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.     

膜技术分离脱脂乳中酪蛋白胶束和乳清蛋白工艺的研究

对膜技术分离脱脂乳酪蛋白胶束和乳清蛋白工艺进行了研究,考察了不同筛分膜的分离效果,并研究了不同操作参数对渗透通量的影响.最终确定用切割分子量300kDa的陶瓷膜进行脱脂乳浓缩分离,酪蛋白胶束和乳清蛋白的分离效果较佳,透过液中乳清蛋白占真蛋白的百分比可达98.91%.确定了膜分离的最适操作压力为0.20MPa,最适操作温度为50℃.     

Effects of milk pH alteration on casein micelle size and gelation properties of milk

The effects of changes in pH above and below the natural pH of milk (ca 6.6) on the casein micelle size and the gelation properties of the pH adjusted and restored samples were investigated. The size of casein micelles increased at pH 7.0 and 7.5, then started to decrease above pH 8.5. It is postulated that at pH below 8.5 the casein micelles swell, while elevated pH causes their dissociation. Conversely, the size of casein micelles decreased on acidification to pH 5.5 and increased when the pH dropped below 5.5, due to the shrinkage of casein micelles at lower pH before their aggregation at pH below 5.5. In response to neutralising treated milk back to normal milk pH of 6.6, it was found that the casein micelle size of treated milk with a narrow range of pH change between 6.0 and 7.0 was reversible, while beyond this range the structure of casein micelles became irreversible. The restoration of casein micelle size was followed by the restoration of some parameters such as soluble calcium, ethanol stability, and milk whiteness. Acid-induced gelation did not change the elastic modulus, while rennet-induced gelation was affected by initial milk pH. In reference to the size of reversible range elastic modulus (G’) of acid or rennet gels made from restored milk, the sizes were similar to control milk after 6 h of gelation.     

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.     

定量分析加热后乳清蛋白与酪蛋白的结合

将复原脱脂乳在 70～ 90℃范围内加热 1 0～ 2 5min后 ,用超速离心分离出酪蛋白微粒 ,并用毛细管电泳法定量分析。结果显示 ,β-乳球蛋白更容易结合到酪蛋白微粒上。当加热条件为 70℃、1 0 min时就有相当多的 β-乳球蛋白发生了这种结合 ,这时酪蛋白微粒中没发现任何 α-乳清蛋白 ,只有当加热温度大于 75℃时才有少量 α-乳清蛋白与酪蛋白微粒结合。复原脱脂乳经 90℃、2 5min加热后几乎所有 β-乳球蛋白都已转入酪蛋白微粒部分 ,而只有近 50 %的α-乳清蛋白转入酪蛋白微粒。     

A kinetic model for whey protein denaturation at different moisture contents and temperatures

The denaturation of whey protein samples that had previously undergone heat-treatment for different times at different temperatures and moisture contents was analysed by differential scanning calorimetry (DSC), using the DSC enthalpy as a measure of residual undenatured protein. Data were fitted to first order irreversible or reversible kinetic expressions, and the resulting rate constants were found to increase with both temperature and moisture content. The whole data set was then fitted as a function of time, temperature and moisture content, with rate constants varying according to either Arrhenius or Williams-Landel-Ferry (WLF) kinetics and with selected fit parameters made empirical functions of moisture content. The best fits were obtained using reversible WLF kinetics, which could be further slightly simplified without loss of accuracy. The model provides a platform for single- and multi-objective drying trajectory optimisation with respect to protein denaturation in dairy products.     

Interactions in heated milk model systems with different ratios of nanoparticulated whey protein at varying pH

To better understand the interactions between nanoparticulated whey protein (NWP) and other milk proteins during acidification, milk model systems were diluted to 0.5% protein concentration and adjusted to pH of 6.0–4.5 following homogenisation and heat treatment. The diluted systems with different concentrations of NWP (0–0.5%) were characterised in terms of particle size, viscosity, surface charge and hydrophobicity. When pH was adjusted to 5.5, aggregation was initiated at levels of NWP (0.25–0.5%) leading to significant increase in particle size and viscosity. Pure NWP (0.5%) showed largest initial surface charge (−27 mv) and higher surface hydrophobicity than the other systems. The results indicated that NWP could self-associate above pH 5.5 and not only the decrease of electrostatic repulsion but also other interactions, such as hydrophobic interaction, play an important role in contributing to the early self-association of NWP.     

Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size

When skim milk at pH 6.55 was heated (75 to 100 degrees C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30-35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of beta-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6.5 to pH 6.7.     

Physicochemical and functional properties of goat milk whey protein and casein obtained during different lactation stages

During lactation, goat milk contains colostrum, transitional milk, mature milk, and end milk. The protein present in goat milk during different lactation periods has different characteristics. This study aimed to characterize the protein profile of goat milk samples obtained at different lactation stages and to identify changes in the physicochemical and functional properties of whey protein and casein from goat milk collected at 1, 3, 15, 100, and 200 d after calving. The results demonstrated that the lactation period had a great influence on the physicochemical and functional properties of goat milk whey protein and casein, especially the protein properties of colostrum on the first day after delivery. The denaturation temperature, hydrophobicity, and turbidity of whey protein were significantly higher on the first day postpartum than at other lactation periods. Correspondingly, the colostrum whey protein also had better functional properties, such as emulsification, oil holding capacity, and foaming properties on the first day postpartum than at other lactation periods. For casein, the turbidity, particle size, water holding capacity, and foaming properties on the first day after delivery were significantly higher than those at other lactation periods, whereas the denaturation temperature, oil holding capacity, and emulsification followed the opposite trend. For both whey protein and casein, the 2 indicators of emulsifying properties, namely, emulsifying activity index and the emulsion stability, also followed an opposite trend relative to lactation stage, whereas the changes in foaming capacity with the lactation period were completely consistent with the change of foaming stability. These findings could provide useful information for the use of goat milk whey protein and casein obtained during different lactation stages in the dairy industry.     

Importance of casein micelle size and milk composition for milk gelation

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their ζ-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

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.     

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.     

Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55 degrees C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20 degrees C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16-18%) and PA (38-62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.     

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.     

Heat and/or high-pressure treatment of skim milk: changes to the casein micelle size, whey proteins and the acid gelation properties of the milk

Skim milk was subjected to heat, pressure or combined processes. In general, higher levels of whey protein denaturation were observed for milk subjected to combined processes than those heat- or pressure-treated only. Heat treatment caused small changes to the casein micelle size. Pressure treatment decreased the casein micelle size; however, the effect was less marked when heat and pressure treatments were combined. Acidification of the skim milks produced gels with a range of firmness, yield stresses and yield strains depending on the treatments applied. These changes in acid gel properties were not related only to whey protein denaturation levels in the milks.     

Controlling heat induced aggregation of whey proteins by casein inclusion in concentrated protein dispersions

Formation of soluble, heat stable whey protein (WP) particles may be achieved via caseins and their chaperone like activity. The aim of this study was to establish effects of caseins on the properties of heat induced WP aggregates. Concentrated dairy protein mixtures (10% total-solids) were prepared with various casein:WP ratios (5:95–30:70) at adjusted pH (5.7–7.5) and heated to 80–120 °C, under a constant shear (500 s⁻¹). Increasing casein fraction at higher pH reduced the average size of WP aggregates and the surface hydrophobicity with simultaneous reduction in apparent viscosity at pH 6.7. WP denaturation was minimal when 30% casein was included in the dispersions. Inhibition of WP denaturation at elevated pH with inclusion of caseins may be attributed to a chaperon like activity of the casein. Thus, by increasing pH and the casein content in the system it was possible to form nano-size whey protein particles.