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.     

Effect of alkalinization and ultra-high-pressure homogenization on casein micelles in raw and pasteurized skim milk

Mechanical and physicochemical treatments of milk induce structural modifications of the casein (CN) micelles, affecting their techno-functional properties in dairy processing. Here, we studied the effect of alkalinization and ultra-high-pressure homogenization (UHPH) on CN micelles in raw skim milk (rSM) and pasteurized skim milk (pSM). The pH of both skim milks (approximately 6.7) was adjusted to 8.5 and 10.5 before UHPH at 100, 200, and 300 MPa. The structural changes of the CN micelles during the treatments were assessed using laser diffraction, transmission electron microscopy, and turbidity measurements. Finally, ultracentrifugation (70,000 × g for 1 h at 20°C) was carried out to evaluate the protein's distribution between the supernatant (serum phase) and the pellet (colloidal phase) by gel electrophoresis and protein concentration measurement. Alkalinization of both skim milks induced a significant reduction in turbidity, whereas an increase of the average particle size was observed, the effect being more severe in pSM than rSM. At alkaline pH, more proteins were recovered in the serum phase, which suggested that the CN underwent major rearrangements into nonsedimentable CN forms of various sizes, as confirmed by transmission electron microscopy. The amount of CN found in the serum phase at pH 8.5 also increased with the UHPH pressure. Although UHPH did not influence the average CN micelle size at pH 6.7 and 8.5, a pressure-dependent decrease was observed at pH 10.5 for both skim milks. The structural changes of the CN micelles observed in this study throughout the combination of alkalinization and UHPH could be of interest for developing new dairy ingredients with improved functionality.     

Casein micelle dissociation in skim milk during high-pressure treatment: Effects of pressure, pH, and temperature

The effect of pH (from 5.5 to 7.5) and temperature (from 5 to 40°C) on the turbidity of reconstituted skim milk powder was investigated at ambient pressure and in situ under pressure (up to 500 MPa) by measurement of light scattering. High-pressure treatment reduced the turbidity of milk for all combinations of pH and temperature due to micelle dissociation. The turbidity profiles had a characteristic sigmoidal shape in which almost no effect on turbidity was observed at low pressures (100 MPa), followed by a stronger pressure dependency over a pressure range of 150 MPa during which turbidity decreased extremely. From the turbidity profiles, the threshold pressure for disruption of micelle integrity was determined and ranged from 150 MPa at low pH to 350-400 MPa at high pH. The threshold pressure diagram clearly showed a relationship between the barostability of casein micelles and pH, whereas almost no effect of temperature was shown. This remarkable pH effect was a consequence of pressure-induced changes in the electrostatic interactions between colloidal calcium phosphate and the caseins responsible for maintaining micellar structure and was explained by a shift in the calcium phosphate balance in the micelle-serum system. Accordingly, a mechanism for high pressure-induced disruption of micelle integrity is suggested in which the state of calcium plays a crucial role in the micelle dissociation process.     

Isolation of caseins from whey proteins by microfiltration modifying the mineral balance in skim milk

The objective of this work was to study the effect of different salts and salt concentration on the isolation of casein micelles from bovine raw skim milk by tangential flow microfiltration. Tangential flow microfiltration (0.22 μm) was conducted in a continuous process adding a modified buffer to maintain a constant initial sample volume. This buffer contained calcium chloride (CaCl₂), sodium phosphate (Na₂HPO₄), or potassium citrate (K₃C₆H₅O₇) in concentrations ranging from 0 to 100 mM. The concentrations of caseins and whey proteins retained were determined by sodium dodecyl sulfate-PAGE and analyzed using the Scion Image software (Scion Corporation, Frederick, MD). A complete isolation of caseins from whey proteins was achieved using sodium phosphate in the range of 10 to 50 mM and 20 times the initial volume of buffer added. No whey proteins were detected at 50 mM but this was at the expense of low caseins being retained. When lower sodium phosphate concentrations were used, the amount of caseins retained was higher but a small amount of whey proteins were still detected by sodium dodecyl sulfate-PAGE. Among the salts tested, calcium chloride at 50 mM and all volumes of buffer showed the higher retention of casein proteins. The highest casein:whey protein ratio was found at 30 mM CaCl₂, but no complete casein micelle isolation was achieved. Potassium citrate was the most ineffective salt because a rapid loss of caseins and whey proteins was observed at all concentrations and with low quantities of buffer added during the filtration process. Our results show the potential of altering the mineral balance in milk for isolation of casein micelles from whey proteins in a continuous tangential flow microfiltration system.     

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。     

The association of lipophilic phospholipids with native bovine casein micelles in skim milk: Effect of lactation stage and casein micelle size

A known biological role of casein micelles is to transport calcium from mother to young and provide amino acids for growth and development. Previous reports demonstrated that modified casein micelles can be used to transport and deliver hydrophobic probes. In this study, the distribution of lipid-soluble phospholipids, including sphingomyelins (SM) and phosphatidylcholines (PC), was quantified in whole raw milk, skim raw milk, and casein micelles of various sizes during early, mid, and late lactation stages. Low-pressure size exclusion chromatography was used to separate casein micelles by size, followed by hydrophobic extraction and liquid chromatography–mass spectrometry for the quantification of PC and SM. Results showed that the SM d18:1/23:0, d18:1/22:0, d18:1/16:0, d16:1/22:0, d16:1/23:0, and d18:1/24:0 and the PC 16:0/18:1, 18:0/18:2, and 16:0/16:0 were dominating candidates appearing in maximum concentration in whole raw milk obtained from late lactation, with 21 to 50% of total SM and 16 to 35% of total PC appearing in skim milk. Of the total SM and PC found in skim milk, 35 to 46% of SM and 22 to 29% of PC were associated with the casein micelle fraction. The highest concentrations of SM d18:1/22:0 (341 ± 17 µg/g of casein protein) and PC 16:0/18:1 (180 ± 20 µg/g of casein protein) were found to be associated with the largest casein micelles (diameter = 149 nm) isolated in milk from late lactation, followed by a decrease in concentration as the casein micelle size decreased.     

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.     

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.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

牛羊乳蛋白组分比较研究

羊乳被国际营养学界誉为"奶中之王",逐渐被人们列为日常生活的营养保健佳品。该文对羊乳和牛乳的蛋白组分(主要是酪蛋白和乳清蛋白)含量、氨基酸组成及变异体等方面的差异进行了综述,并且对两者酪蛋白胶束的差异进行了比较,为羊乳检验和加工提供理论依据。     

Investigation of the microstructure of milk protein concentrate powders during rehydration: Alterations during storage

The aim of this work was to use scanning electron microscopy to investigate the microstructure of rehydrated milk protein concentrate powder (MPC) particles. A sample preparation method for scanning electron microscopy analysis of rehydrated MPC particles is described and used to characterize the time course of dissolution and the effects of prior storage on the dissolution process. The results show that a combination of different types of interactions (e.g., bridges, direct contact) between casein micelles results in a porous, gel-like structure that restrains the dispersion of individual micelles into the surrounding liquid phase without preventing water penetration and solubilization of nonmicellar components. During storage of the powder, increased interactions occur between and within micelles, leading to compaction of micelles and the formation of a monolayer skin of casein micelles packed close together, the combination of which are proposed to be responsible for the slow dissolution of stored MPC powders.     

Dynamics of casein micelles in skim milk during and after high pressure treatment

The effect of high hydrostatic pressure on turbidity of skim milk was measured in situ together with casein micelle size distribution. High pressure (HP) treatment reduced the turbidity of milk with a stronger pressure dependency between 50 and 300 MPa when the temperature was decreased from 20 to 5 °C, while at 30 °C (50–150 MPa) turbidity exceeded that of untreated milk. At 250 and 300 MPa turbidity decreased extremely. During pressurization of milk at 250 and 300 MPa, the turbidity initially decreased, but treatments longer than 10 min increased the turbidity progressively, indicating that re-association followed dissociation of casein micelles. Especially at 40 °C and at 250 and 300 MPa, the turbidity increased beyond untreated milk. Dynamic light scattering was used to investigate casein micelle sizes in milk immediately after long time (up to 4 h) pressurization at 250 and 300 MPa and casein micelle size distributions were bimodal with micelle sizes markedly smaller and markedly larger than those of untreated milk. Pressure modified casein micelles present after treatment of milk at 250 and 300 MPa were concluded to be highly unstable, since the larger micelles induced by pressure showed marked changes toward smaller particle sizes in milk left at ambient pressure.     

Rheological properties of concentrated skim milk: importance of soluble minerals in the changes in viscosity during storage

Properties of condensed milks prior to spray drying dictate to a large extent the functionality of the resulting milk powder. Rheological properties of concentrated skim milk, with total solids content of 45% but different mineral content, were studied as a function of shear rate and storage time at 50 degrees C. These milks are proposed as a model to study the effects of minerals on rheology and age gellation of condensed milk prior to drying. During storage of the concentrated milk, the apparent viscosity, particularly after 4 h, increased markedly at all shear rates studied. The yield stress also increased steeply after 4 h of storage at 50 degrees C. The changes in apparent viscosity of concentrated milk stored for up to 4 h were largely reversible under high shear, but irreversible in samples stored for longer time. The appearance of yield stress suggested the presence of reversible flocculation arising from weak attraction between casein micelles, with a transition from reversible to irreversible aggregation during storage. Particle size analysis confirmed irreversible aggregation and fusion of casein micelles during storage. Gradual reduction of mineral content of concentrated milks resulted in a marked decrease in the apparent viscosity and casein micelle aggregation during storage, while addition of minerals to milk had the opposite effect. The results demonstrated that the soluble mineral content is very important in controlling the storage-induced changes in the rheology of concentrated milks.     

High-pressure-induced interactions between milk fat globule membrane proteins and skim milk proteins in whole milk

The association of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) with milk fat globule membrane (MFGM), when whole milk was treated by high pressure in the range 100 to 800 MPa, was investigated using sodium dodecyl sulfate (SDS)-PAGE under reducing and nonreducing conditions. In SDS-PAGE under reducing conditions, beta-LG was observed in the MFGM material isolated from milk treated at 100 to 800 MPa for 30 min, and small amounts of alpha-LA and kappa-casein were also observed at pressures >600 MPa for 30 min. However, these proteins were not observed in SDS-PAGE under nonreducing conditions. These results indicate that beta-LG and alpha-LA associated with MFGM proteins via disulfide bonds during the high-pressure treatment of whole milk. The amount of beta-LG associated with the MFGM increased with an increase in pressure up to 800 MPa and with increasing time of pressure treatment. The maximum value for beta-LG association with the MFGM was approximately 0.75 mg/g of fat. Of the major original MFGM proteins, no change in butyrophilin was observed during the high-pressure treatment of whole milk, whereas xanthine oxidase was reduced to some extent beyond 400 MPa. In contrast to the behavior during heat treatment, PAS 6 and PAS 7 were stable during high-pressure treatment, and they remained associated with the MFGM.     

Stabilization of milk proteins in acidic conditions by pectic polysaccharides extracted from soy flour

Pectic polysaccharides were extracted from soy flour at either room temperature (SPRT) or 121°C (SPH), and their abilities to stabilize milk proteins in acidic conditions were evaluated. Both SPRT and SPH were found to contain proteinaceous components that were difficult to dissociate from polysaccharide components using size exclusion chromatography, whereas the molar mass of the former was approximately twice that of the latter. Due to the higher molar mass, SPRT was expected to provide stronger steric effects to prevent aggregation between milk proteins in acidic conditions than SPH. Alkaline treatment of SPRT for breaking O-linkages between AA and monosaccharide residues decreased its molar mass by approximately 160 kDa, indicating that they contained naturally occurring conjugates of pectic and proteinaceous moieties. Particle size distributions in simulated acidified milk drink samples containing 0.2% SPRT or SPH showed monomodal distributions with median diameters of around 1.2 μm at pH 4. The presence of large protein aggregates (～5 μm) was detected at 0.2% SPRT and pH 3.2, 0.6 to 0.8% SPRT and pH 4, or 0.2% SPH and pH 3.4. The presence of excess polysaccharide molecules unbound to proteins was detected at 0.2% SPRT and pH 3.2 to 3.4, 0.4 to 0.8% SPRT and pH 4, 0.2% SPH and pH 3.4 to 3.6, and 0.4 to 0.8% SPH and pH 4. The present results suggest that molecular characteristics of pectic polysaccharides vary depending on extraction conditions and hence their functional behavior.     

酪蛋白胶束结构及其对牛乳稳定性的影响

论述了酪蛋白的组成及两类具有代表性的酪蛋白胶束模型—亚胶束模型和内部结构模型,这两种模型可以较好地解释酪蛋白胶束的稳定性。pH值、酶、添加成分、工艺过程等能够影响酪蛋白胶束的稳定性,并直接影响到牛乳的稳定。这一方面有利于乳制品的生产如制作酸奶、干酪,一方面影响乳制品的品质。     

A microfiltration process to maximize removal of serum proteins from skim milk before cheese making

Microfiltration (MF) is a membrane process that can separate casein micelles from milk serum proteins (SP), mainly beta-lactoglobulin and alpha-lactalbumin. Our objective was to develop a multistage MF process to remove a high percentage of SP from skim milk while producing a low concentration factor retentate from microfiltration (RMF) with concentrations of soluble minerals, nonprotein nitrogen (NPN), and lactose similar to the original skim milk. The RMF could be blended with cream to standardize milk for traditional Cheddar cheese making. Permeate from ultrafiltration (PUF) obtained from the ultrafiltration (UF) of permeate from MF (PMF) of skim milk was successfully used as a diafiltrant to remove SP from skim milk before cheese making, while maintaining the concentration of lactose, NPN, and nonmicellar calcium. About 95% of the SP originally in skim milk was removed by combining one 3 x MF stage and two 3 x PUF diafiltration stages. The final 3 x RMF can be diluted with PUF to the desired concentration of casein for traditional cheese making. The PMF from the skim milk was concentrated in a UF system to yield an SP concentrate with protein content similar to a whey protein concentrate, but without residuals from cheese making (i.e., rennet, culture, color, and lactic acid) that can produce undesirable functional and sensory characteristics in whey products. Additional processing steps to this 3-stage MF process for SP removal are discussed to produce an MF skim retentate for a continuous cottage cheese manufacturing process.     

Effect of dietary crude protein degradability and corn processing on lactation performance and milk protein composition and stability

The present study aimed to evaluate the effect of crude protein degradability and corn processing on lactation performance, milk protein composition, milk ethanol stability (MES), heat coagulation time (HCT) at 140°C, and the efficiency of N utilization for dairy cows. Twenty Holstein cows with an average of 162 ± 70 d in milk, 666 ± 7 kg of body weight, and 36 ± 7.8 kg/d of milk yield (MY) were distributed in a Latin square design with 5 contemporaneous balanced squares, 4 periods of 21 d, and 4 treatments (factorial arrangement 2 × 2). Treatment factor 1 was corn processing [ground (GC) or steam-flaked corn (SFC)] and factor 2 was crude protein (CP) degradability (high = 10.7% rumen-degradable protein and 5.1% rumen-undegradable protein; low = 9.5% rumen-degradable protein and 6.3% rumen-undegradable protein; dry matter basis). A significant interaction was observed between CP degradability and corn processing on dry matter intake (DMI). When cows were fed GC with low CP degradability, DMI increased by 1.24 kg/d compared with cows fed GC with high CP degradability; however, CP degradability did not change DMI when cows were fed SFC. Similar interactions were observed for MY, HCT, and lactose content. When cows were fed GC diets, high CP degradability reduced MY by 2.3 kg/d, as well as HCT and lactose content, compared with low CP degradability. However, no effect of CP degradability was observed on those variables when cows were fed SFC diets. The SFC diets increased dry matter and starch total-tract digestibility and reduced β-casein (CN) content (% total milk protein) compared with GC diets. Cows fed low-CP degradability diets had higher glycosylated κ-CN content (% total κ-CN) and MES, as well as milk protein content, 3.5% fat-corrected milk, and efficiency of N for milk production, than cows fed high-CP degradability diets. Therefore, GC and high-CP degradability diets reduced milk production and protein stability. Overall, low CP degradability increased the efficiency of dietary N utilization and MES, probably due to changes in casein micelle composition, as CP degradability or corn processing did not change the milk concentration of ionic calcium. The GC diets increased β-CN content, which could contribute to reducing HTC when cows were fed GC and high-CP degradability diets.     

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.     

葡萄糖对脱脂乳酪蛋白胶束稳定性的影响研究

研究了葡萄糖添加量、体系pH、热处理温度、热处理时间对脱脂牛乳酪蛋白胶束稳定性的影响。研究表明,在接近中性pH条件下,少量葡萄糖分子可降低脱脂乳酪蛋白胶束的稳定性,大量葡萄糖分子可增强其稳定性。添加葡萄糖后,在高温热处理的诱导下,酪蛋白沉淀的pH升高。随着热处理时间的延长,脱脂乳中酪蛋白胶束表现出聚集行为,致使粒径增大,浊度和沉淀率总体呈现上升趋势。该研究结论可为乳品加工提供参考依据。