Factors Determining Yield Stress and Overrun of Whey Protein Foams

ABSTRACT: Foams were formed by whipping whey protein solutions (15% w/v protein) containing NaCl, CaCl₂, lactose, or glycine. Foam overrun and yield stress were determined. Foams made from whey protein ingredients have greater overrun and yield stress if the concentration of β-lactoglobulin is high relative to a-lactalbumin. The presence of 0.4 M CaCl₂ in the foaming solution increases overrun and yield stress for β-lactoglobulin and a-lactalbumin. The high yield stress of β-lactoglobulin and a-lactalbumin foams made from solutions containing CaCl₂ suggests that CaCl₂ is altering rheological properties of the interfacial protein film and/or contributing to protein aggregation or network formation in the lamellae.     

PREPARATION OF ENRICHED FRACTIONS OF α-LACTALBUMIN AND β-LACTOGLOBULIN FROM CHEESE WHEY USING CARBON DIOXIDE

A new process for preparing enriched fractions of α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) from whey protein concentrate was developed. To prepare the fractions, solutions containing from 7% (w/w) to 25% (w/w) whey protein concentrate (WPC75) were sparged with carbon dioxide (CO₂) in a batch reactor. The effects of pressure, temperature, concentration, and residence time on the distribution of the individual whey proteins in each fraction were investigated. Best results were obtained for 7% (w/w) WPC75 solutions, at reactor fill pressures of 4140 kPa and 5520 kPa, temperature of 64C and reactor residence time of 10 min. Gel electrophoresis of unpurified enriched samples showed that recovery of α-La was approximately 55% and that of β-Lg was 78%. The resulting fractions have a pH of 6.0 and contain no added salts.     

Characterization of a thermostable and acidic-tolerable beta-glucanase from aerobic fungi Trichoderma koningii ZJU-T

ABSTRACT:  An extreme thermostable and acidic tolerable β-glucanase was isolated and characterized from aerobic fungi Trichoderma koningii ZJU-T. The optimal reaction temperature and pH for the β-glucanase were 100 °C and pH 2.0, respectively. The β-glucanase showed increased stability at higher temperatures and lower pH values when compared to other β-glucanases. The optimum conditions for the β-glucanase stability were found to be pH 4.0 and 80 °C. Even subjected to 100 °C for 3 h, β-glucanase activity did not show significant reduction. Moreover, K⁺ significantly enhanced β-glucanase activity at the concentration of 1 mM, while EDTA and other metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, Ca²⁺, Fe²⁺, Pb²⁺, and Fe³⁺ inhibited β-glucanase activity. Denaturants, including sodium dodecyl sulfate (SDS) and mercaptoethanol, also inhibited β-glucanase activity at a concentration of 5%. However, in the presence of 7 M urea, residual activity of the β-glucanase still remained 14.5%.     

GEL CHARACTERISTICS OF β-LACTOGLOBULIN, WHEY PROTEIN CONCENTRATE AND WHEY PROTEIN ISOLATE

The gelation characteristics of β-lactoglobulin, whey protein isolate and whey protein concentrate at varying levels of protein (6–11%), sodium chloride (25–400 mM), calcium chloride (10–40 mM) and pH (4.0–8.0) were studied in a multifactorial design. Small scale deformation of the gels was measured by dynamic rheology to give the gel point (°C), complex consistency index (k*), complex power law factor (n*) and critical strain (γ_c). The gel point decreased and turbidity increased with increasing calcium level. The denaturation temperature measured by differential scanning calorimetry was measured at higher pH values. Large scale deformation at 20% and 70% compression was measured using an Instron Universal Testing machine. The true protein level had the largest effect on the stress required to produce 20% and 70% compression and on the consistency (k*) of the gels.     

Caustic-induced gelation of β-lactoglobulin

The gelation kinetics of β-lactoglobulin (βLg) solutions has been determined in the alkaline regime over a wide range of protein concentrations, gelling temperatures and gelation pH (pH_gel), set using NaOH. The behaviour is compared with caustic-induced gelation of whey protein concentrate and the alkaline dissolution of heat-induced whey gels. The gelation time decreases significantly between neutral conditions and pH_gel 9, because of the activation of the free cysteine groups and displacement to the monomeric form, and between pH_gel 10 and 11, due to the base denaturation of βLg. Both transitions are associated with a significant decrease in the activation energy of gelation. At pH_gel >11.5 the gelation time is observed to increase with pH_gel, owing to destruction of interprotein crosslinks. These results are consistent with the recently reported observation that a minimum pH for the dissolution of βLg gels and aggregates exists around 11.6 [ Biomacromolecules 8 (2007) 1162]. This phenomenon has been assigned to the destruction of non-covalent interactions that would inhibit the final percolation of the gel.     

Heat-induced gelation of whey protein at high pH studied by combined UV spectroscopy and refractive index measurement after size exclusion chromatography and by in-situ dynamic light scattering

Summary The interactions between β-lactoglobulin and α-lactalbumin involved in gelation at 67.5 °C at high pH and low salt concentration were studied by size exclusion chromatography, followed by UV and refractive index measurements, and by in-situ dynamic light scattering. This was achieved by choosing whey protein samples with different proportions of the two proteins. The ratio of absorbance at 280 nm to the refractive index was used to demonstrate that α-lactalbumin was incorporated in aggregates and gels and drastically changed the properties of the gel, making them much more turbid than the transparent gels formed by β-lactoglobulin alone at the same pH and ionic strength. At a ratio of 1:2 for α-lactalbumin relative to β-lactoglobulin in the samples, the gel consisted of a 1:1 mixture of the two proteins. The aggregates present after 10 min of heating at 67.5 °C had molar mass of about 6.10⁶ g/mol and a radius of gyration of about 40 nm. After gel formation the field autocorrelation function could be described as a power law over many decades of lag time for all samples, demonstrating selfsimilarity of the gel structure. The only exception to this was for the gel with high content of α-lactalbumin which showed an oscillatory behaviour of the autocorrelation function. Significant amounts of glycosylated caseino-macro-peptide were observed in many of the samples at the position of β-lactoglobulin. However it did not affect gelation as it remains in solution.     

Characteristic properties of equine milk proteins

Summary Properties of three equine milk proteins, lysozyme, α-lactalbumin, and β-lactoglobulin, were investigated and compared with the corresponding ones of bovine milk. The subsite structure similar to that in hen egg lysozyme was assumed in the functional region of lysozyme from interaction with inhibitors. α-lactalbumin has structures similar to those of bovine, but less stability of its intact and 3SS forms are observed than the corresponding bovine irrespective of [Ca²⁺] because of the entropy factors. Although the folding of β-lactoglobulin at acid pH is very similar to that of the bovine protein the conformation of the I strand is different from that of bovine I strand at neutral pH according to NMR data.     

Optimization of Thermal Pretreatment Conditions for the Separation of Native α-Lactalbumin from Whey Protein Concentrates by Means of Selective Denaturation of β-Lactoglobulin

ABSTRACT: In this study a method to obtain native α-lactalbumin with a high degree of purity of 98% (m/m) and recovery of 75% (m/m) by selective denaturation of β-lactoglobulin was developed. To achieve this goal, the thermal pretreatment of whey protein concentrate was optimized varying the composition of the liquid whey protein concentrate in terms of total protein, lactose and calcium content, and pH value. The kinetics of the thermal denaturation of α-la and β-lg were then investigated at predetermined optimal composition (protein content 5 to 20 g/L, lactose content 0.5 g/L, calcium content 0.55 g/L, and pH 7.5). Using the activation energies and reaction rate constants obtained, lines of equal effects for targeted denaturation degrees of α-la and β-lg were calculated. Depending on total protein content, an area of optimal heating temperature/time conditions was identified for each protein concentration level.     

Heat-induced denaturation and aggregation of β-Lactoglobulin: kinetics of formation of hydrophobic and disulphide-linked aggregates

Summary Solutions of a whey protein mixture were subjected to various time/temperature treatments, at pH 6.7. Kinetic and thermodynamic activation parameters for the rates of irreversible denaturation/aggregation of the principal whey protein component—β-lactoglobulin (β-lg) were followed by gel permeation. Fast Protein Liquid Chromatography (non-dissociating, non-reducing conditions) and by SDS-PAGE (dissociating, non-reducing conditions). The rate of loss of native β-lg owing to the formation of disulphide linked protein aggregates (k_sds-page) and the rate of formation of aggregates via both covalent and non-covalent bonds (k_gp-fplc) showed similar biphasic Arrhenius plots. However, the break of the plot occurred at different points. The k_gp-fplc values were higher than values of k_sds-page for all the temperatures examined. There was a similar trend for the thermodynamic activation parameters implying that not all of the β-lg aggregates through thiol–disulphide interactions. Hydrophobically driven associations occur within the aggregates.     

Preparation of β-Lactoglobulin and p-Lactoglobulin-Free Proteins from Whey Retentate by NaCI Salting Out at Low pH

Solubility of the main proteins in 10 x acid and rennet whey retentates was studied in the pH range 2.0 to 4.0 in the presence of NaCI from 2 to 15% (w/v) final concentration, at 20°C to find fractionation conditions suitable for preparing pure β-lactoglobulin and β-lactoglobu-lin-free whey proteins and scaling up. At pH 2.0, 7% NaCI, 20 min holding time, nearly all (3-lactoglobulin remained soluble while a precipitate (PI) containing all other proteins was formed. Pure p-lacto-globulin was quantitatively recovered by salting-out the centrifugation supernatant at 30% NaCI (w/v) final concentration. PI, insoluble at pHs lower than 4.0, was made soluble at any pH by dissolving at pH 9.0, dialyzing against 50 mM formic acid (pH 3.0) and freeze-drying.     

Combined effect of whey protein and aS1-casein genotype on the heat stability of goat milk

Goat milk is characterized by a very low heat stability, which could be related to compositional factors. In this paper, the role of the heat-induced interaction between whey proteins and casein micelles is considered. The effect of the casein micelle structure is evaluated using milk samples with different α_S1-casein genotypes, and the protein interactions are studied at various pH values in order to take into account the strong pH-dependence of the heat stability. The results suggest that the heat-induced interaction of β-lactoglobulin with κ-casein is of minor importance at natural pH, but is promoted at more elevated pH.     

Expression of recombinant wild-type and mutant β-Lactoglobulins in the yeast Pichia pastoris

Summary We have produced β-lactoglobulin gene constructs in the pGAPZαA vector for over expression of recombinant wild-type and a putative monomeric mutant protein in Pichia pastoris . Levels of expression of at least 150 mg L⁻¹ of the wild-type were achieved in shake flask culture. Ion exchange chromatography showed that the expressed recombinant wild-type β-Lg eluted as a heterogeneous mixture of species, some of which may be owing to small variations in the N-terminus and some to ligand binding. The putative monomeric β-lactoglobulin construct gave an expressed protein that was recognized by anti bovine β-lactoglobulin antibody, but we do not know yet if it forms monomers rather than dimers.     

Selective Concentration of Bovine Immunoglobulins and α-Lactalbumin from Acid Whey using FeCl3

Immunoglobulins and α-lactalbumin of acid whey were concentrated in supernatant and precipitate when FeCl₃ was added at pH 4.2 and 2.8, respectively. Optimized conditions of pH 4.2 were preferable because of higher retention of immunochemical activity of immunoglobulins. In acid whey treated with 7.5 mM FeCl₃ at pH 4.2 and 4°C, 90% of β-lactoglobulin coprecipitated with serum albumin while 70% of immunoglobulins (92% immunochemically active IgG) and 95% of α-lactalbumin were retained in the supernatant. More than 98% of added iron was subsequently eliminated as precipitate by holding the treated whey at pH 8-9 and 4°C, without losing immunochemical activity of immunoglobulin G, in addition to retained activity of immunoglobulins A and M.     

ADSORPTION BEHAVIOR OF β-LACTOGLOBULIN ONTO STAINLESS STEEL SURFACES

Nonporous stainless steel particles with an average diameter of 25.5 μm and an average surface area of 215 cm²/g were used to study the adsorption of β-lactoglobulin (β-lg). Scanning electron microscopy of stainless steel particle surfaces with adsorbed β-lg showed an obvious eradication of crevices as protein was adsorbed. The adsorption process for β-lg was very rapid in the first 5 min and essentially reached equilibrium within 10 min. Precipitation of calcium phosphate onto the stainless steel surface was very slow compared to monolayer deposition of β-lg. A monolayer of β-lg adsorbed very tightly to the stainless steel surface and a significant fraction of the protein was irreversibly adsorbed. The amount of adsorbed protein decreased with increasing ratio of surface area to reaction volume. pH in the range 6.0–6.8 did not significantly affect the amount of adsorbed protein.     

Fractionating Soybean Storage Proteins Using Ca2+ and NaHSO3

ABSTRACT:  Individual soybean storage proteins have been identified as having nutraceutical properties, especially β-conglycinin. Several methods to fractionate soy proteins on industrial scales have been published, but there are no commercial products of fractionated soy proteins. The present study addresses this problem by using calcium salts to achieve glycinin-rich and β-conglycinin-rich fractions in high yields and purities. A well-known 3-step fractionation procedure that uses SO_2, NaCl, and pH adjustments was evaluated with CaCl₂ as a substitute for NaCl. Calcium was effective in precipitating residual glycinin, after precipitating a glycinin-rich fraction, into an intermediate fraction at 5 to 10 mM CaCl₂ and pH 6.4, eliminating the contaminant glycinin from the β-conglycinin-rich fraction. Purities of 100%β-conglycinin with unique subunit compositions were obtained after prior precipitation of the glycinin-rich and intermediate fractions. The use of 5 mM SO₂ in combination with 5 mM CaCl₂ in a 2-step fractionation procedure produced the highest purities in the glycinin-rich (85.2%) and β-conglycinin-rich (80.9%) fractions. The glycinin in the glycinin-rich fraction had a unique acidic (62.6%) to basic (37.4%) subunit distribution. The β-conglycinin-rich fraction was approximately evenly distributed among the β-conglycinin subunits (30.9%, 35.8%, and 33.3%, for α', α, and β subunits, respectively). Solids yields and protein yields, as well as purities and subunit compositions, were highly affected by pH and SO₂ and CaCl₂ concentrations.     

Reduction of Streptococcus thermophilus in a whey protein isolate by low moisture extrusion cooking without loss of functional properties

A whey protein isolate powder (WPI) (4–5% water) inoculated with 5x10⁵ viable Streptococcus thermophilus per g, was continuously processed in a twin screw extruder under the following conditions (barrel length = 500 or 1000 mm; screw profile = forward transport and compression elements; moisture content during extrusion = 4–5%; feed rate = 10 kg h^-1; barrel temperature ( T_b ) = 80–204°C; speed of screw rotation = 50 r.p.m.). The minimum residence time determined by pulse injection of erythrosin was 20–25 s (500 mm barrel) or 35–40 s (1000 mm barrel).
Reduction values of viable Streptococcus thermophilus of 10^4.2-fold (500 mm barrel, T_b = 143°C) or 10^4.9-fold (1000 mm barrel, T_b = 133°C) were obtained without any modification of protein solubility or gelling properties. WPI extruded at the highest barrel temperatures (182–204°C) underwent limited browning and reduction of protein solubility. Gel permeation and hydrophobic interaction chromatography of the soluble constituents did not show any aggregates of β-lactoglobulin or α-lactalbumin.
Gels prepared from control or extruded WPI ( T_b 143°C with a barrel length = 500 mm or T_b 133°C with a barrel length = 1000 mm) were identical, as judged by scanning electron microscopy and rheological evaluations.     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

Effect of CaCl2 and KCl on Physiochemical Properties of Model Nutritional Beverages Based on Whey Protein Stabilized Oil-in-Water Emulsions

ABSTRACT: Calcium chloride (0 to 10 mM) and potassium chloride (0 to 600 mM) were added into model nutritional beverage emulsions containing 7% (w/w) soybean oil droplets and 0.35% (w/w) whey protein isolate (pH 6.7). The particle size, surface charge, viscosity, and creaming stability of the emulsions then were measured. The surface charge decreased with increasing mineral ion concentration. The particle size, viscosity, and creaming instability of the emulsions increased appreciably above critical CaCl₂ (3 mM) and KCl (200 mM) concentrations because of droplet flocculation. The origin of this effect was attributed to reduction of the electrostatic repulsion between droplets due to electrostatic screening and ion binding. CaCl₂ promoted emulsion instability more efficiently than KCl because Ca²⁺ ions are more effective at reducing electrostatic repulsion than K⁺ ions.     

Some physico-chemical properties of nine commercial or semi-commercial whey protein concentrates, isolates and fractions

Summary The physico-chemical properties are reported for a group of whey protein powders prepared on a commercial or semi-commercial scale by three companies and chemically characterized as described elsewhere (Holt et al ., 1999). The dependence of the apparent β-lactoglobulin % on the recovered % showed that the nine samples could be placed in three distinct groups with β-lactoglobulin weight % of 70.9 ± 1.1 (Group 1), 62.0 ± 3.4 (Group 2) and 39.5 ± 4.9 (Group 3). Measurements by ¹H-NMR spectroscopy, on 3 of the samples confirmed that the native fold still predominated in the β-lactoglobulin. β-lactoglobulin could be crystallized from all the powders and the normal space group and cell dimensions were determined for the 8 samples that gave crystals of good enough quality for X-ray studies. Differential scanning microcalorimetry of samples dispersed in a phosphate buffer showed a clear difference between Goups 1 and 2 with a more prominent peak due to α-lactalbumin in the Group 2 samples. Light scattering and size exclusion chromatography showed that two types of aggregates were present to a variable extent in all the samples and after a heat treatment, the larger aggregates tended to predominate in Group 2. The rheology measurements, also made in the phosphate buffer, showed a difference of gel stiffness during heat treatment between the Group 1 and Group 2 samples with the exception of the BORCwpc+ sample. Within each group, gel stiffness increased with the degree of lactoslylation of the β-lactoglobulin. Interfacial measurements on samples dispersed in water presented a more complex pattern of behaviour although surface tension measurements at the air water interface of the Group 2 samples showed a two-step pattern of surface tension decrease with time, compared to a single step pattern in the Group 1 samples.     

AN ESTEROLYTIC ACTIVITY FROM A WILD EDIBLE MUSHROOM, LYCOPERDON PERLATUM

Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p -nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn^{2 +} , Fe^{2 +} , Ca^{2 +} and Zn^{2 +} in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p -methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases.

PRACTICAL APPLICATIONS

Esterases catalyzing the cleavage and formation of ester bonds are known α/β-hydrolases (EC 3.1.1.X). Esterases are used for the synthesis of flavor esters for the food industry, modification of triglycerides for fat and oil industry and resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry. Therefore, the search for new enzyme sources is important for the development of new enzymes and applications.