COLLOIDS AND B SURFACES

E L S E V I E R Colloids and Surfaces B: Biointerfaces 10 (1997) 73 85

Electroacoustic determination of size and zeta potential of fat globules in milk and cream emulsions

Theresa Wade, James K. Beattie *

School of Chemistry F11, University of Sydney, Sydney, N.S. W. 2006, Australia

Received 26 May 1997; accepted 6 August 1997

Abstract

Measurements of the zeta potential and droplet size of fat globules in milk and cream emulsions at natural pH have been made using the technique of electroacoustics. This technique requires no dilution or change of environment of the fat globules. Commercial and recombined milk and cream emulsions were studied. A zeta potential of -19 mV was obtained for homogenized fat globules from a commercial milk emulsion, a zeta potential of - 2 2 mV was obtained for natural fat globules from a commercial cream emulsion and a zeta potential of -36 mV was obtained for the fat globules from a recombined milk emulsion. Electroacoustics gives reasonable estimates of median size and zeta potential but at present is unable to give a true indication of the spread of the distribution. © 1997 Elsevier Science B.V.

Keywords: Fat; Electroacoustics; Zeta potential; Emulsion; Homogenize

1. Introduction

Fat in raw milk exists as emulsified globules (natural fat globules) that are surrounded by a thin protective layer described as the milk fat globule membrane ( M F G M ) . Homogenization of milk causes a reduction in the size of fat globules, and since the volume of fat globules remains the same there is an increase in the surface area of the fat globules. This means that the original milk fat globule membrane can no longer completely cover the surface of the fat globules and, as a result, a mixture of proteins are adsorbed from the plasma phase on to the fat globule surface [1-5]. Hence, homogenized fat globules have a surface layer that comprises both the natural membrane and some

* Corresponding author. Tel: (+61) 2 9351 3797; Fax: (+61) 2 9351 3329; e-mail: beattiej@chem.usyd.edu.au

0927-7765/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PH S0927-7765 (97) 00046-5

plasma proteins. Artificial fat globules can be prepared by homogenization of a mixture of anhy- drous milk fat and skim milk to make recombined emulsions. In this case, most of the material of the natural milk fat globules membrane is absent. As a result, the surface layers of the fat globules from recombined emulsions consist predominately of plasma proteins [4, 6, 7].

Since these three different types of fat globule are expected to have different surfaces, an investi- gation of the zeta potentials of the fat globules may be useful. However, to date, the study of the zeta potential of fat globules has been fairly lim- ited [8,9].

The size distribution of fat globules from various milk and cream emulsions has been the subject of many investigations [ 10-13]. Before homogeniza- tion the fat globules range in diameter from less than 0.2 ~tm to about 15 lam. Walstra et al. [12]

74 T. Wade, J.K. Beattie / Colloids Surfaces B." Biointerfaces 10 (1997) 73 85

found that the size distribution obtained depended greatly on the sizing method employed.

In this study, the size and the zeta potential of the milk fat globules of various milk and cream emulsions were investigated with the new technique of electroacoustics. An electroacoustic effect is the generation of electric fields by sound waves or the generation of sound waves by the application of an alternating electric field. The latter is known as the electrokinetic sonic amplitude (ESA). When an alternating electric field is applied to a colloidal suspension, the particles oscillate back and forth and generate pressure disturbances in the sur- rounding liquid in the form of sound waves. This effect is utilized in this study for the determination of fat globule size and zeta potential. The study of size and zeta potential of casein micelles using this technique is described in a previous paper [14].

Most methods used for determining colloidal particle size and zeta potential require dilute samples because they rely on a passage of light through the suspension. Since the electroacoustic effect involves sound rather than light, the ESA measurement can be applied to opaque concen- trated suspensions. By using this technique we have been able to study the three different types of fat globule in their natural environment, even in cream emulsions with fat concentrations of 38%.

2. Materials and methods

2.1. Preparation o f cream and milk emulsions

Four different fat emulsions were investigated: commercial milk, commercial cream, recombined milk and recombined cream. The commercial milk (38 g kg -1 fat, 89 g kg -1 MSNF, United Dairies, Australia) and cream (min. 350 g kg -1 fat, Dairy Farmers, Australia) were used directly. The com- mercial milk had been homogenized at a temper- ature of 45°C with a two-stage homogenizer working at a total pressure of 13.8 MPa with a drop of 3.4 MPa across the second valve. The commercial cream had not been homogenized. Recombined cream (350gkg -1 fat) and milk (40 g kg -~ fat) were prepared by mixing appro- priate amounts of anhydrous milk fat (Unilac, Australia) and commercial skim milk (92 g kg -~

MSNF, United Dairies or Dairy Farmers, Australia) at 50°C. The mixture, whilst being con- tinuously stirred, was recirculated for 1 h through a four-stage homogenizer from a Milko-Tester MK.III (Foss Electric, Denmark) at a total pres- sure of approximately 14 MPa. A total of 1.41 of recombined cream was prepared, whereas only 0.5 1 of recombined milk was prepared. Hence, the recombined cream had significantly fewer passes through the homogenizer than the recombined milk.

2.2. Electroacoustics

Electroacoustic measurements were made using the AcoustoSizer (Matec Applied Sciences, MA, USA). A schematic diagram of the AcoustoSizer measurement cell is given in a previous paper [14].

To determine the particle size and zeta potential a quantity called the "dynamic mobility" is firstly obtained from the ESA signal. Then, from a dynamic mobility spectrum, the size and zeta potential are determined. A description of this procedure is given in a previous paper [14] and a detailed account of the theory of electroacoustics and of the AcoustoSizer is given by O'Brien et al. [15].

For the determination of droplet size and zeta potential the density and the volume fraction of the fat globules must be known. For all emulsions it was assumed that the density of the fat globules was 0.92 g ml - 1 [4]. The volume fractions of the fat globules from the commercial milk and cream emulsions were assumed to be 4% and 38% respec- tively. For the recombined emulsions, the weight of fat was known; hence, the volume fraction could be calculated.

The emulsions were stirred at 300 rev min- 1 and the temperature was maintained at 23°C through- out the experiments. The sampling number of the ESA measurements was increased to four times the default value of 120 for milk, cream and skim milk (plasma) emulsions in order to increase the signal-to-noise ratio.

2.3. Plasma phase

The aim of this work was to measure the ESA signal, and hence determine the zeta potential and

T. Wade, J.K. Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73-85 75

size of the fat globules in both milk and cream emulsions. The ESA signal generated from a milk or cream emulsion is a combination of the ESA signal generated from the fat globules, casein micelles, and the serum phase in which they are dispersed. Hence, the ESA signal generated from the casein micelles and the serum phase (plasma phase) must be subtracted from the ESA signal of the milk or cream emulsion in order to obtain the ESA signal from the fat globules. Consequently, for each emulsion a plasma phase (background) must be obtained.

For the commercial emulsions, a plasma phase was obtained by removing the fat globules by means of centrifugation using a Sorvall RC-5B Refrigerated Superspeed centrifuge (Dupont Instruments). The commercial milk emulsion was centrifuged at 16 100g for 1 h at 4°C. The plasma phase was obtained by carefully placing the needle of a syringe through the fat layer and extracting the skim milk. The remaining solid fat layer was discarded and any pelleted protein was removed from the bottom of the centrifuge tube and was redispersed in the plasma phase by ultrasonicating the plasma phase (Unisonic FX10, 50Hz) for approximately 0.5 h. The cream emulsion was centrifuged at 3330g for 0.5 h. The skim milk suspension was obtained by carefully placing the needle of a syringe through the fat layer and extracting the skim milk.

When anhydrous milk fat is emulsified into skim milk, by homogenization, plasma proteins are forced onto the surface of the fat [6,7]. The recombined milk emulsion contained 40 ml 1 - 1 fat, and hence only a relatively small amount of protein would be adsorbed from the plasma. Hence, the commercial skim milk suspension used in the recombination was used as the plasma phase. In the recombined cream emulsion with 380 ml1-1 fat a greater amount of the protein would be expected to be adsorbed from the plasma phase. Consequently, a plasma phase was obtained by centrifuging the recombined cream emulsion as described for the commercial milk emulsion.

In all cases, an ESA signal was measured on the plasma phase and was subtracted vectorially from the measured ESA signal of the commercial or recombined emulsions to obtain the ESA signal,

and hence the dynamic mobility spectra for the fat globules. A facility for applying such background subtractions is included in the AcoustoSizer software.

2.4. Concentration corrections

As stated in a previous paper [14], for suspen- sions with particle concentrations greater than 2% the effects of particle-particle interactions on the dynamic mobility become significant. The AcoustoSizer software presently applies a semi- empirical concentration correction, but this pro- cedure overestimates the corrections necessary for near-neutrally buoyant systems such as fat glob- ules. A theory has been developed [16] for which the zeta potential and size can be interpreted from the measured dynamic mobility for systems with a volume fraction of particles up to 0.1. This theory is valid for both dense (Ap > 1 g ml- l) and near- neutrally buoyant particles (Ap,~0.1gml-1). Some of the fat globule emulsions studied in this paper have particle concentrations far exceeding 10%, and so a concentrated theory recently devel- oped for near-neutrally buoyant systems was applied. The dynamic mobility in a concentrated suspension of near-neutrally buoyant particles is given by [17]

# = H 1 - ( 2 + ~ ) ( 3 + 3 2 + 22) 2+~b

(1)

where ( is the zeta potential of the particle, q~ is the volume fraction of particles, and e and ~/are the permittivity and viscosity of the solvent respec- tively. The factors H and F are defined by

3 + 32 + 322 H = (2)

3 + 32 + ~22[ 1 + 2(pp/p)]

F = 2{[422I+ ( 1 + 22) e-2ZlJ+ ½} (3)

Here

e ~ J= (4)

1 + 2 + ( 2 2 / 3 )

76 T. Wade, J.IK Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73-85

and

I= [g(r)- l]re -2~" dr (5) 1

where g(r) is the pair distribution function which is dependent on the particle volume fraction. Finally, the parameter 2 is given by

).=(1 +i)N / ~-v (6)

where a is the particle radius, v is the kinematic viscosity of the suspending liquid and o) is the angular frequency of the applied field.

For the commercial and recombined cream emulsions, the volume fraction of the fat globules was equal to 0.38. In order to check the values of zeta potential and size obtained through the appli- cation of O'Brien's concentrated formula, the cream emulsions were diluted to a fat concen- tration where particle-particle interactions did not affect the dynamic mobility spectra. The cream emulsions were diluted in the appropriate plasma phase. The method for obtaining the plasma phase was described Section 2.3. The plasma phase that was used as the diluent was also used as the background for the diluted emulsions.

insignificant, but for particles larger than 1 gm, which includes the fat globules studied here, this effect is important. O'Brien [20] has found that the effect of this extra delay is to change the phase in the mobility by 3ka, that is

# a p p = ~ t r u e e - 3ika (7 )

where /~,pp is the apparent dynamic mobility, #true is the true dynamic mobility, a is the particle radius and k=2~/2, where 2 is the sound wavelength.

2.6. Plasma phase viscosity and density measuremen ts

In order to obtain the zeta potential and size of the particles from the dynamic mobility spectrum it is necessary to know the viscosity of the plasma phase. The viscosity of the plasma phase was measured using an Ostwald viscometer at 25.00 + 0.05°C according to the procedure described by Wilson et al. [21]. In order to deter- mine the viscosity, the density of the plasma phase must also be known. Density measurements were made with a PAAR precision densitometer Model DMA-02C in a DMA 602 remote sensing cell, at a temperature of 25.00_+ 0.01 °C.

2.5. Correction for adsorbed particles on electrodes

When charged particles in a suspension are in the vicinity of an electrode an attractive force is generated by the interaction of the charge on the particle and its image in the electrode [18]. In terms of electroacoustics this means that a number of particles become fixed to the electrodes in the AcoustoSizer which do not move under the influ- ence of the applied field. These particles do not contribute to the ESA signal because they are fixed to the electrode, but they affect the ESA signal by excluding other particles, and so effectively make the length of the measurement rod in the AcoustoSizer longer. This means that the ESA signal generated by the particles in the suspension has further to travel before reaching the transducer at the end of the rod. It has been found experimen- tally [19] that this "extra length" is about three particle radii. For small particles the effect is

3. Results

3.1. Homogenized fat globules: commercial milk (40 ml l - l fat) emulsion

ESA measurements were made on commercial milk emulsion. The conductivity of the commercial milk emulsion at its natural pH of 6.7 was 0.48 S m-1. The measured viscosity of the plasma phase was 1.4 mPa s. The density of the plasma phase was assumed to be that of a commercial skim milk suspension (1.032 gm1-1) which had been measured previously [ 14]. Fig. 1 (a) and 1 (b) respectively show the magnitude and argument of the dynamic mobility as a function of frequency for fat globules in a commercial milk emulsion. The absolute value of the magnitude of the mobil- ity decreases with increasing frequency and the

T. Wade, J.K. Beattie / Co~bids Surfaces B. Biointerfaces 10 (1997) 73-85 77

-0.8 o~

? 07

E ~ -0.6

= "~=0 -0.5

-0.4

(a)

o

o

0

0

0 0

0 0

0

O O

' ' ' ' ' ' '0 '2 0 2 4 6 8 1 1

Frequency (MHz)

f (b)

o

o o

o

o

o o

o o

o o

O O

I I I I I I /

0 2 4 6 8 10 1 2

Frequency (MHz)

Fig. 1. Magnitude (a) and argument (b) of the dynamic mobility as a function of frequency of fat globules from a commercial milk emulsion (40 ml 1 -1 fat ).

phase angles become more negative. This is consis- tent with electroacoustic theory and occurs because the particle inertia becomes more important as the frequency of the applied field increases. Electroacoustic theory predicts that the magnitude and argument of the mobility should be smooth monotonic functions of the frequency [22]. This is true to a good approximation for the magnitude of the mobility (Fig. 1 (a)), and it is also true of the phase angles (Fig. l(b)), although there is some noise in this data. This noise arises because the volume fraction of the fat globules is small, and so the ESA signal from the commercial emul- sion is small and is of a similar magnitude to the ESA signal of the plasma phase. Hence, when the ESA signal of the plasma phase is subtracted from the ESA signal of the commercial milk emulsion the resultant fat globule signal will also be affected

by random noise, which is reflected in the dynamic mobility spectrum.

The zeta potential of the fat globules from the commercial milk emulsion was -18 mV and the median diameter was 1.0 ~tm with a spread of 0.9-1.1 gm. The spread is defined as the size range from the 15th to the 85th percentile. The values of zeta potential and size were reproducible from sample to sample to within 3 mV and 0.1 ~tm respectively. Application of the particle deposition correction to the dynamic mobility spectra made no difference to the values of zeta potential and size. Application of the concentration correction to the dynamic mobility spectra resulted in a zeta potential value of - 19 mV, a median diameter of 1.1 gm and a spread of 1.0-1.2 lam. Hence, at a volume fraction of 0.04 the emulsion can be consid- ered (to a good approximation) to be dilute.

3.2. Natural f a t globules." commercia l cream (380 ml 1 - 1 fa t ) emulsion

ESA measurements were made on a commercial cream emulsion. The conductivity of the commer- cial cream emulsion at its natural pH of 6.7 was 0.26 S m- 1. The measured viscosity of the plasma phase was 1.4 mPa s. The density of the plasma phase was assumed to be that of a commercial skim milk suspension (1.032 g m1-1) which had been measured previously [14]. At a volume frac- tion of 0.38 the effects of particle-particle inter- actions on the dynamic mobility spectra, and hence on the values of zeta potential and size, are signifi- cant. Although there is a formula that takes these interactions into account, the formula was checked by diluting to a concentration where particle-par- ticle interactions are not significant. The commer- cial cream emulsion was diluted down to a fat concentration of 40 ml 1-1 with the skim milk (plasma phase) that was obtained by centrifuging the cream emulsion.

Fig. 2(a) and 2(b) respectively show the magni- tude and phase angles of the dynamic mobility for the fat globules from the 380 mll -I commercial cream emulsion (O) and from the diluted 40 ml 1-1 emulsion (Q). Both the magnitude and phase angles of the dynamic mobility for the cream emulsion decreased monotonically with increasing

78 T. Wade, J.K. Beattie / Colloids Surfaces B." Biointerfaces 10 (1997) 73-85

(a)

-0.8

-0.6

% -0.4

-0.2 I

0

0 •

o 0 • •

0 • O 0

0

I I I i I

2 4 6 8

Frequency (MHz)

o e o • o

1'0 1'2

-10

• !-°

-60

o

oo

& 0

• 0

0

O

0 • 0

• 0

• o • o

• g

. . . . . '0 '2 0 2 4 6 8 1 1

(b) Frequency (MHz)

Fig. 2. Magnitude (a) and argument (b) of the dynamic mobility as a function of frequency of fat globules from a commercial cream (380 ml 1 -1 fat) emulsion (©) and a diluted commercial cream (40 ml 1-1 fat) emulsion (O).

frequency, according to theory. Owing to the high volume fraction of the fat globules, the ESA signal of the emulsion and of the fat globules was signifi- cantly larger in magnitude than the ESA signal of the plasma phase, and as a result no noise was observed in the dynamic mobility spectra. The phase angles of the dynamic mobility for the diluted 40 m l l - ' emulsion show some deviations from electroacoustic theory at higher frequencies for the same reason as described in Section 3.1.

Fig. 2(a) and 2(b) show clearly the effects that particle-particle interactions have on the magni- tude and argument of the dynamic mobilities. The absolute magnitude of the dynamic mobility of the fat globules in the 380 ml 1-1 commercial cream emulsion was substantially less and the phase angles less negative than those of the fat globules

in the 40 ml 1 - 1 diluted emulsion. Hence, the zeta potential and size of the fat globules would be substantially underestimated if calculated from the dynamic mobility of the commercial cream emul- sion by assuming that the cream emulsion was dilute. Note that the dynamic mobility is normal- ized for concentration, that is the ESA signal is divided by the volume fraction of particles in the emulsion. So if, the particle-particle interactions that occur in the 380 ml 1 - 1 emulsion did not affect the dynamic mobility spectra, then the dynamic mobility spectra for the fat globules in the 38% and 4% emulsions would be identical.

A 40 mll-1 emulsion can be considered to be dilute. If there is no agglomeration or disruption of the fat globules upon dilution, it would be expected that the value of zeta potential and size obtained from the dynamic mobility spectra of the diluted 40 ml l -a emulsion would be the same as the values for the fat globules from the original commercial cream (380 ml 1 - ' fat) emulsion. From the dynamic mobility spectra of the diluted 40 ml 1 - 1 emulsion, a zeta potential of - 27 mV was obtained for the fat globules with a median diameter of 3.4 gm and a spread of 3.1-3.8 ~m. The values of zeta potential and size were repro- ducible to within 3 mV and 0.1 gm respectively from sample to sample. Applying the particle deposition correction to the dynamic mobility spectra of the diluted emulsion gave a zeta poten- tial value of -28 mV, a median diameter of 3.7 gm and a spread of 2.4-5.7 gm. Owing to the larger size of the fat globules, the particle deposition of the electrodes has a significant effect on the phase angles, as shown by the change in the spread upon application of the correction.

O'Brien's concentrated mobility formula was applied to the dynamic mobility spectra of the commercial cream emulsion. From the "corrected" mobility spectra the zeta potential of the fat glob- ules was determined to be - 2 2 mV, the median diameter was 3.1 gm with a spread of 2.8-3.4 gm. At present, the correction for particle deposition and the concentration correction cannot be applied together. Hence, it is expected that the spread obtained with the concentration correction for the fat globules from the commercial cream emulsion

T. Wade, J.K. Beattie / Colloids Surfaces B." Biointerfaces 10 (1997) 73-85 79

would be broader if the particle deposition correc- tion had been applied.

3.3. Artificial fa t globules." recombined milk (40 ml 1- l fat) and cream (380 ml 1- l fat) emulsions

ESA measurements were made on a recombined milk emulsion at its natural pH of 6.7; the conduc- tivity of this emulsion was 0.52 S m-1. The mea- sured viscosity of the plasma phase was 1.4 mPa s. It was assumed that the density of the plasma phase would be that of a commercial skim milk suspension (1.032 gm1-1) which had been mea- sured previously [14]. Fig. 3(a) and 3(b) respec- tively show the magnitude and argument of the dynamic mobility as a function of frequency for

-1.6

: ~ -1.0

E -0.8

(a)

% o

O

O

O

O O

O O

O

0

2 4 6 8 1 1

Frequent y (MIk)

-10

~ - 3 0

I

0

(b)

o

g

0

o O

o o 0

O O

O

I I I I / I

2 4 6 8 10 12

Frequency (MI-lz)

Fig. 3. Magnitude (a) and argument (b) of the dynamic mobility as a function of frequency of fat globules from a recombined milk emulsion (40 ml 1-1 fat)

the fat globules from a recombined milk (40 ml 1 - 1 fat) emulsion.

The zeta potential obtained from the mobility spectrum for the fat globules was - 3 5 mV, the median diameter was 1.0 ~tm and the spread was 0.9-1.1 Ixm. The values of zeta potential and size were reproducible from sample to sample to within 1 mV and 0.1 lam respectively. As for the commer- cial milk emulsion, application of the particle deposition correction or concentration correction to the dynamic mobility spectra made no difference to the values of zeta potential and size.

ESA measurements were made on a recombined cream emulsion at its natural pH of 6.7; the conductivity of this cream emulsion was 0.26 S m - 1. The measured viscosity and density of the plasma phase were 1.1 mPa s and 1.026 g ml - 1 respectively. The volume fraction of fat globules in the recombined cream emulsion was 0.38, and so the cream emulsion was diluted to 40 ml l -1 fat so that the dilute approximation could be compared with the values of zeta potential and size obtained with the concentrated formula. The cream emulsion was diluted with the plasma phase. Fig. 4(a) and 4(b) respectively are plots of the magnitude and argument of the dynamic mobility as a function of frequency for the fat globules from the 380ml1-1 recombined cream (O) and from the diluted 40 ml l -1 emulsion (O). The magnitudes and arguments of the dynamic mobility for the recombined cream emulsion decrease smoothly with increasing frequency in agreement with electroacoustic theory. The argu- ment of the dynamic mobility for the diluted 40 ml1-1 emulsion shows some deviations from electroacoustic theory at higher frequencies for the same reasons as described previously.

The zeta potential of the fat globules from the diluted 40 ml 1 - 1 recombined emulsion was - 2 4 mV, the median diameter was 2.5 ~tm and the spread was 2.3-2.8 ~tm. Applying the particle deposition correction gave a zeta potential of - 2 4 m V , a median diameter of 2.5 ~tm and a spread of 2.2-2.7~tm. O'Brien's concentrated mobility formula was applied to the dynamic mobility spectra of the recombined cream emul- sion. From the corrected mobility spectrum the zeta potential of the fat globules was found to be

80 T. Wade, J.K. Beattie / Colloids Surfaces B." Biointerfaces 10 (1997) 73-85

-1.o ' v

m

? ; .o8

E ~ -o.6

• g-0.4 E

(a)

% o

o o • o o o o o o

, I I I I * 110 2 4 6 8

Frequency (MHz)

o

' ln2

?

E

!

(b)

0

-10

. r - 3 0

i . -50

o

o o

o

o o

o 0 O

I I * I a I I ~ 110 I 112 0 2 4 6 8

Frequency (MHz)

Fig. 4. Magnitude (a) and argument (b) of the dynamic mobility as a function of frequency of fat globules from a recombined cream (380 ml 1 - 1 fat ) emulsion (©) and from a diluted recom- bined cream emulsion (40 m l l i fat) (O).

- 19 mV, the median diameter was 2.7 gm and the spread was 2.4-3.0 gin.

4 . D i s c u s s i o n

The results illustrate that the new technique of electroacoustics can be used to measure the zeta potential and the size of fat globules in commercial and recombined milk and cream emulsions. Generally, the dynamic mobility spectra obtained for the fat globules agreed well with electroacoustic theory. The dynamic mobility spectra for the fat globules for the commercial and recombined milk systems showed deviations from electroacoustic theory due to measurement errors in the small ESA signal at low fat concentrations (40 mll-1).

However, the values of zeta potential and size for the recombined and commercial milk emulsions were reproducible, indicating that the deviations were not significant.

Table 1 is a summary of the zeta potentials and sizes obtained for the fat globules of the various commercial and recombined systems that were studied. Table 1 shows the values of zeta potential and size obtained when the concentrated theory was applied, and also the values obtained when the emulsions were assumed to be dilute. For simplicity, no particle deposition correction has been applied to the values shown in Table 1. It was found that this correction was only significant for the fat globules from the commercial cream emulsion and had the effect of widening the spread. It can be seen from Table 1 that, for emulsions which contained only 40 ml 1-1 fat, the concen- trated and dilute formulae gave essentially the same results. The second set of columns in Table 1 illustrates the gross underestimations in zeta poten- tial that occur if the dilute formula is applied to emulsions containing 380 mll -I fat. The under- estimations of droplet size are also significant. However, the dependence on concentration is not as strong for droplet size as it is for zeta potential.

The values of zeta potential obtained for the fat globules from the diluted commercial and recom- bined cream emulsions (40 ml 1-1 fat) were 20% higher in absolute magnitude than values obtained by applying the concentration formula to the dynamic mobility of the original cream emulsions (380 ml1-1 fat). These differences may represent a systematic error in the formula for the concen- tration correction or may be due to a real effect of dilution. The zeta potential and size of concen- trated intravenous fat emulsions which consisted of soybean oil and egg phospholipids have been investigated in this laboratory with electroacoustics [23]. Dilutions of these emulsions were also made so that a comparison with values obtained from the concentrated formulae could be achieved. No difference was found in the zeta potential and size values obtained from the diluted emulsions and the values obtained upon application of the con- centrated formula to the original emulsions [17]. This indicates that the differences observed between the zeta potential of the fat globules from

T. Wade, J.K, Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73-85 81

Table 1 Particle size distributions and zeta potentials ( for fat globules from commercial and recombined emulsions, where ds0, dl~ and d85 represent the median, 15th and 85th percentiles respectively of a log-normal distribution

Emulsion q~ Concentrated formula Dilute formula

((mV) Particle size (~tm) (tmV) Particle size (Inn)

dso d15 dss d~o dis dss

Commercial milk 0.04 - 19 1.1 1.0 1.2 - 18 1.0 0.9 1.1 Commercial cream 0.38 - 22 3.1 2.8 3.4 - 13 1.9 1.7 2.1

0.04 -27 3.5 3.2 3.9 -27 3.4 3.1 3.8 Recombined milk 0.04 - 36 1.1 1.0 1.2 - 35 1.0 0.9 1.1 Recombined cream 0.38 - 19 2.7 2.4 3.0 - 11 1.6 1.4 1.7

0.04 -25 2.6 2.4 2.9 -24 2.5 2.3 2.8

the cream suspensions upon dilution and the values obtained from the application of the concentrated formula are not due to an error in the concentrated formula. In complex systems, such as milk and cream emulsions, dilution could result in changes in the zeta potential of the fat globules. It is also possible that dilution of the cream emulsions to 40 m l l - 1 fat can lead to significant experimental errors. Random noise and systematic errors, such as uncertainties in the exact plasma phase composi- tion, become important.

As can be seen from Table 1, the zeta potential values of the fat globules from the four emulsions studied were all different. It is expected that the surface of the fat globules f rom the different emul- sions would vary in composition. The homoge- nized fat globules from the commercial milk should be surrounded by a membrane that consists of both the natural M F G M and plasma proteins [1- 5]. The zeta potential value ( - 1 9 mV) obtained for the homogenized fat globules from the com- mercial milk emulsion agrees well with zeta poten- tial values obtained by Dalgleish [9] with the technique of laser Doppler electrophoresis. The fat globules were diluted in buffer or ultrafiltrate, and zeta potential values ranging from - 1 3 to - 17 mV were obtained depending on the concen- tration of Ca 2 ÷ in the diluent.

The fat globules in natural unprocessed milk are coated with a membrane derived from the secreting cell. The surface of the fat globules from the commercial cream emulsion should consist solely of this natural membrane. Dalgleish [9] studied

the zeta potential of fat globules from cream emulsions again with the technique of laser Doppler electrophoresis. The cream was diluted in ultrafiltrate or buffer. Dalgleish [9] observed that the mobility of the fat globules after being sus- pended in the diluent increased, until a final zeta potential value of - 1 0 m V was reached after

10 min. Dalgleish [9] suggested that this may be due to a change in the interfacial composition or structure of the fat globules as a result of dilution and found no other obvious time dependence for any other milk constituents suspended in buffer or ultrafiltrate. Payens [8] determined the zeta poten- tial of fat globules in raw milk using microelectro- phoresis and obtained a value of - 13 _+ 2 mV. No experimental details were given, and so it is not known in what the fat globules were diluted. The value of zeta potential obtained with electroacous- tics for the fat globules from the commercial cream emulsion was more negative ( - 2 2 mV) than that obtained by Dalgleish [9] or Payens [8]. I f suspend- ing fat globules in ultrafiltrate or buffer changes the interfacial composition or structure of the fat globules then the zeta potential may also be affected. An experiment was conducted in which the fat globules from the commercial cream emul- sion were diluted in centrifugate, to a concen- tration of 40 ml 1 - t fat. It was found that the zeta potential of the fat globules did not change signifi- cantly upon dilution in centrifugate but that the size of the fat globules obtained with electroacous- tics was larger than the size of the fat globules diluted in the plasma phase. This difference in

82 T. Wade, J.K. Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73-85

droplet size was confirmed with phase contrast microscopy. From the results obtained with elec- troacoustics and phase contrast microscopy it appears as though some change does occur to the fat globules once they are diluted in centrifugate, as was also observed by Dalgleish [9]. However, the difference between the value of zeta potential for the natural fat globules obtained with electro- acoustics and the values obtained by Dalgleish [9] are not due to the use of centrifugate (ultrafiltrate) as the diluent. The difference in the value of the zeta potential may be due to the commercial nature of the sample used for the electroacoustic experi- ment. Payens [8] used raw milk as a source of natural fat globules, and it is not clear as to the history of the cream used by Dalgleish [9] for a source of natural fat globules. It has been found that treatment of milk in a separator can cause some disruption of the fat globules which may result in changes in the membrane [3,4]. Any damage to the fat globule membrane during this process will lead to adsorption of plasma proteins at the damaged spot. The commercial cream emul- sion was cooled to below 4°C immediately after pasteurization, and cooling can lead to the migra- tion of various constituents from the membrane to the plasma phase [4]. The process of pasteuriza- tion can lead to changes in the membrane of the fat globules. McPherson et al. [24] have found that casein and were proteins was incorporated into the membrane matrix of fat globules from a commercial pasteurized emulsion. The different value of zeta potential obtained for the fat globules from the commercial cream sample indicates that changes occurring during processing may cause significant changes in the composition of the sur- face of the natural fat globules.

The artificial fat globules from the recombined emulsions should be surrounded by a membrane consisting only of plasma proteins [4,6,7]. The zeta potential of the fat globules from the recom- bined cream ( - 1 9 m V ) and recombined milk ( - 3 6 mV) are significantly different. There is evi- dence that when milk emulsions are homogenized there is a greater preponderance of casein micelles adsorbed onto the fat surface, though this is depen- dent on conditions of homogenization [ 1,4, 7, 25]. The recombined milk emulsion has an excess of

plasma protein compared with the concentration of fat (40 mll-1). On the other hand, a greater proportion of the plasma protein would be adsorbed onto the surface of the fat globules in the recombined cream emulsion. Hence, if there is a tendency for casein micelles to adsorb onto the fat surface it is possible that the surfaces of the fat globules from the recombined milk and cream emulsions may be different. Indeed, Sharma et al. [26] found that changing the protein/fat ratio changed the concentrations of individual casein components on the surface of recombined milks and that the casein to whey protein ratio adsorbed at the surface decreased as the protein to fat ratio increased. Hence, the protein to fat ratio directly affects the composition of the fat globule surface. The relatively high conductivity of the plasma phase (~0.5 S m -1) means that the magnitude of the electric potential would decrease fairly rapidly with increasing distance from the fat globule sur- face. Relatively small changes in the type and concentration of adsorbed proteins could cause shifts in the shear plane which would cause signifi- cant differences to the value of zeta potential. Dalgleish [9] measured the zeta potential for fat globules homogenized with different amounts of sodium caseinate with the technique of laser Doppler electrophoresis, and found a small but reproducible absolute increase in the zeta potential as the ratio of caseinate to fat was decreased. Dalgleish [9] ascribed the differences to a change in surface properties of the fat globules. The trend in zeta potential with a decreasing caseinate to fat ratio observed by Dalgleish [9] was opposite to the trend observed with electroacoustics for the recombined systems. However, the recombined systems studied with electroacoustics are different to the recombined systems studied by Dalgleish [9]. The results obtained by Dalgleish [9] show that variations in the zeta potential have been observed with differing ratios of fat to casein for recombined systems.

The similarity in the value of zeta potential for homogenized fat globules obtained with electro- acoustics and with laser Doppler electrophoresis by Dalgleish [9] indicates that the technique of electroacoustics can measure the zeta potential of fat globules from various emulsions. The different

72 Wade, J.K. Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73 85 83

zeta potential value obtained for the "natural fat globules" with electroacoustics, compared with values reported in the literature, indicates that separation and pasteurization have a significant effect on the surface of the natural fat globules. The zeta potential values obtained for the various types of fat globule may not give a true indication of the potential at the Stern layer, since the adsorp- tion of plasma proteins may result in displacement of the shear plane far out into the diffuse region. However, the values of zeta potential obtained with electroacoustics confirm observations made in the literature that the different types of fat globule have different surfaces. From the above discussion it is clear that the measurement of zeta potential for these systems would be a useful tool in determining if changes occur to the surface of fat globules during processing and what parame- ters affect the surface of recombined fat globules.

Cornell and Pallansch [10] measured the size of various types of fat globule with a Coulter Counter. The sizes of the fat globules were repre- sented as a cumulative weight distribution, and so a weight median diameter could be extracted from the plots of fat globule size. Median diameters of ~1.8 jam and ~0.8 jam were obtained from the cumulative weight distribution for fat globules from a milk emulsion that had been homogenized at 70kgcm -2 (,,~7MPa) and 176kgcm -2 ( ~ 17 MPa) respectively. The fat globules from the commercial milk emulsion had been homogenized with a two-stage homogenizer at a total pressure of 13.8MPa. The median diameter of 1 jam obtained for these fat globules with electroacous- tics certainly seems reasonable considering the median sizes obtained from the results of Cornell and Pallansch [ 10].

A median diameter of ~4.2jam could be extracted from the cumulative weight distribution curve obtained by Cornell and Pallansch [10] for fat globules from a raw cream emulsion. Walstra [27] studied the fat globule size distributions in milk from Friesian cows. Ten samples were taken from the cows, and an average weight median diameter for fat globules of 3.9 jam was obtained with a range in weight median diameters from 2.9 to 5.5 jam. Walstra states that similar results would be expected for mixed milk but with smaller varia-

tions. Cornell and Pallansch [10] found only slight differences in the size distributions for raw milk and cream. Considering the results obtained by Walstra [27] and Cornell and Pallansch [10], the median diameter obtained with electroacoustics for fat globules from a commercial cream suspen- sion (3.1 jam) seems very reasonable.

The median diameter of the fat globules from the recombined milk emulsion was, as expected, very similar to the median diameter of the homoge- nized fat globules. The median diameter of the fat globules from the recombined cream emulsion was larger and the spread was slightly broader than for the recombined milk emulsion. It has been shown that homogenization becomes less effective, that is there is an increase in the average fat globule diameter, with increasing fat content and with an increasing ratio of fat to plasma proteins [28,29]. The slightly broader spread obtained for the recombined cream emulsion is most likely due to the fact that the recombined cream had fewer passes through the homogenizer than the recom- bined milk emulsion. Mulder and Walstra [4] state that the main effect of repeated homogenization is a narrower size distribution of the fat globules.

A comparison of the size distributions obtained with electroacoustics for the fat globules from the various emulsions with those reported in the lit- erature [10,11, 13,27,30] shows that the size dis- tributions obtained with electroacoustics were sig- nificantly narrower. Investigation of the fat globule size distribution with phase contrast microscopy also showed broader distributions than that obtained with electroacoustics.

Fig. 5 shows the theoretical phase angles of the dynamic mobility for particles with a median diam- eter of 1 jam and the same density as for the fat globules. The open circles represent the particle size distribution having an upper limit (d85) of 1.1 jam, as was the case for the fat globules from a commercial milk emulsion. The closed circles represent the particle size distribution having an upper limit of 2 jam. The difference between the two curves is not substantial, with a difference in the phase angles of about 3 ° at the higher frequen- cies. Some error in the ESA measurement of the fat globules is shifting the phase angles towards more negative values. There are several reasons

84 T. Wade, J.K. Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73-85

,-v b

-10

-20

0

0 •

0 0

0

I I I i I

0 2 4 6 8

Frequency OVII-Iz)

0 0

0

I I

10 12

Fig. 5. Theoretical argument of the dynamic mobility as a func- tion of frequency for particles with the same density as fat globules with a median diameter of 1 ~tm. Upper limit of the spread (d85) of 1.1 ~tm (©); and 2 ~tm ( e ) .

why the phase angles of the emulsions may be more negative than expected, resulting in a nar- rower spread than that observed optically. To obtain the dynamic mobilities for the fat globules, an ESA signal of the plasma phase of the emulsion was measured and subtracted from the ESA signal of the whole emulsion, as explained in Section 2. If the subtracted plasma phase does not represent the true background this may cause errors in the dynamic mobility spectra of the fat globules. The fat globules from the recombined and commercial emulsions have different surfaces, which is reflected in the different ESA signals obtained for the recom- bined and commercial suspensions and their respective plasma phases. Yet the phase angles of the dynamic mobility of the fat globules from the commercial and recombined suspensions are almost identical (Figs. 1 (b) and 3(b) respectively). It seems unlikely that two incorrect plasma phases from two different emulsions could, when subtracted, result in two phase angle spectra that are identical.

Another possibility may lie in the surfaces of the membranes of the fat globules. There is evi- dence that the milk fat globule membrane contains several glycoproteins that are amphipathic in nature [31 ]. The hydrophilic part of the glycopro- tein can protrude from the surface of the mem- brane over a considerable distance, perhaps causing steric repulsion between the fat globules

[32]. Shimizu et al. [33] found that treatment of raw cream with papain, which hydrolyses glyco- proteins, resulted in flocculation of the fat globules. The fat globules from the recombined milk and homogenized milk have membranes that consists of a mixture of adsorbed casein micelles, disrupted casein micelles, serum proteins and, in the case of homogenized fat globules, parts of the natural milk fat globule membrane. There is also evidence that casein micelles have a hydrophilic "hairy" layer which provides steric stabilization [34-37]. It is not known how such an adsorbed layer or how a surface of protruding "hairs" would behave in an alternating electric field.

The other source of error may be in the fact that the milk and cream emulsions are oil-in-water emulsions. The motion of the fluid in the electrical double layer upon application of an alternating electric field may induce movement within a liquid droplet. The extremely small amplitude of the droplets (of the order of 10-12m [16]) upon the application of the alternating applied field may mean that any movement within the liquid droplet or movement of an adsorbed layer or membrane would be significant and could affect the dynamic mobility of the droplets. However, the size and zeta potential of oil droplets in an undiluted intra- venous emulsion have been measured successfully with electroacoustics [23].

The fat globule systems in milk and cream emulsions are extremely complex, and much is still unknown as to the structure of the surfaces of the fat globules. From the above discussion there is also uncertainty as to how these systems behave in an alternating field. Reasonable estimates of the median size and zeta potential of fat globules in various emulsions with no dilution has been achieved with electroacoustics. However, at pre- sent, electroacoustics cannot give a true indication of the spread of the size distribution for these emulsions.

Acknowledgment

This work has been supported by the Australian Dairy Research and Development Corporation through a scholarship to TW. The authors would

T. Wade, J.K. Beattie / Colloids Surfaces B: Biointerfaces 10 (1997) 73-85 85

like to thank Dr. R.W. O'Brien, Dr. R.J. Hunter, Dr. W.N. Rowlands and Dr. M.A. Augustin for helpful advice and discussions.

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