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.     

Thermal Properties of Yak α-Lactalbumin and β-Lactoglobulin: a DSC Study

A differential scanning calorimetry (DSC) method was used to investigate the denaturation temperature of yak α-lactalbumin (α-La), β-lactoglobulin (β-Lg), and a mixture of two proteins and the thermal properties of α-La and β-Lg in the presence of glucose, lactose, sucrose, NaCl, CaCl₂, and at various pH (4.0–10.0). The denaturation temperature (T _d) of α-La increased from 52.1 °C in the absence of β-Lg to 53.9 °C in the presence of β-Lg, while the T _d of β-Lg decreased from 81.4 °C in the absence of α-La to 79.9 °C in the presence of α-La. α-La was thermal stable in the range of pH 4.0–10.0, while β-Lg was more thermal stable in acidic pH than in alkaline pH. Sugars, Na⁺, and Ca²⁺ influenced the stabilization of the two proteins against thermal denaturation with greatly influenced for β-Lg. α-La kept reversibility in the presence of sugars, NaCl, CaCl₂, and over a wide pH range (4.0–10.0), with most of the reversibility values being greater than 90%. In contrast, β-Lg was completely irreversible whether in its native state or in the presence of the additives.     

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.     

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.     

Major whey proteins in ovine and caprine acid wheys from indigenous greek breeds

Acid whey filtrates from the bulk milk of different indigenous greek ovine and caprine breeds were investigated for the quantitative and qualitative characteristics of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG). For comparison reasons acid wheys from bovine milk and from Saanen and Alpine caprine breeds were included. The main characteristic of ovine acid wheys was the low α-LA percentage. The β-LG/α-LA ratio of ovine acid wheys ranged from 3.91 in Chios breed to 6.65 in Boutsiko breed. It was higher than the estimate for bovine acid wheys which ranged from 3.09 to 3.37. The chromatographic and isoelectric focusing profiles of ovine β-LG and α-LA from the different breeds were also variable. The β-LG percentage of caprine acid wheys was lower compared to ovine and bovine acid wheys. Their β-LG/α-LA ratios ranged from 2.02 in Saanen breed to 3.04 in the indigenous breed Skopelos.     

β-Lactoglobulin Gelation and Modification: Effect of Selected Acidulants and Heating Conditions

ABSTRACT: The effect of acidulant selection, heating temperature, and heating rate on the properties of low-pH β-lactoglobulin (β-Lg) gels and powders derived from these gels was investigated by rheological and microscopic techniques. As isothermal gelation temperature was increased from 75 to 85 °C, gels made with hydrochloric and lactic acid showed more rapid gel formation and increased stress at gel fracture. Thickening and water-holding properties of powders derived from these gels also increased with temperature. Increases in gel strength and derivatized powder functionality appeared to plateau above 85 °C. Gels and derivatized powders prepared with phosphoric acid exhibited attributes similar to samples prepared with HCl and lactic acid at lower temperatures. The ion-specific ability of phosphate to increase denaturation temperature was responsible for the shift in properties of gels made with phosphoric acid. Microscopy revealed temperature effects on network building block size, but variations in rheological properties could not be linked to changes in gel micrographs. Alteration of heating rates from 2.0 to 0.2 °C/min during gelation affected the observed gelation temperature, but had little effect on final gel mechanical properties. Acid selection and gelation temperature offer alternatives to control β-Lg gel strength and the functional properties of instant thickening protein ingredients.     

Identification of the genetic variants of κ-casein in milk by isoelectric focusing electrophoresis

The genetic variants of κ-casein (κ-CN) A and B were analysed in milk from Holstein–Friesian (HF) and Jersey cows by means of isoelectric focusing (IEF) electrophoresis. Milk samples were obtained in triplicate from pure-breed HF and Jersey cows (three of each) to estimate the protein content, casein and purify κ-CN. The protein and casein contents in the milk from both breeds were statistically different ( P <  0.05). The κ-CN A migrates first with an isoelectric point (pI) of 7.2–7.3 and then B with a pI of 7.5–7.7. Differences in the expression proportion of both variants were detected.     

Studies on the impact of glycation on the denaturation of whey proteins

It could be shown for technologically relevant whey protein powders that denaturation of β-lactoglobulin (β-Lg) is affected significantly by the extent of covalent modification of lysine residues by lactose. The amount of acid soluble β-Lg as measured via RP-HPLC with UV detection after heating for 10 min at 80 °C increased from 40% (4.6% lysine modification) to 82% (22.4% lysine modification). An increase in glycation leads to a slower denaturation-induced oligomerisation, as shown by SDS-PAGE. Concomitant with an increase in lysine modification, the denaturation temperature increased from 79.5 to 84 °C, as measured by differential scanning calorimetry (DSC). Covalent attachment of lactose to whey proteins during preparation or storage significantly improves the heat stability of whey proteins, which may be of particular importance for the technological use of whey proteins varying in the degree of lysine modification.     

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.     

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.     

Whey protein isolate polydispersity affects enzymatic hydrolysis outcomes

The effects of heat-induced denaturation of whey protein isolate (WPI) on the enzymatic breakdown of α-La, caseinomacropeptide (CMP), β-Lg A and β-Lg B were observed as hydrolysis proceeded to a 5% degree of hydrolysis (DH) in both unheated and heat-treated (80 °C, 10 min) WPI dispersions (100 g L⁻¹). Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. LC–MS/MS identified 23 distinct peptides which were identified in the denatured WPI hydrolysate – the majority of which were derived from β-Lg. The mapping of the detected regions in α-La, β-Lg, and CMP enabled specific cleavage points to be associated with certain serine endo-protease activities. The outcomes of the study emphasise how a combined approach of substrate heat pre-treatment and enzymology may be used to influence proteolysis with attendant opportunities for targeting unique peptide production and amino acid release.     

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.     

Relationship between A and B variants of κ-casein and β-lactoglobulin and coagulation properties of milk (Part II)

The purpose of this study was to relate the genetic variants A and B of κ-casein (κ-CN) and β-lactoglobulin (β-Lg) of the milk of 10 Chilean Holstein–Friesian cows and its coagulation properties (coagulation ability of milk, curd firmness and syneresis of the curd).
The results showed that β-Lg AB phenotypes have a significant effect on the studied properties in comparison with the samples containing the AA phenotype. A and B variants of κ-CN did not have a significant effect on the coagulation properties, although there was a favourable interaction between β-Lg AB and κ-CN B.     

β-Lactoglobulin Separation from Whey Protein Isolate on a Large Scale

A method for obtaining large quantities of β-lactoglobulin (β-Lg) from commercial whey protein isolate (WPI) was developed. β-Lg was separated from the rest of the whey proteins in a solution of 15% (W:W) WPI in distilled water adjusted to pH 2 and 7% NaCl. β-Lg was then separated from NaCl using diafiltration. The results indicate that more than 65% of the β-Lg originally in the WPI solution was recovered. The purity of the β-Lg was greater than 95%.     

Protein composition of milk from Holstein–Friesian dairy cows and its relationship with the genetic variants A and B of κ-casein and β-lactoglobulin (Part I)

The protein composition and the milk yield (morning milking) were studied in 10 Chilean Holstein–Friesian first lactation dairy cows during the spring season. In addition, the genetic variants A and B of κ-casein (κ-CN) and β-lactoglobulin (β-Lg) were also identified. The results showed that the casein content is significantly affected by the presence of B variant of κ-CN. Moreover, positive interactions between the B variant of κ-CN with AB of β-Lg and the combination of A κ-CN with AA of β-Lg for the total protein content, were found.     

Retention of β-galactosidase activity in crude cellular extracts from Lactobacillus delbrueckii ssp. bulgaricus 11842 upon drying

Crude cellular extracts (CCEs) containing active β-galactosidase from Lactobacillus delbrueckii ssp. bulgaricus 11842 were spray-dried at three different outlet air temperatures (45, 55 or 65°C) or freeze-dried, with or without whey proteins, casein, whey or skim milk as drying adjuncts. The use of whey or skim milk resulted in significantly ( P   <  0.05) higher β-galactosidase activity retention in comparison to all other CCEs. This effect was not related to the initial total solids (TS) content (4–10%) of the feedstock solutions, but was presumably caused by the presence of lactose in the whey or skim milk CCE preparations.     

Qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milks using native-PAGE

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milk using whole milk samples as well as their whey protein fraction. Quantification was based on measuring band intensity of bovine β-lactoglobulins in all milk mixtures and bovine α-lactalbumin in caprine/bovine milk blends. Linear relationships were established between the band intensity of bovine β-lactoglobulins and α-lactalbumin vs. volume percentage of added bovine milk in all milk analysed, with the correlation coefficient from 0.9950 to 0.9998. These correlations enabling the quantification of bovine milk percentage within the wide range from 3% or 5% to 90% in caprine/bovine and ovine/bovine milk blends, respectively. The differences between the actual percentages of bovine milk present in the adulterated milk samples and those calculated using the regression lines were less than or equal to 5% for all samples. This method offers a rapid determination combined with unequivocal identification of the bovine whey proteins in almost every caprine/bovine or ovine/bovine milk mixtures.     

Selective Proteolysis of the Glycinin and β-Conglycinin Fractions in a Soy Protein Isolate by Pepsin and Papain with Controlled pH and Temperature

ABSTRACT: Proteolysis of soy protein isolates (SPI) was investigated by using pepsin with a pH of 1.5 to 4.0 at 37°C and papain at a temperature of 37°C to 80°C with pH 7.0. The glycinin fraction in native SPI was selectively hydrolyzed by pepsin in the pH 1.5 to 2.5 range. On the other hand, the p-conglycinin fraction in native SPI was selectively hydrolyzed by papain at 70°C. This selective proteolysis would be significantly correlated with the denaturation of glycinin and β-conglycinin in SPI. A protocol for preparing hydrolysates selectively enriched with glycinin or β-conglycinin was proposed.     

ISOLATION OF β2-CHACONINE, A POTATO BITTERNESS FACTOR

The 4-rhamnoglucoside of alkaloid solanidine, β₂-chaconine, was isolated from a mixture of α-solanine with β₂-chaconine by fractionation from hot ethyl acetate. The mixture of the two glycoalkaloids is obtained by endogenous enzyme action in a homogenate of potato berries and blossoms within a 24–36 h incubation at 37° C, pH 6.00. The identity of β₂-chaconine was ascertained by m.p., [α]D° IR spectrum, TLC of sugar components and reference to partial acid hydrolysis products of α-chaconine.     

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.