Thermal denaturation kinetics of whey proteins at high protein concentrations

A detailed kinetic study of the thermal reaction kinetics of whey protein concentrate was conducted at high protein concentrations. Whey protein solutions with protein concentrations of up to 40% (w/w) were heated at different temperatures for varying periods of times. The denaturation of β-lactoglobulin followed a reaction order of 1.5 and depended strongly on temperature and protein concentration. The rate of denaturation was shown to increase with increasing temperature. This could be explained by the strong influence of the temperature on the unfolding reaction. Furthermore, the protein concentration induced a faster thermal denaturation, most likely due to the increased probability of collision between whey protein molecules with increasing protein concentration which promotes protein aggregation. The results of this study are of industrial relevance for extrusion processes and the production of protein concentrates in evaporators where high protein concentrations are frequently used.     

大豆乳清蛋白的热变性和热聚集的研究

通过SDS-PAGE电泳、差示扫描量热仪(DSC)和体积排阻色谱(SEC-HPLC)研究了大豆乳清蛋白(WSP)的热变性和热聚集.结果表明:大豆乳清蛋白的变性温度为70.6℃和89.4℃,对应于胰蛋白酶抑制剂和大豆凝集素的热变性温度;1%(w/v)的大豆乳清蛋白经80℃和100℃加热30min,会生成分子量约为270万Da的可溶性聚集体.     

High hydrostatic pressure assisted enzymatic hydrolysis of whey proteins

Whey proteins, due to their high nutritional value, are generally hydrolyzed to reduce the allergenicity and used as ingredients in many special products, such as infant formulae, geriatric products, highly energetic supplements or dietetic foods or in foods produced to prevent nutrition related diseases, like food intolerances and allergies.The aim of this work was to assess the applicability of innovative technologies, such as high hydrostatic pressure (HHP) processes, to assist the enzymatic hydrolysis of target proteins, namely whey protein concentrate (WPC-80), in order to modify their antigenicity. Experiments were carried out to verify the effectiveness of HHP technology to accelerate whey protein hydrolysis reaction with selected enzymes (α-chymotrypsin, bromelain), and to affect the protein allergenic power. To this purpose, different HHP treatments were carried out at several pressure levels (100, 200, 300 and 400 MPa) and the untreated whey protein samples were used as control. A defined enzyme/substrate ratio of 1/10 w/w was used in the experiments, while the treatment time was changed from 0 to 30 min (0, 5, 15, or 30 min).The experimental data demonstrated that High Hydrostatic Pressure (HHP) induced WPC-80 unfolding at the highest value of the pressure applied (400 MPa) as indicated by the higher exposure of free sulfhydryl groups. When HHP was used in combination with enzymatic hydrolysis, the degree of hydrolysis increased not only with the pressure level applied but also with the processing time. These results suggested that, even if the exposure of hidden epitopes upon protein unfolding increased the antigenicity of whey proteins, further peptide bonds cleavage also took place after hydrolysis. This effect could modify whey proteins antigenic sequences, and thus their antigenic power.     

The suitability of DSC to obtain thermal reaction kinetics of whey proteins

Differential scanning calorimetry (DSC) provides a method for easily following the progress of reactions upon heating a material. Analysis techniques have been developed which allow reaction kinetics to be determined from thermograms; the two most frequently used, the Borchardt—Daniels and Kissinger methods, both rely on the assumption that only one reaction following Arrhenius kinetics takes place. These assumptions are not true for many systems, especially multi-component systems such as whey protein concentrates. The suitability of DSC for determining thermal reaction kinetics of whey protein concentrates was determined by applying both the Borchardt—Daniels and Kissinger methods to experimental and simulated DSC thermograms. Experimental results showed that kinetic parameters estimated by the Borchardt—Daniels method varied with protein concentration and/or scanning rate, showing that more than one reaction was taking place. The Kissinger method produced inaccurate estimates of the reaction order. Numerical simulations showed that the failure of the Kissinger method was due to sensitivity to random noise in the DSC power signal. The Borchardt—Daniels method was also affected by random noise, but to a lesser extent. It was concluded that the Kissinger method can only be used if the noise in the power signal can be decreased greatly. Kinetic parameters from the Borchardt—Daniels method should only be used with care.     

High pressure and the enzymatic hydrolysis of soybean whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of soybean whey proteins by trypsin, chymotrypsin and pepsin was studied. The experiments were carried out at atmospheric pressure (0.1 MPa, for 30 min, at 37 °C) and at HP (100 and 200 MPa for 15 min at 37 °C) before or during the reaction of hydrolysis. The extent of hydrolysis was measured by OPA method and in the extracts from TCA. The results showed that HP treatments increased the hydrolysis in the three enzymes used and 100 MPa was the better pressure to enhance the hydrolysis. Polyacrylamide gel electrophoreses (SDS–PAGE), showed five peptides lower than 14 kDa after hydrolysis by chymotrypsin and trypsin, and 11 peptides by pepsin. Soybean whey proteins, which are industrially discarded, could be used as a source of peptides, with applications as base in some diets.     

Study of the thermal denaturation of selected proteins of whey and egg by low resolution NMR

The thermal denaturation of solutions of selected components of whey, albumen, and whole chicken albumen was studied during and after a heat treatment at constant temperatures by using low resolution nuclear magnetic resolution (LR-NMR) (on-line and off-line). Ovalbumin and conalbumin were isolated from albumen and β-lactoglobulin from milk, respectively. Pure proteins were studied separately and also in a 1+1 mixture of ovalbumin and β-lactoglobulin. Furthermore, albumen was heated. It was not only possible to record the integral change of molecular mobility due to denaturation processes, but also (a) to distinguish up to four phases of different mobility and to quantify the corresponding fractions and record their temporal evolution during the heat treatment, (b) to determine material-specific temperatures indicating structural changes including denaturation (denaturation temperatures based on data accessible by NMR), and (c) to differentiate, based on a combination of on-line and off-line LR-NMR, reversible and irreversible structural changes due to the thermal treatment as a function of both the process temperature and the duration of the heat treatment (more precisely: the process temperature/time history). Thus, the denaturation temperatures (based on NMR T₂ experiments) of the single proteins, both dissolved alone and as a mixture. By comparison of the T₂-relaxation times in solutions of single proteins (here: ovalbumin and β-lactoglobulin) and of the mixture of ovalbumin and β-lactoglobulin interactions between the proteins were observed. The advantage of NMR experiments is the possibility to perform on-line nondestructive in situ measurements, which is not possible by means of methods based on preparation.     

Controlling denaturation and aggregation of whey proteins during thermal processing by modifying temperature and calcium concentration

Whey protein isolate solutions (8.00 g protein/100 g; pH 6.8) were treated for 2 min at 72, 85 or 85 °C with 2.2 mM added calcium Ca to produce four whey protein systems: unheated control (WPI‐UH), heated at 72 °C (WPI‐H72), heated at 85 °C (WPI‐H85) or heated at 85 °C with added Ca (WPI‐H85Ca). Total levels of whey protein denaturation increased with increasing temperature, while the extent of aggregation increased with the addition of Ca, contributing to differences in viscosity. Significant changes in Ca ion concentration, turbidity and colour on heating of WPI‐H85Ca, compared to WPI‐UH, demonstrated the role of Ca in whey protein aggregation.     

High pressure thermal inactivation kinetics of a plasmin system

A crude plasmin extract was prepared from milk by ultracentrifugation and was partially purified using ammonium sulfate precipitation. Isothermal and high-pressure inactivation of this plasmin system at pH 6.7 could be described by a first-order kinetic model. As expected, the plasmin system displayed a high thermostability. High-pressure treatments were conducted in the 300- to 800-MPa pressure range, combined with temperatures from 25 to 65 degrees C. The plasmin system was very pressure stable at room temperature, but inactivation occurred with combined high-pressure/temperature-treatments. The influence of temperature at different constant pressures on the inactivation rate constant was quantified using the Arrhenius equation. At all temperatures studied, a synergistic effect of temperature and high pressure was observed in the 300- to 600-MPa pressure range. However, an antagonistic effect of temperature and pressure appeared at pressures above 600 MPa.     

Thermal denaturation and coagulation of whey proteins: effect of sugars

The thermal coagulation of unfractionated whey proteins was inhibited by various sugars. The disaccharides, sucrose and lactose, were most effective, and the amino sugar, glucosamine, least effective in this respect. Ultraviolet absorption and light-scattering measurements on the thermal denaturation and coagulation of both unfractionated and individual whey proteins (alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin) showed that sucrose promotes the denaturation of these proteins but inhibits their subsequent coagulation. These results are interpreted in terms of the effect of sucrose on the hydrophobic interactions between solvent and protein.     

High pressure microfluidization treatment of heat denatured whey proteins for improved functionality

Solutions (5% protein) of a whey protein concentrate (WPC) in fresh acid whey or in water, as well as the fresh whey alone, were adjusted to pH 5.8, 4.8 or 3.8, heat treated at 90 °C for 10 min and further exposed to high pressure (150 MPa) microfluidization treatment. The volumes of sediment after centrifugation were recorded as a measure of the degree of insolubility of the proteins. Microfluidization disrupted the heat-induced aggregates into non-sedimenting whey protein polymers so that in some cases, especially at pH 3.8, the products studied were almost completely resistant to sedimentation after the microfluidization treatments. Heat denatured/microfluidized whey proteins reaggregated upon subsequent heating, with the pH having a major impact on the amount of sediment produced. Microfluidization of aqueous WPC solutions heat-treated before spray- or freeze-drying substantially increased the solubility of the powders upon reconstitution. Heat-induced viscoelastic gels were produced from freeze-dried microfluidized samples processed at pH 3.8 and reconstituted to solutions containing 12% (w/w) protein.     

Influence of NaCl concentration and high pressure treatment on thermal denaturation of soybean proteins

In the present work the effect of high pressure (HP) treatment in the presence of NaCl on the thermal behavior of soybean proteins was analyzed by differential scanning calorimetry. The thermograms obtained have shown that NaCl addition increased the thermal stability – increase in temperatures of denaturation (Td) – of both glycinin and β-conglycinin. HP treatments increased thermal stability of glycinin, but decreased that of β-conglycinin. High NaCl concentrations decreased (in glycinin) or inverted (in β-conglycinin) the effects of HP on thermal stability. Cooperativity of denaturation of glycinin was enhanced by NaCl and HP. Cooperativity of denaturation of β-conglycinin was enhanced by HP and also by NaCl at 0.2 mol/L but decreased with the combination of treatments. Salt addition increased the enthalpy, ΔH, of denaturation of glycinin and β-conglycinin, being this effect stronger on glycinin. HP treatment provoked the denaturation of both protein fractions. The presence of NaCl protected glycinin against HP-denaturation at any assayed salt concentration and pressure level, while β-conglycinin was only protected at 200 and 400 MPa, but was more denaturated at 600 MPa in the presence of 0.6 mol/L of NaCl.

Industrial relevance

The knowledge provided by this work may be useful in the handling of high pressure-treated food with high NaCl content (e.g. meat emulsions, smallgoods) where soybean proteins are used as additives, in order to choose high pressure values to achieve their denaturation or predict the effects of ulterior thermal treatments. Thus, this knowledge may be useful to increase the use of high pressure in food industry.     

水牛免疫乳中IgG热变性的研究

本实验采用Ig热变性动力学研究了水牛免疫乳中IgG热变性。结果表明，免疫球蛋白（IgG）对热敏感性高，63℃，30min热处理后，IgG变性率为12．4％，95℃热处理15s和65s后，变性率分别是53．1％和89．5％；在温度为63℃到95℃范围内，IgG热变性反应级数为1．0，表观活化能为140．6kJ／mol；温度为63，75，85，95℃的D值分别为294、89．24、4．54、1．31min，在此温度范围内，Z值为12．77℃。     

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.     

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

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

Effect of hydrocolloids on the thermal denaturation of proteins

The thermal denaturation of bovine serum albumin (BSA), lysozyme and whey protein isolate (WPI) in the presence of hydrocolloids (pectin, guar gum, ι-carrageenan) was investigated. A decrease in the thermal stability of lysozyme was observed in the mixture of protein with ι-carrageenan. The increase in the enthalpy of denaturation (ΔH) of BSA and lysozyme in the presence of hydrocolloids was attributed to the protection of globular proteins against aggregation through blockage of their hydrophobic binding sites by the bulky polysaccharide moeity. Biopolymers had a stabilizing effect on WPI. The thermal stability was the highest in the presence of pectin, whereas the lowest transition temperature was observed in the presence of guar gum. A single transition peak was observed for pure WPI. However, WPI exhibited two transition temperatures when together with pectin and i-carrageenan. WPI was stable against heat denaturation at acidic pH values (pH 4.0), while it was denatured at a low temperature at an alkaline pH (pH 9.0) in the presence of pectin. This was attributed to the formation of extra hydrogen bonding. The increase in the concentration of pectin has little affect on the heat stability of WPI; however, it reduces the cooperativity of transition.     

Influence of pH on the dry heat-induced denaturation/aggregation of whey proteins

The effect of pH on the heat-induced denaturation/aggregation of whey protein isolate (WPI) in the dry state was investigated. WPI powders at different pH values (6.5, 4.5, and 2.5) and controlled water activity (0.23) were dry heated at 100 °C for up to 24 h. Dry heating was accompanied by a loss of soluble proteins (native-like β-lactoglobulin and α-lactalbumin) and the concomitant formation of aggregated structures that increased in size as the pH increased. The loss of soluble proteins was less when the pH of the WPI was 2.5; in this case only soluble aggregates were observed. At higher pH values (4.5 and 6.5), both soluble and insoluble aggregates were formed. The fraction of insoluble aggregates increased with increasing pH. Intermolecular disulphide bonds between aggregated proteins predominated at a lower pH (2.5), while covalent cross-links other than disulphide bonds were also formed at pH 4.5 and 6.5. Hence, pH constitutes an attractive tool for controlling the dry heat-induced denaturation/aggregation of whey proteins and the types of interactions between them. This may be of great importance for whey ingredients having various pH values after processing.     

Immunochemical quantification of heat denaturation of camel ( Camelus dromedarius ) whey proteins

The major whey proteins IgG, serum albumin and alpha-lactalbumin were purified from camel milk using gel permeation and ion-exchange chromatography. Specific antisera against each of them were raised and used to quantify their heat denaturation in early or mature milk over a range of 60-90 degrees C for 10-60 min using the single radial immunodiffusion technique. The heat denaturation midpoints for the mature milk heated 30 min were 67.2, 73.0 and 77.5 degrees C for IgG, albumin and alpha-lactalbumin respectively. The early milk was more sensitive to heat treatment with coagulation at low temperature and heat denaturation midpoints of 64.8, 71.6 and 72.6 degrees C respectively. This difference was related to the high IgG content of the early milk (12.6 mg/ml v. 0.5 mg/ml for the mature milk) and stresses the importance of monitoring the IgG level of milk to assess the absence of colostrum.     

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.     

高压和热结合处理对牛肉蛋白质变性和脂肪氧化的影响

以不同压力(从200～800MPa)和温度(20、40和60℃)结合处理20min对牛肉蛋白质的变性和脂肪氧化的影响进行了研究。差示扫描量热仪(DSC)温度扫描图谱显示胶原蛋白对压力十分稳定,只有在60℃下压力处理才部分变性;而肌球蛋白对热和压力都不稳定,并且在压力变性的同时形成新的结构,这个新的结构对热表现出较低的稳定性。在较高温度(60℃)下,压力对温度引起的蛋白质变性起到部分抑制作用。压力和热以及两者结合处理都能加速脂肪的氧化过程,尤其是在400MPa及其以上压力。处理后的冷藏过程中,除20℃和40℃0.1MPa两个处理外,其它处理的TBA值均呈现快速增加。压力和热处理后蛋白质结构的破坏和变性,导致铁的释放在脂肪氧化过程中起着重要的作用,因铁离子是脂肪氧化的主要催化剂之一。差示扫描量热法(DSC)的扫描结果也显示,在400MPa及其以上压力时大部分蛋白质发生变性。