Gelation of mixed systems whey protein concentrate–gluten in acidic conditions

Gels of whey protein concentrate (WPC)–gluten were prepared by heating WPC–gluten dispersions (10% whey protein/0–5–10% gluten protein, w/w; pH 3.75 or 4.2). Gels were characterized through solubility assays in different extraction solutions, measures of water-holding capacity (WHC), firmness, elasticity and relaxation time, and light microscopy. Differential scanning calorimetry (DSC) of WPC–gluten dispersions was also performed. Gluten increases the firmness and elasticity of gels, mainly at pH 4.2. The WHC also increases with gluten content, being higher at pH 3.75 than at pH 4.2. Solubility assays indicate that electrostatic forces, hydrophobic and H bindings would be involved in maintaining the gel structure of WPC gels at pH 3.75 and 4.2, whereas in mixed gels of WPC–gluten, the principal forces responsible for the maintenance of the gel structure at these pHs would be hydrophobic and H bindings, and in gels prepared at pH 4.2 also disulfide bonds, but in a minor extent. The presence of gluten shifts the apparent transition temperature for whey protein denaturation towards lower temperatures. Gels with gluten present a smooth network with gaps and a more elastic appearance, as observed by light microscopy.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Casein–whey protein interactions in heated milk: the influence of pH

Heat-treatment of milk causes denaturation of whey proteins, leading to a complex mixture of whey protein aggregates and whey protein coated casein micelles. In this paper we studied the effect of pH-adjustment of milk (6.9–6.35) prior to heat-treatment on the distribution of denatured whey proteins in aggregates and coating of casein micelles. Proteins were fractionated using an alternative fractionation technique based on renneting. Acid- and rennet-induced gelation of these milks were used to obtain more information on the characteristics of the milk. Acid-induced gelation appeared to be mainly influenced by the presence of whey protein aggregates. Rennet-induced gelation was determined by the whey protein coating of the casein micelles. Both the quantity of whey proteins present on the surface of the casein micelles and the homogeneity of the coating were determining the renneting properties. These results extend the current knowledge on pH dependent casein–whey protein interactions. In order to present a clear picture of the changes occuring during heat treatment of milk at various pH, the results are summarized in a model. In this model we propose that heating at a pH>6.6 lead to a partial coverage of the casein micelles and the formation of separate whey protein aggregates. Heating at a pH<6.6 lead to an attachment of all whey proteins to the casein micelles. At pH 6.55 the coverage is rather homogeneous but lowering the pH further lead to an inhomogeneus coverage of the casein micelles. Surprisingly small changes of the pH at which the milk was heated had considerable effects on the gelation behaviour both in renneting and in acid gelation.     

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.     

Heat-induced interactions of β-lactoglobulin and α-lactalbumin with the casein micelle in pH-adjusted skim milk

Skim milks, adjusted to pH 6.48, 6.60 or 6.83, were heated for various temperature–time combinations in a pilot-scale ultra-high temperature (UHT) plant. Heat-treated samples were ultracentrifuged and their supernatants analysed by quantitative polyacrylamide gel electrophoresis in order to measure the extent of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) denaturation and their subsequent association with the casein micelle. The activation energy of β-lg denaturation decreased as the pH increased. In contrast, there was no apparent trend for α-la. The extent of β-lg and α-la association with the micelle increased with heating time and temperature. The association of both proteins with the micelle was markedly affected by the milk pH. The rate and extent of association were greatest at pH 6.48, and least at pH 6.83. α-La continued to associate with the micelle although most of the β-lg had already associated. It was possible that α-la interacted at the micelle surface with β-lg that had previously associated with the micelle. A pseudo-first-order mathematical model was used to calculate the apparent rate constant for β-lg association with the micelle.     

Use of micellar casein concentrate and milk protein concentrate treated with transglutaminase in imitation cheese products—Melt and stretch properties

Melt and stretch properties in dairy-based imitation mozzarella cheese (IMC) are affected by the amount of intact casein provided by dairy ingredients in the formulation. Rennet casein (RCN) is the preferred ingredient to provide intact casein in a formulation. Ingredients produced using membrane technology, such as milk protein concentrate (MPC) and micellar casein concentrate (MCC), are unable to provide the required functionality. However, the use of transglutaminase (TGase) has potential to modify the physical properties of MPC or MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC retentates on melt and stretch properties when they are used in IMC and to compare them with IMC made using RCN. The MCC and MPC retentates were produced using 3 different lots of pasteurized skim milk and treated with 3 levels of TGase enzyme: no TGase (control), low TGase: 0.3 units/g of protein, and high TGase: 3.0 units/g of protein. Each of the MCC and MPC treatments was heated to 72°C for 10 min to inactivate TGase and then spray dried. Each MCC, MPC, and RCN powder was then used in an IMC formulation that was standardized to 48% moisture, 21% fat, 20% protein, and 1% salt. The IMC were manufactured in a twin-screw cooker by blending, mixing, and heating various ingredients (4.0 kg). Due to extensive crosslinking, the IMC formulation with the highest TGase level (MCC or MPC) did not form an emulsion. The IMC made from MCC treatments had significantly higher stretchability on pizza compared with their respective MPC treatments. The IMC made from TGase-treated MCC and MPC had significantly lower melt area and significantly higher transition temperature (TT) and stretchability compared with their respective controls. Comparison of IMC made using TGase-treated MCC and MPC to the RCN IMC indicated no difference in TT or texture profile analysis-stretchability; however, the Schreiber melt test area was significantly lower. Our results demonstrated that TGase treatment modifies the melt and stretch characteristics of MCC and MPC in IMC applications, and TGase-treated MPC and MCC can be used to replace RCN in IMC formulations.     

Use of micellar casein concentrate and milk protein concentrate treated with transglutaminase in imitation cheese products—Unmelted texture

The amount of intact casein provided by dairy ingredients is a critical parameter in dairy-based imitation mozzarella cheese (IMC) formulation because it has a significant effect on unmelted textural parameters such as hardness. From a functionality perspective, rennet casein (RCN) is the preferred ingredient. Milk protein concentrate (MPC) and micellar casein concentrate (MCC) cannot provide the required functionality due to the higher steric stability of casein micelle. However, the use of transglutaminase (TGase) has the potential to modify the surface properties of MPC and MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC powders on the unmelted textural properties of IMC and compare them with IMC made using commercially available RCN. Additionally, we studied the degree of crosslinking by TGase in MPC and MCC retentates using capillary gel electrophoresis. Three lots of MCC and MPC retentate were produced from pasteurized skim milk via microfiltration and ultrafiltration, respectively, and randomly assigned to 1 of 3 treatments: no TGase (control); low TGase: 0.3 units/g of protein; and high TGase: 3.0 units/g of protein, followed by inactivation of enzyme (72°C for 10 min), and spray drying. Each MCC, MPC, and RCN was then used to formulate IMC that was standardized to 21% fat, 1% salt, 48% moisture, and 20% protein. The IMC were manufactured by blending, mixing, and heating ingredients (4.0 kg) in a twin-screw cooker. The capillary gel electrophoresis analysis showed extensive inter- and intramolecular crosslinking. The IMC formulation using the highest TGase level in MCC or MPC did not form an emulsion because of extensive crosslinking. In MPC with a high level of TGase, whey protein and casein crosslinking were observed. In contrast, crosslinking and hydrolysis of proteins were observed in MCC. The IMC made from MCC powder had significantly higher texture profile analysis hardness compared with the corresponding MPC powder. Further, many-to-one (multiple) comparisons using the Dunnett test showed no significant differences between IMC made using RCN and treatment powders in hardness. Our results demonstrated that TGase treatment causes crosslinking hydrolysis of MCC and MPC at higher TGase levels, and MPC and MCC have the potential to be used as ingredients in IMC applications.     

Co-precipitation of whey and pea protein – indication of interactions

The aim was to optimise the yield of co-precipitation of whey protein isolate (WPI) and pea protein isolate (PPI) and compare co-precipitates and protein blends with respect to solubility. The yield of co-precipitates was tested with different protein ratios of WPI and PPI in combination with different temperatures and acid precipitation (pH 4.6). The highest precipitation yield was obtained at protein ratios WPI < PPI, high temperature and alkaline protein solvation. The solubility was measured by an instability index and absorption spectroscopy of re-suspended precipitated proteins at pH 3, 7 and 11.5. Co-precipitates had significantly lower solubility than protein blends. Protein ratios WPI > PPI, low precipitation temperature and high pH showed the highest solubility. Differences in protein composition between co-precipitates and protein blends were observed with SDS-PAGE and matrix-assisted laser desorption ionisation time-of-flight, and indicated different protein–protein interaction in samples, which needs further investigations.     

The effect of protein concentration and heat treatment temperature on micellar casein–soy protein mixtures

The objective of this study was to investigate the effect of concentration and temperature on the rheological properties of soy proteins (SP) and micellar casein (MCN) systems. Individual and mixed (1:1) protein systems of 2–15% concentration were prepared and heat treated for 5 min at 40–90 °C. After cooling to 20 °C, their rheological properties were determined using steady-shear rheology. Zeta potential and particle size measurements were also conducted. Both proteins were negatively charged under all experimental conditions, but the absolute values of zeta potential and thus the stability of the protein solutions decreased with temperature and concentration. For SP solutions, viscosity and apparent yield stress increased with concentration. Shear thinning behavior was prevalent, becoming more pronounced with increasing concentration. Heat treatments at T ≥ 80 °C induced glycinin denaturation, followed by aggregation and network formation when C ≥ 7.5%. Heat treatment did not significantly affect viscosity of MCN systems, while increasing concentration resulted in a significant increase in apparent viscosity and apparent yield stress. Most MCN systems exhibited Newtonian flow behavior, with the exception of systems with C ≥ 12.5% treated at T ≥ 80 °C, which became slightly shear thickening. Mixed SP–MCN systems mimicked the behavior of SP, with most values of rheological parameters intermediate between SP and MCN-only systems. Mixtures of 7.5–12.5% concentration treated at 90 °C displayed local phase separation, low viscosity and apparent yield stress, while 15% mixtures treated at 90 °C showed protein aggregation and incipient network formation. The data generated in this study can be used to develop a range of protein based products with unique flow characteristics and storage stability.     

Gel mechanical properties of milk whey protein–dextran conjugates obtained by Maillard reaction

The effect of Maillard reaction on the mechanical properties of whey protein isolate (WPI) heat-induced gels was evaluated. WPI and dextran (15–25 kDa) conjugates were obtained by controlled dry heating during storage at 60 °C and 63% relative humidity for 2, 5 and 9 days. Changes in browning intensity and content of free amino groups were used to estimate the Maillard reaction. A decrease in free amino groups of WPI was observed when increasing polysaccharide concentration and reaction time. An increase in both a* and b* CIE Lab colour parameters indicated the development of a reddish-brown colour, typical of the Maillard reaction. Uniaxial compression and stress relaxation tests were performed to measure the mechanical properties of mixed and conjugate gels. Maillard reaction significantly modified the mechanical properties of WPI/DX gels, and even prevented fracture when conjugate gels were subjected to 80% deformation in uniaxial compression test.     

Effect of β-lactoglobulin A and B whey protein variants on cheese yield potential of a model milk system

Cheese yield mainly depends on the amount and proportion of milk constituents; however, genetic variants of the proteins present in milk may also have an important effect. The objective of this research was to study the effect of the variants A and B of β-lactoglobulin (LG) on cheese yield using a model system consisting of skim milk powder fortified with different levels of a mixture containing α-lactalbumin and β-LG genetic variants (A, B, or A-B) in a 1:2 ratio. Fortified milk samples were subjected to pasteurization at 65°C for 30 min. Miniature cheeses were made by acidifying (pH = 5.9) fortified milk and incubating with rennet for 1 h at 32°C. The clot formed was cut, centrifuged at 2,600 × g for 30 min at 20°C and drained for determining cheese yield. Cheese-yielding capacity was expressed as actual yield (grams of cheese curd per 100 g of milk) and dry weight yield (grams of dried cheese curd per 100 g of milk). Free-zone capillary electrophoresis was used for determining β-LG A or B recovery in the curd during rennet-induced coagulation. The presence of β-LG variant B resulted in a significantly higher actual and dried weight cheese yield than when A or A-B were present at levels ≤0.675% of whey protein (WP) addition. Results of free-zone capillary electrophoresis allowed us to infer that β-LG B associates with the casein micelles during renneting, as shown by an increase in the recovery of this variant in the curd when β-LG B was added up to a maximum at 0.45% (equivalent to 0.675% WP). In general, actual or dried weight cheese yield increased as WP addition was increased from 0.225 to 0.675%. However, when WP addition ranged from 0.675 to 0.90%, a drastic drop in cheese yield was observed. This behavior may be because an increase in the aggregation of casein micelles with a concomitant inclusion of whey protein in the gel occurs at low levels of WP addition, whereas once the association of WP with the casein micelles reach a saturation point at addition levels higher than 0.675%, rearrangements of the gel network result in larger whey expulsion and syneresis. This knowledge is expected to be useful to maximize cheese yield and optimize processing conditions during cheese and cheese analogs manufacturing.     

Evaluation of diacetyl encapsulated alginate–whey protein microspheres release kinetics and mechanism at simulated mouth conditions

In this paper, diacetyl encapsulated alginate–whey protein concentrate (AL–WPC) microcapsule were prepared based on the emulsification/internal gelation method; and diacetyl release was investigated at the simulated mouth condition in different ratios of artificial saliva (0, 1:4 and 1:8) and three various oral shear rates (0, 50 and 100 s-1). The gotten diacetyl release data were fitted to first-order, Korsmeyer–Peppas, Kopcha, and Makoid–Banakar models to evaluate release mechanisms and kinetics. We showed that the shear rate of release media had a significant (p < 0.05) effect on the release of diacetyl from AL–WPC microspheres, but the saliva ratio had no any significant effect. The diacetyl release data fit well to the all kinetic expression with R² values greater than 0.93. It was found that the release kinetics of diacetyl from AL–WPC microspheres followed a classical Fickian diffusion.     

Partitioning of α-lactalbumin and β-lactoglobulin in whey protein concentrate/hydroxypropylmethylcellulose aqueous two-phase systems

Whey protein concentrate (WPC) was fractionated by using hydroxypropylmethylcellulose (HPMC) at pH 6.5. Incompatible mixtures with different proportions of HPMC and WPC were prepared. After phase separation, the protein concentration in both phases was determined by the Kjeldahl method and the proportion of each protein by SDS-PAGE combined with image analysis. The results show that the low molecular weight proteins α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) were retained in high proportion in the upper phase (about 90% compared to 64% of WPC). The most efficient condition to fractionate β-lg and α-lac was the phase separation of an incompatible mixed system with a high initial concentration of WPC and a low initial concentration of HPMC i.e., WPC 20%, wt/wt/HPMC 0.5%, w/w. It can be concluded that the thermodynamic incompatibility which arises from mixing WPC with HPMC could be used as a method for fractionation of whey proteins.     

Effects of protein interactions on properties and microstructure of zein–gliadin composite films

Zein and gliadin are both readily dissolved in aqueous ethanol and have good film-forming property. This article describes an attempt to improve the flexibility of zein films by the addition of gliadin to the zein film-forming solution. The properties of zein–gliadin composite films, i.e., color, transparency, moisture content, water solubility, water vapor permeability, dynamic contact angle which in turn affected the mechanical property, water resistance and glass transition temperature of films were investigated. The contents of second structure were characterized via Fourier transform infrared spectroscopy (FTIR), whereas morphology of films was examined by scanning electron microscopy (SEM). It was observed that the addition of gliadin enhanced the strain at break of zein–gliadin composite films as a result of the increase in the content of α-helix, β-turn structures and decrease in the level of β-sheet structure. The water resistance of films decreased with the content of gliadin increasing. Morphology of composite films showed that gliadin and zein organized a homogeneous material. This work opens a new perspective for zein in flexible food package.     

Size of native and heated casein micelles,content of protein and minerals in milk from Norwegian Red Cattle—effect of milk protein polymorphism and different feeding regimes

Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three different supplementary concentrates, were analysed for genotypes of caseins and whey proteins, the content of different milk salts (Ca²⁺, Ca, Mg and citrate), the content of total protein, casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of α_s1-casein had a statistically significant effect on the content of protein and casein, and the content of whey protein and the casein number were significantly influenced by different feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was significantly influenced by the feeding regimes, genotype of α_s1-casein (native mean size only) and κ-casein, pH and the content of casein, whey protein and casein number. The heat-induced changes in mean micellar size were significantly affected by the calcium ion activity which accounted for approximately 40% of the total variation.     

Integrated production of whey protein concentrate and lactose derivatives: What is the best combination?

In order to model and analyze the techno-economic feasibility of a whey processing unit for the production of whey protein concentrate (WPC) integrated with processing of lactose, the present study utilized the software SuperPro Designer® for modeling of the processes, including risk analysis and study of reduced pollution impacts. Six models were constructed for the production of WPC and processing of lactose, which were (1) WPC 34, (2) WPC 34 and lactose powder, (3) WPC 34 and hydrous ethanol fuel, (4) WPC 80, (5) WPC 80 and lactose powder, and (6) WPC 80 and hydrous ethanol fuel. The economic evaluation was performed by analysis of the Payback Period (PP), Net Present Value (NPV), Breakeven Point (BP) and Internal Rate of Return (IRR). Probability distributions obtained by fitting of historical data for whey prices and the final products were used to perform the risk analysis, submitted to a Monte Carlo simulation using the @Risk software. The project showed to be feasible due to the elevated IRR and NPV values, coupled with low BP and PP. When evaluating the individual production of ethanol, it was verified that the production cost of this product was superior to the sale price, making independent production of ethanol from lactose present in the whey uneconomical. Plants with production of lactose powder were more economically attractive and also presented greater reduction of Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD). The financial indices suggested greater feasibility of WPC 80 compared to WPC 34.     

Influence of shearing on the physical characteristics and rheological behaviour of an aqueous whey protein isolate–κappa-carrageenan mixture

The incompatibility of whey protein isolate (WPI) and κappa-carrageenan (κ-car) in aqueous mixtures has been extensively studied under quiescent conditions; however, the effect of shear on segregative phase separation is still not fully understood. The present work reports for the first time quantitatively the effect of shearing on the segregative phase separation behaviour of these two polymers. Demixing was observed at pH 7.0 and 22 °C, determining the phase diagram and rheological properties of the mixtures. Phase diagrams were derived after heating and cooling mixes at a constant shear rate (28 s⁻¹). The phase behaviour was compared to that of the same mixtures under quiescent conditions. The shearing process affects segregative phase separation, causing a shift in the position of the binodal towards lower concentrations of WPI. The bottom layer contained a higher ratio of WPI while the upper layer was enriched in κ-car. The addition of κ-car to WPI solutions led to a much stiffer heat-induced gel than that prepared with WPI heated in isolation. The height of the plateau of the final elastic modulus G′_∞(t) depends on the position of the system on the phase diagram. Using a selected tie line, the viscosity of different systems measured at 80 °C was more influenced by the amount of WPI, than by the κ-car concentration. Shear treatment of segregative phase separating systems offer a way to modulate the functional properties of the ingredients and the texture of the final product.