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.     

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.     

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.     

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.     

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.     

Adsorption of β-Lactoglobulin variants A and B to the air–water interface

Summary The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of β-lactoglobulin (β-lg) differ by only two amino acids (Asp-64, Val-118 in A, and Gly-64, Ala-118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of β-lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer : monomer equilibrium in the bulk solution.     

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%。     

Interactive Effects of Pressure, Temperature and Time on the Molecular Structure of β-Lactoglobulin

We studied the combined effects of several variables, using temperatures up to 79°C and pressures up to 105 MPa, on β-lactoglobulin at pH 7.0 and pH 5.6 by examination of circular dichroism spectra and application of experimental design methodology. The higher pressures were not sufficient to impart the energy necessary to disrupt the protein structure. When used in combination with temperature and holding time, the applied energy was sufficient to disrupt the tertiary structure of the protein. Processing conditions applied at specific pH therefore could act in combination to cause structural changes.     

反胶团法提取文冠果种仁蛋白的后萃条件研究

以文冠果种仁为原料,利用反胶团法萃取文冠果蛋白,在前萃的基础上研究后萃试验工艺参数.试验考察了后萃条件即后萃水相pH、离子浓度、后萃温度、后萃时间等对文冠果蛋白后萃率的影响.后萃最佳工艺条件为:KCl浓度1.2 moL/L、pH值4.0、后萃时间45 min、后萃温度为60℃.KCl浓度、pH值和温度对后萃率的影响均显...     

Effect of anions on the denaturation and aggregation of β-Lactoglobulin as measured by differential scanning microcalorimetry

Summary Profound differences in the rate of aggregation and denaturation among β-lactoglobulin samples from different sources can be reduced or even eliminated by dialysis of the samples first against a phosphate buffer. Visual evidence and thermograms indicated a reduced rate of aggregation in a phosphate buffered salt solution compared to the salt solution alone. The same effect was not achieved by another divalent tetrahedral ion, sulphate, nor by imidazole-buffered NaCl. Replacement of the background electrolyte by Na acetate had no discernible effect on the thermogram in the phosphate buffer. Phosphate is particularly good at buffering the protein solution at neutral pH at elevated temperature so the main effect is to maintain the net protein charge. In the imidazole and unbuffered media, the protein can move towards its isoelectric point and although the protein is more stable, aggregation is faster. It is speculated that there might also be a specific interaction of the phosphate anion with a cation-rich region of the β-lactoglobulin surface which could also inhibit aggregation at elevated temperatures.     

Enhanced β-galactosidase production by supplementing whey with cauliflower waste

Among the five Kluyveromyces marxianus strains tested for β-galactosidase production, K. marxianus NCIM 3465 showed maximum enzyme activity of 1.62 IU mg⁻¹ dry weight. Different levels (5–25%, w/v) of dried cauliflower waste were incorporated into whey to evaluate the effect of its supplementation on enzyme production. Although a marginal increase in enzyme production was seen by incorporating 5% and 10% cauliflower waste in whey, nearly 15% increase in β-galactosidase production was observed when cauliflower waste level was increased to 20% compared with whey alone. Supplementing whey with 20% cauliflower waste also decreased the production time. Lactose concentration in whey, mainly responsible for increasing the biological oxygen demand load of the effluent water, decreased from 4.2% to nearly 0% at 24 h. Thus, this study demonstrated that both these by-products/residues could be effectively used for β-galactosidase production at commercial scale.     

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.