Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous sections (CEMOVIS)

Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous... Abstract Casein micelles are present in bovine milk as colloidal particles with diameters of 20–600 nm, which are complex macromolecular assemblies composed of four distinct types of casein and colloidal calcium phosphate (CCP). Multiple structural models of casein micelles have been proposed based on their biochemical or physical properties and observed using electron microscopy. However, the CCP distribution and crosslinking structure between CCP and casein remain unclear. Therefore, the internal structure of casein micelles in raw milk was observed using cryo-electron microscopy of vitreous sections (CEMOVIS) with high precision at high resolution. The results confirmed that the average casein micelle diameter was about 140 nm, and that the CCP diameter in casein micelles was about 2–3 nm, with an average diameter of 2.3 nm. The distribution of CCP in casein micelles was not uniform, with an average interval between CCPs of about 5.4 nm. Areas containing no black particles (attributed to CCP) were present, with an average size of about 19.1 nm. Considering previous reports, these areas possibly correspond to pores or cavities filled with water. Based on differences in the density of structures in casein micelles, we estimated that some of the casein aggregates were able to connect with CCP in a string. casein micelle, CEMOVIS, milk, cryo-electron microscopy, colloidal calcium phosphate (CCP), high-pressure freezing Introduction Bovine milk contains all essential nutrients for calf growth and is an important food for humans. Milk contains protein, calcium and phosphorus, which are essential nutrients for bone and muscle growth. Calcium and phosphorus in milk are mostly solubilized, with the structures of casein micelles and colloidal calcium phosphate (CCP) thought to contribute to the solubilization or dispersion of calcium and phosphorus, in addition to preventing their precipitation. Accordingly, CCP and casein micelles have been extensively studied for more than a century [1]. Casein micelles are present as colloidal particles with diameters of 20–600 nm in bovine milk, and are complex macromolecular assemblies composed of four distinct types of caseins, namely, αs1, αs2, β and κ-caseins, and minerals, including calcium and phosphate in the form of CCP. αs1-, αs2- and β-caseins are calcium-sensitive proteins that precipitate in the presence of Ca2+. κ-casein interacts with these caseins and stabilizes casein micelles. Casein micelles are hydrated dynamic structures [2,3] that are heterogeneous in composition and size and polydisperse [4]. When CCP is solubilized due to decreasing pH or calcium removal, the dissociation of casein micelles progresses gradually with a change in the size distribution [5]. Based on the biochemical or physical properties of casein micelles, two kinds of structural models have been proposed. However, the detailed internal structure has yet to be clarified and remains disputed. In the first model (submicelle model) proposed by Schmidt [6], the casein micelles are composed of smaller proteinaceous subunits (submicelles), with CCP cross-linked between them. In the second model (nanocluster model) proposed by Holt [1], there are no submicelles, and nanoclusters of CCP are randomly distributed in a uniform matrix of caseins. Microstructural observation of casein micelles using electron microscopy has also been performed as a powerful method to determine the structural models. Examples using transmission electron microscopy (TEM) include observing casein micelles in reconstituted skim milk using a shadowing method [7], evaluating casein micelle size through negative staining [8], and observing artificial casein micelles using ultrathin sections without staining [9]. An example using scanning electron microscopy (SEM) is the observation of casein micelles in reconstituted skim milk using chemical fixation, followed by dehydration and drying [10]. However, various artifacts are produced during the processes involved in electron microscopy observation methods, such as specimen preparation. These prevent an accurate image of casein micelles being obtained because milk is a colloidal aqueous dispersion and casein micelles have small particle sizes (100–150 nm) [11]. For example, in freeze-fracture replica methods, fine protein structures are coated with a metal, significantly altering the protein surface structure. Negative staining, including an air-drying process, causes specimen shrinkage due to drying. For ultrathin sections, structural alterations preclude accurate observation, crosslinking among proteins occurs due to chemical fixation, shrinking occurs during dehydration using ethanol, and protein loss occurs during infiltration to the resin [12]. Recently, casein micelles have been observed using cryo-electron microscopy by embedding the specimen in vitreous ice to avoid artifacts, and small-angle X-ray scattering (SAXS), including ultra-small-angle X-ray scattering (USAXS). Marchin et al. [13] reported that casein micelles are a complex network of casein polypeptide chains. Trejo et al. [14] reported three-dimensional imaging of inner casein micelles using electron tomography, which showed that casein micelles have porous structures consisting of cavities and water-filled channels. The internal structure of casein micelles has gradually been clarified as the observation techniques described above have been developed. However, the CCP distribution in casein micelles and crosslinking structure between CCP and casein remain unclear, despite CCP being a key structure in casein micelles. Therefore, in this study, cryo-electron microscopy of vitreous sections (CEMOVIS) was employed to observe the internal structure of casein micelles in raw milk with maximum accuracy at high resolution. Materials and methods Materials Bovine raw milk was obtained from the Cheese Research Laboratory (MEGMILK SNOW BRAND Co., Ltd, Japan). Before observation, the milk was kept at 5°C for one day and then incubated at 30°C for 1 h to prepare the specimen for CEMOVIS. Experimental procedure CEMOVIS The specimen was mixed with an equal amount of 40% dextran aqueous solution (dextran from Leuconostoc, Sigma-Aldrich, St. Louis, MO, USA) and agitated using a vortex mixer for 10 s. The specimen was then degassed by storing in a vacuum desiccator (PC-210K, As One, Osaka, Japan) for 1 h. The specimen was cryofixed using two dome-shaped gold-plated copper specimen carriers (Leica Microsystems, Vienna, Austria). One of the carriers was coated with L-α-phosphatidylcholine from egg yolk (Sigma-Aldrich, St. Louis, MO, USA) to allow one side of the carrier to be easily removed later. The carriers were filled with the specimen using a pipette to prevent air interfusion. Freeze fixation was carried out using a high-pressure freezing instrument (EM HPM100, Leica Microsystems, Vienna, Austria). Preparation of vitreous ice sections was performed according to the method described by Okada et al. [15]. Cryo-sections of raw milk in the vitreous phase were prepared using a cryo-ultramicrotome system (EM UC7/FC7, Leica Microsystems, Vienna, Austria) equipped with a static electricity control system (EM CRION, Leica Microsystems, Vienna, Austria), micromanipulator and cryosphere (Leica Microsystems, Vienna, Austria). The specimen was trimmed to 100 μm × 200 μm using a trimming knife (Cryotrim 45°, Diatome Hatfield, PA, USA) at a temperature of −50°C. Feed cryo-sections (25 nm) were cut using a 35° diamond knife (Cryoimmuno 35°, Diatome, Hatfield, PA, USA) with a clearance angle of 6°. The cutting speed was 0.6 nm/s. The sections were transferred using a probe made of animal hair to a holey carbon grid (C-flat CF-2/2-4 C, Protochios, Raleigh, NC, USA) whose hydrophilicity was preliminarily augmented using an ion coater (IB3, Eiko, Tokyo, Japan). The grid was transferred to a transmission electron microscope (JEM-2100: JEOL, Tokyo, Japan) operating at 200 kV, using a cryotransfer holder (914 hightilt cryotransfer system, Gatan, Pleasanton, CA, USA). Vitreous sections were observed at a temperature of −170°C. The focus area was searched using a minimum dose system (JEOL, Tokyo, Japan) and images were recorded using a CCD camera (TemCam-F224: TVIPS, München, Germany) at 60 000× magnification. Measurement of various distances The diameters of the casein micelles and black particles in the images obtained by CEMOVIS were measured, and average values were calculated using image analysis software (Asahi Kasei Engineering Corporation, Tokyo, Japan). In the CEMOVIS images, wrinkles and compression occurred along the cutting direction during cryo-sectioning. Therefore, particle diameters were assumed to be the length of the perpendicular axes in the cutting direction, which was presumably the original particle diameter excluding the influence of compression. Areas where black particles were not present and intervals between black particles were also measured in a similar manner. Results and discussion Low-magnification observation of casein micelles by CEMOVIS Figure 1a–d show low-magnification images of raw milk by CEMOVIS. Most casein micelles were observed as oval shapes elongated orthogonal to the knife mark. As most casein micelles in raw milk are spherical [11], compression along the cutting direction probably deformed the casein micelles into this oval shape. Figure 2 shows data of the casein micelles sizes, as calculated by image analysis of Fig. 1a–d. The average diameter of the casein micelles was about 140 nm, which is within the size range for casein micelles previously reported [8,11]. Fig. 1. View largeDownload slide Low-magnification images of casein micelles in raw milk using CEMOVIS. Small high-density particles are casein micelles. Round circles are apertural areas of holey carbon film on the grid (C-flat), with diameters of 2 μm. Knife marks run from bottom right to top left (triangles indicate casein micelles; arrow, cutting direction; scale bar = 1 μm). (a–d) shows low-magnification cross-sectional images of samples shown in Figs 3a and b and 6a and b, respectively. Fig. 1. View largeDownload slide Low-magnification images of casein micelles in raw milk using CEMOVIS. Small high-density particles are casein micelles. Round circles are apertural areas of holey carbon film on the grid (C-flat), with diameters of 2 μm. Knife marks run from bottom right to top left (triangles indicate casein micelles; arrow, cutting direction; scale bar = 1 μm). (a–d) shows low-magnification cross-sectional images of samples shown in Figs 3a and b and 6a and b, respectively. Fig. 2. View largeDownload slide Distribution of casein micelle sizes, as calculated by image analysis of Fig. 1a–d. The average casein micelle diameter was about 140 nm. Fig. 2. View largeDownload slide Distribution of casein micelle sizes, as calculated by image analysis of Fig. 1a–d. The average casein micelle diameter was about 140 nm. High-magnification observation of casein micelles by CEMOVIS Figure 3a and b shows high-magnification cross-sectional images of the samples shown in Fig. 1a and b, respectively. Numerous black particles were present in the cross-sectional images of the casein micelles. Using electron microscopy, heavier elements are generally observed as darker images. As samples were not stained in this study, the black particles were probably due to phosphorus and calcium, which are heavier elements than those comprising milk proteins. Therefore, black particles observed in the casein micelle cross-section were attributed to CCP [9]. Figure 4 shows data of the black particle sizes as calculated by image analysis of Fig. 3a and b, showing an average black particle diameter of 2.3 nm. Many studies have determined the CCP diameter using electron microscopy. For example, Knoop et al. [9] reported a CCP diameter of 2.5 nm using ultrathin sections, Lyster et al. [16] reported 1.5 nm, and McGann et al. [17] reported 2.5 nm, while, using cryo-electron microscopy, Kuudsen and Skibsted [18] reported 2–3 nm and Marchin et al. [13] reported 2.5 nm. Marchin and McManus [12] reported that the CCP diameter was 2.5 nm using small-angle scattering X-ray diffraction apparatus (SAXS) and 4.6 nm using small-angle neutron scattering (SANS) [19]. Therefore, a CCP size of 2–3 nm was thought to be reasonable, as it was similar to the reported values described above. Fig. 3. View largeDownload slide High-magnification images of casein micelles in raw milk by CEMOVIS. (a) and (b) show high-magnification cross-sectional images of samples shown in Fig. 1a and b, respectively. Numerous black particles were present in the cross-sectional images of casein micelles and appeared to be distributed nonuniformly (60 000× magnification; arrow, cutting direction; scale bar = 100 nm). Fig. 3. View largeDownload slide High-magnification images of casein micelles in raw milk by CEMOVIS. (a) and (b) show high-magnification cross-sectional images of samples shown in Fig. 1a and b, respectively. Numerous black particles were present in the cross-sectional images of casein micelles and appeared to be distributed nonuniformly (60 000× magnification; arrow, cutting direction; scale bar = 100 nm). Fig. 4. View largeDownload slide Distribution of black particle, assigned as CCP, sizes in casein micelles, as calculated by image analysis of Fig 3a and b. The average black particle diameter was 2.3 nm. Fig. 4. View largeDownload slide Distribution of black particle, assigned as CCP, sizes in casein micelles, as calculated by image analysis of Fig 3a and b. The average black particle diameter was 2.3 nm. Figure 5 shows the data for black particle intervals, as calculated by image analysis of Fig. 3a and b, showing average black particle intervals of 5.4 nm. The black particles, assigned as CCP, appeared not to be uniformly distributed in the casein micelle. De Kruif and Holt [4] reported that the CCP interval was 18 nm using small-angle neutron scattering measurements. In this study, it was considered that there was a difference because two of black particles, assigned as CCP, were closest to each other and the intervals of the particles on the perpendicular axes in the cutting direction were measured. Fig. 5. View largeDownload slide Distribution of black particle, assigned as CCP, intervals in casein micelles, as calculated by image analysis of Fig. 3a and b. The average intervals were 5.4 nm. Fig. 5. View largeDownload slide Distribution of black particle, assigned as CCP, intervals in casein micelles, as calculated by image analysis of Fig. 3a and b. The average intervals were 5.4 nm. Figure 6a and b shows high-magnification cross-sectional images of the samples shown in Fig. 1c and d, respectively. The casein micelle surface, as shown in Fig. 6a and b, was not smooth, with some concave regions observed along the interface. An area containing no black particles (assigned as CCP) was present inside all observed casein micelles, including in Figs. 3a and b and 6a and b. Figure 7 shows the size of the area containing no black particles, as calculated in this study, which averaged about 19.1 nm. Recent research has proposed that water-filled or hollow areas are present in casein micelles, with McMahon and Oommen [20] suggesting the existence of cavities within the micelle, while Trejo et al. [14] reported a water-filled area with a diameter of at least 5 nm and 20–30 nm cavities in casein micelles. Dalgleish [21] postulated that hydrophobic interactions of proteins would lead to an uneven distribution of water within the micelle interior and water channel formation. Bouchoux et al. [22] advocated a sponge model with a water-filled area through SAXS analysis. Therefore, the area containing no black particles possibly corresponded to a pore or cavity filled with water, as predicted by these previous reports. We were able to confirm the structure of as somewhat high-density, in addition to the black particles, assigned as CCP, in casein micelle. Differences in density indicate the existence of a structure that appears to have string-like connections to the black particles. Holt et al. [23] suggested that CCP was bound to the phosphate groups of casein based on amino acid analysis in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles. Aoki et al. [24] proved that caseins are cross-linked through the ester phosphate groups of CCP. This suggested that the structure comprised casein aggregates cross-linked with CCP. Fig. 6. View largeDownload slide High-magnification images of casein micelles with concave regions in raw milk using CEMOVIS from samples shown in Fig. 1c and d. Triangles indicate areas containing no black particles inside the casein micelle, which are surrounded by localized black particles (60 000× magnification; short arrow, casein aggregates cross-linked with CCP; large arrow, cutting direction; scale bar = 100 nm). Fig. 6. View largeDownload slide High-magnification images of casein micelles with concave regions in raw milk using CEMOVIS from samples shown in Fig. 1c and d. Triangles indicate areas containing no black particles inside the casein micelle, which are surrounded by localized black particles (60 000× magnification; short arrow, casein aggregates cross-linked with CCP; large arrow, cutting direction; scale bar = 100 nm). Fig. 7. View largeDownload slide Size of area containing no black particles in casein micelles from image analysis, as calculated in this study. The average size of the area was about 19.1 nm. Fig. 7. View largeDownload slide Size of area containing no black particles in casein micelles from image analysis, as calculated in this study. The average size of the area was about 19.1 nm. Based on the above results, the size of casein micelles and size and intervals of CCP were clarified using CEMOVIS by thinning frozen ultrathin sections to a thickness of 25 nm. The CCP distribution in casein micelles was not uniform, with part of the casein aggregates seemingly present to connect with CCP in a string. Therefore, casein micelles were inferred to be hydrated dynamic structures, because they have cavities and concave regions along the interface. No small protein structures, which seem to be submicelles, were found. Possibility of artifacts in CEMOVIS High-pressure freezing conducted in CEMOVIS delays the formation of ice crystals by freezing at 210 MPa, resulting in amorphous ice at the surface region of the specimen with a depth of 200–600 μm. The high pressure reduces the melting point of water to −22°C and maintains its supercooled state, even at around −90°C. High-pressure freezing has been successfully employed to pretreat specimens for observation in various biological fields, including gastric mucosa [25], yeast [26] and pea sprouts [27]. However, the influence of high-pressure freezing on the specimen is not well understood. Gebhardt et al. [28] reported that native micelles disintegrate into small fragments under pressure treatment at 50–250 MPa. Knudsen and Skibsted [29] reported that some large micelles and many small micelles with diameters of 20–50 nm coexist after high hydrostatic pressure treatment at 150–200 MPa, and that the surface of some large micelles appeared smooth and perfectly spherical. Accordingly, the pressure used for freezing is thought to have no influence on the micelle size and shape, because the casein micelles in Figs. 3a and b and 6a and b have major axis lengths of 340–500 nm and non-smooth surfaces. However, CEMOVIS requires dextran to prepare cryo-sections, which might produce artifacts. Structural artifacts, such as wrinkles, compression and crevasses, that occur when cutting cryo-sections are inevitable. Concluding remarks Electron microscopy observations by CEMOVIS were used to determine the internal structure of casein micelles, especially CCP, in raw milk. The results showed that: (i) The average diameter of casein micelles was about 140 nm; (ii) the CCP diameter in casein micelles was about 2–3 nm, with an average diameter of 2.3 nm; (iii) the CCP distribution in casein micelles was not uniform, with average CCP intervals of about 5.4 nm; (iv) an area containing no black particles (thought to be CCP) was present (average size, about 19.1 nm), which, considering previous reports, possibly corresponded to a pore or cavity filled with water and (v) casein aggregates were partially present to connect with CCP in a string. References 1 Holt C ( 1992 ) Structure and stability of bovine casein micelles . Adv. Protein Chem. 43 : 63 – 151 . Google Scholar CrossRef Search ADS PubMed 2 Walstra P ( 1979 ) The voluminosity of bovine casein micelles and some of its implications . J. Dairy Res. 46 : 317 – 323 . Google Scholar CrossRef Search ADS PubMed 3 Snoeren T H M , Klok H J , Van Hooydonk A C M , and Damman A J ( 1984 ) The volumi- nosity of casein micelles . Milchwissenschaft 39 : 461 – 463 . 4 De Kruif C G , and Holt C ( 2003 ) Casein micelle structure, functions and interactions . Adv. Dairy Chem. 1 : 233 – 276 . 5 Dalgleish D G , and Law A J R ( 1989 ) pH-Induced dissociation of bovine casein micelles. II. Mineral solubilization and its relation to casein release . J. Dairy Res. 56 : 727 – 735 . Google Scholar CrossRef Search ADS 6 Schmidt D G ( 1982 ) Association of caseins and casein micelle structure . Dev. Dairy Chem. 1 : 61 – 86 . 7 Kimura T , Taneya S , and Kanaya K ( 1979 ) Observation of internal structure of casein submicelles by means of on beam sputtering . Milchwissenschaft 34 : 521 – 524 . 8 Holt C , Kimber A M , Brooker B , and Prentice J H ( 1978 ) Measurements of the size of bovine casein micelles by means of electron microscopy and light scattering . J. 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Acta 760 : 414 – 420 . 18 Knudsen J C , and Skibsted L H ( 2010 ) High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy . Food Chem. 119 : 202 – 208 . Google Scholar CrossRef Search ADS 19 Kruif C G D , and Holt C ( 2003 ) Casein micelle structure, functions and interactions. In: Fox P F , and McSweeney P L H (eds) Advanced Dairy Chemsitry-1 Proteins. 3rd edition Part A : pp. 233 – 276 ( Kluwer Academy/Plenum Publishers , New York ). Google Scholar CrossRef Search ADS 20 McMahon D J , and Oommen B S ( 2008 ) Supramolecular structure of the casein micelle . J. Dairy Sci. 91 : 1709 – 1721 . Google Scholar CrossRef Search ADS PubMed 21 Dalgleish D G ( 2011 ) On the structural models of bovine casein micelles—review and possible improvements . Soft Matter 7 : 2265 – 2272 . Google Scholar CrossRef Search ADS 22 Bouchoux A , Gésan-Guiziou G , Pérez J , and Cabane B ( 2010 ) How to squeeze a sponge: casein micelles under osmotic stress, a SAXS Study . Biophys. J. 99 : 3754 – 3762 . Google Scholar CrossRef Search ADS PubMed 23 Holt C , Davies D T , and Law A J R ( 1986 ) Effects of colloidal calcium phosphate content and free calcium ion concentration in the milk serum on the dissociation of bovine casein micelles . J. Dairy Res. 53 : 557 – 572 . Google Scholar CrossRef Search ADS 24 Aoki T , Yamada N , Tomita I , Kako Y , and Imamura T ( 1987 ) Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate . Biochim. Biophys. Acta 911 : 238 – 243 . Google Scholar CrossRef Search ADS PubMed 25 Sawaguchi A , and Toyoshima F ( 2012 ) Researches today: ultrastructural study of gastric mucosa by using high-pressure freezing technique . Kenbikyo 45 : 130 – 132 . (in Japanese). 26 Knomi M , Kamasawa N , Takagi T , and Osumi M ( 2000 ) Immunoelectron microscopy of fission yeast using high pressure freezing . Plant Morphol. 12 : 20 – 31 . Google Scholar CrossRef Search ADS 27 Kaneko Y ( 2000 ) Ultrastructure of germinating pea leaves propared by high pressure freezing . Denshikennbikyou 12 : 10 – 19 . (in Japanese). 28 Gebhardt R , Doster W , Friedrich J , and Kulozik U ( 2006 ) Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM . Eur. Biophys. J. 35 : 503 – 509 . Google Scholar CrossRef Search ADS PubMed 29 Knudsen J C , and Skibsted L H ( 2010 ) High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy . Food Chem. 119 : 202 – 208 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. 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Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous sections (CEMOVIS)

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Abstract

Abstract Casein micelles are present in bovine milk as colloidal particles with diameters of 20–600 nm, which are complex macromolecular assemblies composed of four distinct types of casein and colloidal calcium phosphate (CCP). Multiple structural models of casein micelles have been proposed based on their biochemical or physical properties and observed using electron microscopy. However, the CCP distribution and crosslinking structure between CCP and casein remain unclear. Therefore, the internal structure of casein micelles in raw milk was observed using cryo-electron microscopy of vitreous sections (CEMOVIS) with high precision at high resolution. The results confirmed that the average casein micelle diameter was about 140 nm, and that the CCP diameter in casein micelles was about 2–3 nm, with an average diameter of 2.3 nm. The distribution of CCP in casein micelles was not uniform, with an average interval between CCPs of about 5.4 nm. Areas containing no black particles (attributed to CCP) were present, with an average size of about 19.1 nm. Considering previous reports, these areas possibly correspond to pores or cavities filled with water. Based on differences in the density of structures in casein micelles, we estimated that some of the casein aggregates were able to connect with CCP in a string. casein micelle, CEMOVIS, milk, cryo-electron microscopy, colloidal calcium phosphate (CCP), high-pressure freezing Introduction Bovine milk contains all essential nutrients for calf growth and is an important food for humans. Milk contains protein, calcium and phosphorus, which are essential nutrients for bone and muscle growth. Calcium and phosphorus in milk are mostly solubilized, with the structures of casein micelles and colloidal calcium phosphate (CCP) thought to contribute to the solubilization or dispersion of calcium and phosphorus, in addition to preventing their precipitation. Accordingly, CCP and casein micelles have been extensively studied for more than a century [1]. Casein micelles are present as colloidal particles with diameters of 20–600 nm in bovine milk, and are complex macromolecular assemblies composed of four distinct types of caseins, namely, αs1, αs2, β and κ-caseins, and minerals, including calcium and phosphate in the form of CCP. αs1-, αs2- and β-caseins are calcium-sensitive proteins that precipitate in the presence of Ca2+. κ-casein interacts with these caseins and stabilizes casein micelles. Casein micelles are hydrated dynamic structures [2,3] that are heterogeneous in composition and size and polydisperse [4]. When CCP is solubilized due to decreasing pH or calcium removal, the dissociation of casein micelles progresses gradually with a change in the size distribution [5]. Based on the biochemical or physical properties of casein micelles, two kinds of structural models have been proposed. However, the detailed internal structure has yet to be clarified and remains disputed. In the first model (submicelle model) proposed by Schmidt [6], the casein micelles are composed of smaller proteinaceous subunits (submicelles), with CCP cross-linked between them. In the second model (nanocluster model) proposed by Holt [1], there are no submicelles, and nanoclusters of CCP are randomly distributed in a uniform matrix of caseins. Microstructural observation of casein micelles using electron microscopy has also been performed as a powerful method to determine the structural models. Examples using transmission electron microscopy (TEM) include observing casein micelles in reconstituted skim milk using a shadowing method [7], evaluating casein micelle size through negative staining [8], and observing artificial casein micelles using ultrathin sections without staining [9]. An example using scanning electron microscopy (SEM) is the observation of casein micelles in reconstituted skim milk using chemical fixation, followed by dehydration and drying [10]. However, various artifacts are produced during the processes involved in electron microscopy observation methods, such as specimen preparation. These prevent an accurate image of casein micelles being obtained because milk is a colloidal aqueous dispersion and casein micelles have small particle sizes (100–150 nm) [11]. For example, in freeze-fracture replica methods, fine protein structures are coated with a metal, significantly altering the protein surface structure. Negative staining, including an air-drying process, causes specimen shrinkage due to drying. For ultrathin sections, structural alterations preclude accurate observation, crosslinking among proteins occurs due to chemical fixation, shrinking occurs during dehydration using ethanol, and protein loss occurs during infiltration to the resin [12]. Recently, casein micelles have been observed using cryo-electron microscopy by embedding the specimen in vitreous ice to avoid artifacts, and small-angle X-ray scattering (SAXS), including ultra-small-angle X-ray scattering (USAXS). Marchin et al. [13] reported that casein micelles are a complex network of casein polypeptide chains. Trejo et al. [14] reported three-dimensional imaging of inner casein micelles using electron tomography, which showed that casein micelles have porous structures consisting of cavities and water-filled channels. The internal structure of casein micelles has gradually been clarified as the observation techniques described above have been developed. However, the CCP distribution in casein micelles and crosslinking structure between CCP and casein remain unclear, despite CCP being a key structure in casein micelles. Therefore, in this study, cryo-electron microscopy of vitreous sections (CEMOVIS) was employed to observe the internal structure of casein micelles in raw milk with maximum accuracy at high resolution. Materials and methods Materials Bovine raw milk was obtained from the Cheese Research Laboratory (MEGMILK SNOW BRAND Co., Ltd, Japan). Before observation, the milk was kept at 5°C for one day and then incubated at 30°C for 1 h to prepare the specimen for CEMOVIS. Experimental procedure CEMOVIS The specimen was mixed with an equal amount of 40% dextran aqueous solution (dextran from Leuconostoc, Sigma-Aldrich, St. Louis, MO, USA) and agitated using a vortex mixer for 10 s. The specimen was then degassed by storing in a vacuum desiccator (PC-210K, As One, Osaka, Japan) for 1 h. The specimen was cryofixed using two dome-shaped gold-plated copper specimen carriers (Leica Microsystems, Vienna, Austria). One of the carriers was coated with L-α-phosphatidylcholine from egg yolk (Sigma-Aldrich, St. Louis, MO, USA) to allow one side of the carrier to be easily removed later. The carriers were filled with the specimen using a pipette to prevent air interfusion. Freeze fixation was carried out using a high-pressure freezing instrument (EM HPM100, Leica Microsystems, Vienna, Austria). Preparation of vitreous ice sections was performed according to the method described by Okada et al. [15]. Cryo-sections of raw milk in the vitreous phase were prepared using a cryo-ultramicrotome system (EM UC7/FC7, Leica Microsystems, Vienna, Austria) equipped with a static electricity control system (EM CRION, Leica Microsystems, Vienna, Austria), micromanipulator and cryosphere (Leica Microsystems, Vienna, Austria). The specimen was trimmed to 100 μm × 200 μm using a trimming knife (Cryotrim 45°, Diatome Hatfield, PA, USA) at a temperature of −50°C. Feed cryo-sections (25 nm) were cut using a 35° diamond knife (Cryoimmuno 35°, Diatome, Hatfield, PA, USA) with a clearance angle of 6°. The cutting speed was 0.6 nm/s. The sections were transferred using a probe made of animal hair to a holey carbon grid (C-flat CF-2/2-4 C, Protochios, Raleigh, NC, USA) whose hydrophilicity was preliminarily augmented using an ion coater (IB3, Eiko, Tokyo, Japan). The grid was transferred to a transmission electron microscope (JEM-2100: JEOL, Tokyo, Japan) operating at 200 kV, using a cryotransfer holder (914 hightilt cryotransfer system, Gatan, Pleasanton, CA, USA). Vitreous sections were observed at a temperature of −170°C. The focus area was searched using a minimum dose system (JEOL, Tokyo, Japan) and images were recorded using a CCD camera (TemCam-F224: TVIPS, München, Germany) at 60 000× magnification. Measurement of various distances The diameters of the casein micelles and black particles in the images obtained by CEMOVIS were measured, and average values were calculated using image analysis software (Asahi Kasei Engineering Corporation, Tokyo, Japan). In the CEMOVIS images, wrinkles and compression occurred along the cutting direction during cryo-sectioning. Therefore, particle diameters were assumed to be the length of the perpendicular axes in the cutting direction, which was presumably the original particle diameter excluding the influence of compression. Areas where black particles were not present and intervals between black particles were also measured in a similar manner. Results and discussion Low-magnification observation of casein micelles by CEMOVIS Figure 1a–d show low-magnification images of raw milk by CEMOVIS. Most casein micelles were observed as oval shapes elongated orthogonal to the knife mark. As most casein micelles in raw milk are spherical [11], compression along the cutting direction probably deformed the casein micelles into this oval shape. Figure 2 shows data of the casein micelles sizes, as calculated by image analysis of Fig. 1a–d. The average diameter of the casein micelles was about 140 nm, which is within the size range for casein micelles previously reported [8,11]. Fig. 1. View largeDownload slide Low-magnification images of casein micelles in raw milk using CEMOVIS. Small high-density particles are casein micelles. Round circles are apertural areas of holey carbon film on the grid (C-flat), with diameters of 2 μm. Knife marks run from bottom right to top left (triangles indicate casein micelles; arrow, cutting direction; scale bar = 1 μm). (a–d) shows low-magnification cross-sectional images of samples shown in Figs 3a and b and 6a and b, respectively. Fig. 1. View largeDownload slide Low-magnification images of casein micelles in raw milk using CEMOVIS. Small high-density particles are casein micelles. Round circles are apertural areas of holey carbon film on the grid (C-flat), with diameters of 2 μm. Knife marks run from bottom right to top left (triangles indicate casein micelles; arrow, cutting direction; scale bar = 1 μm). (a–d) shows low-magnification cross-sectional images of samples shown in Figs 3a and b and 6a and b, respectively. Fig. 2. View largeDownload slide Distribution of casein micelle sizes, as calculated by image analysis of Fig. 1a–d. The average casein micelle diameter was about 140 nm. Fig. 2. View largeDownload slide Distribution of casein micelle sizes, as calculated by image analysis of Fig. 1a–d. The average casein micelle diameter was about 140 nm. High-magnification observation of casein micelles by CEMOVIS Figure 3a and b shows high-magnification cross-sectional images of the samples shown in Fig. 1a and b, respectively. Numerous black particles were present in the cross-sectional images of the casein micelles. Using electron microscopy, heavier elements are generally observed as darker images. As samples were not stained in this study, the black particles were probably due to phosphorus and calcium, which are heavier elements than those comprising milk proteins. Therefore, black particles observed in the casein micelle cross-section were attributed to CCP [9]. Figure 4 shows data of the black particle sizes as calculated by image analysis of Fig. 3a and b, showing an average black particle diameter of 2.3 nm. Many studies have determined the CCP diameter using electron microscopy. For example, Knoop et al. [9] reported a CCP diameter of 2.5 nm using ultrathin sections, Lyster et al. [16] reported 1.5 nm, and McGann et al. [17] reported 2.5 nm, while, using cryo-electron microscopy, Kuudsen and Skibsted [18] reported 2–3 nm and Marchin et al. [13] reported 2.5 nm. Marchin and McManus [12] reported that the CCP diameter was 2.5 nm using small-angle scattering X-ray diffraction apparatus (SAXS) and 4.6 nm using small-angle neutron scattering (SANS) [19]. Therefore, a CCP size of 2–3 nm was thought to be reasonable, as it was similar to the reported values described above. Fig. 3. View largeDownload slide High-magnification images of casein micelles in raw milk by CEMOVIS. (a) and (b) show high-magnification cross-sectional images of samples shown in Fig. 1a and b, respectively. Numerous black particles were present in the cross-sectional images of casein micelles and appeared to be distributed nonuniformly (60 000× magnification; arrow, cutting direction; scale bar = 100 nm). Fig. 3. View largeDownload slide High-magnification images of casein micelles in raw milk by CEMOVIS. (a) and (b) show high-magnification cross-sectional images of samples shown in Fig. 1a and b, respectively. Numerous black particles were present in the cross-sectional images of casein micelles and appeared to be distributed nonuniformly (60 000× magnification; arrow, cutting direction; scale bar = 100 nm). Fig. 4. View largeDownload slide Distribution of black particle, assigned as CCP, sizes in casein micelles, as calculated by image analysis of Fig 3a and b. The average black particle diameter was 2.3 nm. Fig. 4. View largeDownload slide Distribution of black particle, assigned as CCP, sizes in casein micelles, as calculated by image analysis of Fig 3a and b. The average black particle diameter was 2.3 nm. Figure 5 shows the data for black particle intervals, as calculated by image analysis of Fig. 3a and b, showing average black particle intervals of 5.4 nm. The black particles, assigned as CCP, appeared not to be uniformly distributed in the casein micelle. De Kruif and Holt [4] reported that the CCP interval was 18 nm using small-angle neutron scattering measurements. In this study, it was considered that there was a difference because two of black particles, assigned as CCP, were closest to each other and the intervals of the particles on the perpendicular axes in the cutting direction were measured. Fig. 5. View largeDownload slide Distribution of black particle, assigned as CCP, intervals in casein micelles, as calculated by image analysis of Fig. 3a and b. The average intervals were 5.4 nm. Fig. 5. View largeDownload slide Distribution of black particle, assigned as CCP, intervals in casein micelles, as calculated by image analysis of Fig. 3a and b. The average intervals were 5.4 nm. Figure 6a and b shows high-magnification cross-sectional images of the samples shown in Fig. 1c and d, respectively. The casein micelle surface, as shown in Fig. 6a and b, was not smooth, with some concave regions observed along the interface. An area containing no black particles (assigned as CCP) was present inside all observed casein micelles, including in Figs. 3a and b and 6a and b. Figure 7 shows the size of the area containing no black particles, as calculated in this study, which averaged about 19.1 nm. Recent research has proposed that water-filled or hollow areas are present in casein micelles, with McMahon and Oommen [20] suggesting the existence of cavities within the micelle, while Trejo et al. [14] reported a water-filled area with a diameter of at least 5 nm and 20–30 nm cavities in casein micelles. Dalgleish [21] postulated that hydrophobic interactions of proteins would lead to an uneven distribution of water within the micelle interior and water channel formation. Bouchoux et al. [22] advocated a sponge model with a water-filled area through SAXS analysis. Therefore, the area containing no black particles possibly corresponded to a pore or cavity filled with water, as predicted by these previous reports. We were able to confirm the structure of as somewhat high-density, in addition to the black particles, assigned as CCP, in casein micelle. Differences in density indicate the existence of a structure that appears to have string-like connections to the black particles. Holt et al. [23] suggested that CCP was bound to the phosphate groups of casein based on amino acid analysis in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles. Aoki et al. [24] proved that caseins are cross-linked through the ester phosphate groups of CCP. This suggested that the structure comprised casein aggregates cross-linked with CCP. Fig. 6. View largeDownload slide High-magnification images of casein micelles with concave regions in raw milk using CEMOVIS from samples shown in Fig. 1c and d. Triangles indicate areas containing no black particles inside the casein micelle, which are surrounded by localized black particles (60 000× magnification; short arrow, casein aggregates cross-linked with CCP; large arrow, cutting direction; scale bar = 100 nm). Fig. 6. View largeDownload slide High-magnification images of casein micelles with concave regions in raw milk using CEMOVIS from samples shown in Fig. 1c and d. Triangles indicate areas containing no black particles inside the casein micelle, which are surrounded by localized black particles (60 000× magnification; short arrow, casein aggregates cross-linked with CCP; large arrow, cutting direction; scale bar = 100 nm). Fig. 7. View largeDownload slide Size of area containing no black particles in casein micelles from image analysis, as calculated in this study. The average size of the area was about 19.1 nm. Fig. 7. View largeDownload slide Size of area containing no black particles in casein micelles from image analysis, as calculated in this study. The average size of the area was about 19.1 nm. Based on the above results, the size of casein micelles and size and intervals of CCP were clarified using CEMOVIS by thinning frozen ultrathin sections to a thickness of 25 nm. The CCP distribution in casein micelles was not uniform, with part of the casein aggregates seemingly present to connect with CCP in a string. Therefore, casein micelles were inferred to be hydrated dynamic structures, because they have cavities and concave regions along the interface. No small protein structures, which seem to be submicelles, were found. Possibility of artifacts in CEMOVIS High-pressure freezing conducted in CEMOVIS delays the formation of ice crystals by freezing at 210 MPa, resulting in amorphous ice at the surface region of the specimen with a depth of 200–600 μm. The high pressure reduces the melting point of water to −22°C and maintains its supercooled state, even at around −90°C. High-pressure freezing has been successfully employed to pretreat specimens for observation in various biological fields, including gastric mucosa [25], yeast [26] and pea sprouts [27]. However, the influence of high-pressure freezing on the specimen is not well understood. Gebhardt et al. [28] reported that native micelles disintegrate into small fragments under pressure treatment at 50–250 MPa. Knudsen and Skibsted [29] reported that some large micelles and many small micelles with diameters of 20–50 nm coexist after high hydrostatic pressure treatment at 150–200 MPa, and that the surface of some large micelles appeared smooth and perfectly spherical. Accordingly, the pressure used for freezing is thought to have no influence on the micelle size and shape, because the casein micelles in Figs. 3a and b and 6a and b have major axis lengths of 340–500 nm and non-smooth surfaces. However, CEMOVIS requires dextran to prepare cryo-sections, which might produce artifacts. Structural artifacts, such as wrinkles, compression and crevasses, that occur when cutting cryo-sections are inevitable. Concluding remarks Electron microscopy observations by CEMOVIS were used to determine the internal structure of casein micelles, especially CCP, in raw milk. The results showed that: (i) The average diameter of casein micelles was about 140 nm; (ii) the CCP diameter in casein micelles was about 2–3 nm, with an average diameter of 2.3 nm; (iii) the CCP distribution in casein micelles was not uniform, with average CCP intervals of about 5.4 nm; (iv) an area containing no black particles (thought to be CCP) was present (average size, about 19.1 nm), which, considering previous reports, possibly corresponded to a pore or cavity filled with water and (v) casein aggregates were partially present to connect with CCP in a string. References 1 Holt C ( 1992 ) Structure and stability of bovine casein micelles . Adv. Protein Chem. 43 : 63 – 151 . Google Scholar CrossRef Search ADS PubMed 2 Walstra P ( 1979 ) The voluminosity of bovine casein micelles and some of its implications . J. Dairy Res. 46 : 317 – 323 . 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MicroscopyOxford University Press

Published: Mar 2, 2018

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