preserved the milk's fresh-like characteristics (according to indexes of whey protein denaturation rate, furosine content), and physical stability (turbidity, zeta potential, particle size, color and so on). Therefore, a promising approach is provided for microbial inactivation in dairy industry.

Shelf-stable milk produced by microfiltration and microwave heating: Effects of processing and storage

A customized phase treatment (CPT) process to produce shelf-stable milk with high native whey protein and vitamin contents, and at the same time low plasmin activity was developed. Therefore, skim milk was microfiltered (pore size 0.1 μm) and the obtained retentate was ultra-high temperature (UHT, ϑ = 140 °C, t = 5 s) heated with long pre-heating (ϑ = 95 °C, t = 128 s) while the permeate was microwave heated to 120 °C for 5 s. After sterile mixing and filling the milk was compared to indirectly and directly processed UHT milk. CPT milk resulted in whey protein denaturation, lactulose formation, and thiamine loss ranging in-between direct and indirect UHT milk. CPT and indirectly heated UHT milk had comparable shelf-lives, while direct steam injection resulted in reduced shelf-life with particle formation, sedimentation, and age-gelation accompanied by increased apparent plasmin activity over storage. This study highlights the potential of CPT aiming to achieve eco-friendly and high-quality dairy processes and products. • Novel process for shelf-stable milk: Customized phase treatment (CPT). • CPT uses microfiltration and separate heating of milk, involving microwave heating. • Storage (20 & 30 °C) and evaluation of CPT, directly and indirectly heated UHT milk. • Kinetic modelling of time-temperature integrators. • CPT milk has equal shelf-life with improved quality compared to indirect UHT milk.

Oral retention of thermally denatured whey protein: In vivo measurement and structural observations by CD and NMR

This study investigated structural changes and the in vivo retention in the oral cavity of heated whey protein concentrate (WPC). Heated WPC was shown to have both a higher retention time in the oral cavity compared to unheated whey protein up to 1 min post swallow, and a concomitant increase in free thiol concentration. Nuclear magnetic resonance and circular dichroism demonstrated structural changes in the secondary and tertiary structures of the WPC upon heating. Structural loss of the β-barrel was shown to increase during heating, leading to the exposure of hydrophobic regions. The increase in free thiols and hydrophobic regions are two factors which are known to increase mucoadhesive strength and hence increase oral retention of heated whey protein which may subsequently increase the perception of mouthdrying.

Chemical composition, functional and technological (processing) properties of whey ingredients

The growth in volumes of the milk whey manufacturing has revealed the new field of processing, such as dry whey ingredients production. The authors have made investigations of chemical composition, functional and technological (processing) properties of whey protein concentrates with protein of 35, 55, 80 % in dry matter, whey protein hydrolysate and cheese whey permeate. We used standard methods, generally accepted in research practice. The chemical composition of the tested samples has been determined, including their rehydration properties in terms of wettability, dispersibility and solubility. Heat denaturation of whey proteins during the processing is the reason for the bound groups–SH release and their reactivity enhancement, which provides antioxidant effect of whey ingredients (the antioxidant content in the tested samples is 0.031; 0.058; 0.095; 0.146 and 0.024 mg/g for the whey protein concentrates with protein of 35, 55, 80 % in dry matter, whey protein hydrolysate and whey permeate respectively). The functional and technological properties of whey ingredients make possible their application while producing different product line groups in order to control the technological processes and the quality factors of the enriched products.

Functional properties of skim milk concentrates produced by reverse osmosis filtration and reconstituted commercial powders

Substitution of commercial milk powders by fluid milk concentrates as ingredients in dairy products, may improve product quality and increase sustainability. This study compared the functional properties of skim milk concentrates made by reverse osmosis (SMC) with standard reconstituted high- and low-heat skim milk powders (POW-SMC). SMC had larger Z-average size values, higher viscosities, wider particle size distributions and lower extent of whey protein denaturation than POW-SMC. During storage, SMC showed no acid-induced gelation, but improved rennet-induced gelation compared with POW-SMC. Emulsification activity improved at 20 compared with 10% solids in all concentrates, while emulsion stability was lowest for SMC. Better foaming was observed for high-heat POW-SMC, except after 30 days, where SMC showed highest overrun. In certain dairy products, SMC can thus replace commercial powders to reduce processing costs and improve functionality. SMC showed to be especially advantageous for rennet-gelled products.

Acid gelation of heat-treated recombined milk: Fat globule membrane composition and gelation functionality

Recombined whole milk was prepared by heating skim milk before homogenising with milkfat (HEHO milk) or by homogenising milkfat with unheated skim milk and heating the recombined milk (HOHE milk). Heat treatment caused whey protein denaturation, more at higher temperatures and slightly more in HEHO milk than in HOHE milk. Less protein was adsorbed to the fat globules of the HEHO milk than the HOHE milk; the former had a higher proportion of αS-casein and β-casein and a lower proportion of κ-casein and denatured whey proteins at the interface, indicating a different interaction mechanism. Gelation pH and gelation time were similar for acidified HEHO and HOHE samples and correlated to the denaturation of whey proteins. The final G’ and yield stresses were lower for the HEHO than the HOHE milk. The differences in gelation behaviour were related to the compositions and interactions of the proteins adsorbed to the fat globules.

Effect of β-casein reduction and high heat treatment of micellar casein concentrate on the rennet coagulation properties, composition and yield of Emmental cheese made therefrom

To study the effect of removing β-casein of cheesemilk on cheese manufacture, cheesemilk was formulated from low heat skim milk powder (LH CM), micellar casein concentrate with 1.83% β-casein reduction (MCC CM) and β-casein-reduced micellar casein concentrate with 4.25% β-casein reduction (LB CM) or high heat treatment (HHT; 120 °C, 15 s) LB CM to manufacture Emmental cheeses. MCC- and LB CM had similar rennet coagulation properties and similar compositions and yield in the resultant cheeses. Compared with LB CM, HHT LB CM had an increased level of denatured whey protein and a reduced, yet acceptable, curd firming rate. Higher fat losses, but lower protein losses, from cheesemilk to whey in HHT LB cheeses resulted in a similar yield compared with LB CM cheese. Overall, it was possible to remove β-casein from cheesemilk or apply high heat treatment to whey protein reduced milk without adversely affecting cheese composition and yield.

Impact of heat treatment of milk on acid gelation

Heat treatment of milk is applied in the manufacture of cultured dairy products, primarily to increase viscosity and improve texture. Denatured whey proteins are recognised as mostly responsible for causing these textural changes. The higher isoelectric pH of whey protein triggers earlier acid-induced gelation. Rheological properties of acid gels made from heated milk exhibit significantly higher storage moduli (and lower fracture strain) due to alterations in gel microstructure and the contribution of covalent disulphide bonding. Gels prepared from heated milks also exhibit an increase in the loss tangent parameter due to loss of calcium phosphate crosslinks conferring a greater mobility within casein particles that are already part of the network. Casein interactions are mostly weak in these types of gels, and the balance between inter and intraparticle interactions, as well as the impact of denatured whey protein deposition on the particle surfaces, are discussed for their influence on acid gelation.

Heat-Induced Interaction of Milk Proteins: Impact on Yoghurt Structure.

Heating milk for yoghurt preparation has a significant effect on the structural properties of yoghurt. Milk heated at elevated temperature causes denaturation of whey protein, aggregation, and some case gelation. It is important to understand the mechanism involved in each state of stabilization for tailoring the final product. We review the formation of these complexes and their consequence on the physical, rheological, and microstructural properties of acid milk gels. To investigate the interactions between denatured whey protein and casein, the formation of covalent and noncovalent bonds, localization of the complexes, and their impact on ultimate gelation and final yoghurt texture are reviewed. The information regarding this fundamental mechanism will be beneficial to develop uniform quality yoghurt texture and potential interest of future research.

Heat-induced denaturation and bioactivity changes of whey proteins

As the second most abundant protein in milk, whey contains many bioactive proteins, which play important role in the health benefits and defence system of consumers. Whey proteins usually undergo different heating treatments during dairy processing. Although heat treatment kills pathogens and bacteria to guarantee the safety of dairy products, it also causes changes in the structure and bioactivity of whey proteins. This review first introduces a few abundant whey proteins as well as their biological functions, then describes the changes in the physicochemical properties, Maillard reaction, oxidation, cross-linking and aggregation of whey proteins. Last but not least, we also discuss heat induced the changes in digestibility, allergenicity, and other bioactivity of whey proteins. In summary, the heat induced denaturation of whey proteins may not only influence the nutritional value, but may also change the properties of dairy products, the immunological reaction and bioactivity.

Effect of substituting whey cream for sweet cream on the textural and rheological properties of cream cheese

In the manufacture of cream cheese, sweet cream and milk are blended to prepare the cream cheese mix, although other ingredients such as condensed skim milk and skim milk powder may also be included. Whey cream (WC) is an underutilized fat source, which has smaller fat droplets and slightly different chemical composition than sweet cream. This study investigated the rheological and textural properties of cream cheeses manufactured by substituting sweet cream with various levels of WC. Three different cream cheese mixes were prepared: control mix (CC; 0% WC), cream cheese mixes containing 25% WC (25WC; i.e., 75% sweet cream), and cream cheese mixes with 75% WC (75WC; i.e., 25% sweet cream). The CC, 25WC, and 75WC mixes were then used to manufacture cream cheeses. We also studied the effect of WC on the initial step in cream cheese manufacture (i.e., the acid gelation process monitored using dynamic small amplitude rheology). Acid gels were also prepared with added denatured whey proteins or membrane proteins/phospholipids (PL) to evaluate how these components affected gel properties. The rheological, textural, and sensory properties of cream cheeses were also measured. The WC samples had significantly higher levels of PL and insoluble protein compared with sweet cream. An increase in the level of WC reduced the rate of acid gel development, similar to the effect of whey phospholipid concentrate added to mixes. In cream cheese, an increase in the level of added WC resulted in significantly lower storage modulus values at temperatures <20°C. Texture results, obtained from instrumental and sensory analyses, showed that high level of WC resulted in significantly lower firmness or hardness values and higher stickiness compared with cream cheeses made with 25WC or CC cream cheeses. The softer, less elastic gels or cheeses resulting from the use of high levels of WC are likely due to the presence of components such as PL and proteins from the native milk fat globule membrane. The use of low levels of WC in cream cheese did not alter the texture, whereas high levels of WC could be used if manufacturers want to produce more spreadable products.

Impact of wheat gluten on the denaturation of egg white and whey proteins

AbstractBackground and objectivesWheat gluten (WG) often co‐exists with egg white (EW) and whey proteins in foods. This study investigated the impact of WG on the denaturation of hen egg (S‐)ovalbumin [(S‐)OVA], lysozyme (LYS), and bovine serum albumin (BSA).FindingsWG caused the peak temperature of denaturation of LYS and BSA to increase and that of (S‐)OVA to decrease. It accelerated and decelerated denaturation of BSA and OVA, respectively, and their lowered activation energy. In contrast, the denaturation rate of S‐OVA was higher and the activation energy was not affected by the presence of WG. LYS denaturation was reversible, both in the presence and in the absence of WG. Heating induced changes in the surface hydrophobicity of OVA and BSA and changes in the ζ‐potential of LYS, while the levels of their free sulfhydryl groups did not change.ConclusionsIn model systems, WG impacts both temperatures and kinetics of denaturation of the studied proteins. Hydrophobic interactions between WG and OVA or BSA impact their denaturation kinetics.Significance and noveltyThis study is the first to demonstrate the impact of WG on the denaturation of EW and whey proteins. These insights will be useful in food product design and further elucidating co‐protein effects.

Entrapment of Hydrophilic and Hydrophobic Molecules in Beads Prepared from Isolated Denatured Whey Protein.

The oral route of administration is by far the most convenient route, especially in the treatment of chronic conditions. However, many therapeutics present formulation difficulties which make them unsuitable for oral delivery. Recently, we synthesized a denatured whey protein isolate (dWPI) bead entrapped with insulin. Our present goal was to assess the suitability of this delivery system to the delivery of other potential molecules, both hydrophilic and hydrophobic. Beads of 1.2–1.5 mm in diameter were entrapped with four payloads representing a range of solubilities. The water-soluble payloads were sodium fluorescein (SF) and FITC dextran 4000 Da (FD4), while the hydrophobic ones were Fast Green and curcumin. Encapsulation efficiency (EE) was 73%, 84%, 70%, and 83% for SF, FD4, Fast Green, and curcumin-loaded beads, respectively. The corresponding loading capacity for each bead was 0.07%, 1.1%, 0.75%, and 1.1%, respectively. Each payload produced different release profiles in simulated gastric fluid (SGF) and simulated intestinal fluids (SIF). SF released steadily in both SGF and SIF. FD4 and curcumin release was not substantial in any buffers, while Fast Green release was low in SGF and high in SIF. The differences in release behaviour were likely due to the varying properties of the payloads. The effect of proteolysis on beads suggested that enzymatic degradation of the whey bead may promote payload release. The beads swelled rapidly in SGF compared to SIF, which likely contributed to the release from the beads, which was largely governed by solvent diffusion and polymer relaxation. Our results offer a systematic examination of the behaviour of hydrophilic and hydrophobic payloads in a dWPI delivery system. These beads may be further designed to orally deliver poorly permeable macromolecules and poorly soluble small molecules of pharmaceutical interest.