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The characterization of small emulsion droplets made from milk proteins and triglyceride oil
COLLOIDS AND Colloids and Surfaces A: Physicochemicaland Engineering Aspects 123 124 (1997) 145 153
ELSEVIER
A
SURFACES
The characterization of small emulsion droplets made from milk proteins and triglyceride oil D o u g l a s G . D a l g l e i s h a,., S a r a h J. W e s t a, F. R o s s H a l l e t t b
" Department of Food Science, University of Guelph, Guelph, Ont. N1G 2W1, Canada b Department of Physics, University of Guelph, Guelph, Ont. RIG 2WI, Canada Received 7 May 1996; accepted 25 July 1996
Abstract
Measurements have been made of the light scattering properties of emulsion droplets produced using a Microfluidizer. The droplet size distributions were measured by both Integrated Light Scattering (ILS) over a wide range of scattering angles, and by Dynamic Light Scattering (DLS) at a fixed angle of 90°. From the former, it was possible to derive number distributions, whereas the latter was used to give intensity distributions of the droplets. The calculated distributions from light scattering were also compared with samples studied by transmission electron microscopy. A range of samples was studied, including whole milks, and emulsions prepared from soya oil and stabilized by sodium caseinate; fractions containing the smaller droplets from these emulsions were also collected and studied. The results confirmed that it was possible to isolate a population of small (< 100 nm) particles from microfluidized milk or from the emulsions; larger particles were also present. Both ILS and electron microscopy confirmed these results. DLS also confirmed the presence of the small particles in the emulsions, but principally was useful in the analysis of the separated fractions. To improve the sensitivity of the method, it is necessary to use non-linear channel times in DLS experiments.
Keywords: Particle sizes; Light scattering; Protein stabilized emulsions; Homogenization; Food proteins
1. Introduction
Many prepared foods depend on emulsions for their properties. Products as diverse as coffee creamers, mayonnaises, infant formulae, ice creams and sports beverages all contain emulsified lipids. Moreover, many of these emulsions contain proteins, often milk proteins, which act as surfactants to emulsify the lipid and prevent its recoalescence. It is therefore important for the ideal formulation and production of these materials that the detailed properties (structure, composition, stability) of
* Corresponding author.
emulsion droplets in food materials be understood, and this has been a subject of increasing research over the last 15 years or so. For example, studies have been made of the surface structures of adsorbed proteins I-1-3], as well as their ability to be displaced by other proteins or by smallmolecule surfactants [-4 7]. The properties of these emulsions relevant to stability are perhaps less understood in detail, especially when they are in a shear field, but some information has recently become available [8,9]. If the stability of an emulsion is determined by the detailed structures of the droplets, it becomes important to determine these in detail. One of the factors which are important in defining the droplet
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D. G Dalgleish et al. / Colloids"Surfaees A: Physicochem. Eng. Aspects 123 124 (1997) 145 153
structure is the method of preparation of the emulsion. Traditionally, colloid mills could be used to form relatively coarse emulsions (containing particles up to several microns in diameter), while valve homogenizers could be used to produce finer emulsions, possessing particles with diameters mainly in the range 0.5-1 ktm. Recently, a new family of homogenizing devices has made its appearance, and these devices are capable of producing protein-stabilized emulsions with much smaller average droplet sizes (about 300 nm), and which contain particles with diameters as small as 30 nm. Some studies of the effect of such devices on milk have been made, confirming the small sizes of the particles [ 10]. We have studied several aspects of these fine emulsions in detail, to determine the amounts, structures and properties of the proteins adsorbed to the oil/water interface. During these experiments it became clear that the distribution of particle sizes was very different from that produced by standard homogenization techniques. This paper describes the analysis by light scattering and electron microscopy of the size distributions of particles in emulsions containing milk proteins (caseins), prepared using a Microfluidizer ® [-11]. These preparations used either whole milk, in which the casein is originally in its normal micellar form [ 12], or sodium caseinate, where the micellar structure has been disrupted by the removal of the calcium and phosphate which are contained in the natural micelles.
2. Materials and methods
2.1. Preparation of microfluidized milk Milk was obtained from the University of Guelph Research Farm, and was heat treated at 63 °C for 30 rain in a water bath, to destroy microorganisms and to denature lipases. Samples (100ml) of the milk were tempered at 40°C for 1 h and were homogenized at that temperature using a Microfluidizer M l l 0 S (Microfluidics Corp., Newton, MA). The milk was passed five times through the Microfluidizer at a homogenization pressure of 42 MPa. A single pass caused
bridging flocculation to occur; the flocs were broken up by the second and subsequent passes. The homogenized samples were then stored at 4°C until required. For a comparison material (milk homogenized using a valve homogenizer), we used 3.25%-fat homogenized milk purchased from a local supermarket; this type of milk is typically homogenized in a two-step process with pressures of 3500/500 psi (23.8/3.4 MPa). Homogenized milks can be separated into different fractions using centrifugation [13]. This technique was employed on the milks produced using the Microfluidizer. Samples (20ml) of the microfluidized milks were centrifuged at 86000g for 60 min at 4°C in a Beckman L8-70M ultracentrifuge. Three fractions were collected; a floating layer, a pellet of sinking material, and the serum fraction remaining in the middle of the centrifuge tube. Only the floating and sinking materials were investigated in detail.
2.2. Preparation of emulsions containing caseinate Oil-in-water emulsions (20% soya oil) were prepared using the Microfluidizer at homogenization pressures of 28 MPa (with 1% caseinate) or 70 MPa (with 2% caseinate) in a buffer of 20 mM imidazoleHC1, pH 7.0. The protein solutions were filtered (0.22 p.m) prior to homogenization. The ingredients were weighed into a beaker and a pre-emulsion was made using a hand-held shear-mixing unit (Dia-Med, Mississauga, Ontario). The preemulsion was introduced into the Microfluidizer, and was circulated through the unit five times. All experiments were performed at 25 °C, because the soya oil is liquid at that temperature. Centrifugation of the emulsions was performed in some cases to fractionate the droplets in terms of size. Samples (20 ml) of the emulsions were centrifuged at 15000g for 60 min, and the floating layer of larger particles, and the subnatant, containing the smaller emulsion droplets, were collected. The subnatants were recentrifuged at 40 000g for 30 rain to harvest the smaller droplets.
2.3. Measurement of particle sizes Particle sizes in the emulsions and microfluidized milks were analyzed using two light-scattering
D. G. Dalgleish et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145 153
techniques. Dynamic light-scattering (DLS) was measured at a scattering angle of 90 ° on diluted samples using a Malvern 4700 system attached to a Multi-8 correlator (Malvern Instruments, Inc., Southboro, MA). From the correlation functions, the distributions of particle sizes were calculated using software developed by Hallett et al. [14], which allows the calculation of volume frequencies or the number frequencies of particles in the samples; in this work, the size distributions from DLS are quoted as intensity fractions, i.e. the proportions of the total light-intensity scattered by particles of defined size. Measurements of integrated light scattering (ILS) were also made using multi-angle equipment developed at the University of Guelph, and described elsewhere [15]. These results were analyzed to give number distributions of the suspended particles. In all of these experiments, the homogenized milks or emulsions were diluted at a ratio of 1.5 gl of emulsion per 3 ml of buffer. Samples from milk were suspended in 20 mM imidazole, pH 7.0, containing 5 mM CaCI 2 and 50 mM NaC1, which maintains the aggregated structures of micellar caseins [16], and caseinate emulsions were suspended in 20 mM imidazole, pH 7.0. In the milks, micelles and micellar material were dissociated by suspending the fractions in a buffer of 20 m M imidazole, 6 M urea and 5 mM EDTA. 2.4. Electron microscopy
Transmission electron microscopy (TEM) was performed on the fractions. The method of sample preparation closely followed that of Allan-Wojtas and Kalab [ 17]. Initially the fluid milk or emulsion samples were prepared for thin sectioning by encapsulation in 2% agar tubes [18]. The samples were fixed with a 1.4% glutaraldehyde solution in a buffer of 0.1 M cacodylate, 0.05% CaC12 and 4% polyvinylpyrrolidine, pH 7.4, and postfixed in an imidazole-buffered solution of osmium tetroxide (0.5%). They were dehydrated by treatment with a graduated series of ethanol and propylene oxide, and embedded in Epon 812 epoxy resin. The resin blocks were cut into sections 100 nm thick using a Reichert Ultracut E ultramicrotome. The sections were stained with 4% uranyl acetate in 50% ethanol
147
followed by Reynolds lead citrate solution [ 19] in 1 N NaOH. The sections were mounted on grids and examined using a J E O L 100CX transmission electron microscope with an accelerating voltage of 60 kV. Typically, magnification factors from 15 000 to 100000 were used. Although no problems were experienced in the study of microfluidized whole-milk, the caseinate emulsions proved very difficult to fix. In nearly all cases the larger oil droplets showed a tendency to coalesce during fixation. This was demonstrated by the presence in the micrographs of fat globules which had breaks in their coating of protein, and indeed in some cases it was possible to see pairs of fat globules in the act of coalescing. The behaviour seemed to be mainly confined to the larger globules, since all of the small globules seen in the micrographs had well defined membranes of protein. Confirmation that the coalescence phenomenon was artefactual came from light scattering, which did not show very large particles to be present in the original emulsions, and also from the fact that no creaming occurred during storage of the emulsions for times of one week or greater. Thus it is likely that one of the early stages of the fixation process leads to the coalescence. Because the casein is spread in a monomolecular layer around the fat globules, and because at least some of the large globules appeared to be stabilized by sharing the protein which was on their surfaces, it is possible that the large globules had membranes which were weaker than those of the smaller ones.
3. Results 3.1. Particles in microfluidized milks
The differences between homogenized and microfluidized milks have already been described [20]. In homogenized milks we know that the fat globules have diameters in the range 0.6-1.2 gm, and their surface is formed by the adsorption of intact or partially intact casein micelles and whey proteins [21]. Often, the casein micelles spread over the oil/water interface [22]. From electron microscopy, it is evident that homogenized milk
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contains many intact casein micelles, from which it may be concluded that the stresses encountered when milk passes through the homogenization valve are insufficient to disrupt the casein micelles themselves, which have diameters in the region of 50-250nm. In microfluidized milk, the particles are completely different [-11,23]. Not only are the fat globules much smaller (relatively few are larger than about 300 nm), but their structure is different from those in the homogenized milk, as is shown by a study of the particles in the floating layer after centrifugation (Fig. la). On the larger fat globules, the surface layer of adsorbed protein is clearly different in structure compared with homogenized milk, since adsorbed micelles are almost completely absent, presumably because they are broken up during the process of microfluidization. A layer of protein about 10nm thick is around most of the globules, especially the larger ones. Moreover, some of the larger fat globules are partially coated by small ones, presumably via protein bridges, so that the surface layers of these particles can have a rather complex structure. The microfluidized milk also contains some very small fat globules, with diameters as small as 30nm, which are most clearly seen in the fraction which sinks during centrifugation (Fig. lb). Not only do these small globules appear as individual units, but in some cases, they also appear as clusters associated with particles of protein which may be the remnants of the original casein micelles. These
particles suggest that the fat is adhering to, or is embedded in, the protein matrix. In none of the fractions isolated from microfluidized milk did electron microscopy show the presence of any unaltered casein micelles. Size distributions calculated from light scattering of the floating and sinking fractions of the microfluidized milks are shown in Fig. 2. ILS of the sinking fraction (Fig. 2a) confirms the presence of particles with diameters less than 100 nm, which correspond to the small fat globules seen in the electron micrographs; the larger particles seen in this fraction by electron microscopy are clearly in a minority in terms of number. The intensity distribution of the particles as calculated from DLS (Fig. 2b) shows more clearly the presence of the larger particles in the sinking fraction, and suggests that the volume contribution of these particles (as distinct from their number) may be considerable; the contribution of the smaller particles in terms of weight is clearly small. In contrast, the floating fraction is seen to contain larger particles (up to about 500nm diameter), although the greater volume of particles is in the range 2 0 0 4 0 0 nm in diameter. The difference between the floating and sinking fractions as estimated by DLS is, however, relatively small, although in the sinking fraction it is possible to see some contribution from the smaller particles whose size is of the order of 0.1 gm. The ILS method is rather insensitive to particles less than about 5 0 n m in diameter,
Fig. 1. Electron micrographs of fractions prepared from whole milk treated by microfluidization (42 MPa, 5 passes). (a), sample prepared from the fraction which floats during the centrifugation step; (b), sample prepared from the fraction of particles which sink during the centrifugation step. Scale bars have lengths of (a), 300 nm, and (b), 200 nm.
D. G Dalgleish et al. / Colloids SurJ~tces A: Physicochem. Eng. Aspects 123-124 (1997) 145 153
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0.2 0.4 0.6 0 0.2 0.4 0.6 Particle diameter (~m) Particle diameter (l~m)
Fig. 2. Size distributions from light scattering of particles in microfluidized milk (42 MPA, 5 passes) and fractions. (a) number distributions calculated from ILS for O, sample prepared from the fraction which floats during centrifugation (compare Fig. la); IN, sample prepared from the fraction of particles which sink during the centrifugation step (compare Fig. lb); A, the sinking fraction after treatment with urea and EDTA to dissociate micellar fragments. (b), intensity distributions calculated from DLS; symbols are the same as in (a).
because the scattering of such particles is not greatly dependent on the scattering angle, which is essential for the calculation (i.e. Rayleigh scatterers cannot be detected). Dynamic light-scattering also tends to underestimate the number of the smallest particles, because of the small region of the correlation function in which they can be measured [10]. In both ILS and DLS experiments it was possible to measure the basic sizes of the smaller fat globules by suspending them in a buffer containing urea and EDTA, which dissociates the fragments of casein micelles and leaves the fat globules with only a monolayer coverage of casein. The action of the urea was to decrease the sizes of the particles in the sinking fraction of the microfluidized milk (Figs. 2a and b), and to move the whole distribution of particle sizes to smaller values. These define the sizes of the basic fat droplets in the microfluidized milk, which can be compared with the droplets in the caseinate/soya-oil emulsions described below.
3.2. Emulsions prepared using caseinate The more simple emulsions formed from caseinate and soya oil should, in principle, be interpreted more easily than the microfluidized milks, because
there must be fewer complications arising from the structure of the protein (no casein micelles exist). As with the particles in microfluidized milk, there is clear evidence for the formation in the Microfluidizer of a large number of small particles with diameters less than 100 nm. However, there are significant differences between the milks and the emulsions. In a whole emulsion (20% oil, 1% caseinate) prepared by microfluidization at 28 MPa, ILS showed that there were a few particles with diameters up to about 500 nm, but that the majority had diameters around 200 nm (Fig. 3). After centrifugation, the fraction of the emulsion remaining in the serum contained particles with average diameters in the region of 50 100 nm. The sizes for the whole emulsion and the serum fraction were confirmed by the intensity distributions determined from DLS, although the results for both fractions were somewhat higher, as was to be expected (Fig. 4). The number fractions calculated for the whole emulsion from DLS (not shown) had a maximum at diameter 80 nm and a narrower range than was calculated from ILS. The intensity distribution from DLS was slightly bimodal, showing that both large (diameter 250-450 nm) and small particles (diameter about 100 nm) were present, and not a continuum of particle sizes.
D. G. Dalgleish et aL/ Colloids Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145-153
150 ~ , 0.30
I
I
..~ 0.20
=_o ~ 0.10 E= z 0.00 0.20
0.00
0.40
Particle d i a m e t e r (gm)
Fig. 3. Number distributions of particle sizes in different emulsions made from caseinate and soya oil (20% w/w), measured using ILS. B, whole emulsion prepared using 1% caseinate, at pressure of 28 MPa and 5 passes through the Microfluidizer (compare Fig. 5a); Q, particles remaining in the serum fraction of the emulsion after centrifugation and removal of the larger particles (compare Fig. 5b); A, whole emulsion prepared using 2% caseinate, at pressure of 70 MPa and 5 passes through the Microfluidizer (compare Fig. 5c); II,, particles remaining in the serum fraction of this emulsion after centrifugation and removal of the larger particles (compare Fig. 5d).
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.
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.
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.
.
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Fig. 4. Intensity distributions of particle sizes in different emulsions made from caseinate and soya oil (20% w/w), measured using DLS. Symbols are the same as for Fig. 3.
Electron microscopy of this whole emulsion showed that there were particles with diameters in the range 500-600 nm, as well as confirming the presence of small ones (Fig. 5a). There was evidence
of bridging flocculation; some droplets were in contact and their areas of contact were flattened. No detailed size distributions were found from the electron microscopy, but qualitatively the micrographs agree with the conclusions arrived at from light scattering. Centrifugation to separate the particles gave a serum which contained mostly particles with diameters less than 100nm, with an apparently rather narrow size distribution (Fig. 5b), although there were a few larger particles also present. It is, of course, these particles which have the small sizes which are determined by light scattering. The small droplets neither coalesced or bridged, in contrast to the larger ones. At higher microfluidization pressure and casein concentration (70 MPa; 2% caseinate) the average sizes of the particles decreased, as would be expected from the increase in emulsifier concentration and the severity of the homogenization. Both DLS and ILS showed that the distributions of particle sizes, whether in number or in intensity, were shifted completely to smaller sizes (Figs. 3 and 4). Electron microscopy confirmed the relative absence of large oil droplets (Fig. 5c); indeed, it was difficult to find any in the samples which were studied, mainly because extensive coalescence appeared to have occurred. Measurements of the particles in the serum fraction of the emulsion showed, rather surprisingly, that the number distribution from ILS and the intensity distribution from DLS were shifted to larger particle sizes than the serum fractions of the emulsion made at lower pressure and with lower casein concentration (Figs. 3 and 4). This conclusion was also reinforced by the electron microscopy, where the emulsion droplets were indeed found to be significantly larger than those in the serum of the low pressure emulsion (compare Figs. 5b and d). In all of the oil/caseinate emulsions studied, it was evident from the electron micrographs that the population of very small droplets (less than 50 nm in diameter) was not so apparent as that found in the microfluidized milks. This was also confirmed by light scattering, where both ILS and DLS showed a smaller distribution, especially when the additional protein in the milk was dissociated with the urea (Fig. 2). This result may simply reflect the protein:lipid ratios in the two prepara-
D. G. Dalgleish et al. ' Colloids' Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145-153
151
Fig. 5. Electron micrographs of emulsions (20% w/w soya oil) prepared by microfluidization. (a), whole emulsion containing 1% caseinate, prepared at 28 MPa and 5 passes; (b), particles from the serum fraction of this emulsion after centrifugation; (c), whole emulsion containing 2% caseinate, prepared at 70 MPa and 5 passes; (d) particles from the serum fraction of this emulsion after centrifugation. Scale bars have lengths of (a), 500 nm; (b), 100 nm; (c), 200 nm; (d), 200 nm.
tions; in the milk the casein:fat ratio is about 0.6, whereas in the caseinate emulsions the ratio was either 0.05 or 0.1. In the milk there is more protein to cover the fat surface, although the situation is complicated by the fact that, in milk, the protein is originally micellar, and therefore has an effective concentration less than the true one.
4. Discussion
The observations presented here confirm that the emulsions formed using the Microfluidizer, compared with those formed using a valve homogenizer, contain some very small particles, with diameters of 50 nm or even less. Not all of the lipid droplets are as small as this, but nearly all of them have diameters of less than 500 nm. The sizes of
these droplets also depend on the protein:oil ratio in the emulsion. Emulsions containing 2% caseinate contain overall smaller particles than do emulsions containing 1% caseinate, because the higher protein concentration provides for greater coverage of the interface, and thus an increased surface area of the emulsion. This detailed analysis of lightscattering results allows differences in the sizes of emulsion droplets to be seen when other methods [24] suggest that there is little change in the sizes of the droplets between the two concentrations of casein. The only counter-intuitive result is the larger size of the serum droplets in the emulsion formed using the higher casein concentration and pressure. However, this may at least partly be explained by the fact that much more of the oil is found in small droplets in this emulsion than in the low-pressure
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emulsion. Thus, there is considerably more surface of lipid to be covered in this emulsion, with the result that the droplets will be somewhat larger. The conditions of microfluidization, however, favour the breaking up of larger droplets of oil, so that there are fewer large droplets, more of intermediate size, and fewer small ones than in the other emulsion. Because of the small particle size, the emulsions produced by the Microfluidizer have the advantage of being very stable for creaming, unless flocculation of the droplets occurs, and this is rare if milk proteins are used as the surfactants in the absence of calcium. Because the particles are so small, we may speculate on whether their properties are likely to be significantly altered. One particular problem which may be produced by the size is that a considerable amount of the surface area of the emulsion droplets may be in these small particles. This may be simply expressed by the relation [3Ws
3w1~ / ,
,
where Rs and R l a r e the radii of the large and small particles and ws and wl are their respective weight fractions in the total emulsion, if the area is calculated simply from the average diameter as determined from DLS by the method of cumulants, or from measurement using Fraunhofer diffraction equipment, it is inevitable that the surface area in the emulsion will be considerably underestimated. This in turn will have an effect on determinations of the amount of protein adsorbed to the oil/water interfaces. Estimates of this for caseins range from 0.8 to 3 mg m 2 [25], but changes may have to be made to accommodate the extra area which is neglected by most estimates of particle size. On the positive side, it is evident that the error will be much less if the determination of the adsorbed protein is made on the layer of larger droplets which are harvested when the emulsion is centrifuged [26], because the true particle size will be much closer to the average diameter measured from DLS by the method of cumulants or by Fraunhofer diffraction. A second problem, especially in microfluidized milk, is that of inhomogeneity of the types of
particles. From the electron micrographs, it is clear that the different fat globules possess very different coats of protein. Many of the globules, especially the larger ones, have a fairly even coating of protein, but the smaller ones appear at least partly in aggregates with protein. It follows that the two types of particle may have quite different properties. How these will compare with the properties of homogenized milks has not been established; we know for example that the microfluidized milks share the poor renneting characteristics of homogenized milks, although a limited amount of microfluidization may improve some aspects of cheese milk [27]. Comparisons of heat stability and other effects are not defined; these are tasks for the future now that the structures of the particles in microfluidized milks and emulsions are becoming clear.
Acknowledgements The authors wish to thank Alexandra Smith, Diane Moyles, Lewis Melville and Dan Beniac for assistance with the preparation of samples for the electron microscopy. The research was supported by grants from the Ontario Dairy Council and the Natural Sciences and Engineering Research Council of Canada (to DGD). Sarah West was in receipt of a Rotary International Ambassadorial Scholarship from the United Kingdom.
References E1] D.G. Dalgleish, Colloids Surfaces, 46 (1990) 141. [2] J. Leaver and D.G. Dalgleish, Biochim. Biophys. Acta, 1041 (1990) 217. [3] E. Dickinson, J. Chem. Soc., Farad. Trans., 88 (1992) 2973. [4] E. Dickinson, S.E. Rolfe and D.G. Dalgl~ish, Food Hydrocolloids, 2 (1988) 397. E5] J.-L. Courthaudon, E. Dickinson, Y. Matsumura and A. Williams, Food Structure, 10 (1991) 109. [6] E. Dickinson, S.R. Euston and C.M. Wosken, Prog. Colloid Polym. Sci., 82 (1990) 65. E7] D.G. Dalgleish, M. Srinivasan and H. Singh, J. Agric. Food Chem., 43 (1995) 2351. [8] E. Dickinson and A. Williams, Colloids Surfaces A: Physicochem. Eng. Aspects, 88 (1994) 317. [9] S.O. Agboola and D.G. Dalgleish, Lebensm.-Wiss. U-Technol., 29 (1996) 425.
D.G, Dalgleish et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145 153 1-10] K.B. Strawbridge, E. Ray, F.R. Hallett, S.M. Tosh and D.G. Dalgleish, J. Colloid Interface Sci., 171 (1995) 392. [ l l l O. Robin, N. Remillard and P. Paquin, Colloids Surfaces A: Physicochem. Eng. Aspects, 80 (1993) 211. [12] C. Holt, Adv. Prot. Chem., 43 (1992) 63. [13] D.G. Dalgleish and E.W. Robson, J. Dairy Res., 52 (1985) 539. [14] F.R. Hallett, T. Craig, J. Marsh and B. Nickel, Can. J. Spectrosc., 34 (1989) 63. [15] K.B. Strawbridge and F.R. Hallett, Macromolecules, 27 (1994) 2283. [16] D.G. Dalgleish, J. Agric. Food Chem., 38 (1990) 1995. [17] P. Allan-Wojtas and M. Kalab, Milk Sci. Int., 39 (1984) 323. [18] S. Henstra and D.G. Schmidt, Neth. Milk Dairy J., 24 (1970) 45.
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[19] E.S. Reynolds, J. Cell Biol., 17 (1963) 208. [20] D.G. Dalgleish, S.M. Tosh and S. West, Neth. Milk Dairy J., 50 (1996) 135. [21] H. Oortwijn and P. Walstra, Neth. Milk Dairy J., 33 (1979) 134. [22] H. Oortwijn, P. Walstra and H. Mulder, Neth. Milk Dairy J., 31 (1977) 134. [23] Y. Pouliot, P. Paquin and J. Giasson, Int. Dairy J., 1 (1991) 39. 1-24] Y. Fang and D.G. Dalgleish, Colloids Surfaces B, 1 (1993) 357. [25] Y. Fang and D.G. Dalgleish, J. Colloid Interface Sci., 156 (1993) 329. [26] J.A. Hunt and D.G. Dalgleish, Food Hydrocolloids, 8 11994) 175. [27] A. LeMay, P. Paquin and C. Lacroix, J. Dairy Sci., 77 (1994) 2870.
ELSEVIER
A
SURFACES
The characterization of small emulsion droplets made from milk proteins and triglyceride oil D o u g l a s G . D a l g l e i s h a,., S a r a h J. W e s t a, F. R o s s H a l l e t t b
" Department of Food Science, University of Guelph, Guelph, Ont. N1G 2W1, Canada b Department of Physics, University of Guelph, Guelph, Ont. RIG 2WI, Canada Received 7 May 1996; accepted 25 July 1996
Abstract
Measurements have been made of the light scattering properties of emulsion droplets produced using a Microfluidizer. The droplet size distributions were measured by both Integrated Light Scattering (ILS) over a wide range of scattering angles, and by Dynamic Light Scattering (DLS) at a fixed angle of 90°. From the former, it was possible to derive number distributions, whereas the latter was used to give intensity distributions of the droplets. The calculated distributions from light scattering were also compared with samples studied by transmission electron microscopy. A range of samples was studied, including whole milks, and emulsions prepared from soya oil and stabilized by sodium caseinate; fractions containing the smaller droplets from these emulsions were also collected and studied. The results confirmed that it was possible to isolate a population of small (< 100 nm) particles from microfluidized milk or from the emulsions; larger particles were also present. Both ILS and electron microscopy confirmed these results. DLS also confirmed the presence of the small particles in the emulsions, but principally was useful in the analysis of the separated fractions. To improve the sensitivity of the method, it is necessary to use non-linear channel times in DLS experiments.
Keywords: Particle sizes; Light scattering; Protein stabilized emulsions; Homogenization; Food proteins
1. Introduction
Many prepared foods depend on emulsions for their properties. Products as diverse as coffee creamers, mayonnaises, infant formulae, ice creams and sports beverages all contain emulsified lipids. Moreover, many of these emulsions contain proteins, often milk proteins, which act as surfactants to emulsify the lipid and prevent its recoalescence. It is therefore important for the ideal formulation and production of these materials that the detailed properties (structure, composition, stability) of
* Corresponding author.
emulsion droplets in food materials be understood, and this has been a subject of increasing research over the last 15 years or so. For example, studies have been made of the surface structures of adsorbed proteins I-1-3], as well as their ability to be displaced by other proteins or by smallmolecule surfactants [-4 7]. The properties of these emulsions relevant to stability are perhaps less understood in detail, especially when they are in a shear field, but some information has recently become available [8,9]. If the stability of an emulsion is determined by the detailed structures of the droplets, it becomes important to determine these in detail. One of the factors which are important in defining the droplet
0927-7757/97/$17.00 Copyright© 1997Elsevier Science B.V. All rights reserved PH S0927-7757 (97)03783-1
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structure is the method of preparation of the emulsion. Traditionally, colloid mills could be used to form relatively coarse emulsions (containing particles up to several microns in diameter), while valve homogenizers could be used to produce finer emulsions, possessing particles with diameters mainly in the range 0.5-1 ktm. Recently, a new family of homogenizing devices has made its appearance, and these devices are capable of producing protein-stabilized emulsions with much smaller average droplet sizes (about 300 nm), and which contain particles with diameters as small as 30 nm. Some studies of the effect of such devices on milk have been made, confirming the small sizes of the particles [ 10]. We have studied several aspects of these fine emulsions in detail, to determine the amounts, structures and properties of the proteins adsorbed to the oil/water interface. During these experiments it became clear that the distribution of particle sizes was very different from that produced by standard homogenization techniques. This paper describes the analysis by light scattering and electron microscopy of the size distributions of particles in emulsions containing milk proteins (caseins), prepared using a Microfluidizer ® [-11]. These preparations used either whole milk, in which the casein is originally in its normal micellar form [ 12], or sodium caseinate, where the micellar structure has been disrupted by the removal of the calcium and phosphate which are contained in the natural micelles.
2. Materials and methods
2.1. Preparation of microfluidized milk Milk was obtained from the University of Guelph Research Farm, and was heat treated at 63 °C for 30 rain in a water bath, to destroy microorganisms and to denature lipases. Samples (100ml) of the milk were tempered at 40°C for 1 h and were homogenized at that temperature using a Microfluidizer M l l 0 S (Microfluidics Corp., Newton, MA). The milk was passed five times through the Microfluidizer at a homogenization pressure of 42 MPa. A single pass caused
bridging flocculation to occur; the flocs were broken up by the second and subsequent passes. The homogenized samples were then stored at 4°C until required. For a comparison material (milk homogenized using a valve homogenizer), we used 3.25%-fat homogenized milk purchased from a local supermarket; this type of milk is typically homogenized in a two-step process with pressures of 3500/500 psi (23.8/3.4 MPa). Homogenized milks can be separated into different fractions using centrifugation [13]. This technique was employed on the milks produced using the Microfluidizer. Samples (20ml) of the microfluidized milks were centrifuged at 86000g for 60 min at 4°C in a Beckman L8-70M ultracentrifuge. Three fractions were collected; a floating layer, a pellet of sinking material, and the serum fraction remaining in the middle of the centrifuge tube. Only the floating and sinking materials were investigated in detail.
2.2. Preparation of emulsions containing caseinate Oil-in-water emulsions (20% soya oil) were prepared using the Microfluidizer at homogenization pressures of 28 MPa (with 1% caseinate) or 70 MPa (with 2% caseinate) in a buffer of 20 mM imidazoleHC1, pH 7.0. The protein solutions were filtered (0.22 p.m) prior to homogenization. The ingredients were weighed into a beaker and a pre-emulsion was made using a hand-held shear-mixing unit (Dia-Med, Mississauga, Ontario). The preemulsion was introduced into the Microfluidizer, and was circulated through the unit five times. All experiments were performed at 25 °C, because the soya oil is liquid at that temperature. Centrifugation of the emulsions was performed in some cases to fractionate the droplets in terms of size. Samples (20 ml) of the emulsions were centrifuged at 15000g for 60 min, and the floating layer of larger particles, and the subnatant, containing the smaller emulsion droplets, were collected. The subnatants were recentrifuged at 40 000g for 30 rain to harvest the smaller droplets.
2.3. Measurement of particle sizes Particle sizes in the emulsions and microfluidized milks were analyzed using two light-scattering
D. G. Dalgleish et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145 153
techniques. Dynamic light-scattering (DLS) was measured at a scattering angle of 90 ° on diluted samples using a Malvern 4700 system attached to a Multi-8 correlator (Malvern Instruments, Inc., Southboro, MA). From the correlation functions, the distributions of particle sizes were calculated using software developed by Hallett et al. [14], which allows the calculation of volume frequencies or the number frequencies of particles in the samples; in this work, the size distributions from DLS are quoted as intensity fractions, i.e. the proportions of the total light-intensity scattered by particles of defined size. Measurements of integrated light scattering (ILS) were also made using multi-angle equipment developed at the University of Guelph, and described elsewhere [15]. These results were analyzed to give number distributions of the suspended particles. In all of these experiments, the homogenized milks or emulsions were diluted at a ratio of 1.5 gl of emulsion per 3 ml of buffer. Samples from milk were suspended in 20 mM imidazole, pH 7.0, containing 5 mM CaCI 2 and 50 mM NaC1, which maintains the aggregated structures of micellar caseins [16], and caseinate emulsions were suspended in 20 mM imidazole, pH 7.0. In the milks, micelles and micellar material were dissociated by suspending the fractions in a buffer of 20 m M imidazole, 6 M urea and 5 mM EDTA. 2.4. Electron microscopy
Transmission electron microscopy (TEM) was performed on the fractions. The method of sample preparation closely followed that of Allan-Wojtas and Kalab [ 17]. Initially the fluid milk or emulsion samples were prepared for thin sectioning by encapsulation in 2% agar tubes [18]. The samples were fixed with a 1.4% glutaraldehyde solution in a buffer of 0.1 M cacodylate, 0.05% CaC12 and 4% polyvinylpyrrolidine, pH 7.4, and postfixed in an imidazole-buffered solution of osmium tetroxide (0.5%). They were dehydrated by treatment with a graduated series of ethanol and propylene oxide, and embedded in Epon 812 epoxy resin. The resin blocks were cut into sections 100 nm thick using a Reichert Ultracut E ultramicrotome. The sections were stained with 4% uranyl acetate in 50% ethanol
147
followed by Reynolds lead citrate solution [ 19] in 1 N NaOH. The sections were mounted on grids and examined using a J E O L 100CX transmission electron microscope with an accelerating voltage of 60 kV. Typically, magnification factors from 15 000 to 100000 were used. Although no problems were experienced in the study of microfluidized whole-milk, the caseinate emulsions proved very difficult to fix. In nearly all cases the larger oil droplets showed a tendency to coalesce during fixation. This was demonstrated by the presence in the micrographs of fat globules which had breaks in their coating of protein, and indeed in some cases it was possible to see pairs of fat globules in the act of coalescing. The behaviour seemed to be mainly confined to the larger globules, since all of the small globules seen in the micrographs had well defined membranes of protein. Confirmation that the coalescence phenomenon was artefactual came from light scattering, which did not show very large particles to be present in the original emulsions, and also from the fact that no creaming occurred during storage of the emulsions for times of one week or greater. Thus it is likely that one of the early stages of the fixation process leads to the coalescence. Because the casein is spread in a monomolecular layer around the fat globules, and because at least some of the large globules appeared to be stabilized by sharing the protein which was on their surfaces, it is possible that the large globules had membranes which were weaker than those of the smaller ones.
3. Results 3.1. Particles in microfluidized milks
The differences between homogenized and microfluidized milks have already been described [20]. In homogenized milks we know that the fat globules have diameters in the range 0.6-1.2 gm, and their surface is formed by the adsorption of intact or partially intact casein micelles and whey proteins [21]. Often, the casein micelles spread over the oil/water interface [22]. From electron microscopy, it is evident that homogenized milk
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contains many intact casein micelles, from which it may be concluded that the stresses encountered when milk passes through the homogenization valve are insufficient to disrupt the casein micelles themselves, which have diameters in the region of 50-250nm. In microfluidized milk, the particles are completely different [-11,23]. Not only are the fat globules much smaller (relatively few are larger than about 300 nm), but their structure is different from those in the homogenized milk, as is shown by a study of the particles in the floating layer after centrifugation (Fig. la). On the larger fat globules, the surface layer of adsorbed protein is clearly different in structure compared with homogenized milk, since adsorbed micelles are almost completely absent, presumably because they are broken up during the process of microfluidization. A layer of protein about 10nm thick is around most of the globules, especially the larger ones. Moreover, some of the larger fat globules are partially coated by small ones, presumably via protein bridges, so that the surface layers of these particles can have a rather complex structure. The microfluidized milk also contains some very small fat globules, with diameters as small as 30nm, which are most clearly seen in the fraction which sinks during centrifugation (Fig. lb). Not only do these small globules appear as individual units, but in some cases, they also appear as clusters associated with particles of protein which may be the remnants of the original casein micelles. These
particles suggest that the fat is adhering to, or is embedded in, the protein matrix. In none of the fractions isolated from microfluidized milk did electron microscopy show the presence of any unaltered casein micelles. Size distributions calculated from light scattering of the floating and sinking fractions of the microfluidized milks are shown in Fig. 2. ILS of the sinking fraction (Fig. 2a) confirms the presence of particles with diameters less than 100 nm, which correspond to the small fat globules seen in the electron micrographs; the larger particles seen in this fraction by electron microscopy are clearly in a minority in terms of number. The intensity distribution of the particles as calculated from DLS (Fig. 2b) shows more clearly the presence of the larger particles in the sinking fraction, and suggests that the volume contribution of these particles (as distinct from their number) may be considerable; the contribution of the smaller particles in terms of weight is clearly small. In contrast, the floating fraction is seen to contain larger particles (up to about 500nm diameter), although the greater volume of particles is in the range 2 0 0 4 0 0 nm in diameter. The difference between the floating and sinking fractions as estimated by DLS is, however, relatively small, although in the sinking fraction it is possible to see some contribution from the smaller particles whose size is of the order of 0.1 gm. The ILS method is rather insensitive to particles less than about 5 0 n m in diameter,
Fig. 1. Electron micrographs of fractions prepared from whole milk treated by microfluidization (42 MPa, 5 passes). (a), sample prepared from the fraction which floats during the centrifugation step; (b), sample prepared from the fraction of particles which sink during the centrifugation step. Scale bars have lengths of (a), 300 nm, and (b), 200 nm.
D. G Dalgleish et al. / Colloids SurJ~tces A: Physicochem. Eng. Aspects 123-124 (1997) 145 153
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02
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149
0.2 0.4 0.6 0 0.2 0.4 0.6 Particle diameter (~m) Particle diameter (l~m)
Fig. 2. Size distributions from light scattering of particles in microfluidized milk (42 MPA, 5 passes) and fractions. (a) number distributions calculated from ILS for O, sample prepared from the fraction which floats during centrifugation (compare Fig. la); IN, sample prepared from the fraction of particles which sink during the centrifugation step (compare Fig. lb); A, the sinking fraction after treatment with urea and EDTA to dissociate micellar fragments. (b), intensity distributions calculated from DLS; symbols are the same as in (a).
because the scattering of such particles is not greatly dependent on the scattering angle, which is essential for the calculation (i.e. Rayleigh scatterers cannot be detected). Dynamic light-scattering also tends to underestimate the number of the smallest particles, because of the small region of the correlation function in which they can be measured [10]. In both ILS and DLS experiments it was possible to measure the basic sizes of the smaller fat globules by suspending them in a buffer containing urea and EDTA, which dissociates the fragments of casein micelles and leaves the fat globules with only a monolayer coverage of casein. The action of the urea was to decrease the sizes of the particles in the sinking fraction of the microfluidized milk (Figs. 2a and b), and to move the whole distribution of particle sizes to smaller values. These define the sizes of the basic fat droplets in the microfluidized milk, which can be compared with the droplets in the caseinate/soya-oil emulsions described below.
3.2. Emulsions prepared using caseinate The more simple emulsions formed from caseinate and soya oil should, in principle, be interpreted more easily than the microfluidized milks, because
there must be fewer complications arising from the structure of the protein (no casein micelles exist). As with the particles in microfluidized milk, there is clear evidence for the formation in the Microfluidizer of a large number of small particles with diameters less than 100 nm. However, there are significant differences between the milks and the emulsions. In a whole emulsion (20% oil, 1% caseinate) prepared by microfluidization at 28 MPa, ILS showed that there were a few particles with diameters up to about 500 nm, but that the majority had diameters around 200 nm (Fig. 3). After centrifugation, the fraction of the emulsion remaining in the serum contained particles with average diameters in the region of 50 100 nm. The sizes for the whole emulsion and the serum fraction were confirmed by the intensity distributions determined from DLS, although the results for both fractions were somewhat higher, as was to be expected (Fig. 4). The number fractions calculated for the whole emulsion from DLS (not shown) had a maximum at diameter 80 nm and a narrower range than was calculated from ILS. The intensity distribution from DLS was slightly bimodal, showing that both large (diameter 250-450 nm) and small particles (diameter about 100 nm) were present, and not a continuum of particle sizes.
D. G. Dalgleish et aL/ Colloids Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145-153
150 ~ , 0.30
I
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..~ 0.20
=_o ~ 0.10 E= z 0.00 0.20
0.00
0.40
Particle d i a m e t e r (gm)
Fig. 3. Number distributions of particle sizes in different emulsions made from caseinate and soya oil (20% w/w), measured using ILS. B, whole emulsion prepared using 1% caseinate, at pressure of 28 MPa and 5 passes through the Microfluidizer (compare Fig. 5a); Q, particles remaining in the serum fraction of the emulsion after centrifugation and removal of the larger particles (compare Fig. 5b); A, whole emulsion prepared using 2% caseinate, at pressure of 70 MPa and 5 passes through the Microfluidizer (compare Fig. 5c); II,, particles remaining in the serum fraction of this emulsion after centrifugation and removal of the larger particles (compare Fig. 5d).
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.
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.
.
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.
.
.
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Fig. 4. Intensity distributions of particle sizes in different emulsions made from caseinate and soya oil (20% w/w), measured using DLS. Symbols are the same as for Fig. 3.
Electron microscopy of this whole emulsion showed that there were particles with diameters in the range 500-600 nm, as well as confirming the presence of small ones (Fig. 5a). There was evidence
of bridging flocculation; some droplets were in contact and their areas of contact were flattened. No detailed size distributions were found from the electron microscopy, but qualitatively the micrographs agree with the conclusions arrived at from light scattering. Centrifugation to separate the particles gave a serum which contained mostly particles with diameters less than 100nm, with an apparently rather narrow size distribution (Fig. 5b), although there were a few larger particles also present. It is, of course, these particles which have the small sizes which are determined by light scattering. The small droplets neither coalesced or bridged, in contrast to the larger ones. At higher microfluidization pressure and casein concentration (70 MPa; 2% caseinate) the average sizes of the particles decreased, as would be expected from the increase in emulsifier concentration and the severity of the homogenization. Both DLS and ILS showed that the distributions of particle sizes, whether in number or in intensity, were shifted completely to smaller sizes (Figs. 3 and 4). Electron microscopy confirmed the relative absence of large oil droplets (Fig. 5c); indeed, it was difficult to find any in the samples which were studied, mainly because extensive coalescence appeared to have occurred. Measurements of the particles in the serum fraction of the emulsion showed, rather surprisingly, that the number distribution from ILS and the intensity distribution from DLS were shifted to larger particle sizes than the serum fractions of the emulsion made at lower pressure and with lower casein concentration (Figs. 3 and 4). This conclusion was also reinforced by the electron microscopy, where the emulsion droplets were indeed found to be significantly larger than those in the serum of the low pressure emulsion (compare Figs. 5b and d). In all of the oil/caseinate emulsions studied, it was evident from the electron micrographs that the population of very small droplets (less than 50 nm in diameter) was not so apparent as that found in the microfluidized milks. This was also confirmed by light scattering, where both ILS and DLS showed a smaller distribution, especially when the additional protein in the milk was dissociated with the urea (Fig. 2). This result may simply reflect the protein:lipid ratios in the two prepara-
D. G. Dalgleish et al. ' Colloids' Surfaces A: Physicochem. Eng. Aspects 123 124 (1997) 145-153
151
Fig. 5. Electron micrographs of emulsions (20% w/w soya oil) prepared by microfluidization. (a), whole emulsion containing 1% caseinate, prepared at 28 MPa and 5 passes; (b), particles from the serum fraction of this emulsion after centrifugation; (c), whole emulsion containing 2% caseinate, prepared at 70 MPa and 5 passes; (d) particles from the serum fraction of this emulsion after centrifugation. Scale bars have lengths of (a), 500 nm; (b), 100 nm; (c), 200 nm; (d), 200 nm.
tions; in the milk the casein:fat ratio is about 0.6, whereas in the caseinate emulsions the ratio was either 0.05 or 0.1. In the milk there is more protein to cover the fat surface, although the situation is complicated by the fact that, in milk, the protein is originally micellar, and therefore has an effective concentration less than the true one.
4. Discussion
The observations presented here confirm that the emulsions formed using the Microfluidizer, compared with those formed using a valve homogenizer, contain some very small particles, with diameters of 50 nm or even less. Not all of the lipid droplets are as small as this, but nearly all of them have diameters of less than 500 nm. The sizes of
these droplets also depend on the protein:oil ratio in the emulsion. Emulsions containing 2% caseinate contain overall smaller particles than do emulsions containing 1% caseinate, because the higher protein concentration provides for greater coverage of the interface, and thus an increased surface area of the emulsion. This detailed analysis of lightscattering results allows differences in the sizes of emulsion droplets to be seen when other methods [24] suggest that there is little change in the sizes of the droplets between the two concentrations of casein. The only counter-intuitive result is the larger size of the serum droplets in the emulsion formed using the higher casein concentration and pressure. However, this may at least partly be explained by the fact that much more of the oil is found in small droplets in this emulsion than in the low-pressure
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emulsion. Thus, there is considerably more surface of lipid to be covered in this emulsion, with the result that the droplets will be somewhat larger. The conditions of microfluidization, however, favour the breaking up of larger droplets of oil, so that there are fewer large droplets, more of intermediate size, and fewer small ones than in the other emulsion. Because of the small particle size, the emulsions produced by the Microfluidizer have the advantage of being very stable for creaming, unless flocculation of the droplets occurs, and this is rare if milk proteins are used as the surfactants in the absence of calcium. Because the particles are so small, we may speculate on whether their properties are likely to be significantly altered. One particular problem which may be produced by the size is that a considerable amount of the surface area of the emulsion droplets may be in these small particles. This may be simply expressed by the relation [3Ws
3w1~ / ,
,
where Rs and R l a r e the radii of the large and small particles and ws and wl are their respective weight fractions in the total emulsion, if the area is calculated simply from the average diameter as determined from DLS by the method of cumulants, or from measurement using Fraunhofer diffraction equipment, it is inevitable that the surface area in the emulsion will be considerably underestimated. This in turn will have an effect on determinations of the amount of protein adsorbed to the oil/water interfaces. Estimates of this for caseins range from 0.8 to 3 mg m 2 [25], but changes may have to be made to accommodate the extra area which is neglected by most estimates of particle size. On the positive side, it is evident that the error will be much less if the determination of the adsorbed protein is made on the layer of larger droplets which are harvested when the emulsion is centrifuged [26], because the true particle size will be much closer to the average diameter measured from DLS by the method of cumulants or by Fraunhofer diffraction. A second problem, especially in microfluidized milk, is that of inhomogeneity of the types of
particles. From the electron micrographs, it is clear that the different fat globules possess very different coats of protein. Many of the globules, especially the larger ones, have a fairly even coating of protein, but the smaller ones appear at least partly in aggregates with protein. It follows that the two types of particle may have quite different properties. How these will compare with the properties of homogenized milks has not been established; we know for example that the microfluidized milks share the poor renneting characteristics of homogenized milks, although a limited amount of microfluidization may improve some aspects of cheese milk [27]. Comparisons of heat stability and other effects are not defined; these are tasks for the future now that the structures of the particles in microfluidized milks and emulsions are becoming clear.
Acknowledgements The authors wish to thank Alexandra Smith, Diane Moyles, Lewis Melville and Dan Beniac for assistance with the preparation of samples for the electron microscopy. The research was supported by grants from the Ontario Dairy Council and the Natural Sciences and Engineering Research Council of Canada (to DGD). Sarah West was in receipt of a Rotary International Ambassadorial Scholarship from the United Kingdom.
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[19] E.S. Reynolds, J. Cell Biol., 17 (1963) 208. [20] D.G. Dalgleish, S.M. Tosh and S. West, Neth. Milk Dairy J., 50 (1996) 135. [21] H. Oortwijn and P. Walstra, Neth. Milk Dairy J., 33 (1979) 134. [22] H. Oortwijn, P. Walstra and H. Mulder, Neth. Milk Dairy J., 31 (1977) 134. [23] Y. Pouliot, P. Paquin and J. Giasson, Int. Dairy J., 1 (1991) 39. 1-24] Y. Fang and D.G. Dalgleish, Colloids Surfaces B, 1 (1993) 357. [25] Y. Fang and D.G. Dalgleish, J. Colloid Interface Sci., 156 (1993) 329. [26] J.A. Hunt and D.G. Dalgleish, Food Hydrocolloids, 8 11994) 175. [27] A. LeMay, P. Paquin and C. Lacroix, J. Dairy Sci., 77 (1994) 2870.