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.     

Pilot-scale formation of whey protein aggregates determine the stability of heat-treated whey protein solutions—Effect of pH and protein concentration

Denaturation and consequent aggregation in whey protein solutions is critical to product functionality during processing. Solutions of whey protein isolate (WPI) prepared at 1, 4, 8, and 12% (wt/wt) and pH 6.2, 6.7, or 7.2 were subjected to heat treatment (85°C × 30 s) using a pilot-scale heat exchanger. The effects of heat treatment on whey protein denaturation and aggregation were determined by chromatography, particle size, turbidity, and rheological analyses. The influence of pH and WPI concentration during heat treatment on the thermal stability of the resulting dispersions was also investigated. Whey protein isolate solutions heated at pH 6.2 were more extensively denatured, had a greater proportion of insoluble aggregates, higher particle size and turbidity, and were significantly less heat-stable than equivalent samples prepared at pH 6.7 and 7.2. The effects of WPI concentration on denaturation/aggregation behavior were more apparent at higher pH where the stabilizing effects of charge repulsion became increasingly influential. Solutions containing 12% (wt/wt) WPI had significantly higher apparent viscosities, at each pH, compared with lower protein concentrations, with solutions prepared at pH 6.2 forming a gel. Smaller average particle size and a higher proportion of soluble aggregates in WPI solutions, pre-heated at pH 6.7 and 7.2, resulted in improved thermal stability on subsequent heating. Higher pH during secondary heating also increased thermal stability. This study offers insight into the interactive effects of pH and whey protein concentration during pilot-scale processing and demonstrates how protein functionality can be controlled through manipulation of these factors.     

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.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

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.     

Segregative interactions and competitive binding of Ca in gelling mixtures of whey protein isolate with Naκ-carrageenan

Gelling mixtures of Na⁺κ-carrageenan with whey protein isolate (WPI) at pH 7.0 have been studied rheologically and by differential scanning calorimetry (DSC), with comparative measurements for the individual constituents of the mixtures. The concentration of WPI was held fixed at 10.0 wt% and carrageenan concentration was varied in the range 0.05–3.0 wt%. Ca²⁺ cations, which have been shown previously to be particularly effective in inducing gelation of κ-carrageenan, were introduced as CaCl₂. The concentration of CaCl₂ used in most of the experiments was 8 mM, but other concentrations were also studied. Mixtures were prepared in the solution state at 45 °C, and showed no evidence of either phase separation or complex formation. Rheological changes were monitored by low-amplitude oscillatory measurements of storage modulus, G′, during (i) cooling (1 °C/min) and holding at 5 °C, to induce gelation of the carrageenan in the presence of non-gelled WPI; (ii) heating and holding at 80 °C to dissociate the carrageenan network and induce gelation of WPI; (iii) cooling and holding again at 5 °C, to give composite networks with both components gelled; and (iv) re-heating to 80 °C to dissociate the carrageenan network. Gel structure was characterised further by creep–recovery measurements at the end of each holding period, and by torsion measurements at 5 °C, before and after thermal gelation of WPI.     

Rheology of galactomannan–whey protein mixed systems

The rheological behaviour of whey protein/galactomannan mixtures in aqueous solutions was studied under gelling conditions of the protein component, at neutral pH and at a pH close to the protein isoelectric point. The presence of the neutral polysaccharide had significant effects on the formation and viscoelastic behaviour of the cured gels. This effect was dependent on the structural organisation of the protein network. At pH 7 the galactomannan had a general positive effect on WPI gel formation. It is suggested that under these conditions, the protein network forms a continuous phase that accommodate the polysaccharide chains, acting as a filler of the protein network. The minimum protein concentration for gelation to occur, the gelation temperature and time all decrease in the presence of the galactomannan. Under pH conditions near the whey protein isoelectric point, different effects were observed as a result of the galactomannan addition. At low WPI concentration, the galactomannan had a detrimental influence on the protein network formation, but a negligible effect or even a positive influence on the gelation process at higher concentrations.     

Impact of whey protein – Beet pectin conjugation on the physicochemical stability of β-carotene emulsions

The aim of the present study was to investigate the impact of whey protein isolate (WPI)-beet pectin conjugation on the physical and chemical properties of oil-in-water emulsions incorporating β-carotene within the oil droplets. Covalent coupling of WPI to beet pectin was achieved by dry heating of WPI-beet pectin mixtures of different weight ratios at 80, 90, 100 °C and 79% relative humidity for incubation times ranging from 1 to 9 h. It was confirmed by SDS-polyacrylamide gel electrophoresis that WPI covalently linked to beet pectin. The physical and chemical stability of β-carotene emulsions was characterized by droplet size and distribution, transmission profiles using novel centrifugal sedimentation technique, microstructure and β-carotene degradation during the storage. Compared with those stabilized by WPI alone and unheated WPI-beet pectin mixtures, β-carotene emulsions stabilized by WPI-beet pectin conjugates had much smaller droplet sizes, more homogenous droplet size distribution, less change in centrifugal transmission profiles and obviously improved freeze–thaw stability, indicating a very substantial improvement in the physical stability. Rheological analysis exhibited that emulsions stabilized by WPI-beet pectin conjugates changed from a shear thinning to more like Newtonian liquid compared those with WPI alone and unheated WPI-beet pectin mixtures. Degradation of β-carotene in emulsion during storage was more obviously retarded by WPI-beet pectin conjugate than WPI and unheated WPI-beet pectin mixture, probably due to a thicker and denser interfacial layer in emulsion droplets. These results implied that protein–polysaccharide conjugates were able to improve the physical stability of β-carotene emulsion and inhibit the deterioration of β-carotene in oil-in-water emulsions.     

Barrier and tensile properties of whey protein–candelilla wax film/sheet

Barrier and tensile properties were compared for whey protein isolate- (WPI-) based solution-cast films, extruded sheets and extruded sheets subsequently thinned into films by compression. Solution-cast films were made from mixtures of WPI and glycerol (GLY) in water. Sheets were made by feeding WPI, GLY, and water to a twin-screw co-rotating extruder. In each case, candelilla wax (CAN) was added at 0, 5 or 7.5 g CAN/100 g dry mix to determine the effect on the barrier and tensile properties. Compressed extruded films were made by thinning extruded sheets using a Carver Press equipped with heated platens. Water vapor permeability (WVP), oxygen permeability (OP) and tensile properties were measured. Scanning electron microscopy (SEM) images were also taken.     

Characterization of protein–polyphenol interactions

Dietary polyphenols have received attention for their biologically significant functions as antioxidants, anticarcinogens or antimutagens, which have led to their recognition as potential nutraceuticals. Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein–polyphenol complexes. Questions remain concerning whether and to what extent the protein–polyphenol interaction influences functionality. For example, is the formation of protein–polyphenol complexes an obstacle to the nutritional bioavailability of either species? This article discusses the development of suitable methodologies to investigate the physicochemical basis of protein–polyphenol interactions and the influence of structure–activity relationships on binding affinities.     

Partition and digestive stability of α-tocopherol and resveratrol/naringenin in whey protein isolate emulsions

Co-encapsulation of multiple bioactive components is an emerging field that shows promise as an approach to develop functional foods. Hydrophobic components are generally dissolved in the inner oil phase of protein-stabilised emulsions. Some components may co-adsorb to oil droplet surfaces, due to the ligand-binding properties of proteins. In this study, α-tocopherol and resveratrol/naringenin were co-encapsulated in emulsions stabilised by whey protein isolate (WPI). α-Tocopherol was totally encapsulated and its partitioning inside oil droplets was about 3.3 times that bound by free WPI in the aqueous phase. The total encapsulation efficiency for resveratrol or naringenin was 52% and 58%, respectively. Addition of resveratrol improved digestive stability of α-tocopherol, but naringenin did not. Co-encapsulation with α-tocopherol had no significant influence on the digestive stability of resveratrol/naringenin. The data gathered here should be useful for the delivery of bioactive components with different solubilities.     

Microbiological aspects and challenges of whey powders – I thermoduric,thermophilic and spore-forming bacteria

For dairy processors, spoilage and pathogenic spore-forming bacteria are key sources of concern, not only due to their ability to remain dormant in a desiccated state in powders and to survive heat treatments, but also their ability to form biofilms in the vegetative state that lead to contamination of foods. These include members of the genera Bacillus, Geobacillus, Anoxybacillus, Brevibacillus, Paenibacillus and Clostridium, many of which are associated with food poisoning and spoilage. Here, we review the common bacterial species that form spores in whey powders and their sources and provide insights into their risks and strategies to control them.     

Properties of konjac glucomannan–whey protein isolate blend films

Edible composite packaging has been developed by blending biocomponents for specific applications, aiming to take advantage of complementary functional properties or to overcome their respective flaws. The aim of this work was to study the effect of incorporation of whey protein isolate (WPI) on the properties of konjac glucomannan (KGM) based films. Five aqueous solutions of KGM and/or WPI were prepared by casting and solvent evaporation of 1:0, 0.8:3.4, 0.6:3.6, 0.4:3.8 and 0:4.2 g KGM:g WPI/100 g solution. Glycerol (Gly) was used as a plasticizer at 1.5 and 1.8 g/100 g solution. The result showed that incorporated WPI proportionally increased transparency of KGM-based films. An increase in proportion of WPI resulted in decreased tensile strength and elastic modulus as well as improved flexibility. The incorporation of WPI into the KGM matrix led to an increase in water insolubility which enhanced product integrity and water resistance. Nevertheless, WPI did not improve water vapor barrier of KGM–WPI films. WPI and blend film with the highest concentration of WPI could be heat sealed at 175 °C. Overall, the range of Gly in this study did not apparently affect properties of the films.     

Viscoelastic behavior and microstructure of aqueous mixtures of cross-linked waxy maize starch,whey protein isolate and κ-carrageenan

The viscoelasticity and microstructure of mixtures of cross-linked waxy maize starch (CH10), whey protein isolate (WPI) and κ-carrageenan (κC) at pH 7.0 with 100 mM NaCl were investigated by oscillatory rheometry and confocal laser scanning microscopy (CLSM). Mixtures were heated to 90 °C (1.5 °C/min), held for 10 min at this temperature and cooled. Within the range of concentrations studied, CH10 swollen granules reinforced WPI and κC networks. The mechanical behavior of the three-component mixtures was modified by different WPI concentrations, but κC governed the overall response due to its gelling ability. CLSM images of three-component mixtures showed particulate systems in which swollen starch granules are surrounded by κC and WPI. CH10 granules were immersed in a single phase and a separate phase of κC and WPI, for low and high concentrations of these components, respectively. Therefore, it is possible to obtain two- and three-phase mixtures.     

Evaluation of pea protein–polysaccharide matrices for encapsulation of acid-sensitive bacteria

Protein–polysaccharide capsules containing Bifidobacterium adolescentis were produced and tested in a series of in vitro survival experiments to evaluate capsule protection of the bacterium to simulated stomach conditions, as well as their ability to release the encapsulated bacteria under conditions similar to those found in the lower gut. A protein fraction isolated from peas (pea protein isolate: PPI; 2.0%; w/v) was mixed with each of three different polysaccharides (0.5% (w/v) of either sodium alginate, iota-carrageenan and gellan gum) to produce capsules ranging in size from 2 to 3 mm diameter. All capsule formulations provided significant protection for cells exposed to synthetic stomach juice at 37 °C relative to non-encapsulated bacteria. In addition, PPI-alginate and PPI-iota-carrageenan capsules were found to dissolve in simulated intestinal fluid at 37 °C, releasing 70–79% of their bacteria “payload” within 3 h, with higher cell numbers being released from the freeze-dried capsules. PPI-gellan gum capsules did not dissolve to the same extent and the number of released cells was ~ 26–30% lower. Following a temporal rat feeding study with the test bacterium encapsulated in PPI-alginate, B. adolescentis-specific PCR and qPCR analyses confirmed the presence of DNA from this species in rat feces, but only during the period of capsule intake.     

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.     

Progressive freeze concentration of whey protein–sucrose–salt mixtures

Progressive freeze concentration of whey protein solutions is evaluated. Since solutions in industry are more complex, the effect of the addition of sodium chloride and sucrose on the inclusion behaviour is studied as well. Using a progressive freeze concentrator solutions of whey protein and mixtures of whey protein and/or sucrose and/or sodium chloride were freeze concentrated. At an initial concentration of 4%(w/w), whey proteins were not included in the ice fraction. At higher concentrations the inclusions are caused by the increase in viscosity in the boundary layer, impeding mass transfer. The addition of sucrose caused a similar effect. Presence of sodium chloride causes inclusions through the occurrence of a zone where the solution is locally super-cooled and leads to the formation of dendritic ice which encapsulates pockets of solution in the ice layer. Mixtures of both sucrose and sodium chloride gave no additive effect on solute inclusion but just a concurrent effect.     

Characterization of ‘wet’ alginate and composite films containing gelatin,whey or soy protein

The following study explored how the addition of various proteins (gelatin, soy protein isolate (SPI) and heated/unheated whey protein isolate (WPI)), at two different concentration levels (1% and 2%), affected the mechanical, microstructural and optical properties of calcium cross-linked ‘wet’ alginate films. Additionally, the water holding capacity and textural profile analysis (TPA) properties were determined for the alginate–protein gels. Adding all types of protein significantly (P < 0.05) decreased the force to puncture the ‘wet’ alginate–protein composite films compared to the control alginate film. The tensile test showed significant differences in tensile strength between the various films but interestingly there was no significant difference in the percent elongation at breaks between any of the films. Micrograph images showed that the SPI and heated WPI formed relatively larger protein clumps/regions in the alginate films whereas the gelatin and unheated WPI appeared to be more integrated into the alginate film. The heated WPI films were the least transparent of all the films, followed by the SPI films. Few TPA differences existed between the alginate–protein gels. However, the alginate–gelatin gels did have significantly less water loss than the other alginate–protein gels suggesting that alginate and gelatin may be the most compatible of all the alginate–protein combinations tested.