Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

The two-stage coagulation of milk proteins in the minimum of the heat coagulation time-pH profile of milk: effect of casein micelle size

Milks with casein micelles larger or smaller than control milk were prepared by differential centrifugation. The heat stability of these modified milks increased markedly throughout the pH range 6.4 to 7.1 with decreasing casein micelle size. Within the region of the minimum in the heat coagulation time-pH profile, the control milk coagulated by a two-stage process, but the modified milks, because of their narrower casein micelle size distribution, coagulated by a single-stage process at the pH of minimum stability. The content of kappa-CN and protein hydration increased as the size of the casein micelles decreased, and the level of glycosylation of kappa-CN and protein surface hydrophobicity increased as a function of micelle size. The effect of casein micelle size on the heat stability of milk is likely to be related to changes in the above physico-chemical properties.     

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.     

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.     

Fractionation by microfiltration: Effect of casein micelle size on composition and rheology of high protein,low fat set yoghurt

The effects of casein micelle size on rheological properties of high protein (5.6% crude protein), low fat (≤0.2%) set yoghurt were investigated. Microfiltration with 0.20 μm membranes was used to fractionate skim milk with an average casein micelle size of ∼174 nm into a retentate and a permeate containing “large” (∼183 nm) and “small” (∼129 nm) casein micelles, respectively. The permeate containing the small casein micelles was further concentrated with 0.10 μm membranes. Yoghurt milk bases with large or small casein micelles were subjected to heat treatment at two different temperatures; 95 °C or 75 °C for 5 min. Yoghurt milk base with small casein micelles gave set yoghurts with higher storage modulus (G′) and higher firmness than yoghurt milk base with large casein micelles. Increased gelation capacity can be attributed to an increased amount of κ-casein in the yoghurt milk base containing small casein micelles.     

羊奶酪蛋白热稳定性的研究

以莎能和关中羊奶为原料,通过从羊奶中提取酪蛋白,分别在不同的pH、温度以及添加不同浓度的Ca^2＋、柠檬酸钠、三聚磷酸钠、干酪素的条件下测定酪蛋白的热凝固时间（HCT）,研究其对羊奶酪蛋白热稳定性的影响。结果表明,pH在6．8时酪蛋白的热稳定性最好,高温会降低酪蛋白的热稳定性,钙离子可以降低羊奶酪蛋白的热稳定性,适量的柠檬酸钠或三聚磷酸钠可以有效提高羊奶酪蛋白的热稳定性,干酪素对酪蛋白稳定性影响不明显。     

Gelation mechanism of milk as influenced by temperature and pH; studied by the use of transglutaminase cross-linked casein micelles

Casein micelles in milk are colloidal particles consisting of four different caseins and calcium phosphate, each of which can be exchanged with the serum phase. The distribution of caseins and calcium between the serum and micellar phase is pH and temperature dependent. Furthermore, upon acidification casein micelles lose their colloidal stability and start to aggregate and gel. In this paper, we studied two methods of acid-induced gelation, i.e., 1) acidification of milk at temperatures of 20 to 50 degrees C and 2) decreasing the pH at 20 degrees C to just above the gelation pH and subsequently inducing gelation by increasing the temperature. These two routes are called T-pH and pH-T, respectively. The gelation kinetics and the properties of the final gels obtained are affected by the gelation route applied. The pH-T milks gel at higher pH and lower temperature and the gels formed are stronger and show less susceptibility to syneresis. By using intramicellar cross-linked casein micelles, in which release of serum caseins is prevented, we demonstrated that unheated milk serum caseins play a key role in gelation kinetics and characteristics of the final gels formed. This mechanism is presented in a model and is relevant for optimizing and controlling industrial processes in the dairy industry, such as pasteurization of acidified milk products.     

酸性乳体系中果胶对酪蛋白胶粒稳定作用的研究进展

酪蛋白胶粒在牛乳中可稳定存在,但当pH降低时易发生聚集而使体系失稳。果胶可用于酸性乳饮料中作为稳定剂。在低pH值条件下,果胶吸附于酪蛋白上,由于产生静电排斥作用和空间位阻作用而使乳体系稳定。同时,乳体系中存在的自支持弱网络结构,也对体系有稳定作用。     

Stability of buffalo casein micelles.

Buffalo skim-milk is less heat stable than cow skim-milk. Interchanging ultracentrifugal whey (UCW) and milk diffusate with micellar casein caused significant changes in the heat stability of buffalo casein micelles (BCM) and cow casein micelles (CCM). Buffalo UCW dramatically destabilized CCM, whereas buffalo diffusate with CCM exhibited the highest heat stability. Cow kappa-casein stabilizes alphas-casein against precipitation by Ca better than buffalo kappa-casein. About 90% of alphas-casein could be stabilized by kappa:alphas ratios of 0.20 and 0.231 for cow and buffalo, respectively. Sialic acid release from micellar kappa-casein by rennet was higher than from acid kappa-casein in both buffalo and cow caseins, the release being slower in buffalo. The released macropeptide from buffalo kappa-casein was smaller than that from cow kappa-casein as revealed by Sephadex gel filtration. Sub-units of BCM have less sialic acid (1.57 mg/g) than whole micelles (2.70 mg/g). On rennet action, 47% of bound sialic acid was released from sub-units as against 85% from whole micelles. The sub-micelles are less heat stable than whole micelles. Among ions tested, added Ca reduced heat stability more dramatically in whole micelles, whereas added phosphate improved the stability of micelles and, more strikingly, of sub-micelles. Citrate also improved the heat stability of sub-micelles but not of whole micelles.     

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.     

Protein interactions in heat-treated milk and effect on rennet coagulation

Skim milk was heated at different pH values to cause differential association of whey proteins (WP) with the casein micelles. All of these milk treatments coagulated poorly with rennet. To understand this in more detail, the casein micelles from heated milk were redispersed in unheated serum or unheated micelles were suspended in the sera from heat-treated milk. Systems containing micelles from milk heated at pH 6.7 and 7.1 were marginally better than the heated milk, but that from milk heated at pH 6.3 was not. The sera from milk heated at pH 6.7 and 7.1 impaired the clotting of native micelles but that from the pH 6.3 milk did not. Native casein micelles were suspended in permeates or dialyzed (against unheated milk) sera from heat-treated milk. Permeate systems free of WP/κ-casein complexes produced significantly stronger rennet gels; as did dialyzed systems. The impaired rennet clotting of heat-treated milk was attributed to a synergistic effect of the casein micelles with their heat-modified surfaces, the soluble serum WP/κ-casein complexes, and other dialyzable serum components.     

The effect of ultrasound on casein micelle integrity

Samples of fresh skim milk, reconstituted micellar casein, and casein powder were sonicated at 20 kHz to investigate the effect of ultrasonication. For fresh skim milk, the average size of the remaining fat globules was reduced by approximately 10 nm after 60 min of sonication; however, the size of the casein micelles was determined to be unchanged. A small increase in soluble whey protein and a corresponding decrease in viscosity also occurred within the first few minutes of sonication, which could be attributed to the breakup of casein-whey protein aggregates. No measurable changes in free casein content could be detected in ultracentrifuged skim milk samples sonicated for up to 60 min. A small, temporary decrease in pH resulted from sonication; however, no measurable change in soluble calcium concentration was observed. Therefore, casein micelles in fresh skim milk were stable during the exposure to ultrasonication. Similar results were obtained for reconstituted micellar casein, whereas larger viscosity changes were observed as whey protein content was increased. Controlled application of ultrasound can be usefully applied to reverse process-induced protein aggregation without affecting the native state of casein micelles.     

Comparison of casein micelles in raw and reconstituted skim milk

During the manufacture of skim milk powder, many important alterations to the casein micelles occur. This study investigates the nature and cause of these alterations and their reversibility upon reconstitution of the powders in water. Samples of skim milk and powder were taken at different stages of commercial production of low-, medium-, and high-heat powders. The nature and composition of the casein micelles were analyzed using a variety of analytical techniques including photon correlation spectroscopy, transmission electron microscopy, turbidity, and protein electrophoresis. It was found that during heat treatment, whey proteins are denatured and become attached to the casein micelles, resulting in larger micelles and more turbid milk. The extent of whey protein attachment to the micelles is directly related to the severity of the heat treatment. It also appeared that whey proteins denatured during heat treatment may continue to attach to casein micelles during water removal (evaporation and spray-drying). The process of water removal causes casein and Ca in the serum to become increasingly associated with the micelles. This results in much larger, denser micelles, increasing the turbidity while decreasing the viscosity of the milk. During reconstitution, the native equilibrium between colloidal Ca and serum Ca is slowly reestablished. The reequilibration of the caseins and detachment of the whey proteins occur even more slowly. The rate of reequilibration does not appear to be influenced by shear or temperature in the range of 4 to 40°C.     

A 1H-NMR study of bovine casein micelles; influence of pH, temperature and calcium ions on micellar structure

The influence of pH, temperature and Ca depletion on bovine casein micelle suspensions in D2O containing simulated milk ultrafiltrate was studied by 1H-NMR spectroscopy. In the pH range of 5.8-7.5 the spectrum of the micelles showed very little pH dependence, indicating that no changes occurred in the dynamic behaviour of the proteins constituting the micelle. The NMR spectrum of casein micelles was strongly temperature dependent, particularly in the temperature range of 60-98 degrees C. Increase in temperature resulted in a strong increase in spectral intensity concomitant with changes in the spectral characteristics. In micelle suspensions these changes were reversible, and indicated that at elevated temperatures the rigid structure of the casein micelle started to melt, leading to an increased mobility of appreciable parts of the proteins in the micelle. Ca depletion of the casein micelles by addition of EDTA resulted in an increase in spectral intensity, which arose from the presence of casein components in the serum phase. The spectrum of these serum phase particles resembled closely the spectrum of a solution of total casein in simulated milk ultrafiltrate and was quite different from the spectrum of casein micelles. The implications of these results with respect to models of the structure of bovine casein micelles are discussed.     

Relationship between physical properties of casein micelles and rheology of skim milk concentrate

The properties of casein micelles in milk concentrates are of interest for the use of ultrafiltered (UF) skim milk concentrates in dairy products, and for the general understanding of colloidal stability and behavior of the casein micelle. The rheological behavior of UF skim milk concentrate with a casein concentration of 19.5% (wt/wt) was investigated at different pH and NaCl concentrations by analyzing flow viscometry and small amplitude oscillatory shear measurements. Viscometric flow curves were fitted to the Carreau-Yasuda model with the aim of determining values for the viscosity at infinite high shear rates and thereby estimate the voluminosity of the casein micelles (ν_casein) in the UF concentrate. The voluminosity of the casein micelles increased with addition of NaCl and decreased when pH was decreased from 6.5 to 5.5. At pH 5.2, ν_casein increased because of acid-induced aggregation of the casein micelles. The changes in ν_casein could be interpreted from transmission electron microscopy of freeze-fractured samples of the UF concentrate and partly from dynamic light scattering measurements. Altered interactions between casein micelles due to different pH and NaCl concentrations are proposed to occur due to collapse of the κ-casein layer, changed ionic strength, and altered distance between casein micelles.     

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.     

Particle size determines foam stability of casein micelle dispersions

The role of interfacial properties and size of casein micelles aggregates on foam stability of casein micelle dispersions (CMDs) was examined. CMDs were prepared by redispersing casein micelles pellets obtained by ultracentrifugation. The size of colloidal particles could be controlled by differences in redispersing temperature. CMDs redispersed at 20 °C (CMD_20 °C) and 4 °C (CMD_4 °C) had average particle sizes of around 200 nm (micelles) and 500 nm (micelles and aggregates), respectively. At 3% total protein, the foam half-life, t_½, of CMD_4 °C was significantly higher than that of CMD_20 °C and skim milk. No correlation between foam stability and surface rheological properties or protein composition could be observed. Foam stability was strongly related to the size of colloidal particles present in CMD. This was confirmed by the observation that the foam stability of CMD_4 °C decreased to that of CMD_20 °C when the aggregates were broken down by homogenisation.     

Factors that affect the aggregation of rennet-altered casein micelles at low temperatures

The effects of temperature, CaCl₂ concentration, pH and ionic strength of milk on the aggregation of casein micelles in milk renneted at 15ºC were studied using particle size analysis determined by laser-light scattering. The rate of aggregation of rennet-altered casein micelles became significantly slower on reducing the temperature of renneting from 30 to 10ºC. At 15ºC, the rate of aggregation of rennet-altered casein micelles increased significantly on adding CaCl₂, on reducing the pH of renneted milk or on adding NaCl (up to 50 m m ). These results indicate that particle size analysis can be used successfully to study the aggregation of rennet-altered casein micelles.     

The use of sedimentation field flow fractionation and photon correlation spectroscopy in the characterization of casein micelles

Sedimentation Field Flow Fractionation (SdFFF) was combined with Photon Correlation Spectroscopy (PCS), to characterize changes in the structure of the colloidal particles of reconstituted skim milk of diameter >50 nm (aggregates of casein and calcium phosphate known as casein micelles) with the changes in partitioning (with the addition of salt) of calcium (Ca), inorganic phosphate (Pi) and casein between the serum and colloidal phases of the milk. The number weighted particle size distributions are determined. These are well represented by a log-normal distribution. Methods are presented for estimating the relative contributions of scattering and absorbance to the SdFFF detector signal and for taking both into account when analysing SdFFF data. The values found for the effective density of the casein micelles were in good agreement with the literature and ranged from (1.06-1.08 g cm(-3)) according to the composition of micelles. The changes in the scattering intensity as determined by PCS correlated with the changes in the particle composition. Although the concentrations of colloidal calcium phosphate (CCP) (1.1-3.5 g/kg milk) and micellar casein (18.1-27.2 g/kg milk) varied considerably only small changes in the size distribution of particles >50 nm diameter were observed except for milk to which 30 mmol Pi+10 mmol Ca/kg milk had been added where the particle size distribution shows a swelling of the particles consistent with a lower than expected value for the particle density. These observations suggest that the micelles have the ability to both lose (depleted micelles) and accommodate (enriched micelles) more casein, calcium and inorganic phosphate in their interior, thus confirming the model of the micelles which postulates an open structure allowing freedom of movement of casein and small ions.     

Cryo-transmission electron tomography of native casein micelles from bovine milk

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (∼20 to 30 nm in diameter), channels (diameter greater than ∼5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed.