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Research Note

Observations of casein micelles in skim milk concentrate bytransmission electron microscopy

A.O. Karlsson�, R. Ipsen, Y. Ardo

Dairy Technology, Department of Food Science, Centre for Advanced Food Studies, The Royal Veterinary and Agricultural University, Rolighedsvej 305,

1958 Fredriksberg C, Denmark

Abstract

The microstructure of casein micelles in ultrafiltrated (UF) skim milk concentrate at pH 6.5 and 5.8 was investigated by transmission

electron microscopy using three different preparation methods. The volume fraction of the casein micelles in the UF concentrate was

62.8% (v/v) at pH 6.5 and fixation by glutaraldehyde revealed the close packing of micelles in the UF concentrate as well as a higher

degree of micelle aggregation at pH 5.8. No details of the microstructure of the micellar surface or core could, however, be observed.

Freeze-fracture of cryoprotected, i.e. glycerol, UF concentrate on the other hand, exposed the finer structures of the micellar core but no

pH dependent differences were observed. As cryoprotection includes a dilution of the sample with glycerol, the packing of the micelles in

the UF concentrate could not be observed. Undiluted UF concentrate exposed to rapid freezing using a propane jet followed by freeze-

fracture exhibited development of ice crystals but rough areas on the micrographs were identified as fractured casein micelles. The

micellar core appeared rougher and differences in the micellar core microstructure due to changing pH could be observed when this

preparation method was used.

Keywords: Casein micelles; Ultrafiltration; Microstructure; Transmission electron microscopy

1. Introduction

Casein micelles in bovine milk have a hydrodynamicdiameter of 40–300 nm and can be considered as colloids inan aqueous solute, i.e. serum, at the native pH (6.7) in milk.The casein micelles harbour the main part of the casein inmilk and at native pH they are kept from aggregatingthrough steric and electrostatic stabilization due to thepresence of k-casein molecules on the surface of themicelles. Elucidation of the structure of casein micelleswas initially based on physico-chemical studies (Noble &Waugh, 1965) but development of electron microscopy hasmade it possible to directly observe the microstructure ofsingle micelles (Schmidt, 1982). However, micrographs canbe hard to interpret and sample preparation for any type ofmicroscopy can potentially induce changes in the micro-structure of samples. In the case of bovine casein micelles,

ing author. Tel.: +453528 3253; fax: +45 3528 3190.

ess: aka@kvl.dk (A.O. Karlsson).

different sample preparation techniques for in electronmicroscopy have been shown to induce various changes(McMahon & McManus, 1998) and no optimal techniquefor preparation of casein micelles for electron microscopyexists at present.The vacuum inside an electron microscope requires that

the specimen is dried, frozen or replicated. Traditionalpreparation of casein micelles often includes initial steps ofmixing with agar and fixation with glutaraldehyde. Mixingcasein micelles with agar causes the micelles to appear morecompact, i.e. exhibit dehydration (McMahon & McManus,1998). Glutaraldehyde induces chemical fixation of thesample and Hermansson and Buchheim (1981) reportedthat glutaraldehyde caused gelation of very fine proteinsols. Gastaldi, Lagaude, and Tardodo de la Fuente (1996)reported a ‘‘linkage’’ of casein micelles due to glutaralde-hyde. Solidifying the sample by freezing has to either occurin the presence of a cryoprotector, e.g. glycerol, or underhigh pressure. A cryoprotector can be presumed to affectthe environment (e.g. dehydration or pH change) of the

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micelles. However, differences in size after pressurization ofmicelles in transmission electron microscopy (TEM) havebeen detected also when a cryoprotector has been used(Schrader, Buchheim, & Morr, 1997). Freezing at highpressure requires some mechanical strength of the sampleto avoid destruction. Frozen samples are subsequentlyfractured and a replica of the fractured surface is shapedfrom platinum or gold and observed in the microscope.The quick freezing is supposed to induce minimal changesto the sample but the replicas can be of various quality andsurface structures can be hard to detect (Dalgleish,Spagnuolo, & Goff, 2004).

In this study we will present TEM micrographs ofreplicated casein micelles in ultrafiltrated (UF) skim milk,which have been prepared by a rapid Cryo-jet freezingtechnique. This technique can be used for fluid samples anddoes not require dilution with cryoprotector prior tofreezing thus providing opportunity for observing caseinmicelles in complex food systems without changing thechemical properties of the environment, e.g. pH and ionstrength. Such changes can influence the size and thestructure of the casein micelles. In addition, dilution ofmicelles will result in the sample not reflecting the truedistance between the casein micelles in the original foodsystem. This is of importance in our studied food matrix,i.e. concentrated milk, where the casein micelles are closelypacked in their natural environment. The rapid freezingtechnique is compared with fixation by glutaraldehyde andwith cryofixation by immersion into melting Freon 22(�160 1C) after dilution of the UF skim milk with acryoprotector, i.e. glycerol.

2. Materials and methods

2.1. Preparation of samples of ultrafiltrated skim milk

UF concentrate was produced according to the methoddescribed by Karlsson, Ipsen, Schrader, and Ardo (2005).The ultrafiltration process was stopped when the UFconcentrate had reached a Brix value of 36.21, measuredusing a handheld refractometer (Atago Co., Tokyo,Japan). After production, the UF concentrate was pouredin bottles (100mL), heat treated at 62 1C for 30min,quickly frozen and stored in a freezer (�23 1C).

Prior to use in experiments, UF concentrate andunconcentrated skim milk was thawed in a water-bath(30 1C) for 1 h, preserved with 0.02% (w/w) Thimerosal(Merck, Darmstadt, Germany) and equilibrated for 24 h at30 1C. Glucono-d-lactone (GDL) (Acros Organics, Goel,Belgium) was subsequently added in order to obtain a finalpH of 6.5 or 5.8 after 24 h of storage at 30 1C.

2.2. Chemical analysis of UF concentrate and

unconcentrated skim milk

The pH was measured directly using a Knick Portamess(Knick Elektronische Messgerate; Berlin; Germany)

equipped with a hamilton tiptrode (hamilton instruments;Bonaduz; Switzerland). Total Solids (TS) were determinedaccording to the IDF standard method (InternationalDairy Federation (IDF), 1991). Nitrogen was determinedusing with a Kjeltec System 1026 analyser (Tecator;Hoganas; Sweden). Total nitrogen (TN); noncasein nitro-gen (NCN) and nonprotein nitrogen (NPN) were deter-mined according to the IDF standard methods(International Dairy Federation (IDF), 1993). The proteincontent was estimated by multiplying the nitrogen contentwith a Kjeldahl factor of 6.36 for casein and 6.28 for wheyproteins (van Boekel & Ribadeau-Dumas, 1987). Theanalysis of total solids was performed in triplicate.

2.3. Transmission electron microscopy

Chemically fixed UF concentrate and replicas of UFconcentrate at pH 6.5 and 5.8 were examined using TEM.Three different preparation techniques were used.In the first preparation technique, a small drop (50 mL)

of UF concentrate was fixed in glutaraldehyde (3% w/v) in0.1mol/l acetate buffer for 75min at 20 1C. Post fixationfor 90min (20 1C) was done in osmium tetraoxide (1% w/v)in acetate buffer. The samples of UF concentrate wererinsed twice in acetate buffer and dehydration performed ina series of ethanol solutions. The samples were embeddedin Epon, sectioned (60–90 nm) and stained with uranylacetate (2% w/v) and lead citrate (2.5% w/v). In the secondpreparation technique, UF concentrate was cryoprotectedby mixing 2mL of sample with 1mL of glycerol (87%,w/w) before cryofixation in melting Freon 22 (�160 1C).Freeze-fracture of the samples was done at �120 1C with aBalzers BA 360mol/l unit (Balzers AG, Balzers, Lichten-stein). The replicas were produced by sputtering Pt/C andthen C from an electron gun on the fractured surface. Thereplicas were subsequently cleaned in concentrated hypo-chlorite, distilled water, acetone and finally in distilledwater. In the third preparation technique, a droplet of UFconcentrate was placed on a small gold mesh (BAL-TEC,Witten, Germany). The sample on the mesh was thenencapsulated between two copper plates (BAL-TEC,Witten, Germany). One of the plates had a small cavityproviding space for the sample. The sample was quicklyfrozen by a jet of liquid propane (�180 1C) in a JFD 030Cryo-jet (Balzers AG, Balzers, Lichtenstein). The highdynamic pressure of the jet guaranteed a fast heat exchangeat freezing and the encapsulation protected the fluid samplefrom destruction due to the high dynamic pressure. Afterfreezing the sample was automatically dropped down inliquid propane. The sample was then moved to a tank ofliquid nitrogen (�120 1C). The sample was fractured at�120 1C in a Balzers BA 360mol/l unit (Balzers AG,Balzers, Lichtenstein) by separating the copper plates. Thefractured areas were coated with Pt/C and C. The replicaswere cleaned as described above.Samples were examined in a Philips CM100 transmission

electron microscope (Philips Electronics N.V., Eindhoven,

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Fig. 1. Replicas of casein micelles in ultrafiltrated skim milk of pH 6.5 (a)

and 5.8 (b) observed in TEM. The undiluted ultrafiltrated skim milk was

fixed in glutaraldehyde at 20 1C.

The Netherlands), operated at 60 kV. Micrographs weredigitally recorded.

2.4. Image analysis

By comparing an original micrograph (i.e. in grey scale)of chemically fixed UF concentrate with its binarized image(i.e. black and white) a segmentation procedure formicrographs was developed. The procedure was used toseparate casein micelles from the serum phase and the areafraction of casein micelles was calculated. This procedurewas used for six micrographs of each sample.

In micrographs (i.e. in grey scale) of Cryo-jet frozen UFconcentrate the darkest and the lightest areas representuneven areas of the fracture surface. This made it possibleto perform segmentation between the rough and thesmooth surfaces and area fractions could be calculated.The procedure was performed on six micrographs of eachsample.

The image analysis was preformed using Adobe Photo-shop v. 6.0.1 (Adobe Systems Inc., San Jose, CA, USA)with plug-in functions from The Image Processing Tool Kitv. 5 (Reindeer Graphics Inc., Asheville, NC, USA). Toassure the relevance of the segmentation procedures, everysegmented micrograph was visually compared with itsoriginal.

3. Results and discussion

The batch-wise UF process of skim milk resulted inlower pH (6.5) of the UF concentrate than in theunconcentrated skim milk (pH 6.7). Due to the concentra-tion process of protein in UF, the distance between thecasein micelles was greatly reduced from approximately120 nm in the unconcentrated skim milk (Walstra &Jenness, 1984) to below 10 nm in the UF concentrate with30.5% (w/w) dry matter and 19.5% (w/w) casein (Karlssonet al., 2005). This was observed on the micrographs ofchemically fixed UF concentrate at pH 6.5 and 5.8 (Fig. 1).The casein micelles appear as dark circular close-packedstructures. There were no clear signs of merged caseinmicelles or of pronounced aggregation at pH 6.5 (Fig. 1a).The maximum diameter of the micelles was approximately350 nm. Previously, casein micelles with diameters of up to442 nm have been observed in UF concentrate by TEM(Srilaorkul, Ozimek, Ooraikul, Hadziyev, & Wolfe, 1991).At pH 5.8 the maximum micelle size was somewhat lowerand the borders of the micelles more diffuse (Fig. 1b).There were also signs of merger between micelles. Loss ofmicellar individuality has previously been reported byGastaldi et al. (1996) to occur already at pH 5.8. Thefixation by glutaraldehyde and the sectioning for TEMmade it impossible to detect surface structures on thecasein micelles.

When UF concentrate was cryoprotected by mixing withglycerol the distance between the casein micelles increasedin the freeze-fractured sample and the preparation method

cannot be used for viewing the close packing of micelles inthe UF concentrate. This was well illustrated whencomparing micrographs of replicas from cryoprotectedUF concentrate (Fig. 2) with micrographs of the chemicallyfixed UF concentrate (Fig. 1). Compared to the chemicallyfixed micelles, casein micelles in cryoprotected and subse-quently freeze-fractured UF concentrate did not give theimpression of being larger at pH 6.5. The casein micelles atboth pH levels were judged to be well separated aftermixing with glycerol and indications of aggregation at pH5.8, which was previously observed after chemical fixation(Fig. 1), could not be established.The freeze-fracture proceeds through micelles and clearly

reveals the internal structures but to some extent alsosurface structures of the micelles. However, there is loss offiner details when the fractured surface is coated with metal(McMahon & McManus, 1998). The structures of themicellar core appearing on freeze-fracture TEM micro-graphs has been used to explain the submicelle model of the

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Fig. 2. Replicas of casein micelles from ultrafiltrated skim milk of pH 6.5

(a) and 5.8 (b) observed in TEM. The casein micelles were cryoprotected

by glycerol, fixed in Freon 22 at �160 1C and freeze-fractured at �120 1C.

Fig. 3. Replicas of casein micelles from ultrafiltrated skim milk of pH 6.5

observed in TEM. The casein micelles were cryoprotected by glycerol,

fixed in Freon 22 at �160 1C and freeze-fractured at �120 1C.

casein micelle (Schmidt, 1982; Walstra, 1999). The sub-micelle model has been under debate and been comple-mented by other models. One of the most citied ones is theHorne model, which considers the micellar core as amineralized homogenous cross-linked protein network(Horne, 1998). Recently, Dalgleish et al. (2004) using FieldEmission SEM have obtained micrographs of caseinmicelles absorbed on carbon planchets and chemicallyfixed, i.e. glutaraldehyde and osmium tetroxide. Theirmicrographs show tubular structures on the micellarsurface and they suggested that also the micellar core isorganized into tubular structures. It appeared that the sizeof the rough, raspberry appearance of the micelle surface ofour samples was in the same size range (10–20 nm) asthe tubular structures reported by Dalgleish et al. (2004)(Fig. 3). When pH was changed, no difference in the size ofthe rough structures could be observed (Fig. 2). However,the cryoprotection by glycerol cannot be excluded to affectand erase differences in microstructure. Mainly the micellar

surface but also the size has been observed to change due toaddition of glycerol (Pires, Gatti, Orellana, & Pereyra,1997).When UF concentrate was rapidly frozen by the Cryo-

jet, smooth areas with some small unevenness enclosed in aweb of rough structures were observed (Fig. 4). The roughstructures of the web (Fig. 5) were somewhat larger thanthe structures observed from the micellar interior (Fig. 2)while the uneven structures of the smooth areas were muchsmaller than the structures of the micellar interior ofcryoprotected UF concentrate in Fig. 2. The rough areas ofthe web had an area fraction of 70.1% and 68.7% for UFconcentrate of pH 6.5 and 5.8, respectively. The corre-sponding area fractions of dark areas identified as caseinmicelles in micrographs from chemically fixed UF con-centrate was 62.8% at pH 6.5 and 61.9% at pH 5.8. Bydefinition, the area fraction in a section is equal to thevolume fraction of the bulk (Russ, 1995). From viscometrymeasurements the volume fraction of the same UFconcentrate was previously determined to be 62–64% inthe same pH range (Karlsson et al., 2005). Thus, it can beassumed that the rough areas in Cryo-jet frozen UFconcentrate (Fig. 4) represent the fraction of caseinmicelles. The smooth areas were identified as ice crystals.Although the cryofixation with the Cryo-jet is supposed toassure a high freezing rate, the development of small ice-crystals in the sample cannot be excluded. The sameappearance of ice crystals on micrographs has previouslybeen observed after cryopreparation of boehmite powder(Fauchadour, Pouget, Lechaire, Rouleau, & Normand,1999). The unevenness found in ice crystals can possibly becaused by freeze concentration of lactose and wheyproteins.Interesting is the appearance of the rough structures in

the micellar core when the UF concentrate was quickly

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Fig. 4. Replicas of casein micelles in ultrafiltrated skim milk of pH 6.5 (a)

and 5.8 (b) observed in TEM. The undiluted ultrafiltrated skim milk was

quickly frozen at �180 1C in a propane jet and freeze-fractured at

�120 1C.

Fig. 5. Replicas of casein micelles in ultrafiltrated skim milk of pH 6.5

observed in TEM. The undiluted ultrafiltrated skim milk was quickly

frozen at �180 1C in a propane jet and freeze-fractured at �120 1C.

frozen by the Cryo-jet (Fig. 5). Many of them weresomewhat larger, less smooth and more irregular than inmicrographs of cryoprotected UF concentrate (Fig. 3).Thus, also the rapid freezing cryofixation support micellarmodels, which describe the micellar core as beingstructured, e.g. a micellar core consisting of submicellesheld together by calcium phosphate (Schmidt, 1982).However, with the rapid freezing technique it is possibleto see that pH affects the structures in the micellar core, i.e.larger structures at larger pH (6.5) (Fig. 4). The main effectby lowering the pH from 6.5 to 5.8 is an increasedsolubilization of calcium phosphate bound to the micelles(Walstra & Jenness, 1984). This suggests that calciumphosphate is located in the rough structures and solubiliza-tion affects their size. The calcium phosphate has also beensuggested to be located in the submicelles in a revisedmodel by Walstra (1999). De Kruif and Holt (2003)suggested that structures in the micellar core appearing on

TEM micrographs originated from calcium phosphate/casein nanoclusters. The nanoclusters have been estimatedto have a size of approximately 20 nm by De Kruif andHolt (2003), thus, are smaller than the largest structures inFig. 5. Although, the rapid freezing not clearly can supportany specific casein micelle model it gives valuableinformation by eliminating some artifacts, which diminishmicrostructural differences generated when UF concen-trate is cryoprotected with glycerol. This makes it possibleto not just study micellar size but also changes in themicellar core when environmental factors, e.g. pH, arechanged.

4. Conclusions

Different preparation methods for TEM generatevarious artifacts. Therefore, different sample preparationsshould be compared. In highly concentrated UF concen-trate the casein micelles are interacting over short distancesand this can influence the size distribution of the micelles.Therefore direct observation of undiluted UF concentrateis of interest. In UF concentrate fixed by glutaraldehydeonly the size and not the surface structures nor the core ofcasein micelles could be observed. Replicas of cryopro-tected (i.e. glycerol) and freeze-fractured UF concentraterevealed a core microstructure of the micelles; however, thecryoprotectant cannot be excluded in dehydrating themicelles and effect the observed roughness, and the closepacking of micelles could not been viewed. Replicas ofCryo-jet frozen UF concentrate revealed that even thoughthe freezing was fast ice crystals developed. Rough areasbetween the ice crystals were determined to be caseinmicelles by calculating the area fraction. By this prepara-tion method the structures of the micellar cores appearedsomewhat larger than in micelles of cryoprotected UF

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concentrate and differences in micellar structure of UFconcentrates with pH 6.5 and 5.8 could be observed.Application of the rapid freezing of samples with the Cryo-jet can also be suggested for observation of casein micellesin other food systems, e.g. unconcentrated milk and foams.

Acknowledgements

The work was financially supported by the Danish DairyResearch Foundation and the Danish GovernmentalResearch Program (FØTEK 3). Katrin Schrader (FederalResearch Centre for Nutrition and Food, Kiel, Germany)and Pia Skjødt Pedersen (The Royal Veterinary andAgricultural University, Copenhagen, Denmark) areacknowledged for preparation of samples for TEM.

References

Dalgleish, D. G., Spagnuolo, P. A., & Goff, D. (2004). A possible

structure of the casein micelle based on high-resolution field-emission

scanning electron microscopy. International Dairy Journal, 14,

1025–1031.

De Kruif, C. G., & Holt, C. (2003). Casein micelle structure, functions and

interactions. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced

dairy chemistry, proteins (3rd ed.), Vol. 1 (pp. 233–276). New York,

NY: Kluwer Academic/Plenum Publishers.

Fauchadour, D., Pouget, T., Lechaire, J.-P., Rouleau, L., & Normand, L.

(1999). Evaluation of cryotechniques for TEM observation of sols—

Application to boehmite sols used in catalysts forming. Oils & Gas

Science and Technology, 54, 513–524.

Gastaldi, E., Lagaude, A., & Tardodo de la Fuente, B. (1996). Micellar

transition state in casein between pH 5.5 and 5.0. Journal of Food

Science, 61, 59–64.

Hermansson, A. M., & Buchheim, W. (1981). Characterization of protein

gels by scanning and transmission electron microscopy. A methodol-

ogy study of soy protein gels. Journal of Colloid and Interface Science,

81, 519–530.

Horne, D. S. (1998). Casein interactions: Casting light on the black boxes,

the structure in dairy products. International Dairy Journal, 8,

171–177.

IDF (1991). Sweetened condensed milk: Determination of the total solids

content (reference method). IDF Standard 15B. Brussels, Belgium:

International Dairy Federation.

IDF (1993). Nitrogen content of milk and milk products. IDF Standard

20B. Brussels, Belgium: International Dairy Federation.

Karlsson, A. O., Ipsen, R., Schrader, K., & Ardo, Y. (2005). Relations

between physical properties of casein micelles and rheology of skim

milk concentrate. Journal of Dairy Science, 88, 3784–3797.

McMahon, D. J., & McManus, W. R. (1998). Rethinking casein micelle

structure using electron microscopy. Journal of Dairy Science, 81,

2985–2993.

Noble, R. W., Jr., & Waugh, D. F. (1965). Casein micelles. Formation and

structure. Journal of the American Chemical Society, 87, 2236–2245.

Pires, M. S., Gatti, C. A., Orellana, G. A., & Pereyra, J. (1997). Rennet

coagulation of casein micelles and heated casein micelles: Importance

of steric stabilization after k-casein proteolysis. Journal of Agricultural

and Food Chemistry, 45, 4446–4451.

Russ, J. C. (1995). The image processing handbook (p. 513). Boca Raton,

FL: CRC Press.

Schmidt, D. G. (1982). Association of caseins and casein micelle structure.

In P. F. Fox (Ed.), Developments of Dairy Chemistry—1 (pp. 61–68).

London, UK: Applied Science Publications.

Schrader, K., Buchheim, W., & Morr, C. V. (1997). High pressure effects

on the colloidal calcium phosphate and the structural integrity of

micellar casein in milk. Part 1. High pressure dissolution of colloidal

calcium phosphate in heated milk systems. Nahrung, 41, 133–138.

Srilaorkul, S., Ozimek, L., Ooraikul, B., Hadziyev, D., & Wolfe, F. (1991).

Effect of ultrafiltration of skim milk on casein micelle size distribution

in retentate. Journal of Dairy Science, 74, 50–57.

van Boekel, M. A. J. S., & Ribadeau-Dumas, B. (1987). addendum to the

evaluation of the kjeldahl factor for the conversion of the nitrogen

content of milk and milk products to protein content. Netherlands

Milk and Dairy Journal, 41, 281–284.

Walstra, P. (1999). Casein sub-micelles: Do they exist? International Dairy

Journal, 9, 189–192.

Walstra, P., & Jenness, R. (1984). Dairy Chemistry and Physics.

New York, NY: John Wiley & Sons Inc.