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.     

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 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.     

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.     

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.     

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℃。     

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.     

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.     

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 and thermal inactivation kinetics of polyphenol oxidase and peroxidase in strawberry puree

The objective of this work was to study the thermal and high pressure inactivation kinetics of polyphenol oxidase (PPO) and peroxidase (POD) in strawberry puree. PPO from two strawberry cultivars (‘Festival’ and ‘Aroma’) was found to be highly thermostable in strawberry puree with no significant inactivation even after 30 min treatment at 100 °C. In contrast, POD from the two cultivars displayed very high thermosensitivity with complete inactivation in less than 5 min at 70 °C. The thermal inactivation kinetics of strawberry POD was described by a biphasic model. The activation energies for the inactivation of the stable and the labile fractions were estimated to be 254.9 and 221.6 kJ/mol respectively. Combined high pressure–thermal treatment at pressures ranging from 100 to 690 MPa, temperatures ranging from 24 to 90 °C and treatment times between 5 and 15 min did not have significant effect on PPO while substantial inactivation of POD was observed. The inactivation kinetics of POD during combined high pressure–thermal processing was well described by first-order kinetics probably due to the inactivation of the labile fraction during the pre-heating and the compression phase.Industrial relevanceThe oxidative enzymes polyphenol oxidase and peroxidase cause the degradation of anthocyanins and other polyphenols in strawberry products, leading to discoloration and loss of antioxidant activity. In this work the thermal and high pressure inactivation of strawberry polyphenol oxidase and peroxidase was investigated so as to assess the suitability of high pressure processing as an alternative to thermal processing. Strawberry polyphenol oxidase was found to be highly resistant to both thermal and high pressure inactivation. Thus in order to maintain the quality of processed strawberry products, high pressure processing should be accompanied by additional measures such as exclusion of oxygen, refrigerated storage and the use of natural enzyme inhibitors.     

Functional improvement of milk whey proteins induced by high hydrostatic pressure

High pressure is emerging as a new processing technology that produces particular changes in the molecular structure of proteins and thus gives rise to new properties inaccessible via conventional methods of protein modification. This review deals with the main effects of high hydrostatic pressure on the physicochemical characteristics of milk whey proteins and how modifications in their structural properties contribute to functionality. In this paper the mechanism underlying pressure-induced changes in ss-lactoglobulin, a-lactabumin, and bovine serum albumin is explained, and related to functional properties such as gel-forming ability, emulsifying activity, or foaming capacity. The possibility of using high pressures to favor chemical reactions of proteins with other food components, such as carbohydrates, to produce novel molecules with new food uses is also considered.     

High pressure-induced denaturation of alpha-lactalbumin and beta-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.     

热变性对蛋白质理化性质的影响

本文论述了热变性对蛋白质结构、功能性质以及酶解性质的影响，揭示了蛋白质热变性后理化性质的变化是其二级、三级或四级结构改变的结果，并讨论了其它因素对热变性的影响。     

High hydrostatic pressure modification of whey protein concentrate for improved functional properties

Whey protein concentrate (WPC) has many applications in the food industry. Previous research demonstrated that treatment of whey proteins with high hydrostatic pressure (HHP) can enhance solubility and foaming properties of whey proteins. The objective of this study was to use HHP to improve functional properties of fresh WPC, compared with functional properties of reconstituted commercial whey protein concentrate 35 (WPC 35) powder. Fluid whey was ultrafiltered to concentrate proteins and reconstituted to equivalent total solids (8.23%) as reconstituted commercial WPC 35 powder. Solutions of WPC were treated with 300 and 400 MPa (0- and 15-min holding time) and 600 MPa (0-min holding time) pressure. After HHP, the solubility of the WPC was determined at both pH 4.6 and 7.0 using UDY and BioRad protein assay methods. Overrun and foam stability were determined after protein dispersions were whipped for 15 min. The protein solubility was greater at pH 7.0 than at pH 4.6, but there were no significant differences at different HHP treatment conditions. The maintenance of protein solubility after HHP indicates that HHP-treated WPC might be appropriate for applications to food systems. Untreated WPC exhibited the smallest overrun percentage, whereas the largest percentage for overrun and foam stability was obtained for WPC treated at 300 MPa for 15 min. Additionally, HHP-WPC treated at 300 MPa for 15 min acquired larger overrun than commercial WPC 35. The HHP treatment of 300 MPa for 0 min did not improve foam stability of WPC. However, WPC treated at 300 or 400 MPa for 15 min and 600 MPa for 0 min exhibited significantly greater foam stability than commercial WPC 35. The HHP treatment was beneficial to enhance overrun and foam stability of WPC, showing promise for ice cream and whipping cream applications.     

Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration

Microfiltration and ultrafiltration were used to manufacture skim milks with an increased or reduced concentration of whey proteins, while keeping the casein and milk salts concentrations constant. The skim milks were heated on a pilot-scale UHT plant at 80, 90 and 120 degrees C. The heat-induced denaturation and aggregation of beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA) were quantified by polyacrylamide gel electrophoresis. Apparent rate constants and reaction orders were calculated for beta-lg, alpha-la and BSA denaturation. Rates of beta-lg, alpha-la and BSA denaturation increased with increasing whey protein concentration. The rate of alpha-la and BSA denaturation was affected to a greater extent than beta-lg by the change in whey protein concentration. After heating at 120 degrees C for 160 s, the concentration of beta-lg and alpha-la associated with the casein micelles increased as the initial concentration of whey proteins increased.     

Combined thermal and high pressure inactivation kinetics of tomato lipoxygenase

The combined isothermal (10–60 °C) and isobaric (0.1–650 MPa) inactivation kinetics of lipoxygenase (LOX) extracted from tomatoes and reconstituted in a tomato purée were studied. Thermal inactivation of LOX at atmospheric pressure proceeded in the temperature range of 45–65 °C. LOX inactivation did not follow first order kinetics; the data could be fitted assuming that the two isoforms of LOX with different thermostability were present. Combined thermal and high pressure inactivation occurs at pressures in the range of 100–650 MPa combined with temperatures from 10–60 °C, and followed first-order kinetics. In the high-temperature/low-pressure range, (T≥50 °C and P≤300 MPa) an antagonistic effect is observed, therefore, the Arrhenius and Eyring equation cannot be used over the entire temperature and pressure range. Small temperature dependence is found in the low-temperature/high pressure range. A third degree polynomial model was successfully applied to describe the temperature–pressure dependence of the inactivation rate constants, which can be useful to predict inactivation rate constants of tomato LOX reconstituted in tomato purée in the temperature–pressure range studied.     

Functionality of extrusion--texturized whey proteins

Whey, a byproduct of the cheesemaking process, is concentrated by processors to make whey protein concentrates (WPC) and isolates (WPI). Only 50% of whey proteins are used in foods. In order to increase their usage, texturizing WPC, WPI, and whey albumin is proposed to create ingredients with new functionality. Extrusion processing texturizes globular proteins by shearing and stretching them into aligned or entangled fibrous bundles. In this study, WPC, WPI, and whey albumin were extruded in a twin screw extruder at approximately 38% moisture content (15.2 ml/min, feed rate 25 g/min) and, at different extrusion cook temperatures, at the same temperature for the last four zones before the die (35, 50, 75, and 100 degrees C, respectively). Protein solubility, gelation, foaming, and digestibility were determined in extrudates. Degree of extrusion-induced insolubility (denaturation) or texturization, determined by lack of solubility at pH 7 for WPI, increased from 30 to 60, 85, and 95% for the four temperature conditions 35, 50, 75, and 100 degrees C, respectively. Gel strength of extruded isolates increased initially 115% (35 degrees C) and 145% (50 degrees C), but gel strength was lost at 75 and 100 degrees C. Denaturation at these melt temperatures had minimal effect on foaming and digestibility. Varying extrusion cook temperature allowed a new controlled rate of denaturation, indicating that a texturized ingredient with a predetermined functionality based on degree of denaturation can be created.     

The ‘turbidity’ test as a measurement of thermal denaturation of proteins in wheat

The ‘turbidity’ test, devised to detect heat damage sometimes resulting from the drying of newly harvested damp wheat, has been studied as a routine method for the measurement of heat denaturation in wheat proteins. The ‘turbidity’ test method has been compared with the standard baking test and the results of both methods have been evaluated in terms of denaturation reaction rates. The results have shown that the method is at least as sensitive as the baking test, and, with suitable unheated controls, can be used to measure the denaturation of wheat proteins resulting from the heat treatment of wheat and wholemeal.