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.     

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%.     

β-Lactoglobulin – a three-dimensional perspective

Summary Bovine β-lactoglobulin has been the subject of intense study over the past 60 years by, effectively, every available physicochemical technique, of which one of the most powerful is X-ray crystallography. We present a short review of the X-ray crystallographic work on milk protein together with an overview of the properties as they are seen from the current state of crystallographic analyses. At the present time, structural analyses do not provide any further insights into the possible function of the protein.     

Quantitative X-Ray Diffraction Determination of α-Lactose Monohydrate and β-Lactose in Chocolate

ABSTRACT:  Lactose is a constituent of milk chocolate. During processing and cooling, lactose may precipitate as α-lactose monohydrate and β-lactose. The presence of α-lactose monohydrate has a deleterious effect on the quality of milk chocolate. A quantitative X-ray diffraction method for determination of α-lactose monohydrate and β-lactose in chocolate is described. The α-lactose monohydrate signal at 19.9°2θ with Cu-Kα X-rays is a cubic function of concentration. The β-lactose signal at 20.9°2θ is a linear function of concentration. α-Lactose monohydrate is detectible at about 0.1 weight% and can be quantified at >0.5 weight%. β-Lactose is detectible at about 1 weight% and can be quantified at >3 weight%. About 10 min is required to prepare and run a sample.
Practical Application: The crystalline form of lactose affects the quality of chocolate. A rapid method for quantifying crystalline forms of lactose in chocolate is described. The method can be used for quality control and for improving chocolate quality.     

Specific Disulfide Bond in α-Lactalbumin Infiuences Heat- Induced Gelation of α-Lactalbumin-Ovalbumin-Mixed Gels

The gel strength of ovalbumin mixed with α-lactalbumin (α-La) was determined after heating at 80°C for 15 min at pH 7.0 Gel strength of the mixture of 4%α-La and 4% ovalbumin was two times that of 8% ovalbumin. Modified α-La at Cys6-Cys120 (3 SSα-La) had low enhancement effects on ovalbumin gelation. Competitive ELISA using monoclonal antibody to α-La showed decreased binding reactivities after heating α-La with ovalbumin at specific concentrations. The decrease of total SH groups in the gel mixed with 3SS α-La was much less than when mixed with α-La. A disulfide bond of Cys6-Cys120 in α-La contributed to interactions between ovalbumin and α-La in heat-induced gels.     

Oxymyoglobin and Lipid Oxidation in Phosphatidylcholine Liposomes Retarded by α-tocopherol and β-carotene

An n-3 fatty acid-constructed oxymyoglobin liposome model was used to study the effects of 10 μM α-tocopherol and/or various concentrations of β-carotene on oxymyoglobin and lipid oxidation. Oxidation was delayed by α-tocopherol or β-carotene treatments alone (p < 0.05). β-Carotene at 1.5 μM and 2μM caused greater delay in oxymyoglobin oxidation than α-tocopherol (p < 0.05). For lipid oxidation inhibition, 2 μM β-carotene was similar in effect to α-tocopherol. With α-tocopherol plus 1.5 or 2 μM β-carotene, greater oxidation delaying effect resulted for both oxymyoglobin and lipid oxidations than with 1.5 or 2 μM β-carotene treatment alone (p < 0.05).     

α-Tocopherol, β-Carotene and Ascorbic Acid as Antioxidants in Stored Poultry Muscle

Broilers were fed α-tocopherol or β-carotene for 3 wk or L-ascorbic acid for 24 hr prior to slaughter. α-Tocopherol maintained the redness of unheated meat stored for 8 wk (—20°C). Values for thiobarbituric acid reactive substances in ground, stored meat showed that L-ascorbic acid produced results similar to the control while a-tocopherol produced results lower than the control. Panelists rated meat with added α-tocopherol as different from the control for smell and flavor. β-Carotene was a pro-oxidant compared to the control and other additives. Meat from broilers fed β-carotene had lower α-tocopherol content than the control. Vitamin A in livers of birds fed β-carotene was 64% higher than that of the control.     

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.     

Interactions of (β-Lactoglobulin and High-methoxyl Pectins in Acidified Systems

The interactions that occur when β-lactoglobulin (β-lg) is mixed with a high-methoxyl pectin (HMP) and a modified pectin (mHMP, modified using plant pectinesterase) were examined at pH 3.8. Whereas soluble aggregates formed in β-lg-HMP, β-lg-mHMP precipitated upon mixing. β-lg-HMP mixtures showed soluble aggregates with larger hydrodynamic diameters when heated at 65°C than when heated at 90°C. β-lg-HMP mixtures adjusted to pH 6.0 after heating showed that the aggregates formed at 65°C could be dissociated, but the complexes were not reversible after heating at 90°C. A similar effect also was observed when resuspending the°-lg-mHMP precipitates at pH 6.0. The behavior of the 2 pectins was attributed to their differences in charge distribution.     

Growth and structure of aggregates of heat-denatured β-Lactoglobulin

Summary The aggregation of the globular protein β-lactoglobulin after heat-denaturation was studied in aqueous solution at pH 7 using static and dynamic light scattering. The structure of the aggregates is self-similar with fractal dimension 2.0. Size exclusion chromatography and dynamic light scattering measurements show that the aggregates have a broad size distribution. Initially clusters of about 85 proteins and 15 nm radius are formed which are the elementary units of the larger fractal aggregates. At low ionic strength the formation of the larger aggregates is impeded by electrostatic interactions.
The structure of the aggregates is independent of the concentration and the temperature. The rate of aggregation has an Arrhenius temperature dependence with an activation energy of about 350 kJ/mol weakly dependent on the concentration. The apparent reaction order of the aggregation is 1.5. In the mixture both variants A and B have the same aggregation rate. The gel time increases with decreasing concentration and diverges at about 0.7g L⁻¹. At lower concentration the aggregate growth stagnates when all protein has aggregated.     

Functional Improvement of Alginic Acid by Conjugating with β-Lactoglobulin

Two bovine β-lactoglobulin-alginic acid (β-LG-ALG) conjugates were prepared to improve the function of ALG by using water-soluble carbodiimide and the Maillard reaction. Fluorescence studies suggested that the conformation around Trp had been changed in each conjugate and that the surface of each conjugate was covered with polysaccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around 15Val-29IIe (β -sheet) in each conjugate had changed, while the native structure was maintained around 125Thr-135Lys (α-helix). After conjugating with β -LG, ALG showed retinol-binding and high emulsifying ability. The aggregating property of ALG in acid and in the presence of Ca²⁺ was improved in each conjugate.     

Calcium-Induced Destabilization of Oil-in-Water Emulsions Stabilized by Caseinate or by β-Lactoglobulin

The stability to aggregation of 20% soya oil-in-water emulsions stabilized by 0.3 to 2% sodium caseinate or β-lactoglobulin in the presence of calcium chloride solutions was studied using light scattering and electron microscopy. Stability increased with the amount of protein in the emulsion, and decreased with the concentration of added calcium. Growth of particle size with concentration of Ca²⁺ was more in emulsions containing lower concentrations of protein. Sodium chloride at 50 and 100 mM stabilized both systems to the presence of calcium ions. Microstructure and light scattering showed caseinate emulsions formed clusters even at low concentrations of Ca²⁺ while β-lactoglobulin emulsions formed extensive strands.     

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.     

Relation of β-Lactoglobulin – Sodium Polypectate Aggregation to Bulk Macromolecular Concentration

Aggregation of β-Lactoglobulin β-Lg) solutions, with and without sodium polypectate (SPP), was investigated at pH 6.5 and 3.5 by turbidity measurements and gel permeation chromatography during heating at 1°C/min. The ratio of β-Lg:SPP was maintained at 10:1. At pH 6.5, the transition temperature of β-Lg aggregation decreased linearly with the logarithm of β-Lg concentration. Irrespective of β-Lg concentration, SPP did not affect the rate of β-Lg aggregation during heating at pH 6.5. However, SPP influenced the formation of high-molecular-weight (HMW) β-Lg aggregates during heating at pH 6.5 was related to bulk macromolecular concentration. No thermal aggregation transitions were detected for β-Lg solutions at pH 3.5. SPP interacted with β-Lg at pH 3.5 to form a complex that precipitated on heating.     

Glycation of bovine β-Lactoglobulin: effect on the protein structure

Summary Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60 °C) in an aqueous solution and after a treatment under a restricted water environment (50 °C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non–covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β-LG, confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein.     

Chemical changes in β-Lactoglobulin structure during ageing of protein-stabilized emulsions

Summary Commercial protein-stabilized emulsions are often stored for many months. Reversed phase high performance liquid chromatography (HPLC) analysis of protein displaced from β-lactoglobulin-stabilized oil-in-water emulsions by the competitive adsorption of Tween 20, showed that whereas the retention time of protein displaced from a tetradecane–water interface did not change greatly upon ageing, that of the protein displaced from a soya oil–water interface did. The changes in the retention time of the displaced protein were accompanied by an increase in the mass of the protein. When 2-mercaptoethanol was added prior to emulsification, the rate of modification was significantly slower than in its absence. Tryptic digests of the displaced, modified protein showed that the changes were specific. Analysis of volatile components present in the emulsions showed that emulsification induced the autoxidation of the polyunsaturated fatty acyl chains of the soya oil resulting in the time-dependent formation of low molecular weight compounds. 2-Mercaptoethanol inhibits the process probably by reacting with hydroperoxides and terminating the chain reaction. Aldehydes, particularly enals, are known to react with nucleophilic amino acid residues such as lysine via addition and/or condensation reactions, leading to the observed increase in the mass of the protein.     

Structural and interaction properties of β-Lactoglobulin as studied by FTIR spectroscopy

Summary New aspects of β-Lactoglobulin (BLG) structure which have not previously been found by FT–IR spectroscopy are presented. The conformational changes of BLG and its heat-induced denaturation have been studied at low concentration and different pHs. First, it was found that the spectra of BLG in solution are concentration-dependent. Below 1%, they reveal one component in the 1620–1635 cm⁻¹ region while above this concentration, two components are present. These changes are related to the modifications of the quaternary structure of BLG, i.e. to the monomeric or dimeric forms. As shown at high temperature (85 °C), this concentration (1%) represents the threshold of protein aggregation. Second, the thermal behaviour of BLG at pH 7.4 and 4.4 is compared. The results suggest that the denaturation process and the intermolecular interactions in aggregates are different and lead to two types of aggregate. These observations are in agreement with the formation of two gel microstructures near neutral pH and near the pI of BLG as observed by Stading & Hermasson (1990). Finally, interactions between BLG and two zwitterionic phospholipids have been investigated. Dipalmitoylphosphatidylcholine (DPPC) is unaffected in the presence of BLG, suggesting that no interaction occurs. In contrast, BLG increases the lipid chain conformational disorder of milk sphinglomyelin (SM) as a consequence of hydrophobic interactions of BLG with SM. Since this effect occurs at and above the gel-to-liquid-crystalline phase transition, it is suggested that membrane fluidity plays an important role in these interactions.     

Quenching Mechanisms and Kinetics of α-Tocopherol and β-Carotene on the Photosensitizing Effect of Synthetic Food Colorant FD&C Red No. 3

ABSTRACT: Effects of FD&C Red No. 40, Red No. 3, Yellow No. 5, Yellow No. 6, Green No. 3, Blue No. 1 and Blue No. 2 on 0.03M soybean oil oxidation in acetone at 25 °C under light were studied by measuring headspace oxygen depletion. As Red No. 3 increased from 0 to 5, 20, 100 and 200 ppm, the headspace oxygen was reduced by 2 to 70, 73, 77 and 77%, respectively, for 4 h. Only Red No. 3 acted as a photosensitizer to produce singlet oxygen in the oil. The quenching rates of α-tocopherol and β-carotene for the singlet oxygen by Red No.3 were 4.1 × 10⁷ M⁻¹s⁻¹ and 7.3 × 10⁹ M⁻¹s⁻¹, respectively. When β-carotene was below 1.86 × 10⁻⁶ M, β-carotene quenched singlet oxygen, but it quenched both singlet oxygen and Red No. 3 at or above 3.72 × 10⁻⁶ M. However, α-tocopherol quenched singlet oxygen only.     

反胶团萃取分离葡萄酒下脚料中葡萄皮蛋白质

采用阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)反胶团萃取葡萄皮中的蛋白质。分别考察水相pH值、表面活性剂浓度、离子强度、蛋白质浓度、有机溶剂与助剂种类和比例、萃取温度对前萃取率的影响,并采用二次正交旋转组合的试验方法,考察前萃取过程中离子强度、表面活性剂浓度、水相pH值对前萃取率的影响。结果表明:32.68 mmol/L CTAB/三氯甲烷-正丁醇(体积比4∶1)的反胶团体系用于前萃取水相pH为6.4,包含0.4 mol/L NaCl的葡萄皮中蛋白质的浸提液,前萃取率可达56.54%。理想的前萃取条件是:pH 6.4、NaCl浓度0.4 mol/L、CTAB浓度32.68 mmol/L、蛋白质浓度1 mg/mL、萃取温度30℃。在该条件下,采用pH 3.8、后萃取温度20℃、0.8 mol/L NaCl水相进行后萃取,后萃取率可达71.29%。