Studies on genetic variants of α-lactalbumin and β-lactoglobulin from milk of native Portuguese ovine and caprine breeds

Summary α-Lactalbumin (α-La) and β-lactoglobulin (β-Lg) fractions were obtained from Portuguese native breeds of ewes and goats by preparative gel filtration and further purified by ion exchange; their genetic variants were characterized by isolectric focusing, and β-Lg isolated was further characterized by differential scanning calorimetry. Separation of β-Lg and α-La by molecular exclusion from native whey was relatively easy, whereas β-Lg from both breeds accounted for a single peak via ion exchange under various gradients of NaCl. Isoelectric focusing has indicated that α-La from ovine and caprine wheys appears as a single variant in each case, as well as β-Lg from caprine whey; however, β-Lg from ovine whey appears as two peaks, tentatively denoted as β-Lg A and B. Further comparison with bovine whey made it possible to rank whey proteins by increasing value of pI as follows: bovine β-Lg A, bovine α-La, bovine β-Lg B, ovine and caprine α-La, ovine β-Lg A, and finally ovine β-Lg B and caprine β-Lg. β-Lg from goat's whey showed the highest onset temperature of denaturation in the presence (78–97 °C) or absence (90–100 °C) of NaCl for every pH tested; when NaCl was present, a good correlation between pI and onset temperature of denaturation was obtained for pH values in the range 3.5–7.0.     

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.     

Chemical Pretreatment and Microfiltration for Making Delipidized Whey Protein Concentrate

Chemical pretreatment, microfiltration (MF) and ultrafiltration (UF) were applied to produce delipidized whey protein concentrates (WPC). Processes including both chemical pretreatment and MF resulted in WPC with <0.5% lipids. Low-pH UF and isoelectric point (PI) precipitation were more effective for lipid removal than chemical pretreatment by thermocalcic aggregation. Protein permeation ratios in MF processes were improved by UF preconcentration of whey. Protein permeation and flux were different between the two MF membranes used. Isoelectric point precipitation increased β-Lg contents, but not α-La, in the resulting WPC (B). Minor proteins exhibited lower concentrations in WPC B and MF WPC products.     

Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract

The hydrolysis of bovine whey protein concentrate (WPC), α-lactalbumin (α-La) and caseinomacropeptide (CMP), by aqueous extracts of Cynara cardunculus, was optimized using response surface methodology. Degree of hydrolysis (DH), angiotensin-converting enzyme (ACE)-inhibitory activity and antioxidant activity were used as objective functions, and hydrolysis time and enzyme/substrate ratio as manipulated parameters. The model was statistically appropriate to describe ACE-inhibitory activity of hydrolysates from WPC and α-La, but not from CMP. Maximum DH was 18% and 9%, for WPC and α-La, respectively. 50% ACE-inhibition was produced by 105.4 (total fraction) and 25.6 μg mL⁻¹ (<3 kDa fraction) for WPC, and 47.6 (total fraction) and 22.5 μg mL⁻¹ (<3 kDa fraction) for α-La. Major peptides of fractions exhibiting ACE-inhibition were sequenced. The antioxidant activities of WPC and α-La were 0.96 ± 0.08 and 1.12 ± 0.13 μmol trolox equivalent per mg hydrolysed protein, respectively.     

乳清蛋白的酶法改性研究

采用碱性蛋白酶、风味蛋白酶、木瓜蛋白酶、中性蛋白酶、复合蛋白酶、胰蛋白酶、胃蛋白酶在各自的最适条件下对乳清蛋白粉进行水解,通过HPLC方法测定酶解产物中α-乳白蛋白（α—La）和β-乳球蛋白（β—Lg）的含量。结果表明,碱性蛋白酶对其水解最快,其次为木瓜蛋白酶,而在胃蛋白酶和胰蛋白酶作用下则不易酶解,而且发现α-La比β—Lg更容易水解。     

Fractionation of pasteurized skim milk proteins by dynamic filtration

This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, β-Lactoglobulin (β-Lg) and other large whey proteins, and α-Lactalbumin (α-La). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. α-La and β-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for α-La and 76.6% for β-Lg at a VRR of 5.4. The separation between β-Lg and α-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h^?1 m^?2 at VRR = 5 and 40 °C. α-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. β-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating α-La and β-Lg proteins from skim milk.     

Elimination of β-Lactoglobulin from Whey to Simulate Human Milk Protein

When the pH of cottage cheese whey was adjusted to 4.5 in the presence of 6.7 mM FeCI³, β-lactoglobulin was eliminated from the whey as a precipitate. However, the majority of immunoglobuhns were also coprecipitated. To recover immunoglobulins together with α-lactalbumin, the whey pH was adjusted to 3.0 in the presence of 4.0 mM FeCI³. After centrifugation of the whey, the supernatant contained exclusively β-lactoglobulin; other whey proteins were found in the precipitate. Excess Fe₊₊₊ in the precipitate was removed by ion exchange or by ultrafiltration. This protein concentrate had a protein composition much more similar to that of human milk whey than that of ultrafiltered whey protein concentrate.     

Interfacial and Foaming Properties of Two Types of Total Proteose-Peptone Fractions

Total proteose-peptone (TPP) fractions were extracted from skimmed milk (UHT) and whey protein concentrate (WPC) on a laboratory scale. Protein solutions (0.1 %, 0.5 %, and 1 % w/w) were characterized as function of pH: 4.0, 4.6–4.7 (native pH), and 7.0. Their foaming capacities and stabilities were studied. Beforehand, the surface properties that govern them were investigated, notably the kinetics of adsorption and mechanical properties of monolayer films at the air–water interface involved in the formation and the stability of foams respectively. The TPP extracted from skimmed milk showed the lowest values as well as a significant reduction in surface tension and presented a good mechanically resistant film. The TPP extracted from WPC presented a better foaming capacity and stability which was unexpected. However, foaming properties and surface properties of TPP fractions depended on the pH. The considerable influence of extraction source and method on proteose-peptone’s properties were highlighted.     

Comparison of the gel-forming ability and gel properties of α-lactalbumin,lysozyme and myoglobin in the presence of β-lactoglobulin under high pressure

α-Lactalbumin (α-La) and lysozyme (LZM) each contain four disulfide bonds but no free SH group, whereas myoglobin (Mb) possesses no disulfide bond or free SH group. In this work, the pressure-induced gelation of α-La, LZM and Mb in the absence and in the presence of β-lactoglobulin (β-Lg) was studied. Solutions of α-La, LZM and Mb (1–24%, w/v) did not form a gel when subjected to a pressure of 800 MPa and circular dichroism analysis revealed that both α-La and LZM are pressure-resistant proteins. In the presence of β-Lg (5%, w/v), however, a pressure-induced gel formed for α-La and LZM (each 15%, w/v) but not for Mb (15%, w/v). One- and two-dimensional SDS-PAGE demonstrated the disulfide cross-linking of proteins was responsible for the gelation. Although α-La and LZM are homologous and have the same disulfide bond arrangement, the texture and appearance of the gels formed from α-La/β-Lg and LZM/β-Lg were markedly different even when induced under the same experimental conditions. Microscopic analysis indicated that phase separation occurs during the gelation of LZM/β-Lg but not during the gelation of α-La/β-Lg. NMR relaxation measurement revealed that the association of water molecules with the protein matrix in the α-La/β-Lg gel is tighter compared to that in the LZM/β-Lg gel. These results indicate that the gel-forming ability of a globular protein under high pressure is related to the primary structure of the protein, and that the gel properties depend on the cross-linking reaction and on the phase behavior of protein dispersion under high pressure.     

Fractionation of α-Lactalbumin and β-Lactoglobulin from Whey Protein Isolate Using Selective Thermal Aggregation, an Optimized Membrane Separation Procedure and Resolubilization Techniques at Pilot Plant Scale

An optimized fractionation method in the pilot scale for production of isolated α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) was developed. The method comprises following steps: (1) selective thermal precipitation of α-La, (2) aging of the formed particles, (3) separation of native β-Lg from the precipitate via microfiltration and ultrafiltration, (4) purification of β-Lg, (5) resolubilization of the precipitate, and (6) purification of α-La. The native status of the isolated fractions was confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and differential scanning calorimetry (DSC). Protein fractions with a purity of 91.3% for α-La and 97.2% for β-Lg were produced. These values were based on the native protein detectable in RP-HPLC. High overall yields for α-La between 60.7% and 80.4% and for β-Lg between 80.2% and 97.3%, depending on membrane operation parameters, were achieved. The method offers potential for pilot plant scale and possibly industrial application to produce pure native fractions of α-La and/or β-Lg.     

Influence of fortification with sodium–calcium caseinate and whey protein concentrate on microbiological,textural and sensory properties of set‐type yoghurt

The effect of fortification of yoghurt with sodium–calcium caseinate (SCC) and whey protein concentrate (WPC) on some properties of set‐type yoghurt were investigated. The addition of WPC enhanced the viability of Lactobacillus delbrueckii subsp. bulgaricus more than SCC. The highest firmness values were obtained from SCC‐fortified yoghurts, whereas yoghurts fortified with WPC had the highest water‐holding capacity during storage. The yoghurts fortified with 4% w/w SCC or 4% w/w WPC had the highest viscosity. Yoghurts fortified with 2% w/w SMP, SCC or WPC showed similar taste and overall acceptability scores; however, samples containing 4% w/w SCC or 4% w/w WPC had the lowest scores.     

Heat-Induced Gelation of Individual Whey Proteins A Dynamic Rheological Study

Heat-induced gelation of the bovine whey proteins [serum albumin (BSA), β-lactoglobulin (β-Lg) and α-lactalbumin (α-La)] has been studied individually and in mixture at different conditions by a dynamic rheological method. Values in the shear stiffness modulus (/G*/) appeared on heating at low protein concentration for BSA (～2%) and at intermediate concentration for β-Lg (～ 5%). α-La did not form a heat-induced gel of concentrations up to 20% (w/v). The ratio of viscous to elastic properties (loss factor) at maximum possible measuring temperature was below 0.07 for the BSA gels and 0.1–0.3 for the β-Lg gels. The temperature of gelation was highly dependent on pH. In mixture one protein could not be exchange for another without changing the gelation behavior of the mixture.     

A comparison between acidic and basic protein fractions from whey or milk for reduction of bone loss in the ovariectomised rat

The ability of whey acidic protein fractions to protect against bone loss due to ovariectomy (OVX) in the mature female rat was investigated. The bone bioactivity of these acidic protein fractions, isolated from both mineral acid whey protein concentrate (WPC) and rennet WPC, was compared with that of basic protein fractions isolated from both milk and rennet WPC. Fifty 6-month old rats that had been ovariectomised at 5.5 months were randomised into five groups of ten each. One group remained the OVX control while four groups were each fed one of the acidic or basic protein fractions as 0.3% (w/w) of the diet, for 4 months. Ten sham-operated rats served as a second control group. Sequential measurements of bone mineral density of the spine and femur indicated that the acidic protein fraction prepared from mineral acid WPC reduced bone loss due to OVX, maintaining bone density above OVX levels at week 16 of feeding. Biomechanical data indicated that both acidic fractions tended to increase bone stiffness, and hence resistance against breaking.     

Microencapsulating Properties of Whey Protein Concentrate 75

ABSTRACT Emulsions containing various levels of soya oil dispersed in solutions of whey protein concentrate (WPC) 75 (5% w/v) were spray-dried to yield powders with oil contents ranging from 20% to 75% (w/w). The effect of homogenizing pressure and oil/protein ratio on oil globule size distributions and protein load of the emulsions and the microencapsulation efficiency (ME) and redispersion behavior of the powders were examined. Emulsion oil droplet size decreased with increasing homogenization pressure but was not affected by oil/protein ratio. Emulsion protein load and ME of the powders were negatively correlated with increasing oil/protein ratio. Powders with an oil/protein ratio < 0.75 were least susceptible to destabilization during spray-drying.     

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.     

Coacervate formation of α-lactalbumin–chitosan and β-lactoglobulin–chitosan complexes

α-Lactalbumin (α-La) and β-lactoglobulin (β-Lg) are important whey proteins with isoelectric points of pH 4.80 and 5.34, respectively, and evidence negative charge over a range of pHs. Chitosan exhibits a cationic property under pH 6.5. In an effort to determine the physicochemical properties of mixtures of 0.5% α-La and 0.1% chitosan, and 0.5% β-Lg and 0.1% chitosan, optical structure, turbidity, electrophoresis, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were assessed in a pH range of 2.0–8.0. The results demonstrated that α-La, β-Lg, and chitosan precipitated at pH values of approximately 5.0, 5.0, and 7.0, respectively. The mixtures of α-La and chitosan as well as β-Lg and chitosan coacervated at a pH range of 6.0–6.5. The turbidity of α-La and α-La–chitosan achieved a maximum at pH 5.0, whereas those of β-Lg and β-Lg–chitosan achieved maximum values at a pH of 6.5. The electrophoregram showed a large band with high molecular weight in increasing pH values from 5.0 to 6.0, which suggested that α-La and β-Lg form polymers with chitosan. The denaturation temperature and enthalpy of α-La were shown to increase, whereas those of β-Lg were reduced. The SEM images demonstrated that the α-La was characterized by uneven and associated cluster morphology, whereas that of β-Lg was even, globular, and harbored dense particles, whereas the chitosan evidenced a flat morphology. Our assessment of the complex demonstrated that α-La and β-Lg attached to the surface of the chitosan. The α-La–chitosan and β-Lg–chitosan complexes evidenced opposite charges at a pH range of 5.0–6.0, and formed coacervates. It appears, therefore, that the α-La–chitosan and β-Lg–chitosan coacervates might be applied as a delivery system for foods, nutraceuticals, cosmetics, and drugs.     

High pressure microfluidization treatment of heat denatured whey proteins for improved functionality

Solutions (5% protein) of a whey protein concentrate (WPC) in fresh acid whey or in water, as well as the fresh whey alone, were adjusted to pH 5.8, 4.8 or 3.8, heat treated at 90 °C for 10 min and further exposed to high pressure (150 MPa) microfluidization treatment. The volumes of sediment after centrifugation were recorded as a measure of the degree of insolubility of the proteins. Microfluidization disrupted the heat-induced aggregates into non-sedimenting whey protein polymers so that in some cases, especially at pH 3.8, the products studied were almost completely resistant to sedimentation after the microfluidization treatments. Heat denatured/microfluidized whey proteins reaggregated upon subsequent heating, with the pH having a major impact on the amount of sediment produced. Microfluidization of aqueous WPC solutions heat-treated before spray- or freeze-drying substantially increased the solubility of the powders upon reconstitution. Heat-induced viscoelastic gels were produced from freeze-dried microfluidized samples processed at pH 3.8 and reconstituted to solutions containing 12% (w/w) protein.     

Is the rate of sulfur-disulfide exchange between the native β-Lactoglobulin and PDS related to protein conformational stability?

Summary The sulfhydryl (SH) group of β-lactoglobulin (β-Lg) affects many of its functional properties. Native β-Lg was treated with pyridine disulfide (PDS) at pH 2.6–8.5 (25 °C). SH-disulfide exchange was monitored by spectrophotometry. The kinetics of β-Lg sulphydryl-disulphide exchange was compared with the same reaction for reduced glutathione (GSH). From such results we estimate, ΔG_ex, the free energy change for exposing the SH-group of β-Lg to solvent. The numerical value for ΔG_ex is equal to the free energy change for β-Lg dissociation at pH 7. At neutral pH, the rate of sulphydryl- disulphide exchange appears to be controlled by the dimer-monomer dissociation equilibrium. At other solvent pHs, β-Lg sulphydryl reactivity towards a small disulphide compound like PDS may not involve the dissociation of native β-Lg.     

Influence of storage, heat treatment, and solids composition on the bleaching of whey with hydrogen peroxide

The residual annatto colorant in liquid whey is bleached to provide a desired neutral color in dried whey ingredients. This study evaluated the influence of starter culture, whey solids and composition, and spray drying on bleaching efficacy. Cheddar cheese whey with annatto was manufactured with starter culture or by addition of lactic acid and rennet. Pasteurized fat-separated whey was ultrafiltered (retentate) and spray dried to 34% whey protein concentrate (WPC34). Aliquots were bleached at 60 °C for 1 h (hydrogen peroxide, 250 ppm), before pasteurization, after pasteurization, after storage at 3 °C and after freezing at -20 °C. Aliquots of retentate were bleached analogously immediately and after storage at 3 or -20 °C. Freshly spray dried WPC34 was rehydrated to 9% (w/w) solids and bleached. In a final experiment, pasteurized fat-separated whey was ultrafiltered and spray dried to WPC34 and WPC80. The WPC34 and WPC80 retentates were diluted to 7 or 9% solids (w/w) and bleached at 50 °C for 1 h. Freshly spray-dried WPC34 and WPC80 were rehydrated to 9 or 12% solids and bleached. Bleaching efficacy was measured by extraction and quantification of norbixin. Each experiment was replicated 3 times. Starter culture, fat separation, or pasteurization did not impact bleaching efficacy (P > 0.05) while cold or frozen storage decreased bleaching efficacy (P < 0.05). Bleaching efficacy of 80% (w/w) protein liquid retentate was higher than liquid whey or 34% (w/w) protein liquid retentate (P < 0.05). Processing steps, particularly holding times and solids composition, influence bleaching efficacy of whey. PRACTICAL APPLICATION: Optimization of whey bleaching conditions is important to reduce the negative effects of bleaching on the flavor of dried whey ingredients. This study established that liquid storage and whey composition are critical processing points that influence bleaching efficacy.     

Factors Affecting Rheological Characteristics of Fibril Gels: The Case of β-Lactoglobulin and α-Lactalbumin

ABSTRACT:  Some of the factors that affect the rheological characteristics of fibril gels are discussed. Fibrils with nanoscale diameters from β-lactoglobulin (β-lg) and α-lactalbumin (α-la) have been used to create gels with different rheological characteristics. Values of the gelation time, t_c , the critical gel concentration, c ₀, and the equilibrium value of the storage modulus, G , such as     at long gelation times, derived from experimental rheological data, are discussed. Fibrils created from β-lg using solvent incubation and heating result in gels with different rheological properties, probably because of different microstructures and fibril densities. Partial hydrolysis of α-la with a serine proteinase from Bacillus licheniformis results in fibrils that are tubes about 20 nm in diameter. Such a fibril gel from a 10% (w/v) α-la solution has a higher modulus than a heat-set gel from a 10% (w/w) β-lg, pH 2.5 solution; it is suggested that one reason for the higher modulus might be the greater stiffness of α-la fibrils. However, the gelation times of α-la fibrils are longer than those of β-lg fibrils.