A possible structure of the casein micelle based on high-resolution field-emission scanning electron microscopy

New electron micrographs, produced using the technique of Field Emission Scanning Electron Microscopy, showing the details of the micellar surface, are presented. The images show the micellar surfaces without any coating, and suggest that the surface of the micelle may have a much more complex structure than has previously been demonstrated. Although there appear to be no spherical subunits (submicelles), there is evidence for the organization of the caseins into tubular structures within the micelle. The surface is not smooth, and contains gaps between the substructures. The observations are discussed in terms of published models of micellar structure, where is it suggested how the depiction of the micellar surface can be used to explain certain factors of its reactivity and behaviour.     

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.     

Experimental evidence for previously unclassified calcium phosphate structures in the casein micelle

¹H-³¹P Cross-polarization magic angle spinning (CP-MAS) measurements of 40-d-old Mozzarella cheese and 20 mM EDTA-treated casein micelles revealed that each sample had immobile phosphorus with the same spectral pattern, which did not match that of native casein micelles. To identify the immobile phosphorus bodies, ¹H-³¹P CP-MAS spectra and cross-polarization kinetics measurements were undertaken on native casein micelles, EDTA-chelated casein micelles, and reference samples of β-casein and hydroxyapatite. The results showed that the immobile phosphorus bodies in the mature Mozzarella cheese had the following characteristics: they are immobile phosphoserine residues (not colloidal calcium phosphate); they are not the product of phosphoserine to colloidal calcium phosphate bridging; the phosphate is complexed to calcium; their rigidity is localized to a phosphorus site; their rigidity and bond coupling is unaffected by protein hydration; and the immobile bodies share a narrow range of bond orientations. Combining these observations, the best explanation of the immobile phosphorus bodies is that bonding structures of phosphorus-containing groups and calcium exist within the casein micelle that are not yet clearly classified in the literature. The best candidate is a calcium-bridged phosphoserine-to-phosphoserine linkage, either intra- or inter-protein.     

酪蛋白胶束结构和理化性质的研究进展

酪蛋白是乳中一大类蛋白质的总称,约占乳蛋白质量分数的80%。牛乳中的酪蛋白不是单独存在的,而是和矿质元素相互缠绕在一起而形成的复合物。牛乳的酸凝、酶凝以及液态乳稳定性的变化,从本质上来说都是酪蛋白胶束发生的一系列变化所导致的。本文概述了组成酪蛋白胶束中四种酪蛋白单体各自的结构和理化特征。综述了胶束的组成、胶束的流体力学直径、容积度、密度、水合性、分子间距等几个重要的特征参数。最后总结了核壳模型、双结合模型、亚单元模型和Holt模型等酪蛋白胶束的理论结构模型的特征及各种的缺陷,以期对乳制品的生产和加工提供一些思路。      

酪蛋白胶束酶法修饰研究进展

酪蛋白的稳定性与其结构密不可分。酶法修饰作为一种安全性较高的修饰技术,受到众多研究者的关注。本文综述了近年来国内外关于酪蛋白酶法修饰研究进展,讨论了修饰酪蛋白的稳定性及功能性质,以期为乳制品加工提供参考。      

酪蛋白胶束结构研究方法综述

酪蛋白是乳中蛋白质的主要成分,约占其80%。由于酪蛋白胶束对乳制品的功能特性起重要作用,因此其研究一直备受关注,但酪蛋白胶束的内部结构至今没有确切的定义,而是许多学者提出了各种理论模型。文章综述了酪蛋白胶束结构、解离与聚集的多种研究方法,为以后学者研究酪蛋白提供借鉴。      

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.     

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.     

Activity and nature of plasminogen activators associated with the casein micelle

In fresh milk, plasminogen, the zymogen form of plasmin (PL), is the predominant form. Therefore, plasminogen activators (PA) can contribute significantly to PL activity in milk. Both tissue-type PA (tPA) and urokinase-type PA (uPA) exist in milk; however, contradictory findings have been reported for which type of PA is most closely associated with the casein micelles. Little is known about the factors that might lead to variations in the individual activities of the PA. The objective of this work was therefore to investigate possible factors that might affect the association of tPA and uPA with the casein micelle and their activities thereafter. Plasminogen activators were isolated from milk samples with different somatic cell counts following 2 different isolation protocols. Determination of uPA, tPA, and PL activities was carried out quantitatively following chromogenic assays using 2 different substrates, and qualitatively using specialized sodium dodecyl sulfate-PAGE. Different isolation methods and conditions led to differences in uPA, tPA, and PL activities. Urokinase-type PA activity was significantly higher in PA fractions isolated from milk with high somatic cell counts than from milk with low somatic cell counts. Activity results indicated that in pasteurized milk uPA could dissociate from the somatic cells and bind to casein. Moreover, a high level of PL in isolated PA fractions contributed to significantly enhanced PA activities. Overall, results confirmed the association of both uPA and tPA with the casein micelle; however, their amounts, activities, and molecular weights varied based on the nature of the milk and methods of separation, with uPA being the PA with greater potential to affect plasminogen activation in milk.     

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。     

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.     

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.     

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。     

Evidence for fibril-like structure in bovine casein micelles

The addition of Congo red (CR) dye to diluted raw skim milk resulted in a red shift indicative of the presence of fibril-like structures. Thioflavin T (ThT) is another dye that very specifically binds to protein fibrils, and when added to undiluted raw skim milk, the classic 485 nm fluorescence peak of a ThT-fibril complex was observed. Repeating these experiments with various raw milk components showed that the CR red shift and ThT fluorescence peak were due to the presence of casein micelles, and to a lesser extent, sodium caseinate. Fluorescent peaks were also observed when ThT was added to solutions of purified α_S- and κ-casein, but not β-casein, in 0.5 M HEPES buffer (pH = 6.8). The addition of 25 mM Ca²⁺ had no effect on β-casein fluorescence, and significantly reduced the κcasein peak. However, adding 25 mM Ca²⁺ to α_S-casein produced a turbid solution and a 6-fold increase in fluorescence, indicating that the aggregates formed contain fibril-like structure. Casein micelle images obtained by transmission electron microscopy showed the presence of short (7 to 10 nm) fibers cross-linked by dense aggregate junction zones. The observed fibers closely resemble protofibrils, intermediate structures that are observed during the formation of amyloid fibrils.     

乳蛋白胶粒的稳定性研究及应用

根据蛋白质一级结构原理,选用了一种碱性蛋白质—鱼精蛋白,研究了牛乳蛋白胶粒在适口酸性条件下的稳定性问题。确定出乳蛋白饮料的最佳工艺条件:牛奶含量(v/v)35%,白砂糖12.0%(m/v),柠檬酸含量0.2%(m/v),调酸温度在20℃之内,去离子水定容;以及鱼精蛋白的添加顺序和含量:在调酸前添加稳定效果最佳,含量(m/v)在0.3%即可达到稳定效果。     

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.     

Formation of reconstituted casein micelles with human beta-caseins and bovine kappa-casein

Human beta-casein (CN) is the major protein of the human milk casein fraction (approximately 80%) and exists in six calcium-sensitive forms, having zero to five organic phosphates per molecule. The major forms are the doubly-phosphorylated (beta-CN-2P; approximately 30%) and the quadruply phosphorylated (beta-CN-4P; approximately 35%) forms. Although calcium-insensitive, kappa-CN is known for its role in preventing the precipitation of beta-CN in the presence of Ca+2, but it is not known how the different levels of phosphorylation may affect this. In the present investigation, turbidity, measured at 400 nm, was determined at increasing temperatures (4 up to 37 degrees C) for solutions of beta-CN-2P and beta-CN-4P (3 mg/ml in 0.02 M NaCl, 0.01 M imidazole, pH 7) individually and also mixed with bovine kappa-CN in 6/1 and 3/1 weight ratios of beta/kappa and containing 0, 5, and 10 mM Ca+2. The results indicate that the first step of micelle formation probably leads to polymers of limited size, the only complexes available to beta-CN-2P under most conditions. With beta-CN-4P, these polymers aggregate further to give reconstituted micelles, probably because of the ability to form crosslinks at this phosphorylation level. The formation of reconstituted micelles under various conditions of pH, Ca+2 concentration and kappa-CN content indicates that both hydrophobic interactions and Ca+2 bridges or crosslinks may contribute to protein aggregation and micelle building.     

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.     

牦牛乳酪蛋白胶束琥珀酰化修饰研究

以琥珀酸酐为酰化试剂,牦牛乳酪蛋白为原料,对其进行了酰化修饰。系统研究了多种因素对酰化程度的影响,采用响应面法优化了修饰工艺,研究了酪蛋白乳化性变化。结果显示,牦牛乳酪蛋白胶束琥珀酰化修饰最佳条件为琥珀酸酐与酪蛋白配比0.6∶1(g/g),温度42℃,反应时间49min,pH8.8,酰化程度为86.01%。修饰后,牦牛乳酪蛋白的乳化稳定性和乳化活性分别提高了161.64%和98.54%。酪蛋白酰胺化修饰反应条件温和,易调节,修饰程度高,修饰酪蛋白的乳化性显著改善。该研究可为牛乳酪蛋白的酰化改性提供参考依据。      

3种乳源酪蛋白粒径及胶束结构的差异性

通过纳米粒度分析仪和扫描电子显微镜,分别对水牛乳、牛乳及羊乳中的酪蛋白颗粒直径大小分布情况及酪蛋白胶束结构进行研究。结果表明：水牛乳、牛乳及羊乳中酪蛋白的粒径分布及胶束结构方面存在明显的差异。水牛乳酪蛋白平均颗粒直径为182.3nm,酪蛋白颗粒互相连接成较细长的胶束,胶束之间交联成网络状;牛乳酪蛋白平均颗粒直径为207.4nm,酪蛋白颗粒聚集成直径较大的胶束;羊乳酪蛋白平均颗粒直径为173.8nm,酪蛋白颗粒仅能够形成较短的胶束,也不能交联成网络状。