Denaturation Of Α-lactalbumin Research Articles

Counteraction Effects of Ammonium-Based Ionic Liquids on Urea-Induced Denaturation of α-Lactalbumin: A Comprehensive Molecular Simulation Study.

Ionic liquids (ILs) are known to stabilize protein conformations in aqueous medium. Importantly, ILs can also act as refolding additives in urea-driven denaturation of proteins. However, despite the importance of the problem, detailed microscopic understanding of the counteraction effects of ILs on urea-induced protein denaturation remains elusive. In this work, atomistic molecular dynamics (MD) simulations of the protein α-lactalbumin have been carried out in pure aqueous medium, in 8 M binary urea-water solution and in ternary urea-IL-water solutions containing ammonium-based ethyl ammonium acetate (EAA) as the IL at different concentrations (1-4 M). Attempts have been made to quantify detailed molecular-level understanding of the origin behind the counteraction effects of the IL on urea-induced partial unfolding of the protein. The calculations revealed significant conformational changes of the protein with multiple free energy minima due to its partial unfolding in binary urea-water solution. The counteraction effect of the IL was evident from the enhanced structural rigidity of the protein with propensity to transform into a single native free energy minimum state in ternary urea-IL-water solutions. Such an effect has been found to be associated with preferential direct binding of the IL components with the protein and simultaneous expulsion of urea from the interface, thereby providing additional stabilization of the protein in ternary solutions. Most importantly, modified rearrangement of the hydrogen bond network at the interface due to the formation of stronger protein-cation (PC) and protein-anion (PA) hydrogen bonds by breaking relatively weaker protein-urea (PU) and protein-water (PW) hydrogen bonds has been recognized as the microscopic origin behind the counteraction effects of EAA on urea-induced partial unfolding of the protein.

Denaturation of α-lactalbumin and bovine serum albumin in pressure-treated reconstituted skim milk

The kinetics of α-lactalbumin and bovine serum albumin (BSA) denaturation in pressure-treated reconstituted skim milk samples at pressures of 100–800 MPa and temperatures of 10–40 °C was studied. No denaturation was observed at pressures up to 400 MPa, whereas at 500 MPa or higher, the level of denaturation increased with increasing holding time at each pressure/temperature, with increasing pressure at each holding time/temperature or with temperature at each pressure/holding time. BSA denatured at a faster rate than α-lactalbumin, and denaturation for both proteins could be described as second order. Evaluation of the kinetic and thermodynamic parameters suggested that α-lactalbumin denaturation was an aggregation-limited reaction, whereas for BSA, the denaturation transitioned from aggregation-limited to unfolding-limited as the pressure increased.

The effect of ultrafiltration on the acid gelation properties of protein-standardised skim milk systems

In this study, we investigated the impact of ultrafiltration (UF) on the acid gelation of milk using two protein-standardised milk systems, consisting of either skim milk and retentate (SR) or permeate and retentate (PR), over different seasons in New Zealand. The composition and the physicochemical properties of the two systems before heating were comparable, whereas the levels of heat-induced α-lactalbumin denaturation and the association of the casein micelles with α-lactalbumin were significantly lower in PR than in SR. PR displayed superior acid gelation properties compared with SR, which was most pronounced in the late season. The structural modifications of the whey proteins and casein micelles that were induced by UF and the re-equilibration of calcium in the milk mixtures may have affected the acid gelation properties of the milk by influencing the denaturation and micelle association of the whey proteins. The results suggest that UF has the potential as a tool for tuning the acid gelation properties of milk.

Modifying whey protein concentrate/isolate by heating in the presence of cysteine

Heating whey protein concentrate (WPC) or isolate (WPI) solutions at 63 °C and in the presence of cysteine (CYS) denatured α-lactalbumin. The denaturation of α-lactalbumin was described as a first order reaction, with the rate constant increasing linearly with the log of the CYS concentration. Acidifying the WPC/WPI solutions that were heated in the presence of CYS produced a precipitate of denatured α-lactalbumin and a serum containing the native whey proteins. These precipitates/serums could be separated (centrifugation or ultrafiltration/microfiltration) and dried to produce WPC and WPI powders that were either enriched in β-lactoglobulin/depleted in α-lactalbumin or enriched in denatured α-lactalbumin/depleted in β-lactoglobulin, although the latter was difficult to dry. When added to skim milk before heating and acidification, β-lactoglobulin-enriched WPC markedly increased the stiffness of the acid gels; β-lactoglobulin-enriched WPI slightly increased the stiffness of the gels. The powder enriched in denatured α-lactalbumin decreased the stiffness of the acid gels.

Kinetics of heat-induced denaturation of proteins in model infant milk formulas as a function of whey protein composition

The process of manufacturing infant milk formulas (IMFs) involves heat treatments that can lead to whey protein denaturation. The objective of the study was to determine how protein composition affects the denaturation kinetics of the whey proteins within IMFs. Three model IMFs (1.3% of cow’s milk protein) were produced with a caseins: whey proteins ratio of 40:60, differing only by the whey protein composition. The kinetics of heat-induced denaturation of α-lactalbumin, β-lactoglobulin and lactoferrin were investigated between 67.5 °C and 80 °C by chromatographic quantification of the residual native proteins. Results showed that the heat-denaturation of α-lactalbumin was reduced when β-lactoglobulin was absent. The heat-denaturation of lactoferrin was not affected by the composition of the IMFs but its presence enhanced the heat-denaturation of β-lactoglobulin. This study revealed that, for higher heat treatments (90 °C/15 s, 75 °C/15 min), IMF containing α-lactalbumin and lactoferrin preserved a higher proportion of native whey proteins than IMFs containing β-lactoglobulin.

The effect of thiol reagents on the denaturation of the whey protein in milk and whey protein concentrate solutions

Adding cysteine (CYS-HCl) to skim milk, whey protein isolate (WPI), or milk/WPI solutions promoted the irreversible denaturation of α-lactalbumin at temperatures between 61 and 70 °C, whereas little denaturation of β-lactoglobulin occurred at these temperatures. Above 70 °C, CYS-HCl promoted the irreversible denaturation of both α-lactalbumin and β-lactoglobulin. A greater effect was observed at higher CYS-HCl concentrations at any given pH, or at higher pH at any given CYS-HCl concentration. Similar effects were observed with glutathione and 2-mercaptoethanol. It is proposed that at temperatures between ∼61 and 70 °C, CYS-HCl initiates thiol-disulphide exchange reactions with the disulphide bonds on unfolded α-lactalbumin, but not with those on the native β-lactoglobulin, thus α-lactalbumin irreversibly aggregates. At higher temperatures, CYS-HCl and the free thiol on unfolded β-lactoglobulin initiates thiol-disulphide exchange reactions with the disulphide bonds on both α-lactalbumin and β-lactoglobulin, rapidly aggregating both proteins via disulphide bonds and non-covalent interactions.

Influence of heating treatment and membrane concentration on the formation of soluble aggregates

This study investigated the effect of thermal denaturation on milk concentrated by ultrafiltration (UF) with or without diafiltration (DF) at various time–temperature combinations. The non-sedimentable protein fractions present in milk after heating were analyzed using size exclusion chromatography and their composition was determined. The extent of whey protein denaturation and the composition of the soluble complexes depended not only on heating temperature and time, but also on the protein concentration. There was a greater extent of protein aggregation in concentrated milk compared to 1× milk, and there was a significant difference in the size and composition of the aggregates present in the non-sedimentable fraction depending on whether the milk was concentrated only by UF or if DF was also used. DF milk contained a higher concentration of non-sedimentable protein compared to UF milk. In all samples, κ-casein and β-lactoglobulin were the main proteins present in the soluble aggregates, but higher temperatures and longer heating times resulted in greater extents of denaturation of α-lactalbumin. This work clearly identified differences in the extent of aggregation between milk concentrated by UF or UF and DF, and unconcentrated milk, with important consequences on the processing functionality of caseins.

Denaturation of α-lactalbumin and myoglobin by the anionic biosurfactant rhamnolipid

Glycolipid biosurfactants (GBS) are promising environmentally friendly alternatives to chemical surfactants. Surfactants interact with proteins in many applications, often leading to significant changes in protein properties. Given GBS' marked difference in structure compared to traditional chemical surfactants, it is of interest to investigate their impact on protein structure and stability. Here we combine spectroscopic and calorimetric studies to analyze the interactions between the anionic GBS rhamnolipid (RL) and two model proteins α-lactalbumin in the Ca2+-free apo-form (αLA) and myoglobin (Mb), whose interactions with traditional surfactants are well known. RL denatures αLA at sub-cmc concentrations (0.1–1mM) while Mb is only denatured above the cmc, i.e. in the presence of RL micelles. Denaturation leads to increased α-helicity, similar to the effect of SDS. The proteins bind approximately the same amount of RL by weight as SDS. However, RL employs a denaturation mechanism which combines features from non-ionic surfactants (very slow unfolding kinetics and few unfolding steps) with those of SDS (unfolding below the cmc in the case of αLA and the ability to unfold stable proteins in the case of Mb). We ascribe these features to RL's weakly acidic carboxylic head group and complex hydrophobic tail, which lead to a low cmc and low protein affinity. These features restrict the concentration range where RL monomers can bind and denature proteins while still allowing micelles to bind and denature to a significant extent.

Assessing the heat induced changes in major cow and non-cow whey proteins conformation on kinetic and thermodynamic basis

In this study, the RP-HPLC technique was used as the main analytical method to measure the residual native β-lactoglobulin and α-lactalbumin concentration after heat treatment in raw milk from three different species (goat, sheep and cow). Further, a detailed comparative kinetic study of β-lactoglobulin and α-lactalbumin denaturation was carried out at temperature ranging from 72.5 to 90°C.Kinetic studies showed that the thermal denaturation of β-lactoglobulin followed biphasic behavior, resulting in activation energy of 91.68±13.18kJmol−1, 137.13±25.25kJmol−1 and 62.11±3.26kJmol−1 for the denaturated fraction in goat, sheep and cow milk and 307.91±61.29kJmol−1, 158.99±23.64kJmol−1 and 170.18±43.61kJmol−1 for the native fraction in milk samples. α-Lactalbumin denaturation followed the first-order kinetics, resulting in activation energy values of 202.65±1.42kJmol−1, 155.56±5.53kJmol−1 and 140.44± 6.14kJmol−1 respectively in goat, sheep and cow milk. The heat-induced changes in protein structure were outlined after running molecular dynamics simulations at different temperatures, supporting the experimental observations. These experiments were conducted only for cow and goat α-lactalbumin and were limited by the lack of protein structures from databases.

Effect of Denaturation of α-Lactalbumin on the Formation of BAMLET (Bovine α-Lactalbumin Made Lethal to Tumor Cells)

A complex of alpha-lactalbumin with oleic acid, also known as HAMLET/BAMLET (human/bovine alpha-lactalbumin made lethal to tumor cells), causes apoptosis-like death in tumor cells but has little effect on healthy differentiated cells. The aim of this study was to examine whether irreversible denaturation of alpha-lactalbumin is detrimental to the formation and cytotoxicity of BAMLET. Commercial bovine holo alpha-lactalbumin (1-4% w/v) was heated at 80 degrees C for up to 100 min. With an increasing concentration of protein, the denaturation of alpha-lactalbumin proceeded faster, and aggregation became more extensive. Native and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a high proportion of the aggregates was linked by disulfide bonds. BAMLET was prepared from native and heat-treated alpha-lactalbumin according to a previously described chromatographic method. Despite the high content of denatured and aggregated alpha-lactalbumin in the heat-treated samples, their conversion into BAMLET was not negatively affected, resulting in BAMLET complexes partly composed of covalently linked aggregates of alpha-lactalbumin. The cytotoxicity of all prepared BAMLET samples was comparable to that of the control sample prepared from native alpha-lactalbumin (LD(50) = 34.6 +/- 2.7 mumol L(-1)). It was concluded that alpha-lactalbumin is not required to be in its native conformation for the conversion into its biologically active BAMLET complex.

Effect of pH at pressure treatment on the acid gelation of skim milk

Abstract Skim milk at pH between 6.4 and 7.3 was pressure treated at 200–600 MPa for 30 min and then slowly acidified with glucono-δ-lactone to form acid gels. Milks at low pH produced acid gels with low elastic moduli (final G ′) and yield stresses and those at higher pH produced acid gels with higher final G ′ and yield stresses. Pressure treatment disrupted the casein micelles at all pH and transferred high levels of casein to the serum phase. Denaturation of α-lactalbumin occurred at a pressure of 600 MPa only, and the level of denaturation increased with increasing pH. Denaturation of β-lactoglobulin (β-LG) occurred at all pressures, with the level of denaturation increasing with the magnitude of the pressure treatment and with pH. The denaturation of the whey proteins and the disruption of the casein micelles could not entirely account for the changes in the rheological properties of the acid gels, as denaturation of up to 50% of the whey proteins produced acid gels with very low final G ′ and yield stresses. It is proposed that the pH and the magnitude of the pressure treatment affect the interactions of the denatured β-LG with the casein proteins in the pressure-treated milks, and that this affects the ability of the denatured β-LG to participate in the acid gel structures. Industrial relevance The control and manipulation of the firmness of acid skim milk gels is important in many dairy food applications such as yogurts and some types of cheeses. This study has demonstrated that acid gel firmness can be substantially manipulated when the milk is pH adjusted and pressure treated before acidification, and that these effects are different to those obtained through heating. The commercial uptake of high pressure processing in the dairy industry is dependent on this technology producing unique functional properties in milk when compared with traditional processing. The results of this study indicates that high pressure processing of milk may offer unique functional properties in acid gel applications which could be used for the development of new or improved dairy products.

Effect of high-pressure treatment at various temperatures on activity of indigenous proteolytic enzymes and denaturation of whey proteins in ovine milk

The effect of high-pressure (HP) processing (200, 450 or 650 MPa) at various temperatures (20, 40 or 55 °C) on the total plasmin plus plasminogen-derived activity (PL), on plasminogen activators (PA), on protease activity (which seemed to be equivalent to cathepsin D), and on denaturation of major whey proteins in ovine milk was investigated. There were synergistic effects between pressure and temperature at 450 and 650 MPa for partial inactivation of PL, PA and cathepsin D. In contrast, processing at 200 MPa did not affect the activity of these enzymes. Treatment at 650 MPa was necessary for the denaturation of α-lactalbumin, and denaturation was enhanced with increasing temperature. In contrast, denaturation of β-lactoglobulin occurred at all combinations of HP and temperature studied. In conclusion, HP affected the activity of various indigenous proteolytic enzymes and the denaturation of major whey proteins of ovine milk, which may influence its behaviour during the manufacturing of dairy products.

Predictions of some product parameters based on the processing conditions of ultra-high-temperature milk plants

The temperature–time profiles of 22 Australian industrial ultra-high-temperature (UHT) plants and 3 pilot plants, using both indirect and direct heating, were surveyed. From these data, the operating parameters of each plant, the chemical index C ∗, the bacteriological index B ∗ and the predicted changes in the levels of β-lactoglobulin, α-lactalbumin, lactulose, furosine and browning were determined using a simulation program based on published formulae and reaction kinetics data. There was a wide spread of heating conditions used, some of which resulted in a large margin of bacteriological safety and high chemical indices. However, no conditions were severe enough to cause browning during processing. The data showed a clear distinction between the indirect and direct heating plants. They also indicated that degree of denaturation of α-lactalbumin varied over a wide range and may be a useful discriminatory index of heat treatment. Application of the program to pilot plants illustrated its value in determining processing conditions in these plants to simulate the conditions in industrial UHT plants.

Effect of milk solids concentration on whey protein denaturation, particle size changes and solubilization of casein in high-pressure-treated skim milk

Skim milk (10–20% total solids, TS) was high-pressure-treated at 200–600 MPa for 0–60 min at 20 °C. At 200 MPa, and to a lesser extent at 300 MPa, the denaturation of β-lactoglobulin was reduced on increasing the %TS of the milk. However, at higher pressures, the level of denaturation of β-lactoglobulin was essentially the same at all milk concentrations. Denaturation of α-lactalbumin occurred at pressures ⩾500 MPa and was independent of milk concentration. For the 10% TS milk, the particle size decreased by ∼45% on increasing the pressure from 200 to 400 MPa, with little further change at up to 600 MPa. As the milk solids concentration increased, the effect of pressure on casein micelle size was less pronounced. An unusual behaviour was observed on treatment at 300 MPa, where the particle size distribution broadened; this effect was more pronounced at higher milk solids concentrations, and the size distribution changed from monomodal to bimodal. Much casein was non-sedimentable after pressure treatment; however, the concentration of milk solids only affected levels of non-sedimentable casein at 200 MPa, and less so at 300 MPa. Most of the whey protein remained in the milk serum even after treatment at the highest pressures and the longest holding times.

Effects of high pressure on some constituents and properties of buffalo milk

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100-600MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by approximately 35%. Levels of non-micellar alpha(S1)and beta-caseins were increased by treatment > or = 250MPa, and were highest after treatment at 400-800MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from approximately 82 for untreated milk to approximately 65 for milk treated at 800 MPa. Milk pH was increased by approximately 0.07 units after treatment at 100-800 MPa, with no significant difference between treatment pressures. Denaturation of alpha-lactalbumin occurred at pressures > or = 400 MPa, and reached >90% after treatment at 800 MPa, whereas beta-lactoglobulin (beta-Ig) was denatured > 100 MPa, reaching approximately 100% after treatment at 400MPa; after treatment > or = 400MPa, all beta-Ig was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250-800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.

Potential applications of high pressure homogenisation in processing of liquid milk

Studies of the potential of high pressure homogenisation (HPH) for the combined pasteurisation/ homogenisation of raw bovine milk were undertaken. Raw milk was preheated to 45 degrees C and HPH-treated at 150, 200 or 250 MPa; milk outlet temperature at these pressures were 67, 76.8 and 83.6 degrees C, respectively, with a holding time of approximately 20 s. Raw and commercially pasteurized and homogenized (CPH) milk samples were analysed as controls. Fat globules in HPH samples were approximately half the size of those in CPH samples, although differences were not significant (P>0.05). beta-Lactoglobulin was denatured at pressures > or =150MPa, although little denaturation of alpha-lactalbumin was observed. Numbers of psychrotrophic bacteria in raw milk were reduced by 2.73 log cycles by HPH at 150 MPa and were uncountable following HPH at 200 or 250 MPa. Mesophilic bacterial counts were reduced by 1.30, 1.83 and 3.06 log cycles by HPH at 150, 200 or 250 MPa, respectively. No viable Staphylococcus aureus nor coliform cells remained in any HPH milk samples. HPH did not affect the colour of milk and HPH samples did not cream during refrigerated storage. The activities of plasmin, alkaline phosphatase and lactoperoxidase in milk were all greatly reduced by HPH. Pseudomonas fluorescens, inoculated into milk (approximately 10(6) cfu/ml), was reduced to undetectable levels by HPH at 200MPa (milk inlet temperature, approximately 10 degrees C); however, Ps. fluorescens proteinase was quite resistant to HPH under such conditions. Overall, owing to the significant increase in temperature and the possibility of varying the holding time, there may be potential applications for HPH as a novel liquid milk processing technique, combining many advantages of conventional homogenization and pasteurization of milk in a single process.

High pressure-induced denaturation of α-lactalbumin and β-lactoglobulin in bovine milk and whey: a possible mechanism

In this study, high pressure (HP)-induced denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in dairy systems was examined. In both milk and whey, beta-lg was less baroresistant than alpha-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of alpha-la and beta-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KlO3, to milk or whey increased HP-induced denaturation of beta-lg, but reduced the denaturation of alpha-la. Denaturation of both alpha-la and beta-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of alpha-la and beta-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of alpha-la and beta-lg significantly. The higher level of HP-induced denaturation of alpha-la and beta-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of alpha-la and beta-lg in milk.

High pressure treatment of bovine milk: effects on casein micelles and whey proteins.

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.

Determination of the heat treatment undergone by milk by following the denaturation of alpha-lactalbumin with a biosensor.

Dairy industries are interested in knowing the heat treatment undergone by milk so as to control the quality of drinking milks or to control their heating systems. Among the different techniques available to characterize the heat treatment of milk, estimation of the denaturation of proteins has been widely used. However, because the concentration of the proteins in raw milk can fluctuate significantly, determining only the concentration of a native protein without knowing its concentration in the raw milk before undergoing heat treatment can lead to significant imprecision. The objective of this study was to develop, on Biacore 3000, a biosensor assay for determining the denaturation index of alpha-lactalbumin by quantifying separately the native and "heat-denatured" forms of alpha-lactalbumin with specific monoclonal antibodies. alpha-Lactalbumin denaturation index is independent of the concentration of alpha-lactalbumin in the original raw milk. The technique developed is discriminating, fast, repeatable, fully automated, and requires no pretreatment of the milk sample.

Effects of high pressure treatment on the yield of cheese curd from bovine milk

Cheesemaking properties of milk are affected by high pressure (HP) treatment; this study focussed on HP-induced changes in curd yield. Yield was not influenced by treatment at pressures ≤250 MPa, but at higher pressures, curd yield was increased, by up to 25% after treatment at 600–800 MPa. In parallel with increased curd yield, increased curd moisture content and reduced protein content in the whey were observed after treatment at such pressures. Denaturation of α-lactalbumin or β-lactoglobulin occurred at >400 or >100 MPa, respectively. Curd yield and curd moisture content increased and protein content in the whey decreased with increasing treatment time (0–30 min of treatment at 400 or 600 MPa). Increased curd yield is probably a result of both the incorporation of denatured whey proteins and enhanced moisture retention in the curd. HP-induced increases in curd yield may indicate possible applications of HP treatment of milk for cheese manufacture.