Properties of whey proteins obtained from different whey streams

The physio-chemical characteristics of whey proteins (WPs) in sweet, salty, native and acid whey were investigated and compared. WPs from acid whey were characterised by hydrophobically and covalently driven protein aggregation. Covalent aggregation in acid whey consisted of both thiol/disulphide and non-thiol/disulphide mediated reactions. Fourier transform infrared data characterised this protein aggregation as a β-sheet attraction causing subtle changes in the secondary structure. In contrast, WPs in salty whey aggregated via van der Waals, hydrogen, electrostatic interactions and covalent bonds. Both thiol/disulphide and non-thiol/disulphide interactions led to cross-linked β-sheets, disrupting the secondary protein structures. This aggregation exposed hydrophobic segments while oxidising a high number of free thiol groups. The absence of these types of WP aggregation in sweet or native whey highlighted the fact that elevated salt concentration in salty whey or heat treatment applied during production of acid whey are largely responsible for structural differences.     

Review: Milk Proteins as Nanocarrier Systems for Hydrophobic Nutraceuticals

Milk proteins and milk protein aggregates are among the most important nanovehicles in food technology. Milk proteins have various functional properties that facilitate their ability to carry hydrophobic nutraceutical substances. The main functional transport properties that were examined in the reviewed studies are binding of molecules or ions, surface activity, aggregation, gelation, and interaction with other polymers. Hydrophobic binding has been investigated using caseins and isolated β‐casein as well as whey proteins. Surface activity of caseins has been used to create emulsion‐based carrier systems. Furthermore, caseins are able to self‐assemble into micelles, which can incorporate molecules. Gelation and interaction with other polymers can be used to encapsulate molecules into protein networks. The release of transported substances mainly depends on pH and swelling behavior of the proteins. The targeted use of nanocarrier systems requires specific knowledge about the binding mechanisms between the proteins and the carried substances in a certain food matrix.     

The use of whey protein particles in gluten-free bread production,the effect of particle stability

Wheat dough has unique properties for bread making due to its elastic and strain hardening behaviour. A mesoscopically structured whey protein particle system possesses those elastic and strain hardening properties when mixed with starch to a certain extent. However, the extensibility is lower and the particles are more stable than gluten particles upon kneading, probably due to a too high degree of internal crosslinking. This study describes the relation between the number of disulphide bonds of a mesoscopic whey protein particle suspension blocked by NEM treatment and the resulting properties of a dough and bread prepared with that suspension. This study shows that the properties of the particle network are influenced by the ability to form disulphide bonds. Our study shows that a certain amount of disulphide bonds is essential, but too many disulphide bonds can lead to too stiff dough and poorer bread properties.     

Effects of non-covalent interactions between the milk proteins on the rheological properties of acid gels

When skim milk (SM) and whey-protein-enriched skim milk (WPE-SM) were heated (80 °C, 30 min) with N-ethylmaleimide (NEM, 0–0.8 mm), the levels of residual native whey proteins increased to ∼70%, whereas the levels of disulphide-bonded whey proteins decreased to <10%. Acid gels prepared from heated SM with added NEM had slightly lower firmness than those made from control heated SM because the former gels lacked intermolecular disulphide bonds. In contrast, acid gels made from heated WPE-SM with added NEM had higher firmness than those prepared from control heated WPE-SM. This implies that, even without intermolecular disulphide bonds, non-covalent interactions can be sufficient to produce acid gels with firmness higher than gels with disulphide bonds. Nevertheless, disulphide interactions can be more important than non-covalent interactions in influencing the yield properties of the gels because acid gels without disulphide bonds can be fractured more easily than those with disulphide bonds.     

Acid gelation properties of fibrillated model milk protein concentrate dispersions

Whey proteins in milk are globular proteins that can be converted into fibrils to enhance functional properties such gelation, emulsification, and foaming. A model fibrillated milk protein concentrate (MPC) was developed by mixing micellar casein concentrate (MCC) with fibrillated milk whey proteins. Similarly, a control model MPC was obtained by mixing MCC with milk whey proteins. The resulting fibrillated model MPC and control model MPC contained 5% protein and a ratio of casein to whey proteins similar to milk. The objective of the current study was to understand the rheological characteristics of fibrillated and control model MPC during acid gelation, using Förster resonance energy transfer (FRET) to assess small amplitude oscillation and casein–whey protein interaction. The results from the FRET index images showed greater interactions between caseins and whey proteins in fibrillated model MPC compared with the moderate and uniform interactions in control model MPC gels. Rheological study showed that the maximum storage modulus of acid gel of fibrillated model MPC was 546.9 ± 15.5 Pa, which was significantly higher than acid gel made from control model MPC (336.9 ± 11.3 Pa), indicating that fibrillated model MPC produced a firmer gel. Therefore, it can be concluded that acid gel produced from fibrillated model MPC was stronger than control model MPC. Selective fibrillation of the whey protein fraction in MPC can be used to improve gelation characteristics of acid gel type products.     

FUNCTIONAL PROPERTIES OF HIGH HYDROSTATIC PRESSURE-TREATED WHEY PROTEIN

Surface hydrophobicity, solubility, gelation and emulsifying properties of high hydrostatic pressure (HHP)‐treated whey protein were evaluated. HHP treatment of whey protein buffer or salt solutions were performed at 690 MPa and initial ambient temperature for 5, 10, 20 or 30 min. Untreated whey protein was used as a control. The surface hydrophobicity of whey protein in 0.1 M phosphate buffers treated at pH 7.0 increased with an increase in HHP treatment time from 10 to 30 min. HHP treatments of whey protein in salt solutions at pH 7.0 for 5, 10, 20 or 30 min decreased the solubility of whey proteins. A significant correlation was observed between the surface hydrophobicity and solubility of untreated and HHP‐treated whey protein with r = ?0.946. Hardness of HHP‐induced 20, 25 or 30% whey protein gels increased with an increase in HHP treatment time from 5 to 30 min. An increase in the hardness of whey protein gels was observed as whey protein concentration increased. Whey proteins treated in phosphate buffer at pH 5.8 and 690 MPa for 5 min exhibited increased emulsifying activity. Whey proteins treated in phosphate buffer at pH 7.0 and 690 MPa for 10, 20 or 30 min exhibited decreased emulsifying activity. HHP‐treated whey proteins in phosphate buffer at pH 5.8 or 7.0 contributed to an increase in emulsion stability of model oil‐in‐water emulsions. This study demonstrates that HHP treatment of whey protein in phosphate buffer or salt solutions leads to whey protein unfolding observed as increased surface hydrophobicity. Whey proteins treated in phosphate buffers at pH 5.8 and 690 MPa for 5 min may potentially be used to enhance emulsion stability in foods such as salad dressings, sausage and processed cheese.     

Chemistry of gluten proteins

Gluten proteins play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten proteins can be divided into two main fractions according to their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partially closely related protein components characterized by high glutamine and proline contents. Gliadins are mainly monomeric proteins with molecular weights (MWs) around 28,000-55,000 and can be classified according to their different primary structures into the alpha/beta-, gamma- and omega-type. Disulphide bonds are either absent or present as intrachain crosslinks. The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have a varying size ranging from about 500,000 to more than 10 million. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. Based on primary structure, glutenin subunits have been divided into the high-molecular-weight (HMW) subunits (MW=67,000-88,000) and low-molecular-weight (LMW) subunits (MW=32,000-35,000). Each gluten protein type consists or two or three different structural domains; one of them contains unique repetitive sequences rich in glutamine and proline. Native glutenins are composed of a backbone formed by HMW subunit polymers and of LMW subunit polymers branched off from HMW subunits. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are important for the aggregation of gliadins and glutenins and implicate structure and physical properties of dough.     

Influence of pH on the dry heat-induced denaturation/aggregation of whey proteins

The effect of pH on the heat-induced denaturation/aggregation of whey protein isolate (WPI) in the dry state was investigated. WPI powders at different pH values (6.5, 4.5, and 2.5) and controlled water activity (0.23) were dry heated at 100 °C for up to 24 h. Dry heating was accompanied by a loss of soluble proteins (native-like β-lactoglobulin and α-lactalbumin) and the concomitant formation of aggregated structures that increased in size as the pH increased. The loss of soluble proteins was less when the pH of the WPI was 2.5; in this case only soluble aggregates were observed. At higher pH values (4.5 and 6.5), both soluble and insoluble aggregates were formed. The fraction of insoluble aggregates increased with increasing pH. Intermolecular disulphide bonds between aggregated proteins predominated at a lower pH (2.5), while covalent cross-links other than disulphide bonds were also formed at pH 4.5 and 6.5. Hence, pH constitutes an attractive tool for controlling the dry heat-induced denaturation/aggregation of whey proteins and the types of interactions between them. This may be of great importance for whey ingredients having various pH values after processing.     

Enhancement of emulsifying properties of whey proteins by controlling spray-drying parameters

Whey protein concentrate is the main source of globular proteins in food products which are principally used as emulsifying, foaming and gelling ingredients. These whey proteins are commonly used in powder form obtained by a spray-drying process. It is well known that β-lactoglobulin, the major protein component in whey, is greatly affected by heat treatments, with consequences on its adsorption properties at fluid–fluid interfaces. This study concerned four whey protein powders obtained using spray-drying at four different air inlet temperatures (from 170 to 260 °C), leading to different levels of protein solubility, denaturation and end-use properties. After evaluation of the protein denaturation by HPLC, the emulsifying properties were studied through particle size parameters and rheological properties in relation with spray-drying parameters. Our results indicated that oil-in-water emulsions, stabilized by 5% (w/w) protein samples, exhibited a shear-thinning flow behaviour, and the harsher the spray-drying conditions (the higher the protein denaturation), the less viscous were the emulsions. The apparent viscosity of emulsions measured at 20 °C and 50 s⁻¹ shear rate was around 0.08 Pa s when containing whey proteins before drying, and around 0.05–0.018 Pa s after drying at air inlet temperatures from 170 to 260 °C. These differences in emulsion rheological properties were related to particle size effects, in regards to analysis of particle size distributions which showed a finer emulsion according to spray-drying intensity. Our results will be presented and discussed in terms of optimization of spray-drying process relative to globular protein surface activity.     

Use of Whey Protein Soluble Aggregates for Thermal Stability—A Hypothesis Paper

Forming whey proteins into soluble aggregates is a modification shown to improve or expand the applications in foaming, emulsification, gelation, film‐formation, and encapsulation. Whey protein soluble aggregates are defined as aggregates that are intermediates between monomer proteins and an insoluble gel network or precipitate. The conditions under which whey proteins denature and aggregate have been extensively studied and can be used as guiding principles of producing soluble aggregates. These conditions are reviewed for pH, ion type and concentration, cosolutes, and protein concentration, along with heating temperature and duration. Combinations of these conditions can be used to design soluble aggregates with desired physicochemical properties including surface charge, surface hydrophobicity, size, and shape. These properties in turn can be used to obtain target macroscopic properties, such as viscosity, clarity, and stability, of the final product. A proposed approach to designing soluble aggregates with improved thermal stability for beverage applications is presented.     

Addition of crude tiger nut protein extract affects stiffness of enzymatically cross‐linked dairy proteins

The influence of crude tiger nut protein extract on the gel properties of enzymatically cross‐linked dairy proteins was investigated. Enzymatic cross‐linking of dairy proteins in the presence of crude tiger nut proteins caused the formation of larger casein polymers and increased the degree of polymerisation. Gel stiffness of acidified products containing whey proteins was higher when cross‐linking occurred in the presence of crude tiger nut proteins. The results are relevant for improving the textural characteristics of acidified aqueous tiger nut extract (tiger nut milk) enriched in dairy proteins.     

Chemical changes in bovine milk fat globule membrane caused by heat treatment and homogenization of whole milk

The effects of heat treatment and homogenization of whole milk on chemical changes in the milk fat globule membrane (MFGM) were investigated. Heating at 80 degrees C for 3-18 min caused an incorporation of whey proteins, especially beta-lactoglobulin (beta-Ig), into MFGM, thus increasing the protein content of the membrane and decreasing the lipid. SDS-PAGE showed that membrane glycoproteins, such as PAS-6 and PAS-7, had disappeared or were weakly stained in the gel due to heating of the milk. Heating also decreased free sulphydryl (SH) groups in the MFGM and increased disulphide (SS) groups, suggesting that incorporation of beta-Ig might be due to association with membrane proteins via disulphide bonds. In contrast, homogenization caused an adsorption of caseins to the MFGM but no binding of whey proteins to the MFGM without heating. Binding of caseins and whey proteins and loss of membrane proteins were not significantly different between milk samples that were homogenized before and after heating. Viscosity of whole milk was increased when milk was treated with both homogenization and heating.     

Proteins recovered from milks heated at alkaline pH values

Approximately 95% of available nitrogen can be precipitated from milk on adjustment to pH 4.6 after heating at 90°C × 15 minutes at its natural pH and pH 7.5, while 89% can be precipitated after heating at pH 10.0 at 60°C × 3 minutes. Non-recovered protein includes some serum albumin, β-lactoglobulin, α-lactalbumin and proteose peptones. Protein isolates precipitated from milk heated at pH >7.0 are more soluble in the pH range 6.0–7.0 than those precipitated from milk heated at its natural pH. Whey proteins complex onto the casein micelles after heating milk at its natural pH, while on heating at pH >7.0 whey proteins appear to interact with k-casein in the serum phase. When N-ethylmaleimide is present in milk during heating the percentage protein recovered on pH 4.6 precipitation is decreased, confirming that disulphide linkage is involved in complex formation. However, addition of β-mercaptoethanol to recovered isolates did not result in dissociation of the casein/whey protein complex, suggesting that forces other than disulphide bonding are also involved in maintaining the complex.     

Water Holding as Determinant for the Elastically Stored Energy in Protein‐Based Gels

To evaluate the importance of the water holding capacity for the elastically stored energy of protein gels, a range of gels were created from proteins from different origin (plant: pea and soy proteins, and animal: whey, blood plasma, egg white proteins, and ovalbumin) varying in network morphology set by the protein concentration, pH, ionic strength, or the presence of specific ions. The results showed that the observed positive and linear relation between water holding (WH) and elastically stored energy (RE) is generic for globular protein gels studied. The slopes of this relation are comparable for all globular protein gels (except for soy protein gels) whereas the intercept is close to 0 for most of the systems except for ovalbumin and egg white gels. The slope and intercept obtained allows one to predict the impact of tuning WH, by gel morphology or network stiffness, on the mechanical deformation of the protein‐based gel. Addition of charged polysaccharides to a protein system leads to a deviation from the linear relation between WH and RE and this deviation coincides with a change in phase behavior.     

High pressure-induced changes in milk proteins and possible applications in dairy technology

This paper reviews the newest information on the effects of high pressure (HP) on whey proteins, caseins and milk enzymes, and discusses their influence on milk properties. HP treatments cause substantial modification to milk proteins and to the mineral balance of milk. Casein micelles disaggregate into smaller particles or aggregate, depending the intensity and the temperature of the HP treatment. Whey proteins are denatured, possibly interacting with the remnants of the casein micelles, and give aggregated forms different from those produced by heat treatment. These events influence rennet coagulation properties of milk, with micellar disintegration favoring coagulation and whey protein denaturation hindering the aggregation of renneted micelles and enhancing cheese yield. HP treatment of milk favors acid coagulation and produces acid gels whose structure is greatly determined by the different micellar sizes attainable and the degree of whey protein denaturation. Milk gels can also be formed from concentrated milk under HP, providing new structures inaccessible via conventional methods.     

Newtonian viscosity behavior of dilute solutions of polymerized whey proteins. Would viscosity measurements reveal more detailed molecular properties?

Dilute solution Newtonian viscosity of whey protein polymers at different temperatures has not been assessed in literature. In this work, a thermally-treated whey protein solution at 8% w/w and pH 6.8 was prepared. Dilute solution viscometry was investigated at different temperatures from 30 °C to 65 °C. Intrinsic viscosity and voluminosity data indicate slight shrinkage in molecular size upon temperature increase. The temperature dependence of viscosities was expressed by the Arrhenius–Frenkel–Eyring equation and the thermodynamic parameters of viscous flow of polymer solutions were calculated. Results show a positive entropy change of viscous flow, indicating ordered structures. Chromatographic separation results prove that although disulphide bonds form the polymer backbone chain, both hydrophobic associations and hydrogen bonding still play a role in molecular structuring, even at very low protein concentrations. The calculated shape factor indicates spherical polymer molecules over the entire investigated temperature range.     

Influence of sucrose on NaCl-induced gelation of heat denatured whey protein solutions

A solution of heat-denatured whey proteins was prepared by heating 10 wt.% whey protein isolate at pH 7.0 to 80°C for 10 min in the absence of salt. This treatment caused the globular protein molecules to partially unfold and aggregate. When the heat-denatured whey protein solution was cooled to room temperature and mixed with 200 mM NaCl it formed a gel. The influence of sucrose (0 to 10 wt.%) in the protein solutions prior to NaCl addition on the gelation rate was investigated. At relatively low concentrations (0–8 wt.%) sucrose decreased the gelation rate, presumably because sucrose increased the aqueous phase viscosity. At higher concentrations (> 8 wt.%) sucrose increased the gelation rate, probably because it decreased the thermodynamic affinity of the globular proteins for the aqueous solution, which increased the attraction between proteins. This data has important implications for the application of cold-setting whey protein ingredients in sweetened food products such as deserts and beverages.     

Protein–protein interactions of a whey–pea protein co-precipitate

The aim of this study was to investigate the mechanisms behind protein–protein interactions in a co-precipitate of whey protein isolate (WPI) and pea protein isolate (PPI). A co-precipitate and blend, consisting of 80% WPI and 20% PPI, were compared. Covalent disulphide interactions were studied by blocking of free thiols with N-Ethylmaleimide (NEM), while electrostatic interactions were studied in systems with 0.5 m NaCl and hydrophobic interactions with 0.2% SDS. Protein solubility, stability and secondary, tertiary and quaternary protein structures were analysed. Co-precipitation did not introduce different protein–protein interactions than the direct blending of proteins. SDS affected solubility (P < 0.05), secondary and tertiary structure. However, the effects of NEM and NaCl were significant greater (P < 0.05) on the same parameters and thermal stability, especially when combined (P < 0.01). Thus, the protein–protein interactions in a whey–pea co-precipitate and protein blend consisted of disulphide bonds and electrostatic interactions.     

CHARACTERISATION OF A COMMERCIAL SOY ISOLATE BY PHYSICAL TECHNIQUES

The mechanical features of a partially denatured commercial soy isolate (PP 500E) have been explored for comparison with data available on the gelation characteristics of native globular proteins and to improve the understanding of its textural properties as a structuring ingredient in the production of low fat products. The soy sample was reconstituted at 30C and networks were developed either during cooling to 5C or on heating to 90C (complete denaturation of the protein) followed by cooling to 5C. Throughout the course of experimentation, dynamic oscillatory (time, temperature, frequency and strain sweeps) and creep testing (in aqueous or urea solutions) measurements were recorded. Reduction in the thermal energy of the system causes a monotonic increase in storage modulus (G′) whereas the temperature rise results in equilibrium G′ values well below the elastic response observed at 30C. The absence of a positive thermal transition, observed in the gelation of native globular proteins, indicates a different mechanism for structure formation in commercial soy isolates. Application of the cascade treatment to the concentration dependence of the storage modulus argues that the heated and cooled networks possess a higher degree of bond permanency than their cooled-only counterparts. Mechanical spectra in combination with the pattern of network breakage at high deformations suggests that disulphide bonds participate in the network formed by totally denatured soy protein (heated and cooled samples). Inclusion of urea in the aqueous preparations destabilises the predominantly physical forces of attraction in the unheated gels. By contrast, the heated and cooled samples achieve an equilibrium deformation whose storage modulus can be employed in the constitutive equation of rubber elasticity theory. On that basis the number of disulphide bridges per molecule was found to vary between 2.0 and 2.03. This result is consistent with the “string of beads” model proposed for the three-dimensional structure of globular protein gels, where a dendric network is built by the occasional cross-linking of corpuscular strands.     

Effects of heating rate and pH on fracture and water-holding properties of globular protein gels as explained by micro-phase separation

The effect of heating rate and pH on fracture properties and held water (HW) of globular protein gels was investigated. The study was divided into 2 experiments. In the 1st experiment, whey protein isolate (WPI) and egg white protein (EWP) gels were formed at pH 4.5 and 7.0 using heating rates ranging from 0.1 to 35 °C/min and holding times at 80 °C up to 240 min. The 2nd experiment used one heating condition (80 °C for 60 min) and probed in detail the pH range of 4.5 to 7.0 for EWP gels. Fracture properties of gels were measured by torsional deformation and HW was measured as the amount of fluid retained after a mild centrifugation. Single or micro-phase separated conditions were determined by confocal laser scanning microscopy. The effect of heating rate on fracture properties and HW of globular protein gels can be explained by phase stability of the protein dispersion and total thermal input. Minimal difference in fracture properties and HW of EWP gels at pH 4.5 compared with pH 7.0 were observed while WPI gels were stronger and had higher HW at pH 7.0 as compared to 4.5. This was due to a mild degree of micro-phase separation of EWP gels across the pH range whereas WPI gels only showed an extreme micro-phase separation in a narrow pH range. In summary, gel formation and physical properties of globular protein gels can be explained by micro-phase separation. PRACTICAL APPLICATION: The effect of heating conditions on hardness and water-holding properties of protein gels is explained by the relative percentage of micro-phase separated proteins. Heating rates that are too rapid require additional holding time at the end-point temperature to allow for full network development. Increase in degree of micro-phase separation decreases the ability for protein gels to hold water.