aggregation, viscosity measurements and direct observation of protein coated latex particles under...

13
Aggregation, viscosity measurements and direct observation of protein coated latex particles under shear Lars Hamberg, Pernilla Walkenstro ¨m, Mats Stading, Anne-Marie Hermansson * SIK, The Swedish Institute for Food and Biotechnology, PO Box 5401, SE-402 29 Go ¨teborg, Sweden Received 28 July 2000; revised 8 September 2000; accepted 9 November 2000 Abstract The aggregation under shear, of latex particles coated with whey protein isolate was monitored, in a continuous phase with a complex behaviour in relation to temperature dependence and shear thinning. The monitoring was done with viscosity measurements and microscopy. An aggregating dispersion of whey coated polystyrene latex particles, salt, sucrose and gelatine was sheared in a rheometer at shear rates between 0.05 and 5 s 21 . The viscosity was monitored as a function of time during a temperature increase from 30 to 608C. The viscosity curves were interpreted with the aid of additional information from light microscopy micrographs. The aggregation was clearly visible as an increase in viscosity. Aggregation was observed to initiate at a temperature between 40 and 508C. Unbound protein, i.e. protein not a part of particle coating, was found to be essential for the aggregation of latex particles. After aggregation, a shear thinning behaviour was detected. This was due to two phenomena: structural changes of the aggregates and shear thinning behaviour of the dispersion. The build-up of the aggregates was followed by direct observation in a confocal laser scanning microscope. A sequence of micrographs was taken, in an unstopped 3-D flow field generated in a four-roll mill, which showed the evolution of the size of the aggregates. The micrographs were in good agreement with the viscosity measurements. This showed that the four-roll mill and a confocal laser scanning microscope is a useful tool for studying aggregation in an undisturbed 3-D flow. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Whey protein; Aggregation; Flocculation; Coated particles; Shear; Confocal laser scanning microscope 1. Introduction Aggregating particle systems in general have been of interest for a long time in a wide area of fields, such as waste-water treatment, optical properties of ink, pulp pump- ing, mineral recovery, nuclear material in power plants, blood coagulation and food production (Amirtharajah & O’Melia, 1990; Al-Jabari, Weber, & van de Ven, 1996; Isaksson, Rigdahl, Flink, & Forsberg, 1998; Mathur, Singh, & Moudgil, 2000). Particles coated with protein can aggregate under certain conditions, which makes them of special interest for several technological, immunological and clinical applications (Horbett, 1982; Colowick & Kaplan, 1987; Ortega-Vinuesa, Molina-Bolı ´var, & Hidalgo-A ´ lvares, 1996). In this investigation of the aggre- gation of protein coated latex particles, whey protein isolate was chosen for the coating. Common to nearly all of the fields mentioned above is that the aggregation usually occurs in a flow field. The flow promotes the aggregation constantly by introducing dynamic forces that act on the particles, e.g. rotation, break up and reconstruction (Russel, 1980; van de Ven, 1982; West, Melrose, & Ball, 1994). The flow, more speci- fically the shear flow, also facilitates the aggregation by increasing the rate of encounters between particles, which increases the rate of aggregation (Walstra & Jenness, 1984). It is not only the rate of flow that is important: different types of flow, such as elongation, hyperbolic and shear flow, also introduce various effects on the aggregation and break up (Higashitani & Iimura, 1998). One way to generate various flow types is to use a four-roll mill, 4-RM, (Taylor, 1934). The 4-RM can produce mixed flow fields varying from shear flow to hyperbolic flow. There is also a stagnation point where the flow is slow, but where elongation and shear forces act on the aggregates. Flow is known to affect the aggregation of whey protein by introducing coarser aggre- gates, when the suspension is sheared than there are in unsheared suspension. Also the size of whey protein aggre- gates is affected by shear and decreases with rising shear rate (Taylor & Fryer, 1994; Walkenstro ¨m, Panighetti, Wind- hab, & Hermansson, 1998a; Walkenstro ¨m, Windhab, & Hermansson, 1998b; Walkenstro ¨m, Nilsen, Windhab, & Hermansson, 1999). Food Hydrocolloids 15 (2001) 139–151 0268-005X/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0268-005X(00)00060-6 www.elsevier.com/locate/foodhyd * Corresponding author. Tel.: 146-31-335-5600; fax: 146-31-83-3782. E-mail address: [email protected] (A.-M. Hermansson).

Upload: lars-hamberg

Post on 16-Sep-2016

213 views

Category:

Documents


0 download

TRANSCRIPT

Aggregation, viscosity measurements and direct observation of proteincoated latex particles under shear

Lars Hamberg, Pernilla WalkenstroÈm, Mats Stading, Anne-Marie Hermansson*

SIK, The Swedish Institute for Food and Biotechnology, PO Box 5401, SE-402 29 GoÈteborg, Sweden

Received 28 July 2000; revised 8 September 2000; accepted 9 November 2000

Abstract

The aggregation under shear, of latex particles coated with whey protein isolate was monitored, in a continuous phase with a complex

behaviour in relation to temperature dependence and shear thinning. The monitoring was done with viscosity measurements and microscopy.

An aggregating dispersion of whey coated polystyrene latex particles, salt, sucrose and gelatine was sheared in a rheometer at shear rates

between 0.05 and 5 s21. The viscosity was monitored as a function of time during a temperature increase from 30 to 608C. The viscosity

curves were interpreted with the aid of additional information from light microscopy micrographs. The aggregation was clearly visible as an

increase in viscosity. Aggregation was observed to initiate at a temperature between 40 and 508C. Unbound protein, i.e. protein not a part of

particle coating, was found to be essential for the aggregation of latex particles. After aggregation, a shear thinning behaviour was detected.

This was due to two phenomena: structural changes of the aggregates and shear thinning behaviour of the dispersion.

The build-up of the aggregates was followed by direct observation in a confocal laser scanning microscope. A sequence of micrographs

was taken, in an unstopped 3-D ¯ow ®eld generated in a four-roll mill, which showed the evolution of the size of the aggregates. The

micrographs were in good agreement with the viscosity measurements. This showed that the four-roll mill and a confocal laser scanning

microscope is a useful tool for studying aggregation in an undisturbed 3-D ¯ow. q 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Whey protein; Aggregation; Flocculation; Coated particles; Shear; Confocal laser scanning microscope

1. Introduction

Aggregating particle systems in general have been of

interest for a long time in a wide area of ®elds, such as

waste-water treatment, optical properties of ink, pulp pump-

ing, mineral recovery, nuclear material in power plants,

blood coagulation and food production (Amirtharajah &

O'Melia, 1990; Al-Jabari, Weber, & van de Ven, 1996;

Isaksson, Rigdahl, Flink, & Forsberg, 1998; Mathur,

Singh, & Moudgil, 2000). Particles coated with protein

can aggregate under certain conditions, which makes them

of special interest for several technological, immunological

and clinical applications (Horbett, 1982; Colowick &

Kaplan, 1987; Ortega-Vinuesa, Molina-BolõÂvar, &

Hidalgo-AÂ lvares, 1996). In this investigation of the aggre-

gation of protein coated latex particles, whey protein isolate

was chosen for the coating.

Common to nearly all of the ®elds mentioned above is

that the aggregation usually occurs in a ¯ow ®eld. The ¯ow

promotes the aggregation constantly by introducing

dynamic forces that act on the particles, e.g. rotation,

break up and reconstruction (Russel, 1980; van de Ven,

1982; West, Melrose, & Ball, 1994). The ¯ow, more speci-

®cally the shear ¯ow, also facilitates the aggregation by

increasing the rate of encounters between particles, which

increases the rate of aggregation (Walstra & Jenness, 1984).

It is not only the rate of ¯ow that is important: different types

of ¯ow, such as elongation, hyperbolic and shear ¯ow, also

introduce various effects on the aggregation and break up

(Higashitani & Iimura, 1998). One way to generate various

¯ow types is to use a four-roll mill, 4-RM, (Taylor, 1934).

The 4-RM can produce mixed ¯ow ®elds varying from shear

¯ow to hyperbolic ¯ow. There is also a stagnation point

where the ¯ow is slow, but where elongation and shear

forces act on the aggregates. Flow is known to affect the

aggregation of whey protein by introducing coarser aggre-

gates, when the suspension is sheared than there are in

unsheared suspension. Also the size of whey protein aggre-

gates is affected by shear and decreases with rising shear

rate (Taylor & Fryer, 1994; WalkenstroÈm, Panighetti, Wind-

hab, & Hermansson, 1998a; WalkenstroÈm, Windhab, &

Hermansson, 1998b; WalkenstroÈm, Nilsen, Windhab, &

Hermansson, 1999).

Food Hydrocolloids 15 (2001) 139±151

0268-005X/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.

PII: S0268-005X(00)00060-6

www.elsevier.com/locate/foodhyd

* Corresponding author. Tel.: 146-31-335-5600; fax: 146-31-83-3782.

E-mail address: [email protected] (A.-M. Hermansson).

The chemical properties of the continuous phase

surrounding the particle are critical for the mechanism and

the outcome of the aggregation. Most of the work to inves-

tigate the mechanism of aggregation, has monitored it by

light scattering under static conditions for differing ion

concentrations, pH and temperatures (Quesada, Puig,

Delgado, Peula, Molina, & Hidalgo-AÂ lvarez et al., 1997;

Molina-BolõÂvar, Galisteo-GonzaÂlez, & Hidalgo-AÂ lvarez,

1998; Molina-BolõÂvar, Galisteo-GonzaÂlez, & Hidalgo-

AÂ lvarez, 1999a). In general the aggregates formed during

the aggregation of protein coated particles were found to be

fractal and the stability of the aggregates to be in agreement

with the DLVO (Derjaguin-Landau-Verwey-Overbeek)

theory. At low ion concentration, below 0.1 M, aggregation

increases with increasing concentration. Between concen-

trations of 0.1 M and around 0.5 M, the ion concentration

does not alter the aggregation; however at high concentra-

tion, above 0.5 M, the aggregation slows with rising ion

concentration (Molina-BolõÂvar, Galisteo-GonzaÂlez, &

Hidalgo-AÂ lvarez, 1999b).

The conditions that affect the protein are also important

for the aggregation. Effects of pH on the aggregation of the

protein b-lactoglobulin, the main protein in whey, have

been investigated after gelation. The gels have been found

to be strongest, and are de®ned as aggregate or particle gels,

at a pH interval between 4 and 6 (Stading & Hermansson,

1990, 1991). The rate of heat increase in thermally induced

aggregation of whey protein has also been reported to play a

role in aggregation and structuring of gels by altering the

size of aggregated particulate protein (Stading, Langton, &

Hermansson, 1993; Langton & Hermansson, 1996). Shear

in¯uenced thermal aggregation of whey protein and its

effects have been found for gel structures and viscoelastic

properties (WalkenstroÈn & Hermansson, 1996; Walken-

stroÈm et al., 1998a,b, 1999).

The surface properties, in particular the protein behaviour

at the surface, are another key factor for the aggregation.

Work has been done on the system of oil-in-water dispersion

with protein present at the surface and recently reviewed

speci®cally for milk proteins (Dickinson, 1999). With

milk protoins, the aggregation of oil drops stabilised by

caseinate under shear in an oil-in-water emulsion has been

investigated both by monitoring the viscosity with time and

by measuring the size of the aggregates (Dickinson & Gold-

ing, 1997a,b; Schokker & Dagleish, 1998). The investiga-

tions have shown that the amount of unadsorbed protein in

the continuous phase determines the aggregation and the

result of viscosity measurements. With protein present in

the continuous phase, they found that depletion ¯occulation

was the mechanism underlying the aggregation.

The traditional way to trace aggregation under shear is to

measure the viscosity. Doing this can sometimes lead to

dif®cult interpretations and often complimentary type of

measurement of aggregate size is required. To do this the

aggregates in the dispersions have to be withdrawn from the

¯ow, hence the dynamic of the forces and the balance from

forces acting on the aggregate in the ¯ow are disrupted. If

the aggregates are fragile, this treatment is critical and the

size measurements uncertain (Schokker & Dagleish, 1998).

However, simply placing a part of a process or a selected

dynamic condition under the microscope removes the

necessity to disrupt the dynamics. To our knowledge, moni-

toring the aggregation of particles under shear in micro-

scopes has been performed only for two-dimensional

surface ¯ow or at interfaces (Roussel, Camoin, & Blanc,

1989; Liu & Glasgow, 1997; Velikov, Durst, & Velev,

1998; Hansen & BergstroÈm, 1999a; Hansen, Malmsten,

BergenstaÊhl, & BergstroÈm, 1999b).

The aim of this work is to monitor the aggregation under

shear of latex particles coated with whey protein isolate in a

continuous phase with a complex behaviour in relation to

temperature dependence and shear thinning for example.

The continuous phase included gelatine, sucrose, salt and

water. The strategy was to monitor the aggregation not only

by viscosity measurements, but also to by direct observa-

tions with microscopy. The bene®t of this dual approach

was to enable a more accurate microstructural interpretation

of the viscosity measurements. The system was designed to

be similar to real systems, but also to have the advantages of

a model system. The particles were introduced to make it

possible to monitor the ¯ow in the microscope, as well as to

enlarge the result of the forces introduced by the ¯ow, on the

aggregates. The particles also needed to have a size of

.5 mm to allow the early stages in the aggregation to be

monitored at a low magni®cation. To look at the aggrega-

tion in a three-dimensional ¯ow ®eld, a Confocal Laser

Scanning Microscope, CLSM, was used, which made it

possible to depict a focal plane from inside the ¯owing

suspension.

2. Materials and methods

2.1. Materials

Whey protein isolate, (WPI) speci®cally LACPRODANw

DI-9224: .88% protein, ,6.0% moisture, ,4.5 ash,

,0.2% fat, ,0.2% lactose and ,2.1% minerals), was

obtained from MD Foods Ingredients Amba, Viby,

Denmark. Monodisperse polystyrene latex particles, of

5 mm, known as SOURCE 5RPC, were acquired from

Amersham Pharmacia Biotech, Uppsala, Sweden. The gela-

tine, (bloom strength 250, MW 118 kD) was obtained from

Extraco, Klippan, Sweden. The contrast agent Rhodamin 6

G was obtained from E. Merck AG Darmstadt, Germany.

2.2. Dispersion preparation

The dispersion of whey coated polystyrene latex particles

was prepared by dissolving these particles in a small volume

of a water/ethanol mixture, that contained 25% ethanol. To

reduce the amount of ethanol, the dispersion was stirred and

heated to 908C; this temperature was maintained for 15 min.

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151140

After cooling, sodium chloride, rhodamin dissolved in

water, and whey protein concentrate were dissolved

cautiously in the dispersion. Rhodamin was used to form a

stronger contrast in the microscope micrographs by colour-

ing the protein. The pH was then adjusted to 5.4 with small

amounts of HCl.

A solution containing 10% gelatine, 10% sucrose,

sodium chloride at three concentrations (0.05, 0.125

and 0.2 M), and water was prepared in a small beaker.

The solution was heated to 608C to dissolve the gelatine

and reduce the amount of dissolved gases in the solu-

tion. The gases were reduced to minimise bubbles later

on in the measurements. After 15 min, the temperature

was reduced to around 308C by placing the beaker with

the solution in a temperate water bath. As a ®nal step,

the particle dispersion was mixed with the same amount

of gelatine solution. The dispersion was regulated and

kept at 308C for 10 min during stirring to avoid gelation

of the gelatine. The concentration in the ®nal dispersion

is presented in Table 1.

The reason for adding sucrose to the dispersion was to

increase the density of the continuous phase, thereby to

reduce sedimentation of the particles during the measure-

ments. To raise the viscosity and thereby have more stable

¯ow conditions, gelatine was added to the particle system.

Another bene®t of introducing gelatine was that this would

automatically ®x the aggregates for the microscopy. The

®xation was brought about by gelation of the gelatine

when the temperature was lowered below 308C.

2.3. Experimental design

The experimental design was constructed with two design

variables: salt and shear rate. For the salt, two levels, 0.05

and 0.2 M, and a centre-point were chosen. The salt concen-

tration for the centre-point was 0.125 M. The different

concentrations of salt were used to investigate whether the

aggregation time could be in¯urnced this way (Molina-BolõÂ-

var et al., 1998). For the shear rate, three levels were chosen:

0.05, 0.5 and 5 s21. The middle level was also used to the

centre-point. The levels were chosen with respect to an

assumed logarithmic dependence between viscosity and

shear rate.

2.4. Viscosity measurements

The viscosity measurements were made in a Bohlin VOR

Rheometer (Bolin Rehology, Chichester, UK) equiped with

the Millennium software (Bholin Reologi, OÈ ved, Sweden).

The measuring system consisted of a cup and bob with

double-gap geometry. The inner and outer walls (diameter

21.7 and 27.4 mm) rotated during measuring while the bob

(diameter 23.9±24.9 mm) was ®xed. During the measure-

ments, the Taylor numbers were always in the region of

0.3±6 £ 1027; thus they were far below the critical number

of 3400 for Taylor vortices (Chandrasekher, 1961).

Next, 10 ml of the ®nal dispersion was poured into the

measuring system and covered with a thin layer of low

viscosity silicone oil, 10 mPas, to prevent evaporation

during the measuring. Steady continuous-shear viscosity

measurements were made during a temperature pro®le.

The pro®le started at 308C and rose to 608C with a slope

of 28C min21. The temperature was then held at 608C for

75 min. The three different shear rates were used for the

continuous-shear were 0.05, 0.5 and 5 s21.

2.5. Light microscopy and preparation

Directly after the viscosity measurements, the cup was

taken from the rheometer and gently tilted to the side so

that the silicone oil could be removed and the warm disper-

sion dripped onto four pre-heated slides and immediately

covered with a cover slip. For the ®rst slide a drop was taken

from the top of the cup. For the second, the drop was taken

from the middle, and for the two remaining, the drops were

taken from the very bottom of the cup. When the tempera-

ture fell below 308C, the gelatine in the drops gelled and

®xed the aggregates formed by particles. Three micrographs

from each drop were then taken at a 4 £ magni®cation with

a digitalised light microscope, Microphoto-FXA (Nikon

Corp., Tokyo, Japan); these images were stored digitally.

2.6. Direct microscopy under dynamic conditions

The direct microscopy studies under dynamic conditions

were performed in situ, directly in an unstopped ¯ow-®eld,

without any sample preparation for the microscope. The

microscopy was conducted on a Leica TCS 4D Confocal

Laser Scanning Microscope, CLSM, (Leica, Heidelberg,

Germany) in combination with a four-roll mill 4RM,

made by the workshop at ETH, EidgenoÈssische Technische

Hochschule, Zurich, Switzerland. The light source for the

CLSM was an argon-krypton laser with emissions maxima

at 488, 467 and 647 nm; the signal from the sample was

collected with a Nile red ®lter. The 4-RM, Fig. 1, has a

sample chamber consisting of a double-bottomed, ¯at,

open, dish with a diameter of 200 mm and a height of

20 mm. Four rolls, with a diameter of 45 mm and a height

of 18 mm, are symmetrically attached in a square, with a

centre distance of 55 mm. Two motors control the speeds of

the rolls; each motor controls a diagonally opposite pair of

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151 141

Table 1

Final contents of the prepared dispersion of whey coated polystyrene latex

particles

Material Amount

Particles 0.5 w/w%

WPI 1.0 w/w%

Gelatine 5.0 w/w%

Sucrose 5.0 w/w%

NaCl 0.05, 0.125, 0.2 M

Ethanol , 1 w/w%

rolls. In the middle, there is an optic window at the bottom

of the dish. Warm and cold water are circulated in the

hollow bottom of the dish to control the temperature.

By letting one pair of rolls rotate in one direction and the

other pair in the other direction, a hyperbolic ¯ow-®eld can

be generated in the centre of the chamber. This induces a

stagnation-point at the centre. By placing the scanning laser

at the stagnation-point where the ¯ow is slow, the dispersion

in motion can be observed. If the ¯ow is laminar, the ¯ow

speed is always low near the stagnation-point, irrespective

of the speed of the rolls. A typical ¯ow-®eld at the centre is

shown in Fig. 2. The investigations were made at a roll

speed of at 4 rpm. The dispersion was prepared in exactly

the same way as for the viscosity measurements, it

contained only half of concentration of particles and whey

protein. The sample chamber was ®lled with 300 g of the

dispersion and the surface was covered with a thin layer of

silicone oil. The total depth of the dispersion in the chamber

was 12 mm. The 4RM was placed under the CLSM so that

the laser, could scan near the stagnation point which had a

focal depth of 1.250 mm measured from the oil/dispersion

interface.

3. Results and discussion

3.1. Viscosity as a function of time and temperature

Measurements of viscosity of the dispersion were

continued for a period of 5000 s. During the ®rst

900 s, the temperature was ramped from 30 to 608C.

The temperature was then held at 608C until the end

of the measurements. The viscosity and temperature

were sampled for 10 s every 30 s to facilitate monitor-

ing their evolution. By following viscosity with time, it

is possible to gain understanding of aggregates and their

evolution. The aggregates are built up of the WPI

coated polystyrene particles; typical aggregates are

presented in Fig. 3.

The average curves for the time dependence of the visc-

osity for the three different shear rates 0.05, 0.5 and 5 s21 are

shown in Fig. 4. The curves are point to point averages for a

set of independent series. Salt concentration was varied

within the experimental set, but since no clear difference

was found for the three levels of salt contents, the averaged

series are averaged for these as well. The relatively large

error-bars at the start of the 0.05 s21 curve were due to

oscillation in the measurements at the beginning of the

time series at low shear rates.

All three curves start at different values but show the

same behaviour. Shortly after the start, the viscosities for

all of the measurements decrease to reach a local minimum.

An increase of the viscosity then starts and after a while the

increase levels out, and the viscosity reaches a stable value

towards the end.

The mutual relationship for viscosity, h, between the

curves with different shear rates, _g is well described by a

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151142

Fig. 2. A typical laminar ¯ow-®eld in the centre of the 4-roll mill.

Fig. 3. LM-micrograph of solution taken after the viscosity measurements.

(1% WPI, 0.125 M NaCl, shear rate 0.5 s21).

Fig. 1. A schematic picture over the 4-Roll Mill. (a)±(d) rolls, (e) sample

chamber, (f) optic window.

power law relationship such as:

h � m _g �n21�: �1�

The value of n is the shear thinning index n� 1 for a

Newtonian ¯uid with no shear thinning. The coef®cient m,

normally depends on temperature and concentration

(Macosko, 1994). Extracting the index n as a function of

time from the curves in Fig. 4 gives an initial value of n at

around 0.6. The shear thinning index, n, develops further

with time at around 500 s it reaches its maximum of 0.7,

after which the value starts to decrease. After 1500 s, n is

stable at around 0.3 and the value remains the same until the

end of the measurements. This means that the increase in

shear thinning behaviour of the dispersion does not start

until the temperature has reached approximately 608C.

For the curves in Fig. 4, it is possible to detect three

regions for which all three curves have the same behaviour.

The ®rst is the signi®cant decrease just after the start. The

second region is the rising one until the third region takes

over at the end, when the viscosity levels out and reaches a

stable value. The time intervals of the regions differ for the

three curves. Each of them is discussed in more detail

below.

To study the interior of the aggregates, LM-micrographs

at a high resolution were recorded by ¯uorescent techni-

ques: the protein was stained with rhodamin. In Fig. 5, the

whey protein stained with rodamin is bright, the darker areas

are the continuous phase, and the circular objects are latex

particles. The bright parts of in the micrograph can be seen

both between and edging the circular forms, hence whey

protein is present both as coated protein on the surface of

the latex particles and as free protein aggregates in the

dispersion.

While some of the latex particles clearly touch each other,

with only the coating protein between them, other particles

are solitary within the aggregate with just free protein aggre-

gates connecting them to the rest of the aggregate. This

supports the conclusion that both the coating protein and

the free protein aggregates contribute to the binding

between the particles. From the picture it seems reasonable

to believe that the free protein also forms small aggregates

with a spherical shape in the size order a tenth of the latex

particles. Both the coating protein and the free protein

aggregates should be rather similar and have the same prop-

erties, since hard proteins, such as b-laktoglobulin (the main

protein in whey) have a tendency to adsorb at interfaces

with few or no structural changes (Malmsten, 1998). That

free protein aggregates are essential for the aggregation has

also been found for the rather similar system consisting of

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151 143

Fig. 4. Viscosity shown as a function of time. The viscosity describes the aggregation of polystyrene spheres covered with WPI under shear for three shear

rates, £ 0.05, A 0.5, X 5.0 s21. The temperature increases from 30 to 608C during the ®rst 900 s and is then held at 608C to the end of the measurements.

Fig. 5. LM-micrograph of a small part of an aggregate taken with ¯ores-

cence technique. The light areas between the particles are WPI coloured

with Rhodamin.

drops of oil covered with sodium caseinate (Dickinson &

Golding, 1997a). In dispersions with free protein aggregates

present, the mechanism at early stages of the aggregation,

when two particles form a doublet, is expected to be deple-

tion ¯occulation (Dickinson & Golding, 1997a).

3.1.1. Onset of aggregation

The early decrease in viscosity exhibited by all three

curves characterises the initial region. Most of the decrease

takes place during the temperature ramp from 30 to 608Cduring the ®rst 900 s of the measurements. The ®rst region

of the three curves in Fig. 4 differs in time required for the

decrease. For the upper curve, with a shear rate of 0.05 s21,

the time from the start of measurements to the point of

minimum viscosity, is 490 s. For the middle curve, with

the shear rate 0.5 s21, the time is 720 s, and for the bottom

curve, at a shear rate of 5 s21, the time is 960 s. The devia-

tions of decrease also correspond to different temperatures.

For the top curve, the minimum viscosity is at 468C, for the

middle curve 548C and for the bottom curve 960 s which

means that the temperature has already reached the ®nal

608C.

LM-micrographs taken of a sample at the start tempera-

ture, 308C, Fig. 6(a), show that at the beginning the disper-

sion consists mostly of single latex particles, but that a small

number of doublets are also present. In LM-micrographs of

samples from the 4-RM, at 508C, after 600 s, Fig. 6(b), most

of the particles have formed aggregates consisting of two,

three or more particles. This indicates that the aggregation

starts between 30 and 508C, although the denaturation

temperature of sheared whey protein is around 708C (Ju,

Hettiarchchy, & Kilara, 1999; WalkenstroÈm et al., 1998a,b).

Many examples found in literature show that an aggrega-

tion or an increase in aggregate size increases the viscosity

(Dickinson & Golding, 1997b; Krieger, 1972; Campeanella,

Dorwood, & Singh, 1995; Barnes, 1994) and accurate physi-

cal models have been constructed (Sonntag & Russel, 1986,

1987; Potanin & Uriev, 1991; Potanin, 1991, 1992). This

phenomenon is due to entrapment of a certain volume of the

continuous phase within the aggregated structure; it

increases the effective hydrodynamic volume fraction of

aggregates, which in turn results in an increased apparent

viscosity. Nevertheless, at the same time as these small

aggregates are formed, the total viscosity is decreasing.

The fall in viscosity in the continuous phase is caused by

the well-known Arrenius type of decrease with temperature

and can be expressed by (Macosko, 1994):

h0 / eEh=RT �2�

Since time dependence is unlikely in the temperature

motivated Arrenius decrease in Eq. (2), and the aggregation

of whey protein is known to be triggered by temperature, the

differences in time to minimum viscosity have to be related

to the shear rate. Shear is known to affect aggregation by the

mechanism that greater shear increases the number of

encounters between particles (Walstra & Jenness, 1984)

which raises the rate of formation for new aggregates.

However, it also is known that shear decreases the overall

aggregate growth by introducing more and more break-up

and rearrangements (West et al., 1994). This combination is

the explanation to the shift in minimum viscosity time

towards later times for higher shear rates. Break-up and

rearrangements disrupt the early aggregation of particles

and the effect of this phenomenon increases with shear

rate. Hence, the rise in viscosity by the initial aggregate

build-up is weaker for large shear rates: for longer time

periods, the increase in viscosity at this early state is

outmatched by the decrease of the viscosity in the contin-

uous phase.

Consequently, the time to minimum viscosity would not

reveal the temperature when the aggregation starts, but it

does make it possible to determine the forces between the

particles. The longer time to minimum viscosity, together

with the LM-micrographs, point to the explanation that the

incipient aggregates are fragile in the beginning. The forces

between the particles, due to interactions between proteins

below 608C, have to be relatively weak or of the same order

as the forces acting on the particles due to shear. By measur-

ing and subtracting the viscosity decrease from the contin-

uous phase, it should be possible to obtain information on

when the aggregation starts. When this starting time is

known and, thus, the temperature, as well as the viscosity

for the system, it should be possible to calculate the forces

between the particles, by implementing the assumption that

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151144

Fig. 6. Two micrographs taken with the LM-microscope: (a) at start 308C,

and (b) after 600 s at 50C8. Samples are taken from the 4-RM.

when the aggregation starts, the forces between particles

should equal the drag forces from the surrounding shear.

To investigate and verify the effect of temperature,

experiments have been done with exactly the same

substances and procedure, but with a temperature ramp

between 30 and 408C (instead of the ramp between 30 and

608C). The result from such an experiment is shown in

Fig. 7. The inset curves show the time evolution of the

viscosity at a shear rate of 0.5 s21. In the inset ®gure, a

curve for a particle dispersion with whey protein, and a

temperature rise up to 608C, is compared with a similar

low-temperature dispersion with a temperature rise up to

just 408C. Both the 30±608C and the 30±408C curve start

with an initial decrease of the viscosity. For the 30±408Ccurve, the decrease is smaller than for the 30±608C curve.

After the initial fall, there is only a small rise with time

for the 30±408C curve in comparison with the large and

steep rise that occurs for the 30±608C curve. The differ-

ence in viscosity towards the end and the absence of the

steep rise lead to the conclusion that no aggregation of

particles occurs: hence, the onset of the protein aggrega-

tion is between 40 and 508C. In the literature it was found

that an interesting early aggregation of b-lactoglobulin,

the main protein in whey, occurred even in the tempera-

ture interval of around 408C (Stading & Hermansson,

1990).

3.1.2. Aggregation

The middle region of the viscosity curves in Fig. 4 is

distinguished by the large, steep increase in viscosity that

occurs after the minimum. In this region, the three curves

show more or less the same behaviour; the error-bars are

smaller than in the ®rst region. The viscosity rise for the

curves is relatively steep at ®rst, but levels out with time. At

around 2000 s, the increase has levelled off and viscosity

has reached a stable value. The steep rise of the viscosity

occurs mainly after the dispersion has reached 608C. The

increase in viscosity is probably due to an accelerated aggre-

gation of the whey coated particles. When the temperature

has reached 608C, the forces that hold the particles together

seem to be strong enough to let the aggregates overcome the

resistance even from high shear, and to grow.

To investigate whether or not the aggregation is due to the

presence of protein in the dispersion, experiments with and

without whey protein were made. The dispersion and set-up

were identical as to the centre-point condition, 0.125 M salt,

and the shear rate 0.5 s21, both with and without whey

protein, with the exception that additional salt was used to

compensate for the salt content of the WPI. Viscosity curves

for the measurements, both with and without whey are

shown in Fig. 7. The inset ®gure, displaying the time evolu-

tion of the viscosity at the shear-rate of 0.5 s21, shows that

the curves with and without whey differs even at the begin-

ning. The curve without WPI starts from a lower value and

has only a small decrease in the beginning. In contrast the

curve with whey has its steep increase around 1000 s, but

only a very small increase in viscosity is visible for the

curve without whey. This absence of the characteristic

steep rise in viscosity in the middle region leads to the

conclusion that no aggregation, or very limited aggregation,

occurs without protein. After rising both curves reach a

steady state. The three steady state viscosities are presented

as bars in Fig. 7. In this ®gure bars are given for all three

shear rates, and for the dispersion with whey as well as that

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151 145

Fig. 7. The bars shows steady-state viscosity as a function of shear-rate for three dispersions: S (black) whey 30±608C, B (dark grey) without whey 30±608C

and K (light grey) whey 30±408C. The inset ®gure shows the time evolution of the viscosity for the same dispersions at the shear-rate of 0.5 s21.

without whey protein. For all three shear rates, the bars for

the dispersions with and without whey are clearly different.

The same is true for a micrograph taken of a sample with-

drawn from the dispersion without whey at the end of the

measurements, Fig. 8, in compersion with another micro-

graph taken of a sample from the dispersion with whey, Fig.

3. In the micrograph from the dispersion with whey protein,

large aggregates consisting of 20±200 particles are found,

but in the micrograph without whey protein only singles,

doublets and some triplets are found.

Although the aggregation process could be driven and

controlled by temperature, shear could also have an in¯u-

ence. To investigate whether shear is critical to the process,

experiments were made with no shear present during the

®rst part. The temperature pro®le was the same, with the

increase from 30±608C during the ®rst 900 s. Three curves

with different times for the start of shear are compared in

Fig. 9. The shear, which starts at 0 s, at 900 s and at 1800 s,

has a rate of 0.5 s21. The two curves that start after 900 s and

1800 s correspond well to the curve for shear present from

the start. At the end, all three curves are more or less at the

same level, which indicates that shear and aggregation are

relatively independent. This supports the conclusion that the

in¯uence of increased encounters due to shear seems to be

minor and that the actual aggregation process seems to be

driven and controlled by the temperature. This is surprising,

since the in¯uence of shear on the aggregation is expected to

dominate over temperature by factors of 4, 40 and 400 for

the three shear rates 0.05, 0.5, and 5 s21 respectively

(Walstra & Jenness, 1984). The basis of this expectation

is a simple energy comparison between kinetic and thermal

energy, translated into encounters between the particles.

The theory uses quantities such as viscosity, h , temperature,

T, Boltzmann's constant, k, shear rate, _g , and primary parti-

cle size, d. The numbers above are calculated for latex

particle aggregation in a dispersion at 608C and with

numbers for the viscosity taken from the measurements

according to Eq. (3).

Shear

Temp/ d3h _g

2kT�3�

If the theory is tested with the assumption instead that

the free protein aggregates, i.e. the protein not coated on

the latex particle, control the aggregation, hence using the

protein as the primary particle, changes the result. For the

free protein aggregates, which is expected to be at least 10

times less than that of the latex particles (Langton &

Hermansson, 1996), the same energy comparison instead

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151146

Fig. 8. LM-micrograph of the dispersion taken after the viscosity measure-

ments: 0% WPI, 0.125 M NaCl, shear rate 0.5 s21.

Fig. 9. Viscosity as a function of time. The measurements were not started at the same time in the temperature pro®le: (A) At the beginning, 308C; ( £ ) after

900 s, 608C; and (X) at 1800 s from start, 608C. For all curves the shear rate is 0.5 s21.

demonstrates that the in¯uence of temperature is greater

by factors of 400, 40 and 4.

Both the aggregate build-up and the previously

mentioned shear thinning exponent, n, seem to occur during

the same time period and have an inverse appearance. When

the aggregates increases, the exponent decreases; it is there-

fore possible that the phenomena could be closely connected

in such a way that part of the shear thinning behaviour is due

to aggregate build-up.

3.1.3. Steady state viscosity

In the ®nal region of the viscosity pro®le, measurements

presented in Fig. 4, between 2000 and 5000 s, the viscosity

maintains steady state values. The steady state viscosity

values are 130 ^ 27 mPas for 0.05 s21, 23.1 ^ 2.7 mPas

for 0. 5 s21 and 5.16 ^ 0.40 mPas for 5 s21.

To analyse the phenomena underlying the differences in

viscosity, some dispersions were measured at other shear

rates after 5000 s. The additional viscosity measurements

were also performed at the shear rates of 0.05, 0.5 and

5 s21; the results are presented in Fig. 10. In the ®gure the

bars are shaded according to the shear rate their pro®les had

for the ®rst 5000 s. The shear rate before 5000 s is later in

the text is called pre-shear rate, the shear rates after 5000 s

are called measuring-shear rate. The grouping in the ®gure

are made according to the measuring-shear rates. For all

different pre-shear rates, the viscosity falls with increasing

shear rate. Also for all different measuring-shear rates, visc-

osity falls with increasing pre-shear rate. Comparing the

viscosities within the group with the same measuring

shear rate reveals a viscosity difference that is due to struc-

tural changes in the dispersion. If the viscosities with the

same pre-shear rates were compared instead, this would

reveal the differences due to shear thinning. Hence, the

conclusion is that the clear difference in viscosity value

for the differing shear rates is due to two separate effects:

the shear thinning in the continuous phase and the structural

changes of aggregates.

3.2. Viscosity measurements in a dispersion

When viscosity measurements are made on dispersions, it

is vital to check for any known problems that could affect

the measurements. For measurements as long as 5000 s,

settling could be a problem. Settling is usually omitted if

the time for an aggregate to migrate 10% of the height of a

concentric cylinder is more than the time needed for the

measurements (Macosko, 1994). The time can be obtained

by using of Stokes' law for an aggregate with a radius, a

(2.5 mm), in a concentric cylinder of the height h (55 mm),

under the in¯uence of gravity, g:

t10%h � 0:45hsh

rp 2 rs

��� ���a2g�4�

where r p (1050 kgm23) and r s (1080 kgm23) are the

density for the particles and the continuous phase disper-

sion, and h s is the viscosity of the dispersion. Taking

values for the properties according to the worst case

yields a maximum aggregate radius of 20 times the parti-

cle radius without a settling problem. In reality, more

realistic values allow the aggregate radii to be around

100 times larger than the particle radius. The conclusion

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151 147

Fig. 10. The bars show the steady state viscosity after different shear-histories. They are coloured according to the shear rate of their measurements for the ®rst

5000 s: ( £ ) (black) 0.05 s21, (A) (dark grey) 0.5 s21 and (X) (light grey) 5 s21. The grouping is according to the shear rate of the measurements after the time

5000 s. The inset ®gure shows the time evolution of the viscosity.

drawn from this is that only the very largest aggregate

will be affected. This does not seem to affect the viscos-

ity, since in Fig. 4 viscosity shows no tendency to

decrease with time in the steady state region.

A large aggregate can also alter the velocity ®eld

around itself, which can cause deviations in the viscosity

measurements. To omit this, a particle Reynolds number

should be smaller than 0.1 (Lin, Perry & Schowalter,

1970).

Rep � a2rs _g

hs

! 0:1 �5�

For the shear rates, _g , in the measurements, 0.05, 0.5 and

5 s21, and with the other values as above, Eq. (5) shows that

the ¯ow is not affected by small aggregates for any of the

shear rates. 0.05 s21 gives Re� 2 £ 1029, 0.5 s21 gives

Re� 1 £ 1027 and 5 s21 gives Re� 6 £ 106. However,

when the aggregates become large, e.g. 100 times a particle

radius, the Reynolds numbers become, for 0.05 s21

Re� 2 £ 1025, for 0.5 s21 Re� 1 £ 1023 and for 5 s21

Re� 6 £ 1022. This indicates that the ¯ow could deviate

for the highest shear rate. The result of a deviating ¯ow

®eld is only that the measured viscosity is shifted to a

slightly higher value than for undisturbed conditions. This

will have little effect on the conclusions: it is the evolution

of the viscosity that is of most interest here. Nevertheless

this phenomenon decreases the measured shear thinning

slightly for the highest shear rate, 5 s21.

Another known effect of particles during viscosity

measurements is the particle distribution in the gap (of the

cup and bob) due to wall effects. The particles have a

tendency to migrate towards the centre of the gap if the

Brownian motion cannot keep the particles uniformly

distributed. For equal distribution, K needs to be smaller

than 0.1 (Ho & Leal, 1974), in the following equation:

K � rp �y2a4

bkT, 0:1 �6�

where b is the gap (1.1 mm), T the temperature (608C), k

is Boltzmann's constant and �v is 12

vmax � R _g =2 where R

is the cup radius (12 mm). The value of K exceeds 0.1

for all shear rates when aggregates consist of ®ve parti-

cles or more. The varying distribution and the resulting

higher aggregate concentration in the middle of the gaps

can be expected, since the system was designed to

suppress the Brownian motion. The varying distribution

slightly alters the ¯ow pro®le by a small increase in

shear rate close to the walls and a small decrease in

shear rate at the middle of the gaps. Since this deviation

from ¯ow pro®le increases with shear rate, the actual

differences in shear rate are smaller than those between

the theoretical shear rates of 0.05, 0.5 and 5 s21. This

can have only a minor effect on the shear-thinning

index.

3.3. Direct observations under dynamic conditions

The actual build-up process has been followed in direct

observations under dynamic conditions and visual

evidence of the build-up is presented in Fig. 11, which

is a sequence of CLSM-micrographs taken in the 4-RM.

In these six views the particles are slightly oval because

of motion disturbance due to the continuous ¯ow and also

because the aggregates move during the scanning proce-

dure. The micrographs are from the centre of the 4-RM

where the ¯ow speed is automatically reduced; hence, at

the moment when the aggregates are exposed to lower

stresses than average. The aggregates have free mobility

in all directions and the surface effects are reduced, since

the micrographs are from a focal plane 1.25 mm below

the interface between the dispersion and the silicone oil.

In the ®rst image, a, only singles and occasional doublets

are present as large dots; this one was just after the start

when the temperature in the dispersion is 308C. There is

no notable change from micrograph a±b, where the

temperature is increased to 408C. There are still only

singles and some doublets which is expected since the

protein does not aggregate below 408C. This is also in

good agreement with the explanation that the viscosity

decreases in the beginning are due to viscosity changes

in the continuous phase; they are not due to any structural

changes among the particles. In micrograph c, at 508C, a

weak tendency towards aggregation is visible, since a

number of loose aggregates consisting of 5±10 particles

are visible. In micrograph d, taken after 900 s, when the

temperature had just reached 608C, the aggregates are

larger than in the previous micrograph and they consist

of 10±30 particles. Between the aggregates there are still

many singles. After 1200 s, the aggregates have become

even larger and consist of 50±200 particles. In the ®nal

micrograph, f, the aggregates are of the same order of

magnitude as in e. Although singles are still present,

most of the particles are incorporated in the large aggre-

gate. The evolution of the size of the large aggregates in

the micrograph sequence is in good agreement with the

viscosity pro®le in regions 2 and 3. The good agreement

between the micrographs and pro®le shows that the new

combination of the 4RM together with the CLSM is a

powerful tool for studying the aggregation mechanism

in a continuous undisturbed 3-D ¯ow. The new tool is

useful for situations where the changes in viscosity due

to the structural changes are smaller than the viscosity

changes in the continuous phase. It is also useful when

the aggregates are fragile and the preparation could affect

the aggregates or when the shapes of the aggregates are in

dynamic equilibrium with the undisturbed ¯ow. When

computers become even faster than now, the option to

perform faster scans will become available, so that a

higher resolution becomes possible in the time dimension.

Then, with this technique, it would be possible to follow

smaller and/or faster movements within the dispersion.

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151148

One can also combine the 4-RM and the CLSM with

image analysis and perform quantitative measurements

in the micrographs. This can shift the set-up further,

from a qualitative microscope towards a measurement

tool for dynamic in situ condition.

Acknowledgements

This work is a part of the LiFT program (Future Tech-

nologies for Food Production), ®nanced by the SSF (Swed-

ish Foundation for Strategic Research).

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151 149

Fig. 11. CLSM-micrographs of the dispersion taken during heating and ¯owing: 0.15% particles, 0.30% WPI, 0.125 M NaCl; (a) 308C, 0 s; (b) 408C, 300 s; (c)

508C, 600 s; (d) 608C, 900 s; (e) 608C, 1200 s; (f) 608C, 2000 s.

References

Al-Jabari, M., Weber, M. E., & van de Ven, T. G. M. (1996). Modeling

®nes elutriation from a sprouted bed of pulp ®bers. Chemical Engineer-

ing Communications, 148-150, 465±476.

Amirtharajah, A., & O'Melia, C. R. (1990). Coagulation processes: desta-

bilisation, mixing, and ¯occulation. In F. W. Pontius, Water quality and

treatment (pp. 268±365). New York: McGraw-Hill, Inc.

Barnes, H. A. (1994). Rheology of emulsionsÐa review. Colloids and

surfaces A: Physicochemical and Engineering Aspect, 91 (1), 89±95.

Campeanella, O. H., Dorwood, N. M., & Singh, H. (1995). A study of the

rheological properties of concentrated food emulsions. Journal of Food

Engineering, 25 (3), 427±440.

Chandraskhar, S. (1961). Hydrodynamic and hydromagnetic stability,

London: Oxford University Press.

Colowick, S. P., & Kaplan, N. O. (1987). Methods in enzymology, San

Diego: Academic Press Vol. 136.

Dickinson, E., & Golding, M. (1997a). Depletion ¯occulation of emulsions

containing unadsorbed sodium caseinate. Food Hydrocolloids, 11 (1),

13±18.

Dickinson, E., & Golding, M. (1997b). Rheology of sodium caseinate

stabilised oil-in-water emulsions. Journal of Colloid and Interface

Science, 191 (1), 166±176.

Dickinson, E. (1999). Adsorbed protein layers at ¯uid interfaces: Interac-

tions, structure and surface rheology. Colloids and Surfaces B: Bioin-

terfaces, 15 (2), 161±176.

Hansen, P. H. F., & BergstroÈm, L. (1999a). Perkinetic aggregation of

alkoxylated silica particles in two dimensions. Journal of Colloid and

Interface Science, 218 (1), 77±87.

Hansen, P. H. F., Malmsten, M., BergenstaÊhl, B., & BergstroÈm, L. (1999b).

Orthokinetic aggregation in two dimensions of monodisperse and bidis-

perse colloidal systems. Journal of Colloid and Interface Science, 220

(2), 269±280.

Higashitani, K., & Iimura, K. (1998). Two-dimensional simulation of the

break-up process of aggregates in shear and elongational ¯ows. Journal

of Colloid and Interface Science, 204 (2), 320±327.

Ho, B. P., & Leal, L. G. (1974). Inertial migration of rigid spheres in two

dimensional unidirectional ¯ows. Journal of Fluid Mechanics, 65, 365±

400.

Horbett, T. A. (1982). In S. L. Cooper & N. A. Peppas, Biomaterials:

interfacial phenomena and applications ACS. Advances in chemistry

series (p. 233), Vol. 199. Washington, DC: American Chemical

Society.

Isaksson, P., Rigdahl, M., Flink, P., & Forsberg, S. (1998). Aspects of the

elongational ¯ow behaviour of coating colours. Journal of Pulp and

Paper Science, 24 (7), 204±209.

Ju, Z. Y., Hettiarchchy, N., & Kilara, A. (1999). Thermal properties of

whey protein aggregates. Journal of Dairy Science, 82 (9), 1882±

1889.

Krieger, I. M. (1972). Rheology of monodisperse latices. Advances in

Colloid and Interface Science, 3, 111±136.

Langton, M., & Hermansson, A. -M. (1996). Image analysis of particulate

whey protein gels. Food Hydrocolloids, 10 (2), 179±191.

Lin, C. -J., Perry, J. H., & Schowalter, W. R. (1970). Simple shear ¯ow

round a rigid sphere: inertial effects and suspension rheology. Journal

of Fluid Mechanics, 44, 1±17.

Liu, S. X., & Glasgow, L. A. (1997). Aggregate disintergration in turbulent

jets. Water, Air and Soil Pollution, 95, 257±275.

Macosko, C. R. (1994). Rheology-principles, measurements and applica-

tions, New York: VCH Publishers Inc.

Malmsten, M. (1998). Formation of adsorbed protein layers. Journal of

Colloid and Interface Science, 207 (2), 186±199.

Mathur, S., Singh, P., & Moudgil, B. M. (2000). Advances in selective

¯occulation technology for solid-solid separations. International Jour-

nal of Mineral Processing, 58 (1-4), 201±222.

Molina-BolõÂvar, J. A., Galisteo-GonzaÂlez, F., & Hidalgo-AÂ lvarez, R.

(1998). Cluster morphology of protein-coated polymer colloids. Jour-

nal of Colloid and Interface Science, 208 (2), 445±454.

Molina-BolõÂvar, J. A., Galisteo-GonzaÂlez, F., & Hidalgo-AÂ lvarez, R.

(1999a). Colloidal aggregation in energy minima of restricted depth.

Journal of Chemical Physics, 110 (11), 5412±5420.

Molina-BolõÂvar, J. A., Galisteo-GonzaÂlez, F., & Hidalgo-AÂ lvarez, R.

(1999b). Development of a high sensitivity lgG-latex immunodetection

system stabilised by hydration forces. Polymer International, 48 (8),

685±690.

Ortega-Vinuesa, J. L., Molina-BolõÂvar, J. A., & Hidalgo-AÂ lvares, R. (1996).

Particle enhanced immunoaggregation of F(ab 0)2 molecules. Journal of

Immunological Methods, 190 (1), 29±38.

Potanin, A. A., & Uriev, N. B. (1991). Microrheological models of aggre-

gated suspensions in shear ¯ow. Journal of Colloid and Interface

Science, 142, 385±395.

Potanin, A. A. (1991). On the mechanism of aggregation in shear ¯ow

of suspensions. Journal of Colloid and Interface Science, 145,

140±157.

Potanin, A. A. (1992). On the microrheological modelling of aggregat-

ing colloids. Journal of Dispersion Science and Technology, 13,

527±548.

Quesada, M., Puig, J., Delgado, J. M., Peula, J. M., Molina, J. A., &

Hidalgo-AÂ lvarez, R. (1997). A simple kinetic model of antigen-anti-

body reactions in particle-enhanced light scattering immunoassays.

Colloids and Surfaces B: Biointerfaces, 8 (6), 303±309.

Roussel, J. -F., Camoin, C., & Blanc, R. (1989). Image analysis of kinetics

of aggregation. Journal de Physique France, 50, 3259±3267.

Russel, W. B. (1980). Review of the role of colloidal forces in the rheology

of suspensions. Journal of Rheology, 24 (3), 287±317.

Schokker, E. P., & Dagleish, D. G. (1998). The shear-induced destabilisa-

tion of oil-in-water emulsions using caseinate as emulsi®er. Colloids

and Surfaces A: Physicochemical and Engineering Aspects, 145 (1-3),

61±69.

Sonntag, R. C., & Russel, W. B. (1986). Structure and break-up of ¯ocs

subjected to ¯uid stresses I. Shear experiments. Journal of Colloid and

Interface Science, 113, 399±413.

Sonntag, R. C., & Russel, W. B. (1987). Structure and break-up of ¯ocs

subjected to ¯uid stresses II. Theory. Journal of Colloid and Interface

Science, 115, 378±389.

Stading, M., & Hermansson, A-M. (1991). Large deformation proper-

ties of (b-lactoglobulin gel structures. Food Hydrocolloids, 5 (4),

339±352.

Stading, M., & Hermansson, A-M. (1990). Viscoelastic behaviour of

(b-lactoglobulin gel structures. Food Hydrocolloids, 4 (2), 121±135.

Stading, M., Langton, M., & Hermansson, A. -M. (1993). Microstructure

and rheological behaviour of particulate b-lactoglobulin gels. Food

Hydrocolloids, 7 (3), 195±212.

Taylor, G. I. (1934). The formation of emulsions in de®nable ®elds of ¯ow.

Proceedings Royal Society, A146, 501±523.

Taylor, S. M., & Fryer, P. J. (1994). The effect of temperature/shear on the

history of thermal gelation of whey protein concentrates. Food Hydro-

colloids, 8 (1), 46±61.

WalkenstroÈm, P., & Hermansson, A. -M. (1996). Effects of shear on pure

and mixed gels of gelatine and particulate whey protein. Food Hydro-

colloids, 12 (1), 77±87.

WalkenstroÈm, P., Nilsen, M., Windhab, E., & Hermansson, A. -M. (1999).

Effects of ¯ow behaviour on the aggregation of whey protein suspen-

sions. Pure and mixed with xanthan. Journal of Food Engineering, 42

(1), 15±26.

WalkenstroÈm, P., Panighetti, N., Windhab, E., & Hermansson, A. -M.

(1998a). Effects of ¯uid shear and temperature on whey protein

gels, pure or mixed with xanthan. Food Hydrocolloids, 12 (4),

469±479.

WalkenstroÈm, P., Windhab, E., & Hermansson, A. -M. (1998b). Shear-

induced structuring of particlate whey protein gels. Food Hydrocol-

loids, 12 (4), 459±468.

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151150

Walstra, P., & Jenness, R. (1984). Diary chemistry and physics, New York:

John Wiley & Sons.

van de Ven, T. G. M. (1982). Interactions between colloidal particles in simple

shear ¯ow. Advances in Colloid and Interface Science, 17, 105±127.

Velikov, K. P., Durst, F., & Velev, O. D. (1998). Direct observation of

dynamics of latex particles con®ned inside thinning water-air ®lms.

Langmuir, 14 (5), 1148±1155.

West, A. H. L., Melrose, J. R., & Ball, R. C. (1994). Computer simulations

of the break-up of colloid aggregates. Physical Review E, 49 (5), 4237±

4249.

L. Hamberg et al. / Food Hydrocolloids 15 (2001) 139±151 151