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.     

Effects of feed composition,protein denaturation and storage of milk serum protein/lactose powders on lactosylation

During whey powder production, the feed is subjected to several heat treatments which can cause lactosylation of proteins. In this study, lactosylation of whey proteins was evaluated in spray-dried powders before and after storage by varying the native protein fraction as well as the serum protein/lactose ratio in the powders. The lactosylation of native α-lactalbumin and β-lactoglobulin in the powders before storage was not affected to a large extent by the protein denaturation or if the feed had been heat treated in a high or low lactose environment. After storage (relative humidity of 23.5%, 30 °C, 25 days), the kinetic of lactosylation tended to increase with increasing native protein fraction and bulk protein content in the powders. An explanation could be that proteins dissolved in the lactose glassy structure might have a lower reactivity, while proteins present in the protein glassy structure with dissolved lactose may display higher lactosylation reactivity.     

Effect of high-pressure treatment on denaturation of bovine β-lactoglobulin and α-lactalbumin

The effect of high-pressure treatment on denaturation of β-lactoglobulin and α-lactalbumin in skimmed milk, whey, and phosphate buffer was studied over a pressure range of 450–700 MPa at 20 °C. The degree of protein denaturation was measured by the loss of reactivity with their specific antibodies using radial immunodiffusion. The denaturation of β-lactoglobulin increased with the increase of pressure and holding time. Denaturation rate constants of β-lactoglobulin were higher when the protein was treated in skimmed milk than in whey, and in both media higher than in buffer, indicating that the stability of the protein depends on the treatment media. α-Lactalbumin is much more baroresistant than β-lactoglobulin as a low level of denaturation was obtained at all treatments assayed. Denaturation of β-lactoglobulin in the three media was found to follow a reaction order of n = 1.5. A linear relationship was obtained between the logarithm of the rate constants and pressure over the pressure range studied. Activation volumes obtained for the protein treated in milk, whey, and buffer were −17.7 ± 0.5, −24.8 ± 0.4, and −18.9 ± 0.8 mL/mol, respectively, which indicate that under pressure, reactions of volume decrease of β-lactoglobulin are favoured. Kinetic parameters obtained in this work allow calculating the pressure-induced denaturation of β-lactoglobulin on the basis of pressure and holding times applied.     

Effect of dynamic high-pressure microfluidization at different temperatures on the antigenic response of bovine β-lactoglobulin

The antigenic response of β-lactoglobulin (β-Lg), treated by dynamic high-pressure microfluidization (DHPM) at different temperatures, was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from rabbit serum. DHPM treatment causes changes in the protein structure and may influence the antigenicity of β-Lg. DHPM treatment of β-Lg at 90 °C showed significant effects with the antigenic response of 5.2 μg mL⁻¹ (untreated), 45 μg mL⁻¹ (40 MPa), 79 μg mL⁻¹ (80 MPa), 132 μg mL⁻¹ (120 MPa), and 158 μg mL⁻¹ (160 MPa). In combination with temperature treatment (70–90 °C), the antigenic response enhanced as the temperature increased at 160 MPa. The β-Lg antigenicities were about 14, 108, and 158 μg mL⁻¹ at 70, 80, and 90 °C, respectively. However, the influence of DHPM pressures on the antigenic response of β-Lg standards was different. DHPM modified β-Lg standards showed a remarkable increase in antigenicity when treated to 80 MPa. Above 80 MPa, the antigenic response decreased.     

The effect of temperature and shear upon technological properties of whey protein concentrate: Aggregation in a tubular heat exchanger

Microparticulation of whey proteins at low concentration (2%, w/v), was examined in a pilot plant tubular heat exchanger (THE). Turbulent flow in combination with moderate temperatures (≤85 °C) was used in the heating section to prevent fouling, whereas the flow was varied from laminar to turbulent in the holding section of the THE. The logarithm of the formal rate of denaturation of β-lactoglobulin (β-Lg) k_f was −5.4 to −2.5 depending on the temperature. Variation of flow velocity in the holding section had a negligible impact on denaturation degree of β-Lg and particle size of agglomerates. A high increase of elastic modulus, G′, of agglomerates was combined with only bisection of water holding capacity. Advanced modifications of particle structure and properties are supposed to be achievable by more freedom in control of flow character at a heating section of a THE for example through application of direct heat transfer principles.     

Microwave irradiation under different pH conditions induced a decrease in β-lactoglobulin antigenicity

Whey proteins adjusted at pH values 2, 4.6, 9 and at the natural milk pH (pH 6.8) were subjected to microwave irradiation at 300 W for 20 min or 700 W for 10 min. The protein composition of treated and native whey proteins were evaluated by Lowry’s method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The ability of treated whey to bind IgG polyclonal antibody was determined by an enzyme-linked immunosorbent assay (ELISA) using sera obtained from rabbit immunized to β-Lactoglobulin (β-Lg). Significantly higher losses in soluble protein concentrations were observed for microwave irradiated whey proteins at pH value 4.6 (35.6% at 300 W and 44.33% at 700 W) (P < 0.0001) compared with those irradiated at the natural milk pH (26% at 300 or 700 W). The electrophoretic patterns of these proteins revealed a considerable decrease in intensity of the band corresponding to α-lactalbumin but only a slight modification was observed for the electrophoretic profiles of β-lactoglobulin. The data obtained with a rabbit anti-β-lactoglobulin immunoglobulin indicated a low antigenic response for microwave-irradiated whey proteins at the natural milk pH (up to 29.32% as well as 300–700 W) (P < 0.001). The lowest antigenicity was observed for samples adjusted to pH 4.6 followed by microwave irradiation at 300 or 700 W (46.99% at 300 W and 41.16% at 700 W) (P < 0.0001).     

Effect of pre- and post-heat treatments on the physicochemical,microstructural and rheological properties of milk protein concentrate-stabilised oil-in-water emulsions

The effect of heat treatment on the physical stability of milk protein concentrate (MPC) stabilised emulsions was investigated; 3% (w/w) MPC dispersions were preheated at 90 °C for 5 min at neutral pH prior to emulsification. Heat-treated (120 °C, 10 min) emulsions stabilised by preheated MPC had slightly fewer droplet–droplet interactions than that stabilised by unheated MPC because the whey proteins were pre-denatured (∼90% denaturation of the total whey proteins), which led to a reduction in subsequent heat-induced droplet–droplet and droplet–protein interactions. Emulsions stabilised by calcium-depleted MPC were also investigated. The presence of some non-micellar casein fractions gave better emulsification and may have conferred a protective stabilising effect on whey protein aggregation, in both the dispersed phase and the continuous phase during the secondary heat treatment. It was concluded that calcium manipulation and thermal modification of MPC can be utilised to control the functionality in oil-in-water emulsions.     

Influence of processing conditions on reducing γ-aminobutyric acid content during fortified milk production

The work studied the effects of processing conditions on the γ-aminobutyric acid (GABA) loss during fortified milk production. Bovine milk or their proteins/lactose fractions (0.66% whey protein and 2.6% casein or 4.9% lactose, w/v) containing 0.05–1.0% added γ-aminobutyric acid (w/w, based on bulk milk or these fractions) were subjected to a simulated milk technological process as following the sequential preheating (25–60 °C), homogenization (0–20 MPa), and pasteurization (62 °C/30 min, 72 °C/15 s, 95 °C/5 min, and 138 °C/2 s) or their unit processes to treat GABA. The resulting samples were characterized through GABA and lactose concentrations under various processing conditions. The amine and carboxyl groups and the structural characteristics of the resulting protein (lactose) were also examined through their concentrations (for lactose) and mass/spectral analyses, respectively. The results showed that the increase in temperature significantly promoted a reduction in GABA content. Whey protein fractions than caseins were primarily responsible for inducing GABA, whereas lactose had no remarkable effect on it. The rationale for GABA reduction is potential reactions with milk proteins/lactose, which preliminarily confirmed by the measurement of protein modification and lactose mass spectrometry.     

Yak milk whey protein denaturation and casein micelle disaggregation/aggregation at different pH and temperature

At the natural pH of yak milk (pH 6.6), a low level (<30%) of κ-casein (κ-CN) was found in the serum phase after heating at 95 °C for 30 min, indicating that as much as 70% of the β-lactoglobulin (β-Lg) and κ-CN complexes is associated with the micelle colloidal particles. The β-Lg and κ-CN levels increased from 13.2% and 2.6% at pH 6.0 to 35.2% and 60.1% at pH 7.0, respectively, when yak milk was heated at 95 °C for 30 min. At pH 6.0–6.4, the denatured whey proteins were associated with the caseins in the colloidal phase, resulting in milk gelation upon heating. The distribution of β-Lg and κ-CN complexes increased in the serum phase, demonstrated by the increasing levels of both β-Lg and κ-CN with increasing pH; at high pH (6.6–7.0), large proportions of β-Lg and α-lactalbumin were lost, presumably forming complexes in the colloidal phase.     

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.     

Available lysine and fluorescence in heated milk proteins/dextrinomaltose or lactose solutions

In order to predict and compare the effects of dextrinomaltose and lactose on available lysine loss by the Maillard reaction, six model systems were prepared by mixing casein, laboratory whey protein or commercial whey protein with dextrinomaltose or lactose. The solutions were prepared at concentrations similar to those used in enteral and infant formula processing and were heated at 100, 120 or 140 °C for 0–30 min. The progress of the Maillard reaction in these model systems was followed by monitoring free fluorescence intermediary compounds. Model systems with lactose showed higher available lysine less than the model systems with dextrinomaltose; linear lysine losses were obtained between 0 and 20 min at 100 and 120 °C. At sterilization temperature and time (120 °C/10 min), lysine losses of milk proteins with dextrinomaltose as reducing sugar were 6.1% for casein, 4.1% for laboratory whey protein and 13.4% for commercial whey protein. Available lysine showed correlation with furosine in model systems prepared with lactose and casein or laboratory but not commercial whey protein at 100 and 120 °C. The initial fluorescence value obtained by mixing casein or laboratory whey protein with lactose or dextrinomaltose was low (between 3.8 and 5.7), whereas the value obtained when commercial whey proteins were used was close to 9. At 120 °C/10 min, there was only a small increase of fluorescence in casein and laboratory whey protein but a large increase in commercial whey protein (threefold the initial value). Fluorescence measurement is useful for finding the extent of the Maillard reaction in commercial whey protein (thermally damaged protein). An absolute value greater than 10 may indicate that products were prepared with thermally damaged proteins.     

Improving the structure and rheology of high protein,low fat yoghurt with undenatured whey proteins

The objective of this study was to investigate the effects of whey protein denaturation and whey protein:casein-ratio on the structural, rheological and sensory properties of high protein (8% true protein), low fat (<0.5% fat) yoghurt. Yoghurt milk bases were made by adding undenatured whey proteins from native whey protein concentrate (NWPC) to casein concentrate in different whey protein:casein-ratios. The degree of whey protein denaturation was then controlled by the temperature treatment of the yoghurt milk bases. Addition of NWPC in low (whey protein:casein-ratio 25:75) or medium levels (whey protein:casein-ratio 35:65) in combination with heat treatment at 75 °C for 5 min gave yoghurts with significantly lower firmness, lower storage modulus (G′), and better sensory properties (less coarse and granular and more smooth), compared with corresponding yoghurts produced from yoghurt milk bases heat-treated at 95 °C for 5 min or with control yoghurts (no addition of NWPC).     

Fractionation of casein micelles and minor proteins by microfiltration in diafiltration mode. Study of the transmission and yield of the immunoglobulins IgG,IgA and IgM

There is little information concerning the fractionation by microfiltration (MF) of casein micelles and immunoglobulins plus other minor whey proteins with ceramic gradient membranes. The order of transmission was α-lactalbumin (α-La, 2.3 nm)> β-lactoglobulin (β-Lg; 4.2 nm)>IgG (10.7 nm)> lactoperoxidase (8.2 nm)> IgA (18.1 nm)> IgM (23.8 nm)> lactoferrine > blood serum albumin (7.8 nm), irrespective of the applied transmembrane pressure (0.6–3 bar) and equal to 55% > 50% > 47% > 41%>39% > 32% > 22% > 19%. Including preconcentration, it was possible to obtain 90% of the initial IgG, IgA and IgM within 85, 119, and 160 min, based on 1 m² of membrane area and 50 L of skim milk volume. The long-term process exposure at 50 °C did not affect α-La and IgG but β-Lg (3–5% denaturation), which, however, was selectively retained by the MF. In conclusion, MF is not only suitable for fractionation of the major whey proteins and caseins, but also for the minor and far bigger immunoglobulins.     

A Kinetic Study on the Heat-Induced Changes of Whey Proteins Concentrate at Two pH Values

Whey protein concentrate (WPC) is used as food ingredients due to their commercially important functional properties. The effects of heat treatment on the components of milk are very important for the final product character, since they undergo modifications that affect sensorial and nutritional quality of milk. The heat-induced changes on dispersions of whey proteins concentrate were monitored by measurement of thiol availability, protein solubility, and turbidity at pH 6.6 and 7.5. The fractional conversion model was used to quantitatively describe the effect of different temperature–time combination on denaturation mechanism. The results demonstrate that heat-induced changes of WPC greatly influence their solubility, expressed as degree of denaturation at pH 4.6 and were related to the heating conditions. The denaturation mechanism involved a number of consecutive conformational changes in the molecules. A curvature in Arrhenius plots was observed around 75 °C, indicating changes in the reaction mechanism. The deflection of Arrhenius plot reflects the generally accepted two-step denaturation/aggregation process of whey proteins.     

The thermal denaturation of the total whey protein in reconstituted whole milk

The kinetic parameters for thermal denaturation of the total whey proteins in whole milk were determined. Denaturation was a second‐order reaction, and an Arrhenius plot showed a change in slope at ~85 °C. At 70–85 °C, the activation energy, enthalpy and entropy were in the range expected for denaturation processes, whereas at 85–115 °C, these parameters were typical for chemical reactions such as aggregation. Equations to predict the denaturation after heating were developed and tested on a range of independently prepared milk samples. There was a good agreement between the predicted and the experimentally determined denaturation levels.     

Optimization of β-carotene production by a mutant of the lactose-positive yeast Rhodotorula acheniorum from whey ultrafiltrate

The present work investigated on carotenogenesis with high β-carotene content by a new isolated high-activity strain-producer Rhodotorula acheniorum mutant MRN in cheese whey ultrafiltrate. After a serial of UV, ethymethanesurfonate (EMS), and nitrosoguanidine (NTG) mutagenesis, a mutant named MRN of the red lactose-positive yeast strain R. acheniorum was obtained. Then, the effects of different growth medium factors on carotenoid production by this mutant at batch-scale level were identified and optimized by means of response surface methodology (RSM) in order to achieve high-level production of β-carotene. The optimum conditions required to achieve the highest level of β-carotene (262.12±1.01 mg/L) were determined as follows: whey ultrafiltrate (WU) lactose concentration 55 g/L, pH 5.85, ammonium sulfate concentration 3.5 g/L, temperature 23°C, and aeration rate 1.56 vvm. The medium optimization resulted in a 6.45-fold increase in volumetric production (262.12±1.01 mg/L) and a 4.62-fold increase in the cellular accumulation (10.69±0.19 mg/g) of β-carotene.     

Glycation compounds in peanuts

In the present study, the extent of the Maillard reaction in peanuts was investigated, using N-ε-fructosyllysine (ε-Fru-Lys, determined via furosine) as an indicator for the early stage, and pyrraline and N-ε-carboxymethyllysine (CML) as representatives for advanced glycation. In commercial samples, ε-Fru-Lys ranged between 1.5 and 13.3 mmol/mol lysine. Pyrraline was found in amounts between not detectable (below 0.3 mmol/mol lysine) and 9.0 mmol/mol lysine, and CML between 0.8 and 2.7 mmol/mol lysine. Lysine modification by glycation products was very low in cooked and fried peanuts (below 1%). In laboratory-scale roasting experiments, the amount of ε-Fru-Lys reached maximum values of 12.0 mmol/mol lysine after 20 min at 160 °C, whereas pyrraline increased up to 38.5 mmol/mol lysine after 25 min at 170 °C. Amount of CML was up to 1.8 mmol/mol lysine in peanuts roasted for 25 min at 170 °C. Such high amounts of pyrraline have not yet been described for any other food item. Only about one tenth of the totally observed lysine modification of up to 50% can be explained by the three glycation compounds, indicating that currently unknown reactions occur during peanut roasting. Reactions between proteins and carbonyl compounds, most likely originating from oxidative degradation of lipids, may play an important role for lysine derivatization in peanuts and should be analyzed more in detail.     

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.     

Usefulness of some Maillard reaction indicators for monitoring the heat damage of whey powder under conditions applicable to spray drying

The Maillard reaction (MR) rate was observed according to available lysine loss, furosine, hydroxymethylfurfural (HMF), furfural, and brown colour during the heating of freeze-dried nano-filtered whey at 60, 75, and 90 °C and water activities of 0.11, 0.33, 0.43, and 0.73. The physical state of lactose was measured and associated with MR rate. The values obtained for available lysine, furosine, HMF and browning index ranged between, respectively, 11.3 and 1.63 (g 100 g⁻¹ protein), 0.44 and 11.1 (g 100 g⁻¹ protein), not detected and 57.7 (mg 100 g⁻¹ protein) and 0.0104 and 0.1707. The greatest heat damage occurred with low moisture content and high temperature. The MR rate was influenced by the physical state of lactose, heating temperature and the moisture content of the whey. This occurred to a greater extent during the initial and intermediate stages of the MR as opposed to during the formation of coloured compounds.     

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).