Fundamental study of physical and biochemical alterations ...

Fundamental Study of Physical and Biochemical

Alterations to Casein Micelles during Milk

Evaporation and Ultrafiltration

Zhe Liu

Submitted in total fulfilment of the requirements for the degree of

Doctor of Philosophy

December 2013

Department of Chemical & Biomolecular Engineering

The University of Melbourne

Victoria, Australia

i

Abstract

Casein represents 80% of the protein in milk, which is predominantly present in the

form of hydrated colloidal assemblies known as casein micelles. It is one of the most

important components in milk system that has significant influence on the properties of

dairy products. It is also the main component of several highly value-added dairy

products (such as milk protein concentrate (MPC) and milk protein isolate (MPI)). The

objective of this work was to investigate the physico-chemical alterations of skim milk

(specifically casein and casein micelles) during processing (evaporation and

ultrafiltration) and to understand the processes at a fundamental level in order to

determine the effect of the processing parameters on the properties of casein micelles

and milk products to ultimately improve the efficiency of dairy processing.

Evaporation in the dairy industry is used for concentrating products such as milk and

whey while it is also used as a preliminary step to drying. Milk products intended for

milk powder are normally concentrated to a final concentration of 40 – 50 % total solids

before going to the spray dryer. To reduce heat impact on the heat sensitive components

in milk system, evaporation takes place under vacuum at relatively low temperature. In

this study, skim milk was concentrated by vacuum evaporation to concentration 12 – 45 %

total solids content. The hydration, composition and size of the casein micelle were

investigated. The results show that during evaporation, both inter and intra micelle

water were removed from the milk system and preferentially the serum water. The

alterations were rapidly reversible but not completely.

Ultrafiltration is a process in which semi-permeable membranes are used to separate

components in a fluid on the basis of size. It is widely used in the dairy industry for

recovering, fractionating and concentrating proteins in milk process streams. In this

study, skim milk was concentrated by ultrafiltration up to four folds at different

processing temperatures. The changes of milk and caseins were discussed and different

processing conditions were compared.

Casein micelles and colloidal calcium phosphate are affected contemporaneously by the

system temperature. A comprehensive physico-chemical investigation of the dynamic

responses of casein micelles to changes in temperature was performed in the range

ii

10 °C – 40 °C in real time. The temperature effects on casein micelles were consistent

with the results of the ultrafiltration processing at different temperature.

Lactose is an important energy source in milk system, and it also regulates the water

content of milk during secretion. In evaporation and UF concentration, micellar water

behaved differently: the micelle hydration decreased during evaporation, whilst

increased during UF concentration. Lactose is highly responsible for this difference, and

the lactose effects on casein micelles were studied in this work.

This study investigates the temperature dependent dynamics of casein micelles, and

physico-chemical behaviours under different lactose concentrations, evaporation or

ultrafiltration conditions. The results show that casein micelles and colloidal calcium

phosphate (CCP) are dynamic entities which are significantly affected by processing.

The results have important implications for improving milk processing in dairy industry.

iii

Declaration

This is to certify that

the thesis comprises only my original work towards the PhD,

due acknowledgement has been made in the text to all other material used,

the thesis is less than 100,000 words in length, exclusive of tables, maps,

bibliographies and appendices.

Dylan Zhe Liu

iv

Preface

Sections of this thesis have been published in the following journal papers:

1 Liu, D. Z., Dunstan, D. E., & Martin, G. J. O. (2012). Evaporative concentration of

skimmed milk: Effect on casein micelle hydration, composition, and size. Food

Chemistry, 134 (3), 1446-1452.

2 Liu, D. Z., Weeks, M. G., Dunstan, D. E., & Martin, G. J. O. (2013). Temperature-

dependent dynamics of bovine casein micelles in the range 10 – 40 °C. Food Chemistry,

141 (4), 4081-4086.

3 Liu, D. Z., Weeks, M. G., Dunstan, D. E., & Martin, G. J. O. (2014). Alterations to the

composition of casein micelles and retentate serum during ultrafiltration of skim milk at

10 °C and 40 °C. International Dairy Journal, 35 (1), 63-69.

Note: Further journal paper presenting the remainder of the work in this thesis is

currently in preparation as:

Liu, D. Z., Weeks, M. G., Dunstan, D. E., & Martin, G. J. O. (2014). Lactose effects on

casein micelles: The regulator of micelle hydration.

v

Acknowledgements

First of all, I want to express my heartfelt gratitude to my supervisors, Professor David

Dunstan and Dr. Greg Martin, for giving me direction, invaluable advice, support and

encouragement. The knowledge and experience I gained from this study was invaluable.

Also, I like to express my deepest gratitude to Dr. Ian Mckinnon for his help, advice and

providing access to the DWS and UF system.

I would like to thank Dr. Michael Weeks of Dairy Innovation Australia for advice and

feedback and the regular meetings we had for keep me on track. I am also thankful to Dr.

Lydia Ong for her advice and help. I would like to thank Dr. Jayani Chandrapala who

provided a lot of help. I would also like to thank Ms. Alita Anastasia Aguiar for her

assistance with the AAS. Also, I thank Annmaree Sharkey for sorting out my progress

forms.

Equally important are my fellow lab- and office-mates, who made the three years here

more interesting and less stressful. Thanks a lot for their help and support.

Thanks to DIAL, Australian Research Council (ARC), Department of Chemical and

Biomolecular Engineering and the University of Melbourne for financial assistance.

Finally, I would like to thank my family and friends who helped and encouraged me on

this journey.

vi

Table of Contents Abstract .............................................................................................................................. i

Declaration....................................................................................................................... iii

Preface ............................................................................................................................. iv

Acknowledgements .......................................................................................................... v

Index of Figures ................................................................................................................ x

List of Tables .................................................................................................................. xii

Nomenclature................................................................................................................. xiii

1 Introduction ................................................................................................................... 1

2 Literature Review .......................................................................................................... 4

2.1 Properties of Milk ................................................................................................... 4

2.1.1 Water ............................................................................................................... 4

2.1.2 Lactose ............................................................................................................. 5

2.1.3 Milk Fat ........................................................................................................... 6

2.1.4 Vitamins .......................................................................................................... 6

2.1.5 Minerals ........................................................................................................... 7

2.1.6 Proteins ............................................................................................................ 8

2.2 Casein and Casein micelles .................................................................................. 11

2.2.1 Caseins ........................................................................................................... 11

General Information ............................................................................................... 11

Casein Properties .................................................................................................... 14

2.2.2 Casein Micelles ............................................................................................. 16

Formation and digestion of casein micelles ........................................................... 17

Casein micelle structure.......................................................................................... 18

Casein micelle properties........................................................................................ 23

Alterations to casein micelle during processing ..................................................... 28

2.3 Summary ............................................................................................................... 34

vii

3 Experimental Techniques and Applications ................................................................ 35

3.1 Overview of experimental approach..................................................................... 35

3.2 Milk viscosity and the determination of casein micelle volume faction .............. 36

3.3 Turbidity ............................................................................................................... 38

3.4 Dynamic Light Scattering ..................................................................................... 40

3.5 Ultracentrifugation................................................................................................ 42

3.6 Calcium measurement .......................................................................................... 48

3.7 Lactose measurement ........................................................................................... 49

3.8 SDS-PAGE and Native-PAGE ............................................................................. 49

4 Evaporative concentration of skim milk: Effect on casein micelle hydration,

composition, and size ..................................................................................................... 50

4.1 Introduction .......................................................................................................... 50

4.2 Materials and methods .......................................................................................... 51

4.2.1 Skim milk and concentrated skim milk samples ........................................... 51

4.2.2 Casein micelle hydration rate and mass balance ........................................... 52

4.2.3 SDS-PAGE .................................................................................................... 52

4.2.4 Calcium measurement ................................................................................... 52

4.2.5 Turbidity and viscosity measurements .......................................................... 53

4.2.6 Particle size analysis ...................................................................................... 53

4.3 Results .................................................................................................................. 54

4.3.1 Direct measurements of concentrated milk ................................................... 54

4.3.2 Measurements of re-diluted concentrated milk ............................................. 57

4.4 Discussion ............................................................................................................. 62

4.4.1 Hydration of casein micelles ......................................................................... 62

4.4.2 Composition of casein micelles ..................................................................... 63

4.4.3 Size of casein micelles ................................................................................... 64

4.5 Conclusions .......................................................................................................... 66

viii

5 Temperature-dependent dynamics of bovine casein micelle in the range 10-40 °C ... 67

5.1 Introduction .......................................................................................................... 67


5.2.1 Skim milk samples ........................................................................................ 71

5.2.2 Fraction of casein by centrifugation .............................................................. 71

5.2.3 Casein micelle hydration and serum calcium measurement .......................... 71

5.2.4 Determination of casein micelle volume fraction.......................................... 72

5.2.5 Dynamic light scattering ................................................................................ 72

5.2.6 Turbidity and pH measurement ..................................................................... 72

5.2.7 Temperature kinetic studies ........................................................................... 73

5.3 Results and discussion .......................................................................................... 73

5.3.1 Micelle equilibrium as a function of temperature ......................................... 73

5.3.2 Micelle dynamics ........................................................................................... 78

5.4 Conclusions .......................................................................................................... 80

6 Alterations to the composition of casein micelles and retentate serum during

ultrafiltration of skim milk at 10 °C and 40 °C .............................................................. 81

6.1 Introduction .......................................................................................................... 81


6.2.1 Skim milk samples ........................................................................................ 83

6.2.2 Ultrafiltration and pH measurements ............................................................ 83

6.2.3 Casein micelle separation and hydration ....................................................... 84

6.2.4 Protein and calcium quantification ................................................................ 84


6.2.6 Determination of lactose content ................................................................... 85


6.4 Conclusions .......................................................................................................... 95

7 Lactose effects on casein micelles: The regulator of micelle hydration...................... 96

ix

7.1 Introduction .......................................................................................................... 96


7.2.1 Skim milk and MPC solutions ....................................................................... 97

7.2.2 Volume fraction and pH ................................................................................ 97

7.2.3 Casein micelle hydration ............................................................................... 97


7.2.5 Casein and Calcium quantification ................................................................ 98

7.2.6 Determination of lactose content ................................................................... 98


7.3.1 Lactose effects on casein micelles in MPC solutions .................................... 98

7.3.2 Lactose effects in skim milk ........................................................................ 101

7.3.3 Lactose effects during milk processing ....................................................... 104

7.4 Conclusions ........................................................................................................ 105

8 Conclusions and recommendations ........................................................................... 106

8.1 Conclusions ........................................................................................................ 106

8.2 Recommendations for Future Research .............................................................. 110

References .................................................................................................................... 112

Appendix: Physical properties of whole bovine milk (Sherbon, 1988; Singh et al., 1997;

Walstra et al., 1984) ...................................................................................................... 126

x

Index of Figures

Figure 1.1 Application of membrane separation processes in dairy industry .................. 2

Figure 2.1 Amino acid sequences of the bovine αs1-, αs2-, β- and κ-caseins. ................. 12

Figure 2.2 Protocols for casein whey co-precipitates ..................................................... 16

Figure 2.3 SEM image of a casein micelle ..................................................................... 17

Figure 2.4 Formation of casein micelles (CM) in Golgi vesicles (G). Top, a Golgi

vesicle discharged its contents into the alveolar lumen; Bottom, One casein micelle

presents in lumen. ........................................................................................................... 18

Figure 2.5 Representation of the submicelle model of casein micelles proposed by

Walstra ............................................................................................................................ 20

Figure 2.6 Schematic diagram of casein micelle assembly according to dual-binding

model reproduced from (Horne, 2003) ........................................................................... 21

Figure 2.7 Representation of the nanocluster model ...................................................... 22

Figure 2.8 Schematic picture of casein micelle sponge model....................................... 23

Figure 2.9 Outline of some processes used to produce milk protein products ............... 29

Figure 3.1 Composition of milk, evaporative concentrates and UF retentates separated

by ultracentrifugation ..................................................................................................... 36

Figure 3.2 Simplified schematic of dynamic light scattering set-up .............................. 41

Figure 3.3 Geometry of Ultracentrifuge rotor 90 Ti ....................................................... 43

Figure 3.4 The amount of soluble caseins in the supernatants of original skim milk after

different period of ultracentrifugation. ........................................................................... 45

Figure 3.5 The amount of soluble caseins in the supernatants of ultrafiltered CF4

retentates and evaporative concentrated skim milk (42% TS).. ..................................... 47

Figure 4.1 Analyses performed on evaporative concentrates ......................................... 51

Figure 4.2 Hydration and water content of casein micelles during evaporation. ........... 55

Figure 4.3 Total serum calcium and casein in the supernatant during evaporation. ...... 56

Figure 4.4 Turbidity and average micelle size of rediluted evaporative concentrated milk

........................................................................................................................................ 58

Figure 4.5 Total serum calcium and casein in the supernatants of rediluted evaporative

concentrates.. .................................................................................................................. 59

xi

Figure 4.6 Relative alterations of average micelle size, viscosity, pH, turbidity and

soluble caseins of rediluted evaporative concentrates. ................................................... 61

Figure 4.7 Casein micelle alterations during skim milk evaporative concentration and

re-dilution. ...................................................................................................................... 66

Figure 5.1 Analysis performed on fresh skim milk as a function of temperature .......... 70

Figure 5.2 Equilibrium state of skim milk as a function of temperature. ....................... 74

Figure 5.3 SDS-PAGE image of soluble milk proteins in ultracentrifugation (75940 g)

supernatants at different temperatures. ........................................................................... 75

Figure 5.4 Relative amount of casein present as soluble casein, ‘small’ casein micelles

(CM), ‘medium’ CM, or ‘large’ CM in skim milk as a function of temperature as

determined by different centrifugation. .......................................................................... 77

Figure 5.5 Dynamics of skim milk warmed and cooled between 10 and 20 °C (A), and

10 and 40 °C (B).. ........................................................................................................... 79

Figure 6.1 Analyses performed on UF retentates and permeates ................................... 83

Figure 6.2 Native-PAGE analysis of UF permeates and the supernatants of the

ultracentrifuged retentates (60 min ultracentrifugation). ................................................ 86

Figure 6.3 Native-PAGE analysis of whey protein content in the serum of retentates

from ultrafiltered skim milk.. ......................................................................................... 87

Figure 6.4 Alterations to the casein micelle and serum compositions of UF retentates as

a function of volumetric concentration factor (CF).. ...................................................... 89

Figure 6.5 Alterations to skim milk after UF concentration to CF2 at 10 °C or 40 °C

followed by concentration to CF3 at 40 °C or 10 °C.. ................................................... 93

Figure 6.6 Casein micelle alterations during UF ............................................................ 95

Figure 7.1 Pelletization of casein micelles and composition of the serum of MPC 85

disolved in water (A, B) and SMUF (C, D) as a function of lactose concentration. ...... 99

Figure 7.2 Relative turbidity of MPC 85 solutions as a function of lactose concentration.

...................................................................................................................................... 101

Figure 7.3 Alterations of casein micelles and serum of skim milk as a function of

lactose concentration. ................................................................................................... 103

Figure 7.4 Micelle hydration alterations in response to lactose concentration. ........... 105

Figure 8.1 A schematic overview of the effect of evaporative concentration and

ultrafiltration on casein micelles as revealed through this study .................................. 106

xii

List of Tables

Table 2.1 Composition of bovine milk (Walstra & Jenness, 1984) ................................. 4

Table 2.2 Distribution of salts between the soluble and colloidal phase of milk (20 °C,

pH 6.77) (Davies & White, 1960) .................................................................................... 7

Table 2.3 Major bovine milk proteins and some of their biological functions (adapted

from (Korhonen & Pihlanto, 2007)) ................................................................................. 8

Table 3.1 Pelleted micelle hydration of skim milk after different period of

ultracentrifugation .......................................................................................................... 46

Table 3.2 Pelleted micelle hydration of concentrated milk samples after different period

of ultracentrifugation ...................................................................................................... 48

Table 4.1 Rehydration of casein micelles upon redilution of concentrated skim milk.

Comparison of pelleted solids, pelleted water, and pellet hydration for ultracentrifuged

skim milk and a corresponding sample of concentrate (34% TS) rediluted to 8.7% TS.57

Table 5.1 Fractionation of definite sized micelles .......................................................... 71

Table 6.1 Micelle and mineral alterations during UF. .................................................... 91

Table 7.1 Micellar lactose and micelle hydration in MPC 85 ...................................... 100

Table 7.2 Micellar lactose and micelle hydration in Skim milk ................................... 102

xiii

Nomenclature

A Baseline at infinite time

a Particle radius

aw Water activity

Abs Spectrophotometer value of absorbance

B Amplitude

Csca Light scattering cross-section

cvi Volume concentration

D Translational diffusion coefficient

d Effective Stokes diameter

dave Average pelleted casein micelle size

d (H) Hydrodynamic diameter

g Gravitational acceleration

H Effective setting height

Have Average centrifugation path length of casein micelles

I Incident light intensity

I0 Transmitted light intensity

k Boltzmann’s constant

L Pathlength of the light through the scattering suspension

Lc Pathlength of the light through cuvette

n Number concentration

Qsca Scattering coefficient

xiv

q Scattering vector

T Absolute temperature

t Time delay

te Effective setting time

Vg Terminal setting velocity

Vi Voluminosity

τ Turbidity

τa Turbidity for mono dispersed spherical particles with radius ‘a’

τshear Shear stress

τsample Turbidity of the re-diluted milk concentrate sample

τskim Turbidity of original skim milk

τsample fat Turbidity of the re-diluted milk concentrate sample diluted in

EDTA multiplied by 10 to account for the dilution

τskim fat Turbidity of the original skim milk diluted in EDTA

multiplied by 10 to account for the dilution

ηc Coefficient of viscosity

η Viscosity

ηcp Viscosity of the continuous phase

η0 Coefficient of viscosity of the portion in the fluid

consisting of water and low molecular mass species

φ Volume fraction

φd Volume fraction of all dispersed particles

xv

φmax Hypothetical maximum volume fraction

φf Volume fraction of fat

φc Volume fraction of casein

φw Volume fraction of whey proteins

φl Volume fraction of lactose

φ spherical Volume fraction of spherical particle

γ Shear rate

θ Light scattering testing angle

Δρ Density difference between the solid particle

and suspending fluid

AAS Atomic absorption spectroscopy

BSA Bovine serum albumin

CCP Colloidal calcium phosphate

CF Concentration factor

CM Casein micelle

DLS Dynamic light scattering

MF Microfiltration

MPC Milk protein concentrate

MPI Milk protein isolate

NF Nanofiltration

PCS Photon correlation spectroscopy

RI Refractive index

RO Reverse osmosis

xvi

SEM Scanning electron microscopy

SMP Skim milk powder

TCA Trichloroacetic acid

TEM Transmission electron microscopy

TS Total solids

UF Ultrafiltration

UHT Ultra high temperature processing

WPC Whey protein concentrate

1

1 Introduction

Fresh milk and dairy products are widely used high nutritional value human food. Since

the first cows arrived in Australia in the 1700’s, the Australian dairy industry has

developed both its size and range of products with a large export component. Today,

with a gross value of over $4 billion annually (in 2011), it is the third largest

agricultural industry in Australia (PricewaterhouseCoopers, 2011). Australia exports

nearly half of its milk products, which accounts for 10% of the global market. This

makes Australia the third largest exporter in the world (PricewaterhouseCoopers, 2011).

Importantly, there are still significant opportunities for growth of the Australian dairy

industry as the global market demand continues to increase (DairyAustralia, 2013).

As most milk products are transported and stored, proper processing is needed to meet

microbiologically stable requirement. Reducing water activity by concentration and

drying is one of the most widely used techniques. Since the nineteenth century,

evaporation has been used in the food industry for liquid concentration. In the dairy

industry, evaporation is an important process used for concentration of different milk

products and it is also used as a preliminary step to spray drying (Henning, Baer,

Hassan & Dave, 2006). As most milk products are heat sensitive, evaporation is

conducted at low temperatures under vacuum for short times in order to reduce thermal

degradation and flavour changes (Heldman & Hartel, 1998). However, alterations of

physicochemical state and functional properties of milk products still take place during

milk evaporation.

Membrane filtration (Figure 1.1) is a more recent technology used to fractionate and

concentrate solutes on the basis of size. Since the beginning of the 1970s, the following

categories of membrane filtration have been adapted for the dairy industry to serve

different purposes (TetraPak, 1995):

Reverse Osmosis (RO) is used for dehydration of whey, ultrafiltration permeates and

retentates by removal of water.

Nanofiltration (NF) is used for desalination of whey, ultrafiltration permeate or retentate

by partial removal of monovalent ions like sodium and chlorine.

2

Ultrafiltration (UF) is typically used for concentration and standardization of milk

proteins which is intended for cheese, yoghurt and other products.

Microfiltration (MF) is primarily used for removing bacteria from skim milk, whey and

brine, although sometimes it is also used for whey defatting.

Figure 1.1 Application of membrane separation processes in dairy industry (adapted

from Dairy processing handbook (TetraPak, 1995))

Among these filtration techniques, UF is used widely in the dairy industry to produce

several highly value-added products such as milk protein concentrate (MPC) and milk

protein isolate (MPI). In the application of UF, water, soluble minerals and lactose pass

through membranes thereby concentrating whey proteins and casein to achieve a final

protein concentration. However, the physical and biochemical alterations of milk during

UF are not yet understood in sufficient detail.

Casein micelles comprise most of the protein in milk system. Four different casein

proteins and nanoclusters of colloidal calcium phosphate (CCP) exist as micelles in a

state of dynamic equilibrium (Walstra, 1999) with the proteins and mineral species in

milk serum (Holt, 2004). The properties of the casein micelles (such as micelle

hydration and size, and protein distribution), and their interactions with other milk

components are sensitive to alterations in process variables such as temperature, pH and

concentration (Martin, Williams & Dunstan, 2007). However, there is currently only

3

limited knowledge about how casein micelles behave during milk evaporation and UF

(Karlsson, Ipsen & Ardö, 2007; Martin, Williams & Dunstan, 2010; Singh, 2007).

Lactose, a disaccharide of β-1,4-linked glucose and galatose, is the major carbohydrate

in milk. The presence of lactose regulates the final concentration of caseins in milk

during secretion in the Golgi vesicles (Jenness & Holt, 1987) and also alters the casein

micelle structure (Farrell Jr, Malin, Brown & Qi, 2006). During evaporation and UF,

lactose is either concentrated or removed from milk, however, detailed information

about the lactose effects on casein micelles is currently limited.

The major theme of this thesis is the systematic study of casein micelle alterations

during milk evaporation and UF processing to investigate the effects of casein and

lactose on the physico-chemical properties of casein micelles. The data and information

presented here sheds new light on the physicochemical behaviour of casein micelles

during milk processing, which will importantly help improve the performance of milk

evaporation and UF and the functional properties of the final products.

In this thesis, Chapter 2 provides general information about milk and reviews the

existing literatures on casein protein and casein micelles. Emphasis is placed on the

casein micelle structure and casein micelle alterations in response to processing and

changes in environmental conditions. The experimental techniques and protocols

applied in this work are described in Chapter 3. The first experimental section, Chapter

4, investigates the evaporation effects on casein micelle hydration, composition and size

distribution, and their reversibility on re-dilution back to the native concentration. In

Chapter 5, the dynamics of the temperature effect on casein micelles are investigated in

a comprehensive study using real-time measurement of casein micelle properties in the

range 10 - 40 °C. In Chapter 6, the effects of UF concentration at different processing

temperatures on the hydration, composition and size of casein micelles were

investigated and the composition of retentate serum and micelles were compared,

providing new understanding of the temperature effects on skim milk UF and the

resulting retentates. In Chapter 7, the lactose effects on casein micelles were studied in

different milk protein systems. The thesis concludes with a general discussion of the

main outcomes of these studies and the implications of the findings (Chapter 8).

4

2 Literature Review

2.1 Properties of Milk

Milk is a biological fluid which is secreted by mammals to provide nutrition to the

neonate including energy, amino acids, water, inorganic elements, essential fatty acids

and vitamins (Fox & McSweeney, 1998a). Bovine milk contains hundreds of molecular

species; however, most are at trace levels. The typical concentration of the major

components is outlined in Table 2.1.

Table 2.1 Composition of bovine milk (Walstra & Jenness, 1984)

Component Typical Content Range

[wt %] [wt %]

Water 87.3 85.5~88.7

Solids-not-fat 8.8 7.9~10.0

Fat in dry matter 31.0 21.0~38.0

Lactose 4.6 3.8~5.3

Fat 3.9 2.4~5.5

Protein 3.25 2.3~4.4

Casein 2.6 1.7~3.5

Mineral substance 0.65 0.53~0.80

Organic acids 0.18 0.13~0.22

Miscellaneous 0.14 0.10~0.19

2.1.1 Water

The water content of dairy products ranges from 2.5 to 94% (Fox et al., 1998a) and cow

milk contains about 87% of water. The most important practical aspect of water in dairy

5

products is its effect on their chemical and physical stability. Biochemical changes such

as the denaturation and re-organization of proteins, lipid oxidation, crystallization of

lactose, Maillard reaction and loss of certain vitamins are influenced by water content

and water activity. (Water activity is defined as the ratio between the water vapour

pressure exerted by the water in a food system and that of pure water at the same

temperature. Due to the presence of various solutes, the water exerted vapour pressure

in milk system is always less than that of pure water.)

2.1.2 Lactose

Lactose is the predominant carbohydrate in milk (Fox et al., 1998a). It is a reducing

disaccharide of linked glucose and galactose, which is responsible for the Maillard

reaction when milk is heated to a high temperature.

Lactose plays a distinctive role in milk synthesis, as it is one of the major osmole in

milk. In mammary gland, the lactose synthesis process regulates the final water content

in milk by drawing water into it (Jenness et al., 1987). Although 30% of calories is

provided by lactose in bovine milk, disaccharides cannot be absorbed in intestine.

Therefore, lactose is hydrolysed by β-galctosidase during digestion (Thompson, Boland

& Singh 2009). Most mammalian species are able to produce adequate level of lactase,

however, as they age, the secretion declines and eventually becomes inadequate for

complete hydrolysis lactose. Lactose intolerance frequency and intensity vary from

nearly 100% in Southeast Asia to around 5% in Northwest Europe (Ingram, Mulcare,

Itan, Thomas & Swallow, 2009; Thompson et al., 2009).

Lactose is important in dairy processing. In the absence of nuclei and agitation, the

solubility of lactose is temperature dependent. Before spontaneous crystallization,

lactose solutions are capable of being highly saturated (Fox et al., 1998a). Cooling a

saturated solution or continuous concentrating beyond the saturation point can lead to

super saturation of lactose, in which case crystallization does not occur readily (Fox et

al., 1998a). During the manufacture of concentrated milk products, the insolubility of

lactose and its capacity to form supersaturated solution are of considerable practical

importance. In the mean time, lactose is also significant in manufacturing fermented

dairy products such as yogurt. The lactose metabolize ability of lactic acid bacteria

6

making them have a competitive advantage over many spoilage and pathogenic

organisms (Fox et al., 1998a).

2.1.3 Milk Fat

Milk fat is the most compositionally variable component in milk and it is the major lipid

source used by the mammalian newborn for accumulating body adipose tissue. Milk fat

consists of numerous different lipids, however, is largely present in milk (98 to 99 wt%)

as droplets of triacylglycerides surrounded by a interfacial membrane (8 to 10 nm in

thickness) comprised of phospholipid and proteins (Shipe et al., 1978). The majority of

these liquid fat droplets range from 0.1 to 15 µm in diameter, with the membrane

stabilizing the fat globules in an emulsion within the aqueous environment of milk.

When exposed to moderate mechanical treatment-pumping and flow in pipes, the fat

globules can change their shapes easily without being released from their membranes

(TetraPak, 1995). The remaining 1 to 2 wt% of milk lipid comprises cholesterol,

phospholipids, sterol, carotenoids, fat-soluble vitamins A, D, E, and K, and some other

trace fatty acids (Walstra et al., 1984).

2.1.4 Vitamins

Vitamins are organic chemicals required in trace amounts. The chemical structures of

the vitamins have no relationship with each other. Milk contains all the major vitamins

as it is the only source of nutrients for the neonatal mammal until weaning (Fox et al.,

1998a). The fat soluble vitamins A, D, E, and K are found mainly in milk fat, while the

water soluble B group vitamins (thiamine, riboflavin, niacin, biotin, panthothenate,

folate, pyridoxine, and related substances), cobalamin and its derivatives, and ascorbic

acid are found in the aqueous phase of milk.

Riboflavin is one of the most important vitamins as it is part of two significant

coenzymes involved in numerous metabolic pathways. It also contributes to DNA

reparation in the ribonucleotid reductase pathway (Haug, Høstmark & Harstad, 2007).

In milk products, riboflavin is responsible for the off flavour when exposed to light,

which is caused by photo-oxidation and the production of methional (Fox et al., 1998a;

Hand & Sharp, 1939). Light exposure is the most important parameter affecting the

7

stability of riboflavin in dairy products (Fox et al., 1998a; Hand et al., 1939). The

fluorescence of vitamins (specifically riboflavin in this study) can affect the analysis of

milk products as discussed in section 5.2.5.

2.1.5 Minerals

Milk contains a large variety of minerals and salts present in a complex state of

speciation. The total concentration of minerals in milk is less than 1 wt%. The milk

minerals are mainly the chlorides, citrates, sulphates, carbonates, bicarbonates and

phosphates of sodium, potassium, magnesium and calcium. Trace amount of other

elements are also found in milk including iron, silicon, zinc, copper and iodine. Milk

and milk products are important mineral source, in particular in the uptake of sufficient

quantities of calcium to ensure healthy bones and teeth, and prevention of hypertension

(Insel, Turner & Ross, 2004). Sufficient intake of magnesium is also important, being

involved in more than 300 reactions in the body (Haug et al., 2007).

Various minerals are interrelated and the interrelationships are affected by pH either

directly or indirectly. Milk salts exist in two forms (Table 2.2): diffusible (soluble) and

non-diffusible (colloidal) which are bound to proteins. The mineral equilibrium between

soluble and colloidal phase and the role of calcium phosphate in casein micelle structure

will be discussed in the next chapter.

Table 2.2 Distribution of salts between the soluble and colloidal phase of milk (20 °C,

pH 6.77) (Davies & White, 1960)

Constituent Diffusible Colloidal

Total calcium

Magnesium

Sodium

Potassium

Total phosphorus

Citrate (as citric acid)

Chloride

30.5%

65.5%

98.0%

93.9%

45.6%

93.5%

100%

69.5%

34.5%

2.0%

6.1%

54.4%

6.5%

0%

8

2.1.6 Proteins

Normal bovine milk contains 30-35 g protein/L which serves as a source of essential

amino acid, enzymes, antibodies and immune stimulants (Clare & Swaisgood, 2000;

Korhonen, Pihlanto-Leppäla, Rantamäki & Tupasela, 1998) (Table 2.3). Milk proteins

can be categorised as either caseins or whey proteins, with the caseins defined as those

proteins that precipitate on lowering of the pH to 4.6. Minor proteins also exist in the

milk serum and in the fat globule membrane (Walstra et al., 1984). Another important

difference between casein and whey proteins is that caseins are heat insensitive, whilst

whey proteins denature at high temperatures (Visser & Jeurnink, 1997).

Table 2.3 Major bovine milk proteins and some of their biological functions (adapted

from (Korhonen & Pihlanto, 2007))

Protein

Average

concentration

in milk (g/L)

Molecular

weight

Major biological Functions

α-Lactalbumin 1.2 14200 Lactose synthesis in mammary gland

β-Lactoglobulin 3.3 18400 Retinol carrier, antioxidant and vitamin-

A-binding protein

Bovine serum albumin 0.3 66300 Peptide source

Immunoglobulin 0.5 – 1.0 150000 -

1000000

Immune protection

Lactoferrin 0.1 80000 Antimicrobial, antioxidant and anti-

inflammatory

Caseins 28 19000 -

25000

Mineral carrier and peptide source

Whey Proteins

Milk whey proteins, which are classified as globular proteins, are those remaining in

solution at pH 4.6. The group consists of 4 types of proteins: α-lactalbumin, β-

lactoglobulin, proteose-peptones, and blood proteins such as serum albumins and

immunoglobulins. Some of them are only mammary secretory cell synthesized proteins

(e.g. α-lactalbumin and β-lactoglobulin).

9

α-lactalbumin (α-LA) is a protein unique to milk whey and is present in the milks of all

mammalian subdivisions: eutherians, marsupials and monotremes. It is the regulatory

protein of the lactose synthesis enzyme system that catalyses and regulates the synthesis

of lactose in the lactating mammary gland (Walstra et al., 1984). It is a small, relatively

stable and pressure resistant protein (Considine, Patel, Anema, Singh & Creamer, 2007),

which exists in two genetic variants, A and B. The structure of α-LA consists of 4 α-

helices contained in a bundle like form. This general structure can change to three

different forms: calcium bound, calcium free, and low pH form, which results from its

ability to bind to calcium and other ions (Creamer & MacGibbon, 1996). The binding of

calcium to native α-LA is very strong and associated (Kronman, Sinha & Brew, 1981;

Schaer, Milos & Cox, 1985) and indicates the presence of the calcium binding sites on

the protein (Kuwajima, 1996; Kuwajima, Mitani & Sugai, 1989). It is not required for

activation of α-LA (Kronman et al., 1981; Musci & Berliner, 1985), but has strong

effect on molecular stability (Ikeguchi, Kuwajima & Sugai, 1986). In the reduced

denatured protein, calcium binding is also necessary for native disulfide bond formation

and refolding (Ewbank & Creighton, 1991; Rao & Brew, 1989). For denatured α-LA,

more than two orders of magnitude of the rate of refolding can be accelerated by

calcium while maintaining the general pathway of the transition (Forge et al., 1999;

Kuwajima et al., 1989). The folding rate of α-LA is dependent on the calcium

concentration, while on removal of calcium, the protein is more susceptible to

denaturation (Walstra et al., 1984).

β-lactoglobulin (β-LG) which represents around half of the whey proteins (Thompson et

al., 2009) has been studied extensively. Consequently, its amino acid sequence and

cross-linking mechanisms have been known for some time. The primary function of β-

lactoglobulin is not nutritional, as it is very resistant to proteolysis in its native state

(Thompson et al., 2009). The structure of β-LG shows the main features are an eight-

stranded β-barrel and a three turn helix lying parallel to three β-strands (Creamer et al.,

1996; Visser et al., 1997). The amino acid sequence of the entire structure contains five

cysteine residues, of which four are bound via disulphide linkages, and the fifth, at room

temperature is buried within a group of hydrophobic residues between the helix and the

β-strands (Creamer et al., 1996; Sawyer et al., 2002; Visser et al., 1997; Walstra et al.,

1984). The free thiol group plays a significant role in the changes of β-LG and its

interactions with other milk proteins during thermal processing. At elevated

10

temperatures, β-LG unfolds, exposing the reactive thiol group, resulting in irreversible

aggregation with itself and other molecules such as κ-casein and α-LA (Visser et al.,

1997).

The structure and aggregation state of β-LG is strongly dependent on pH (Creamer et al.,

1996; Sawyer et al., 2002; Visser et al., 1997), and the effect of temperature varies as a

function of pH. Below pH 2.0, β-LG exists as a monomer; when closer to its isoelectric

point (pH=5.1), it exists as an octamer at room temperature. Between pH 5.5 and 6.5, β-

LG exists as a dimer at room temperature. Dissociation to a monomer occurs at

temperature above 50 °C. Further heating to 65 °C results in unfolding of the structure

and subsequent exposure of the free SH group, which is followed by aggregation.

Bovine serum albumin (BSA) is a large protein and it makes up approximately 10 to 15%

of total whey protein in bovine milk. BSA consists of three domains and nine sub-

domains (Krisdhasima, Vinaraphong & McGuire, 1993). It can irreversibly adsorb onto

either hydrophobic or hydrophilic surfaces regardless of the surface charge (Frateur,

Lecoeur, Zanna, Olsson, Landolt & Marcus, 2007; Fukuzaki, Urano & Nagata, 1995;

Nasir & McGuire, 1998), with the presence of divalent cations in milk (Ca2+

and Mg2+

)

enhancing this adsorption (Zanna, Compère & Marcus, 2006).

Immunoglobulin has multiple functions, including bacterial opsonisation, agglutination

and complement activation (Thompson et al., 2009). The protein is glycosylated at

different sits, and β-sheet structures make up both the heavy chain and the light chain

predominantly.

Lactoferrin is a monomeric Fe3+

binding glycoprotein (Strom, Svendsen & Rekdal,

2000). The calcium bound state of bovine lactoferrin is more stable to chemical

denaturants and heat (Rossi et al., 2002).

Caseins

About 80% of milk proteins are caseins and there are four casein variants (αs1-, αs2-, β-

and κ-casein) in bovine milk system. The main functions of caseins in milk system are

to safely transport calcium and phosphate to the mammary gland without precipitation

(Creamer et al., 1996). In order to do this, caseins form multi-molecular complex

11

structure called “casein micelle”, which is the colloidal phase of milk. A detailed review

of the known information about caseins and casein micelles will be presented in the

following chapters.

Nutritional value of milk proteins and peptides

It is known that both the mineral binding and cytomodulatory peptides in bovine milk

are health enhancing, which are claimed to be able to reduce the risk of getting diseases

and improve physiological functions (Meisel & FitzGerald, 2003). For example, some

milk peptides have antihypertensive effects both due to inhibition of angiotensin-

converting enzyme and binding minerals (Jauhiainen & Korpela, 2007).

2.2 Casein and Casein micelles

The overall theme of this thesis is the understanding of the effects of processing on the

physicochemical properties of casein micelles. This section provides a comprehensive

review of the current literature on casein, casein micelles, and casein micelles during

processing that is necessary for the research performed in the thesis.

2.2.1 Caseins

General Information

The caseins are phosphoproteins (Farrell Jr et al., 2004) that constitute 76 to 86% of the

total protein in milk system. There are four casein variants (αs1-, αs2-, β-, and κ-casein)

in bovine milk which can be categorized into calcium sensitive caseins (αs1-, αs2-, β-

caseins) and calcium insensitive κ-casein. The primary structures are shown in Figure

2.1. The polar and apolar residues of all caseins are not uniformly distributed but occur

in clusters, giving hydrophobic and hydrophilic regions (Thompson et al., 2009).

12

P02662| αs1-Casein

MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAME

DIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKE

GIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTT

MPLW

P02663| αs2-Casein

MKFFIFTCLLAVALAKNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEVVRNANEEEYSIG

SSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIVLNPWDQVKRNAVPITPTLNR

EQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQKAMKPWIQ

PKTKVIPYVRYL

P02666| β-Casein

MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQT

QSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSL

TLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQE

PVLGPVRGPFPIIV

P02668| κ-Casein

MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVA

LINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEI

PTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV

Figure 2.1 Amino acid sequences of the bovine αs1-, αs2-, β- and κ-caseins. (Swiss-Prot

accession number, proteins names and sequences for the caseins. The database

sequences are for the B variant of αs1-casein, the A variant of αs2-casein, the A2 variant

of β-casein and κ-casein A.) Confirmed phosphorylation sites are shown in bold type in

αs1-, αs2- and β-casein sequences, potential phosphorylation and glycosylation sites are

indicated in bold in κ-casein sequence (modified from (Thompson et al., 2009)).

13

The four bovine caseins do not appear to have highly organized secondary structure

suggesting only short-length structures are present (Kumosinski, Brown & Farrell, 1993;

Kumosinski & Farrell, 1994). The fairly uniformly distributed proline residues and the

unphosphorylated and unglycosylated polypeptides can form regular structures, giving

the caseins a type of helix loop conformation (Fox & McSweeney, 1998b; Holt &

Sawyer, 1988). Considering the biological function of the caseins, these conformations

on the calcium sensitive caseins are suited to modulating the precipitation of calcium

phosphate in milk by either acting as sites for nucleation or binding rapidly to calcium

phosphate nuclei (Holt et al., 1988).

α-caseins stabilize milk proteins by both refolding properties and chaperone-like

activity (Sakono, Motomura, Maruyama, Kamiya & Goto, 2011). The solubility of α-

caseins is marginally influenced by temperature but strongly influenced by pH (Post,

Arnold, Weiss & Hinrichs, 2012).

αs1-casein which has two predominantly hydrophobic regions and one highly charged

polar zone (Fox et al., 1998a), exists as a loose flexible polypeptide chain. αs1-casein A

is soluble at calcium concentrations up to 0.4 M below 33 °C, whilst αs1-casein B and C

are insoluble and form coarse precipitates when calcium is present at concentrations

above 4 mM (Singh & Flanagan, 2006). The presence of αs1-casein A modifies the

behaviour of αs1-casein B, which is then soluble in 0.4 M calcium at low temperature

(Singh et al., 2006).

αs2-casein is more varied than αs1-casein and is generally present as a mixture of four

phosphor forms with 10-13 phosphates (Thompson et al., 2009). It has a dipolar

structure where the N terminus is negatively charged, and the C terminus is positively

charged (Fox et al., 1998a). αs2-casein is insoluble in calcium solution over 4 mM

(Singh et al., 2006).

β-casein has an unordered structure, which has a high degree of segmental motion

(Nasir et al., 1998). It has a strong negatively charged N terminus and an uncharged

hydrophobic tail (Krisdhasima et al., 1993). It can stabilize the calcium phosphate in

solutions (Holt, 2004) but in a temperature-dependent manner (Singh et al., 2006). This

could be because β-casein undergoes a temperature-dependent conformational change in

which the content of poly-proline helix decrease as a function of temperature (Singh et

al., 2006).

14

By following a sequential process, β-casein is able to form micelle-like aggregates

(O'Connell, Grinberg & de Kruif, 2003) and this is primarily attributed to diffusive

motion, long range concentration fluctuations (de Kruif & Grinberg, 2002) and

hydrophobic interactions (Pierre & Brule, 1981). The experiments related to β-casein

micelle formation fit well with the thermodynamics underlying the shell model of

casein micelle structure, which has been well discussed by many researchers (Kegeles,

1979; Mikheeva, Grinberg, Grinberg, Khokhlov & de Kruif, 2003).

κ-casein has a flexible structure which includes a positively charged hydrophobic

regions, negatively charged polar regions, and connecting domains (Fox et al., 1998a).

It contains high amount of β-sheet structure which can form close association with

phosphorylated caseins (Farrell, Cooke, Wickham, Piotrowski & Hoagland, 2003). κ-

casein is markedly different from the other three caseins in terms of its solubility

characteristics. It is insensitive to calcium and is soluble in calcium solutions at all

concentrations up to those at which general salting out occurs (Singh et al., 2006). It is

able to stabilize and absorb onto other caseins, terminating casein aggregation so that no

further casein protein adsorption can be achieved, even in the presence of calcium. This

is consistent with the understanding that it forms the outer layer of casein micelles,

helping to stabilize the colloidal particles (Nagy, Váró & Szalontai, 2012). The

proportion of κ-casein in casein micelles has been shown to be inversely related to their

size (Davies & Law, 1983).

Casein Properties

Solubility

Solubility is an important functional property for proteins in fluid and it is also essential

for other functionalities since insoluble proteins cannot perform useful properties. In a

calcium free system, the solubility of individual caseins is pH and temperature

dependent (Bingham, 1971). The caseins are insoluble at their isoelectric point (pH 4.6)

and the insolubility range becomes wider with increasing temperature (Fox et al.,

1998a). Due to the open structure and relatively high water binding capacity of caseins,

they are able to form high viscous solutions; however, not more than 20% casein protein

can be dissolved even at elevated temperature (Fox et al., 1998a).

15

Gelation

In milk, caseins undergo gelation when the environment is changed in one of several

ways. The most important of these are rennet induced coagulation for cheese or rennet

casein manufacture and acidification to the isoelectric point. In addition, caseins may

also be gelled or coagulated by organic solvents or extremely severe thermal conditions

(Fox et al., 1998a). Studying the gelation process can provide crucial information on the

casein micelle structure (Horne, 2002a).

Surface activity

As far as their functionality is concerned in food system, the surface activity of caseins

is an important property that makes them good foaming agents and emulsifers (Fox et

al., 1998a). To be an effective emulsifying agent, a molecule should be relatively small

and capable of adsorbing onto oil-water or air-water interface. αs1- and β-casein which

have relatively high surface hydrophobicity meet these requirements very well

(Dickinson, 1989).

Co-precipitation with whey proteins

Casein and whey protein co-precipitation can be achieved by acidification of denatured

whey proteins to pH 4.6 or by addition of calcium at 90 °C (Figure 2.2) (Fox et al.,

1998a). The functionality of the caseins is not adversely affected when adjusting the

milk to an alkaline pH before denaturing the whey proteins and then co-precipitating

them with casein acidification at pH 4.6. This might be because the denatured whey

proteins do not form complex with the casein micelles at elevated pH (Mulvihill, 1992).

16

Figure 2.2 Protocols for casein whey co-precipitates (Mulvihill, 1992)

2.2.2 Casein Micelles

It has been known for a long time that casein in milk exists predominantly in the form

of complex, approximately spherical aggregates (Figure 2.3) with a diameter in the

range 50 – 500 nm (Fox et al., 1998a) that are termed casein micelles. Casein micelles

scatter light and the white colour of the skim is largely due to the presence of casein

micelles. The micelles are very stable; they can withstand heat treatment, commercial

homogenization and high calcium concentration (Thompson et al., 2009). The casein

micelles are able to keep high concentrations of calcium phosphate in colloidal form

making them essential for development of bones and teeth in the sucking infant, as it is

able to deliver these supersaturated minerals to mammary gland (Thompson et al.,

2009). At the same time, micelles are also known as important amino acid source

(Thompson et al., 2009).

Milk pH Adjustment or adding Calcium

Heat treatment

Precipitation

Casein and whey protein co-precipitation

17

Figure 2.3 SEM image of a casein micelle (Dalgleish & Corredig, 2012)

Formation and digestion of casein micelles

In the Golgi apparatus, caseins appear to be spherical complexes with a diameter of 10

nm (Farrell Jr et al., 2006). Calcium transport starts from the phosphorylation of caseins

by calcium activated kinase which binds with the Golgi membrane (Bingham & Farrell,

1974) and followed by delivery of calcium to the casein vesicles with a membrane

associated ATPase (Bingham, McGranaghan, Wickham, Leung & Farrell Jr, 1993). The

step-by-step interactions of caseins, calcium and phosphate gradually form complex

structures which lead to the casein micelles (shown in Figure 2.4, top) and insure the

effective transport of the vital minerals. The formed casein micelles are then secreted by

reverse pinocytosis (shown in Figure 2.4, bottom) (Farrell Jr et al., 2006). After milk

ingestion, casein micelles are precipitated due to acidification in stomach, which delays

the entry of proteins into the small intestine and improves the digestibility (Fox &

Brodkorb, 2008). The micelles are then digested by pepsin in the stomach followed by

trypsin digestion in the intestine (Ono, Takagi & Kunishi, 1998).

18

Figure 2.4 Formation of casein micelles (CM) in Golgi vesicles (G). Top, a Golgi

vesicle discharged its contents into the alveolar lumen; Bottom, One casein micelle

presents in lumen.(Farrell et al., 2003)

Casein micelle structure

The structure of casein micelles has attracted the attention of scientists for a

considerable time. Knowledge of micelle structure is important because the stability and

behaviour of the micelles are central to many dairy processing operations. Since the

pioneering work of some researchers (Shimmin & Hill, 1964; Waugh, 1958), numerous

investigations and a number of models have been made and refined. The following is a

summary of some of the important attributes of casein micelles that are generally

accepted in the literature:

(1) Casein micelles have a porous structure in which protein occupies only about 25%

of the total volume (Fox et al., 1998a). The micelles are highly hydrated, having a water

content of 2 to 4 gram of water per gram of protein (de Kruif, 1999; Morris, Foster &

Harding, 2000; Walstra, 1979). Cryo-TEM studies have revealed the presence of water

filled cavities and channels inside casein micelles (Trejo, Dokland, Jurat-Fuentes &

Harte, 2011).

(2) κ-casein, which represents about 15% of total casein, covers part of the casein

micelle surface (Dalgleish, 1998). It stabilizes the calcium sensitive caseins, which

19

represent about 85% of total casein (Fox et al., 1998a). The content of κ-casein in casein

micelles is inversely proportional to the micelle size (Dalgleish, Horne & Law, 1989;

Davies et al., 1983).

(3) Some casein is able to shift between the serum and the casein micelles, particularly

as a result of changes in temperature (Creamer, Berry & Mills, 1977; Udabage,

McKinnon & Augustin, 2003). Upon cooling, β-casein is liberated from large micelles

and κ-casein is solubilised from small micelles (Ono, Murayama, Kaketa & Odagiri,

1990). The released caseins have been reported to be fully reversible after incubation at

room temperature (Davies et al., 1983). At high temperature (i.e. above 70 °C), micelle

size increases due to heat induced protein aggregation and attachment of denatured

whey proteins (Anema & Li, 2003).

(4) The calcium and phosphate within the micelles form high density colloidal calcium

phosphate (CCP) nanoclusters (Choi, Horne & Lucey, 2011; McMahon & Oommen,

2008; Rose & Colvin, 1966; Trejo et al., 2011), which are associated with the

phosphorylated amino acid residues of caseins through electrostatic interactions (de

Kruif, Huppertz, Urban & Petukhov, 2012b). Calcium and phosphate are known to

exchange between the serum and the micelles (McMahon & Brown, 1984). CCP is

more stable at high temperature (Morr, 1967) and removal of CCP will not change the

size distribution and protein composition of casein micelles (Griffin, Lyster & Price,

1988; Lin, Leong, Dewan, Bloomfield & Morr, 1972).

Modified from the models presented by Waugh (1958) and Schmidt (1982), the well

known submicelle model was developed (Walstra, 1990; Walstra, 1999) (shown in

Figure 2.5). In this model, researchers proposed that casein sub-micelle units build up

the internal structure and form roughly spherical casein micelles which have a hairy

outer layer (comprising mostly κ-casein) on the surface. The sub-micelles vary in

composition (one type consists of α-casein and β-casein primarily, and the other type

consists of α-casein and κ-casein) and they can be linked together by calcium phosphate

clusters between them (Walstra, 1999). Early X-ray scattering study and electron

microscopy suggested the existence of sub-micelles around 15 nm in diameter (Pessen,

Kumosinski & Farrell, 1989; Schmidt, 1982), however, more recently this has been

determined to be an artefact due to sample preparation (such as casein separation and

dehydration, chemical fixation as well as metal coating, which lead to protein

20

coagulation and crosslinking and altered micelle original state) (McMahon &

McManus, 1998; McMahon et al., 2008).

Figure 2.5 Representation of the submicelle model of casein micelles proposed by

Walstra (1999)

Although the sub-micelle model is able to explain many of the physico-chemical

reactions and the principal features of casein micelles, it has never enjoyed unanimous

support. The main challenge is that inadequate evidence has been found to support the

presence of sub-micelles from cryo-EM (McMahon et al., 1998). As alternative, Visser

(1992) described a randomly aggregated casein micelle structure held together by

hydrophobic bonds and amorphous calcium phosphate, with κ-casein at the outer

surface layer, however, this model retains the key features of the sub-micelle model.

Several models have been proposed as alternatives. The dual-binding model was first

developed by Horne (1998). In this model, the assembly and growth of casein micelles

take place by a polymerization process involving the links between calcium phosphate

nanoclusters and through hydrophobic regions of caseins. Each casein molecule remains

intact and functions as a block copolymer effectively as shown in Figure 2.6. The

hydrophobic regions (shown in Figure 3.6) on caseins offer the possibility of multiple

interactions. κ-casein is the most important molecule in the dual-binding model, which

is able to link through its hydrophobic block with the growing chains, whilst the

21

hydrophilic part cannot sustain the growth. The other calcium sensitive caseins, contain

phosphoserine clusters and have multiple functionalities for cross-linking. αs1-casein

can associate through the hydrophobic blocks; αs2-casein, which is only a small fraction

of total bovine casein, has both hydrophobic regions and phosphoserine clusters and is

able to sustain growth through all its blocks; β-casein contains hydrophilic and

hydrophobic blocks can also form polymer links in the network. The dual-binding

model describes casein micelles as a dynamic system and the presence of these weak

interactions are able to lead to breaking and recombining on a continuous basis

(Thompson et al., 2009).

Figure 2.6 Schematic diagram of casein micelle assembly according to dual-binding

model reproduced from (Horne, 2003)

(Blue parts represent the hydrophilic regions, Red parts represent the hydrophobic

regions, C on the κ-casein represents the C-terminal)

Holt (1992; Holt, 2004) proposed the nanocluster model, which describes the micelle to

be a gel-like structure with tangled web of flexible caseins and the calcium phosphate

nanoclusters dispersed as small ‘cherry stones’ (as shown in Figure 2.7) (de Kruif et al.,

2012b). The presence of the nanoclusters in casein micelles is to avoid calcification of

mammary gland. This model is very similar to the dual-binding model, which suggests

22

that the proteins assemble through a collection of weak interactions (including hydrogen

bonding, ion bonding, hydrophobic interactions, weak electrostatic interactions and

other factors) (de Kruif et al., 2012b).

Figure 2.7 Representation of the nanocluster model (de Kruif & Holt, 2003)

The sponge model is a recent description of casein micelle as shown in Figure 2.8

(Bouchoux, Gésan-Guiziou, Pérez & Cabane, 2010). It presents the casein micelle as a

soft micro-emulsion-like object when responding to environment changes (i.e. water

removal) (Sørensen, Pedersen, Mortensen & Ipsen, 2013). The internal structure of the

micelles contains hard regions (including CCP nanoclusters) which are difficult to

compress and regions that can be at least partly merged with other parts upon

compression. This is an alternative model to those based on individual sub units.

23

Figure 2.8 Schematic picture of casein micelle sponge model (Bouchoux et al., 2010)

More recently, the internal structure of casein micelles was studied by small-angle

neutron and X-ray scattering, and the results indicate that the nanocluster model predicts

the distribution of CCP nanoclusters successfully with an average distance of 18.6 nm

(de Kruif et al., 2012b; Holt, Carver, Ecroyd & Thorn, 2013). Meanwhile, with the help

of high-resolution TEM, a sponge-like structure was also observed with CCP

nanoclusters linking the proteins together (Dalgleish, 2011b; Dalgleish et al., 2012;

McMahon et al., 2008). In summary, casein micelles are complex and somewhat

dynamic entities. There is no precise description of the micelle internal structure to date

and each of the models is in various ways oversimplification. The casein micelle models

presented here will be discussed in relation to the results presented in the following

sections which will aid in the interpretation of the results and help achieve a better

understanding of the micelle structure.

Casein micelle properties

To study the casein micelle alterations during milk processing, it is essential to have a

detailed understanding of casein micelle properties and their alterations upon

environmental changes.

Mineral equilibrium

24

As mentioned above, milk contains a large variety of minerals. Except chloride, all the

major ionic species in milk are distributed between the soluble and colloidal phases

(Table 2.2). However, the principal colloidal salt is calcium phosphate. About 67% and

57% of the total calcium and phosphate are in the colloidal phase respectively (Fox et

al., 1998a), associated with the caseins, incorporated into the casein micelles as CCP.

The chemical composition of CCP is yet to be determined with certainty. Based on the

assumption that the amount of calcium bound directly to casein is equivalent to the

number of ester phosphate groups present, Pyne (1960) first suggested that CCP has an

apatite structure with the formula: 3Ca3(PO4)2·CaHCitr- or

2.5Ca3(PO4)2·CaHPO4·0.5Ca3Citr2-. The hypothesis from Schmidt (1982) suggested

that CCP is most likely to be amorphous calcium phosphate [Ca9(PO4)6] ion clusters

linked through calcium bridges to the phosphoseryl residues of caseins. Based on this

information, a more detailed physical study on the structure of CCP has been

undertaken using various forms of X-ray spectroscopy, it was concluded that the most

likely form of CCP is brushite (CaHPO4·2H2O), which has also been identified in bone

and other calcified tissues (Holt, Davies & Law, 1986; Holt et al., 1989). The difference

between the calcium and phosphate ratio (from 1.0 to 1.6) found by analysis is

presumably due to the ability of phosphate moiety of phosphoserine to substitute a

brushite type lattice on their surface sites (Fox et al., 1998a).

The CCP does not precipitate out of milk because of physical protection and chemical

association with caseins inside the micelles. Although CCP represents only about 6 %

of the dry weight of the casein micelles, as mentioned in the nanaocluster model, it

plays essential role in the structure and function of casein micelles and therefore has

major effects on the properties of milk. The concentration of calcium and phosphate

affects casein micelles coagulation during rennet (Choi, Horne & Lucey, 2007;

Dalgleish, 1983; Dalgleish et al., 2012; Lucey & Fox, 1993) and the heat and calcium

stability of the caseins (Fox & Hoynes, 1975; Rose, 1961; Singh & Fox, 1985; Singh &

Fox, 1987).

The equilibrium between the soluble and colloidal minerals of milk is influenced by

many factors including changes in temperature, dilution or concentration, and addition

of salts. For instance dilution reduces the concentration of serum minerals and leads to

solubilisation of some CCP, making the milk more alkaline (Fox et al., 1998a). The

25

details of these relationships and other important effects relating to mineral balance are

now discussed below in the context of alterations to casein micelles in response to

environmental factors.

The effects of pH and minerals on casein micelles

Casein micelles formed at low pH have more compact structure due to low charge on

the proteins (Liu & Guo, 2008). The content and surface hydrophobicity of micelles can

also be changed by acidification (Jean, Renan, Famelart & Guyomarc'h, 2006; Law,

1996). A progressive solubilisation of CCP and other colloidal salts from the micelles

takes place at low pH regardless of the overall ionic strength (Le Graët & Gaucheron,

1999). All of the inorganic phosphate is known to solubilise at pH 5.2, whilst most of

the remaining calcium ions are solubilised when pH reaches 4.6, with the disappearance

of the CCP nano-clusters confirmed by small angle X-ray scattering (Marchin, Putaux,

Pignon & Léonil, 2007). The addition of alkali results in the dissociation of κ-casein

which increases with temperature above pH 6.5 (Anema & Klostermeyer, 1997). At

about pH 11 almost all the calcium phosphate is present in the colloidal phase and these

changes are not reversible on subsequent dialysis against untreated milk (Fox et al.,

1998a). When milk is heated at pH values over 6.9, the amount of α- and β-casein

liberated into serum increases as a function of temperature up to 60 °C and also above

100 °C (Park, Nakamura & Niki, 1996; Singh, McCarthy & Lucey, 1997) but decreases

in the range between 60 to100 °C (Anema & Li, 2000). The CCP can also be removed

from casein micelles by adding EDTA, citrate or oxalate (Horne, 2002b), which might

combine with the release of soluble caseins (Griffin et al., 1988; Lin et al., 1972).

Casein micelles have a sufficiently open structure to allow movement of casein proteins

and the exchange of mineral ions (Udabage et al., 2003). The voluminosity of the core

of the casein micelles is dependent on the hydration and relative partitioning of

exchangeable minerals and casein between the micelles and serum. The overall

(hydrodynamic) voluminosity of the casein micelles also depends on the thickness of

the κ-casein hairy layer, which increase with the addition of NaCl (Kapsimalis & Zall,

1981). Adding calcium has more complicated effects. It increases the calcium activity

(McGookin & Augustin, 1991) which decreases the soluble casein concentration

(Famelart, Le Graet & Raulot, 1999; Sood, Graind & Dewan, 1979). It also reduces the

26

negative charge of caseins (Anema et al., 1997; Anema, Lowe & Stockmann, 2005) and

weakens the κ-casein hairy layer (Müller-Buschbaum, Gebhardt, Roth, Metwalli &

Doster, 2007) thereby modifying the hydrophobic interactions and electrostatic

interactions between casein micelles (Van Hooydonk, Hagedoorn & Boerrigter, 1986).

The addition of magnesium and ferric iron can reduce micelle heat stability (Philippe,

Le Graët & Gaucheron, 2005), while the addition of secondary Na or K phosphate

causes the precipitation of CCP, with concomitant decrease in the concentration of

soluble calcium and calcium ion (Fox et al., 1998a). Adding citrate reduces the

concentration of CCP, and increases the soluble calcium, soluble phosphate and the pH

(Fox et al., 1998a).

Temperature induced alterations of casein and casein micelles

Temperature has a large influence on the state of the milk system and milk viscosity

decreases as a function of temperature (Fernández-Martín, 1972). The caseins have

marked heat stability and there are two main reasons for this. Firstly, being amorphous

unstructured proteins the caseins do not denature and aggregate on heating (Fox et al.,

1998a). Secondly, the structure of casein micelles is such that the calcium sensitive

caseins are located inside the micelles and are protected from further aggregation at

elevated temperature by the outer layer of κ-casein (Griffin, Price & Martin, 1986). The

high heat stability of caseins allows heat sterilized dairy products to be produced

without major changes in physical properties. The heat stability of unconcentrated milk

is almost always sufficient to withstand the temperatures associated with normal heat

treatment processes. However, the heat stability of milk decreases sharply on

concentration (Fox et al., 2008) and is usually inadequate to withstand in-container or

UHT processing unless certain adjustments and/or treatment are made (Fox &

Morrissey, 1977; Horne & Muir, 1990).

Soluble casein is known to shift from serum into the micelles when increasing the

temperature (Creamer et al., 1977; Rose, 1968), which consequently leads to a compact

internal micelle structure and decrease of the apparent voluminosity of casein micelles

(Nöbel, Weidendorfer & Hinrichs, 2012). The effects of heat treatment on the

partitioning of caseins between serum and micelle phase are reversible on cooling

(Creamer et al., 1977; Downey & Murphy, 1970; Pouliot, Boulet & Paquin, 1989b). β-

casein is able to shift from large micelles to serum (Creamer et al., 1977) and κ-casein is

27

solubilised from small micelles (Ono et al., 1990) at low temperature. Loss of these

temperature sensitive caseins from micelles leads to additional releases of caseins to

serum (Rose, 1968).

At high temperature, interactions between denatured whey proteins and caseins can

occur (Donato, Guyomarc'h, Amiot & Dalgleish, 2007; Jovanovic, Barac, Macej, Vucic

& Lacnjevac, 2007; Oldfield, Singh & Taylor, 2005; Oldfield, Singh, Taylor & Pearce,

2000), due to the formation of inter-molecular disulfide bonds (Jang & Swaisgood,

1990; Lowe, Anema, Bienvenue, Boland, Creamer & Jiménez-Flores, 2004). During

heating, whey proteins denature, exposing free thiol groups that then form disulfide

bonds with κ-casein on the surface of the casein micelles. This can result in a significant

size increase as well as surface modification of the casein micelles (Anema et al., 2003;

Park et al., 1996; Singh et al., 1997). β-lactoglobulin is essential in this whey protein-

casein reaction (Considine et al., 2007; Corredig & Dalgleish, 1999; Elfagm &

Wheelock, 1978). It forms aggregates with α-lactalbumin in a fixed ratio before

interacting with caseins (Guyomarc'h, Law & Dalgleish, 2003). The addition of β-

lactoglobulin reduces the level of soluble α- and β-casein at temperature above 60°C

(Anema et al., 2000), presumably due to the formation of whey protein-casein complex.

The exchange of minerals between the serum and colloidal phases is also temperature

dependent, and soluble calcium concentration is known to decrease at high temperature

(Rose & Tessier, 1959) due to decrease of solubility (Morr, 1967) while the distribution

of Na, K, Mg and citrate is not affected (Rose et al., 1959). Cooling on the other hand,

increases the concentrations of soluble calcium and phosphate at the expense of CCP

(Fox et al., 1998a; Pouliot et al., 1989b). The mineral composition of milk is also

important to the zeta potential of casein micelles, which decreases as a function of

temperature (Darling & Dickson, 1979).

As discussed above, much is known about the temperature effects on casein and casein

micelles. However, most of the studies have investigated the casein micelles in an

equilibrium state and the dynamic of casein micelles have not been fully elucidated,

which is clearly important. In Chapter 6 of this thesis, both the equilibrium and kinetic

aspect of casein and mineral alterations in response to temperature was examined.

28

Alterations to casein micelle during processing

Of the various skimmed milk components, the casein micelles arguably have the

greatest impact on properties of different milk products. As discussed above,

environmental conditions can have a considerable influence on the properties and

structure of casein micelles in milk. During processing (as illustrated in Figure 2.9),

milk is exposed to a range of different conditions that can cause various alterations to

the physico-chemical properties of casein micelles. In this section, the current

understanding of these processes is reviewed and the key research questions for the

thesis indentified in this context.

29

Figure 2.9 Outline of some processes used to produce milk protein products

30

Casein micelle alterations during evaporation

Highlighted in Figure 2.9, concentration of skim milk by evaporation is an important

unit operation in dairy processing. In particular, the manufacture of spray dried products

such as skim milk powder (SMP) requires evaporative concentration of milk (usually

performed under vacuum at temperature in the range of 45 to 50 °C) up to

approximately 40 to 50% total solids (TS) prior to spray drying. Casein micelles are

highly hydrated, having in the order of 2-4 g water per gram of protein, depending on

the method of measurement (de Kruif, 1999; Morris et al., 2000; Walstra, 1979).

Although, as discussed above, their overall structure is quite sound over a wide range of

processing conditions, the combination of heating and water removal occurring during

evaporative concentration would be expected to influence the hydration, composition

and size of the casein micelles.

As mentioned above, literature covers the effect of pH and high temperature on casein

micelles extensively. Apart from the information above, it is known that the micellar

phosphate shifts to serum at low pH (Tsioulpas, Lewis & Grandison, 2007; Walstra,

1990). The decrease of soluble caseins at high temperature was also confirmed (Davies

et al., 1983; Tsioulpas et al., 2007; Walstra, 1990). These alterations are in part because

the CCP is in dynamic equilibrium with the minerals contained in the serum, which are

in a complex state of speciation (Holt, 2004). While studies have investigated the pH

and temperature effects on casein micelles in detail, (Ali, Andrews & Cheeseman, 1980;

Creamer et al., 1977; Davies et al., 1983; Downey et al., 1970; Rose, 1968; Tsioulpas et

al., 2007; Visser, Minihan, Smits, Tjan & Heertje, 1986; Walstra, 1990), surprisingly,

information on the effect of concentration on casein micelles is comparatively sparse

(Singh, 2007).

It has been shown that both calcium and phosphate increasingly shift from the serum to

the micellar phase during evaporative concentration of skim (Le Graët & Brule, 1982;

Vujicic & Deman, 1966) and full-cream milk (Nieuwenhuijse, Timmermans & Walstra,

1988). However, the hydration and size of the micelles and the partitioning of casein

between the serum and micelles were not examined. Other studies have investigated the

effect of concentration on macroscopic rheological properties of milk and suggested that

the viscosity of milk concentrates increases as a function of total solids content

(Bienvenue, Jiménez-Flores & Singh, 2003; Vélez-Ruiz & Barbosa-Cánovas, 1998).

31

Collapse (Bouchoux et al., 2010) and aggregation of casein micelles (Bienvenue et al.,

2003; Vélez-Ruiz & Barbosa-Cánovas, 2000) were also observed at high concentration

and during storage, but these investigations have not thoroughly examined the effects on

casein micelle composition. Other studies have also been performed using concentrated

suspensions of reconstituted skim milk powder (SMP) to examine the effects of high

pressure treatment (Huppertz, Fox, de Kruif & Kelly, 2006) and acidification (Anema,

1998; Ward, Goddard, Augustin & McKinnon, 1996), however, not on the casein

micelle structure. In addition, Martin et al., (2007) have previously shown that the

casein micelles in reconstituted SMP are different to those in fresh skim milk. Although

not thoroughly investigated in this previous study, it appeared that some of the observed

alterations to casein micelles were incurred by the evaporative concentration process

(Martin et al., 2007). While it appears significant alterations to casein micelles can

occur during evaporative concentration, a comprehensive study has yet to be performed.

In order to provide detailed information about casein micelle alterations during

evaporation, in Chapter 4 of this thesis, the properties of casein micelles in evaporative

concentrates were investigated, including the size, hydration and composition of casein

micelles. The extent and rate of reversibility were also examined by re-diluting

concentrated samples with water.

Casein micelle alterations during ultrafiltration

Ultrafiltration (UF), as outlined in Figure 2.9, is an important processing step in the

manufacture of cheese and dairy ingredients and in milk standardization (Gésan-

Guiziou, 2013). During UF of skim milk, water, soluble minerals and lactose pass

though the membrane into the permeate while proteins are concentrated in the retentate

(Mistry & Maubois, 2004).

Casein is an important component of skim milk to be considered during UF. The casein

micelles have been reported to be responsible for concentration polarization and gel

layer that form during UF of skim milk, directly influencing flux and processing

efficiency at different pH (Bouzid, Rabiller-Baudry, Paugam, Rousseau, Derriche &

Bettahar, 2008; Rabiller-Baudry, Gesan-Guiziou, Roldan-Calbo, Beaulieu & Michel,

2005) as well as the cleaning efficiency (Rabiller-Baudry, Bégoin, Delaunay, Paugam &

Chaufer, 2008). High zeta potential of the casein micelles will lead to high processing

32

flux at neutral pH (Bouzid et al., 2008; Rabiller-Baudry et al., 2005). In the mean time,

casein micelles are dynamic entities that can themselves be affected by UF processing.

A fundamental understanding of casein micelle behaviour during UF, could therefore

lead to improvements in processing efficiency (David, Pignon, Narayanan, Sztucki,

Gesan-Guiziou & Magnin, 2008; Rabiller-Baudry et al., 2005), the use of UF retentates

for standardizing cheese milk, and the quality of products such as milk protein

concentrate (MPC) powders (Martin et al., 2010). Despite this, to date only a limited

number of studies have been devoted to understanding the effect of UF on the

composition, size or structure of casein micelles (Singh, 2007). In one analysis of

transmission electron microscopy (TEM) images, a decreased average casein micelle

size was observed in UF retentates at volume concentration factors of 3 and 5 compared

with the unconcentrated skim milk (Srilaorkul, Ozimek, Ooraikul, Hadziyev & Wolfe,

1991). The size changes were attributed to compositional changes in the retentate;

however the composition of the micelles was not directly investigated. In another study

employing TEM, the size of casein micelles was observed to remain relatively

unchanged during UF but become progressively swollen on subsequent diafiltration

(McKenna, 2000). In a more recent study, different methods were employed to compare

casein micelles in fresh skim milk to those in commercial UF retentates and

reconstituted milk protein concentrate powders (MPC). The average size of casein

micelles was found to be similar in all these samples as measured by dynamic light

scattering (Martin et al., 2010). Rabiller-Baudry et al. (2005) and Karlsson et al. (2007)

have investigated the effect of pH and mineral content on the physical properties of

skim milk during membrane filtration and found that the casein micelle voluminosity

appeared to increase in the presence of added NaCl and decrease when the pH was

decreased from 6.5 to 5.5. In studies on the functional properties of UF treated milk,

alterations to the bulk mineral composition caused by UF have been shown to affect the

renneting process (Ferrer, Alexander & Corredig, 2011; Martin et al., 2010; Sandra &

Corredig, 2013). It was also found that on rennet induced gelation of casein micelles,

stiffer gels could be formed from UF concentrated skim milk than unconcentrated milk

presumably due to an increase in the network bonds (McMahon, Yousif & Kaláb, 1993;

Sandra, Cooper, Alexander & Corredig, 2011). While these studies showed that UF

processing can affect the functional properties of casein micelles, alterations to the

composition of the casein micelles were not investigated. In addition, as discussed

above, the temperature effects on casein micelles are very important. However, the

33

alterations of casein micelles during UF at different temperatures have not been

examined thoroughly (section 6.1). This information will lead to a better understanding

of UF processing of skim milk and the properties of the resulting retentates.

Casein micelle alterations during other processing steps

During processing, milk can be extensively concentrated and the casein micelles still

behave like a dispersion of hard spheres (Bouchoux, Cayemitte, Jardin, Gésan-Guiziou

& Cabane, 2009; Mezzenga, Schurtenberger, Burbidge & Michel, 2005; Sandra et al.,

2011). However, further concentration leads to expulsion of the internal water and

micelle fusion (Bouchoux, Debbou, Gésan-Guiziou, Famelart, Doublier & Cabane,

2009). During the manufacture of milk powder, irreversible shift of small casein

particles towards larger casein micelles takes place (Martin et al., 2007) and whey

proteins attachment to casein micelles during heat treatment occurs, which will continue

in the water removal processes (Martin et al., 2007; Oldfield, Singh, Taylor & Pearce,

1998). In the cheese making process, κ-casein molecules on the micelles breakdown and

leads to the decrease of repulsion (Panouillé, Nicolai & Durand, 2004). These allow

micelles to approach each other and form gels (Castillo, Lucey & Payne, 2006; Gastaldi,

Trial, Guillaume, Bourret, Gontard & Cuq, 2003; Li & Dalgleish, 2006; Tranchant,

Dalgleish & Hill, 2001).

Other treatments such as pressure treatment and ultrasonication have also been

investigated. Aggregation of casein micelles has been reported as a function of pressure

below 300 MPa (Anema, 2008; Anema et al., 2005). Ultrahigh pressure processing

(over 300 MPa), leads to micelle size decrease initially (Gaucheron, Famelart, Mariette,

Raulot, Michela & Le Graeta, 1997; Gebhardt, Doster, Friedrich & Kulozik, 2006;

Huppertz, Fox & Kelly, 2004) and follows by re-association for longer processing time

(Orlien, Knudsen, Colon & Skibsted, 2006). Compared to static high pressure treatment,

there are few changes in the micelles during dynamic high pressure processing (i.e.

homogenization) (Sandra & Dalgleish, 2007). Casein micelle is also very stable in milk

system during the exposure to ultrasound while reducing the size of fat globules and

breaking up whey protein aggregates (Chandrapala, Martin, Zisu, Kentish &

Ashokkumar, 2012).

34

2.3 Summary

Evaporation and ultrafiltration are important processing steps in the manufacture of

different dairy products. It has been shown from current literature that casein micelles

have great impact on the properties of milk system, quality of the products and the

processing efficiency, however, little is currently known about how the

physicochemical properties of casein micelles change in response to evaporative

concentration and ultrafiltration. In this study, the physicochemical behaviours of casein

micelles under different evaporative concentration (Chapter 4) or ultrafiltration (Chapter

6) conditions were investigated by studying alterations of both the serum and casein

micelle phase.

Temperature is an important parameter in milk processing that affects a wide range of

physicochemical properties. While many studies are available on the temperature effects

on casein micelles in an equilibrium state, detailed information about casein micelle

kinetic response to heating and cooling are yet to be obtained. In Chapter 5 of this thesis,

the temperature induced kinetic changes of casein micelles and mineral equilibrium

were examined by using close to real-time pH and turbidity measurements. Based on

this information, the effects of ultrafiltration temperature on casein micelles were

further studied in Chapter 6.

In addition, while only water was removed during evaporative concentration, lactose,

minerals and water pass membrane in skim milk UF process. The casein micelle

hydration was different in these two processing units (discussed in Chapter 4, 6 and 7).

The lactose effect on casein micelle hydration was studied in Chapter 7, as lactose is

known to regulate the water content of milk during secretion.

The thorough studies in this thesis have important implications for improving

processing efficiency as well as the properties of the resulting products.

35

3 Experimental Techniques and Applications

A systematic study using a number of complementary techniques was employed in this

study to investigate the alterations of casein micelles during processing. The general

experimental approach, methodology and principles of the analytical techniques that

were used will be presented in this chapter. Specific information about the materials and

methods pertaining to each experimental chapter will then be presented separately.

3.1 Overview of experimental approach

In order to study alteration to the casein micelles and the partitioning of components

between the micelles and the serum, ultracentrifugation was used to separate the

micelles from the serum. The supernatant and pellet fractions were then analysed to

obtain information about the serum and casein micelles respectively. This enabled direct

observation of changes at the actual concentration to be performed, with other

techniques that require dilution (e.g. DLS) performed as supplementary techniques.

Figure 3.1 shows how the components of milk are separated during evaporation and UF

and how the evaporative concentrates and UF retentates were separated for analysis by

ultracentrifugation. A more detailed summary of the analyses performed on evaporative

concentrates (Chapter 4), fresh skim milk as a function of temperature (Chapter 5) and

UF retentates and permeates (Chapter 6) are shown in each chapter respectively.

36

Figure 3.1 Composition of milk, evaporative concentrates and UF retentates separated

by ultracentrifugation (sCN, soluble casein)

3.2 Milk viscosity and the determination of casein micelle volume faction

The volume faction of casein micelle is an important parameter that was investigated in

this work. The relationship between casein micelle and its volume fraction is important,

both for viscosity to be used as a measure of micelle volume fraction, and conversely,

changes in the volume faction of casein micelles will affect the viscosity and therefore

impact on processing. Milk is a dilute emulsion of fat globules and a colloidal

suspension of casein micelles suspended in an aqueous solution of lactose and minerals.

The physical properties of milk are similar to those of water but are modified by the

presence of various solutes (proteins, salts and lactose and to a lesser extent the residual

fat droplets) in the continuous phase and by the alterations of the casein micelle

colloidal suspension (see Appendix for specific properties). Data on the physical

properties of milk are important as such parameters influence the design and operation

of dairy processing. It also can be used to determine physicochemical changes of

specific components in milk such as the total volume fraction occupied by the casein

micelles.

37

Under certain conditions, milk behaves as a Newtonian fluid, as described by this

equation:

(3.1)

Where: τshear is the shear stress,

γ is the shear rate,

ηc is the coefficient of viscosity.

The coefficient of viscosity for a Newtonian fluid is independent of shear rate but is

influenced by temperature and pressure. The coefficient of viscosity for whole milk and

skim milk are 2.127 mPa·s and 1.790 mPa·s respectively, for water and milk serum are

1.002 and 1.68 mPa·s respectively at 20 °C (Fox et al., 1998a).

The viscosity of milk and Newtonian milk products is influenced by composition,

concentration, pH, temperature, thermal history and processing operations. In skim

milk, casein is one of the principal contributors to the viscosity as it represents the

majority of the particulate volume fraction, with whey proteins and low molecular mass

species occupying less (Jeurnink & de Kruif, 1993). Einstein equation was used to

described the volume fraction of solid non-interacting spheres in highly dilute

dispersion, however, in more concentrated dispersions (such as skim milk), Eiler’s

equation is known to well describe the relationship between viscosity and volume

fraction (Anema, Lowe & Li, 2004; Krishnankutty Nair, Alexander, Dalgleish &

Corredig, 2014; Walstra et al., 1984):

(3.2)

Where: η0 is the coefficient of viscosity of the portion in the fluid consisting of water

and low molecular mass species,

φd is the volume fraction of all dispersed particles.

The volume fraction of any component is given by

(3.3)

Where: Vi is the voluminosity of component i,

38

cvi is the volume concentration of the component in the product.

The volume fraction of different components in milk system is given by

(3.4)

Where: φf, φc, φw, and φl are the volume fractions of fat, casein, whey proteins and lactose

respectively.

φmax in equation 3.2 is the hypothetical maximum volume fraction. It is the volume

fraction that could be occupied by the close packing of the representative particles. To

apply Eiler’s equation to milk system, it is necessary to have a reasonable estimate of

φmax, which has been found to be 0.79 (Snoeren, Damman & Kolk, 1982) and used for

original and concentrated milk systems (Krishnankutty Nair et al., 2014; Snoeren &

Damman, 1984; Snoeren et al., 1982). The relative viscosity measurements were

performed by using a capillary viscometry as described in section 4.2.5.

3.3 Turbidity

The turbidity is a reliable and repeatable technique which can detect real-time

alterations of milk samples in the native concentration (i.e. measurements can be

obtained without requiring dilution). To interpret turbidity data in meaningful way, it is

necessary to understand what contributes to the turbidity. Due to absorption and

scattering of light by the suspended particles in solution, turbidity by definition is the

extinction or reduction in intensity of light transmitted through a suspension of length L.

Sometimes, when light with a particular wavelength hits particles in solution and

converted into heat instead of being propagated or scattered, absorption occurs.

Compared to light scattering, in the turbidity measurements the absorption is assumed to

be negligible. The turbidity is defined by Lamber-Beer law as below (Kerker, 1969):

(3.5)

Where: I is the incident light intensity,

I0 the transmitted light intensity,

τ is the turbidity,

39

L is the pathlength of the light through the scattering suspension.

For a uniform particle:

(3.6)

Where: n is the number concentration,

Csca is the light scattering cross-section.

And the Csca for spherical particles can be described as:

(3.7)

Where: Qsca is the scattering coefficient which depends on the relative refractive index,

a is the particle radius.

As the volume fraction of a spherical particle is:

(3.8)

The turbidity for mono-dispersed spherical particles with radius ‘a’ is

(3.9)

Based on the discussion above, the turbidity is dependent on the concentration, as well

as the size, number and relative refractive index of the particles. In skim milk system,

casein micelle is the only significant contributor to turbidity (Martin, 2008), therefore,

various investigations on casein micelles alterations have been done by monitoring

turbidity changes (Huppertz & de Kruif, 2007; Mizuno & Lucey, 2005; Orlien et al.,

2006; Regnault, Thiebaud, Dumay & Cheftel, 2004).

An analytical spectrophotometer was used for turbidity measurement in this study.

Longer wavelength results in less light scattering allowing measurements to be

performed at higher concentrations, and avoids absorbance by proteins that occurs at

lower wavelengths (Martin, 2008). As the upper wavelength limit (900 nm) of the

available spectrophotometer did not operate stably, 860 nm was selected. Skim milk

samples were investigated in a relative short pathlength 2 mm cuvette to minimize the

effect of multiple scattering from the dense suspension. The standard spectrophotometer

40

data is absorbance, and it can be converted to turbidity (with a unit of length-1

) by using

the following equation when assuming light absorbance is negligible compared to

scattering.

(3.10)

Where Lc is the pathlength of light through the suspension in cuvette,

Abs is the spectrophotometer value of absorbance.

3.4 Dynamic Light Scattering

Dynamic light scattering (DLS) or known as Photon Correlation Spectroscopy (PCS) is

a technique for measuring particle size in a solution. As shown in Figure 3.2, a DLS

system comprises of several components including laser, attenuator, detector and

collector. The laser (with a single wavelength) is used to illuminate the samples and the

attenuator is used to adjust the intensity. The size of a particle can be calculated by

using the Stokes-Einstein equation:

(3.11)

Where: d (H) is the hydrodynamic diameter,

D is the translational diffusion coefficient,

k is the Boltzmann’s constant,

T is the absolute temperature,

η is the viscosity.

The translational diffusion coefficient D can be determined by correlating the

movement of particles with time according to the following equation (Finsy, 1994):

(3.12)

Where: A is the baseline at infinite time,

B is the amplitude,

41

q is the scattering vector,

D is the translational diffusion coefficient,

t is the time delay.

Unique correlation function with a single exponential decay can be generated from each

mono-disperse population of particle sizes. An algorithm is then used to trial different

sets of correlation functions and the best set corresponding to a particle size distribution

is then presented as the result.

Figure 3.2 Simplified schematic of dynamic light scattering set-up

As casein micelles undergo Brownian motion in milk system, the hydrodynamic size

can be measured by DLS. To avoid a faster decay of the autocorrelation function, which

42

results from multiple-scattered signal in concentrated samples (Finsy, 1994), it is

necessary to dilute milk samples in DLS measurements. Therefore, different diluting

buffers were used in this study including UF permeate (Anema et al., 2005), simulated

milk ultrafiltrate (SMUF) (K2HPO4 1.58 g·L-1

, K2SO4 0.18 g·L-1

, K2CO3 0.30 g·L-1

, K3

citrate · H2O 1.2 g·L-1

, CaCl 0.74 g·L-1

, KCl 0.6 g·L-1

, Na3 citrate·2H2O 1.79 g·L-1

,

MgCl2·6H2O 0.65 g·L-1

which was adjusted to pH 6.6 using KOH) (Jenness & Koops,

1962; Regnault et al., 2004) and simplified simulated ultrafiltrate buffer (Tris base 0.02

mol·L-1

, NaCl 0.05 mol·L-1

, CaCl2 0.003 mol·L-1

which was adjusted to pH 6.7 using

0.1 mol·L-1

HCl) (Martin et al., 2007) to minimize the dissociation of casein micelles

during dilution due to ionic strength alterations. (Large fat globules are removed from

skim milk samples, and the remaining small fat globules only scatter little light and

create minor interference (Martin, 2008).) An intensity-based size distribution was used

in this study, which tends to emphasise relative large particles as they scatter more

lights than small ones.

3.5 Ultracentrifugation

As discussed above, ultracentrifugation was employed thoroughout this work as the

mean of separating casein micelles from the serum to determine alterations to casein

micelles and the partitioning of different micelle components. It is particularly powerful

for this purpose as it can be applied to milk and milk concentrates in their native state

(i.e. temperature and concentration). It has been reported that casein micelles can be

fractionated by ultracentrifugation as a pellet (Corredig & Dalgleish, 1996; Huppertz et

al., 2007; Park et al., 1996; Philippe et al., 2005) from serum by using different

centrifuge regimes. Although exact determination of the sedimentation times of

different micelles was not possible owing to the complex geometry, Stokes law can be

used to provide estimates of the sedimentation times of micelles of different sizes:

(3.13)

Where: Vg is the terminal setting velocity of a particle,

H is the effective setting height for the particle,

d is the effective Stokes diameter of the particle,

43

te is the effective setting time for the particle,

Δρ is the density difference between the solid particle and suspending fluid,

(the density of casein micelle: 1253.1 Kg·m-3

, the density of casein free serum:

1026.4 Kg·m-3

at 25 °C (Ford, Ramsdell & Alexander, 1959; Martin, 2008))

g is the gravitational acceleration,

ηcp is the viscosity of the continuous phase (the viscosity of the milk ultrafiltrate:

0.001143 Kg·m-1

·s-1

at 25 °C (Martin, 2008)).

The effective setting time for a particle can be calculated as:

(3.14)

Figure 3.3 Geometry of Ultracentrifuge rotor 90 Ti (Beckman Instruments Inc., Palo

Alto, CA, USA) used in this study (rmin=34.2 mm, rav=55.4 mm, rmax=76.5 mm)

The average pelleted casein micelle size for a certain period of centrifugation time is

dependent on the density, viscosity, gravitational acceleration, and average

centrifugation travel path length. The average pelleted casein micelle size can be

calculated by:

44

(4.15)

Where: dave is the average pelleted casein micelle size,

Have is the average centrifugation path length of casein micelles.

Exact separation of the casein micelles from milk serum by ultracentrifugation is not

straightforward, and many different combinations of centrifugation speed and time

(from 60,000 g 40 min to 100,000 g 90 min) have been reported in the literature for this

purpose (Corredig et al., 1996; Huppertz et al., 2007; Park et al., 1996; Philippe et al.,

2005). If insufficient centrifugation force is applied incomplete palletisation of the

micelles will be achieved, whereas if the centrifugation force is too great, the micelles

will be compressed and true measures of micelle hydration will not be obtained. In this

study 35,000 rpm was used in the rotor shown in Figure 3.3 with a centrifugation force

of 75,940 g on average (ca 76,000 g). To verify and determine the most effective

micelle pelletisation conditions, a series of ultracentrifugation tests were performed as a

function of centrifugation time. Comparison of the pelletisation and investigation of the

potential compression of casein micelles during ultracentrifugation were performed to

ensure that a consistent evaluation could be applied across the different temperatures

and concentrations of the milk samples to be tested. The soluble casein concentration in

the supernatant and the pelleted micelle hydration were measured. The protein

concentration in the supernatant was determined by SDS-PAGE (see section 4.2.3) and

the hydration of the pelleted micelle phase was measured by mass balance of the pellet

before and after drying (105 °C, 24 h) (see section 4.2.2). As the upper temperature

limit for ultracentrifugation is 40 °C, original skim milk samples were ultracentrifuged

at 10, 20 and 40 °C respectively. As shown in Figure 3.4, at both 10 °C and 20 °C, at

least 60 min of centrifugation was required to achieve complete pelletisation of micellar

caseins. However, at long centrifugation times, compression of casein micelles was

evident as a reduction in the pelleted water (Table 3.1). At 40 °C, after 39 min of

ultracentrifugation, the consistent pelletisation was achieved throughout the range of

times investigated (Figure 3.4), with no evidence of micelle compression (Table 3.1)

due to the already relatively dehydrated states of the micelles at this temperature (which

will be discussed in Chapter 5).

45

In order to pellet the micellar materials from the serum of original milk and get

comparable hydration results, a centrifugation time of 60 min was used in Chapter 4,

Chapter 6 and Chapter 7. In chapter 5 the only variable that was tested was the

temperature. As such, the ultracentrifugation time were adjusted to account for the

change in viscosity of the solvent as function of temperature. To fractionate the casein

micelles into broadly different sizes based on different centrifugation (details discussed

in section 5.2.2), ultracentrifugation was performed for 78 min at 10 °C and 39 min at

40 °C at different rotational speeds in accordance to Stokes law.

Figure 3.4 The amount of soluble caseins in the supernatants of original skim milk after

different period of ultracentrifugation at 10 °C (), 20 °C () and 40°C () relative to

the 90 min ultracentrifuged milk samples. Error bars represent the standard deviation of

duplicate measurements of a single sample.

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

20 40 60 80 100

Re

lati

ve a

mo

un

t o

f so

lub

le c

ase

ins

in

sup

ern

atan

t

Centrifugation time (min)

46

Table 3.1 Pelleted micelle hydration of skim milk after different period of

ultracentrifugation

Sample (Temperature, centrifugation time) Micelle Hydration (g water/g solids)

10 °C-30 min 3.22±0.23

10 °C-60 min 2.91±0.14

10 °C-78 min 2.89±0.19

10 °C-90 min 2.57±0.16

20 °C-30 min 2.72±0.21

20 °C-60 min 2.50±0.13

20 °C-78 min 2.49±0.18

20 °C-90 min 2.27±0.15

40 °C-30 min 2.08±0.10

40 °C-39 min 2.04±0.07

40 °C-60 min 2.05±0.05

40 °C-90 min 2.04±0.06

Errors represent the standard deviation of duplicate measurements of a single sample

During vacuum evaporation and ultrafiltration, milk is concentrated and its viscosity

increases. To verify the ultracentrifugation condition (most importantly the

centrifugation time), the micelle hydration and the soluble protein concentration in the

supernatant were measured for a 42 % total solids (TS) content evaporative

concentrated milk (30 min, 60 min and 90 min of ultracentrifugation respectively at

40 °C) and four-fold concentrated (CF4) UF retentates generated at 10 °C and 40 °C UF

respectively (30, 60, 78, and 90 min for 10 °C ultracentrifugation and 30, 40, 60 and 90

min for 40 °C ultracentrifugation).

As shown in Figure 3.5 and Table 3.2, after 60 min ultracentrifugation, the soluble

casein concentration in the supernatant of both UF retentates and evaporative

concentrated milk was stable up to 90 min ultracentrifugation. This indicates that the

casein micelles were properly pelleted after 60 min of centrifugation. The hydration

showed no change at 40 °C after 60 min of centrifugation, and it decreased at 10 °C

centrifugation as a function of time, which suggested a compression of the casein

micelles at long centrifugation times. These results show that 60 min of

ultracentrifugation is able to separate micellar caseins from concentrated milk samples

47

and get comparable hydration trends. In order to separate casein micelles from milk

system and to study the micelle hydration changes, milk concentrates were

ultracentrifuged for 60 min in both Chapter 4 and Chapter 6.

Figure 3.5 The amount of soluble caseins in the supernatants of ultrafiltered CF4

retentates and evaporative concentrated skim milk (42% TS). CF4 retentate at 10 °C ()

and 40°C () and evaporative concentrated skim milk (42% TS) at 40 °C () after

different period of ultracentrifugation relative to the 90 min ultracentrifuged milk

samples. Error bars represent the standard deviation of duplicate measurements of a

single sample.

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

20 40 60 80 100

Re

lati

ve a

mo

un

t o

f so

lub

le c

ase

ins

in

sup

ern

atan

t

Centrifugation time (min)

48

Table 3.2 Pelleted micelle hydration of concentrated milk samples after different period

of ultracentrifugation

Sample (Temperature, centrifugation time) Micelle Hydration (g water/g solids)

CF4-10 °C-30 min 3.43±0.17

CF4-10 °C-60 min 3.30±0.11

CF4-10 °C-78 min 3.26±0.10

CF4-10 °C-90 min 3.12±0.08

CF4-40 °C-30 min 2.24±0.15

CF4-40 °C-39 min 2.25±0.07

CF4-40 °C-60 min 2.23±0.09

CF4-40 °C-90 min 2.25±0.06

42% TS-40 °C-30 min 1.39±0.12

42% TS -40 °C-60 min 1.21±0.07

42% TS -40 °C-90 min 1.20±0.09

Errors represent the standard deviation of duplicate measurements of a single sample

3.6 Calcium measurement

As mentioned in section 2.1.5, milk contains colloidal minerals and soluble minerals in

the system. It has been shown that calcium exists as micellar calcium and serum

(soluble) calcium in milk (Wahlgren, Dejmek & Drakenberg, 1990; Yamauchi, Yoneda,

Koga & Tsugo, 1969). The micellar calcium is the calcium bound to the casein micelles,

and it is assumed to be associated with phosphate as CCP, whilst the serum (soluble)

calcium includes ionic calcium, calcium-anion complexes and calcium associated with

serum caseins (Martin, 2008; Wahlgren et al., 1990). In this study, changes in the

partitioning of calcium between serum and micelle phase were well investigated.

Atomic absorption spectroscopy (AAS) is now a routinely and widely employed

technique for trace analysis of complex liquid samples and has been widely used for

dairy products (Jeng, Lee & Lin, 1994; Murthy & Rhea, 1967; Murthy & Rhea, 1968).

In this thesis, the calcium content of different milk samples was determined by using

AAS (see section 4.2.4). Lanthanum is a releasing agent which releases calcium from

phosphates, and it was added to overcome the interferences of phosphates (Welch,

Hamar & Fettman, 1990).

49

3.7 Lactose measurement

The lactose content in milk samples and in casein micelles was measured by using

enzymatic method. Skim milk samples and resuspended ultracentrifugation pelleted

casein micelles were deproteinated following the ISO standard (ISO 5764/IDF 79-part

2). Carrez reagents which have proven to be very effective in removing turbid

impurities and in breaking up emulsions (Kleyn, 1985) were used to remove proteins.

Lactose in these pre-treated samples was then hydrolysed to glucose and galactose by

using lactase. The galactose was subsequently oxidized and generate colour (OD 570

nm). Lactose amount was then plotted verses absorption readings. Background control

was necessary to correct the free galactose reading in the samples.

3.8 SDS-PAGE and Native-PAGE

Polyacrylamide gel electrophoresis (PAGE) is a widely used technique to separate

proteins. It is based on the different mobility of macromolecules through the gel, which

is depending on the size, conformation and charge of the proteins. In sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE), SDS is used to denature,

linearize and impact charge on the proteins. In Native-PAGE, the conformation of

proteins or protein complexes remains intact. In this study, both SDS-PAGE (see

section 4.2.3) and native-PAGE (see section 6.2.4) were used in this study to determine

the relative concentration of proteins in milk samples in both denatured and native states.

50

4 Evaporative concentration of skim milk: Effect on casein micelle

hydration, composition, and size

(This Chapter has been published as Liu, D. Z., Dunstan, D. E., & Martin, G. J. O.

(2012). Evaporative concentration of skimmed milk: Effect on casein micelle hydration,

composition, and size. Food Chemistry, 134 (3), 1446-1452.)

4.1 Introduction

As discussed in Chapter 2, evaporative concentration is an important step in milk

processing, which is used to reduce the content of water economically. It alters the

physicochemical state of the milk, affecting process relevant properties such as viscosity,

and influencing the functional properties of the end products. As mentioned above, the

casein micelles which consist of four different proteins (αs1-, αs2-, β-, and κ-casein), and

colloidal calcium phosphate (CCP) (Bouchoux et al., 2010; Dalgleish, 2011a), have the

greatest impact on the macroscopic and functional properties of skim milk. However,

little is currently known about how the physicochemical properties of the casein

micelles change in response to evaporative concentration.

The casein micelles have highly hydrated internal structures which consist

predominantly of αs1-, αs2-, and β-casein and nanoclusters of colloidal calcium

phosphate, while the κ-casein is located preferentially on the micelle surface (Dalgleish,

2011a). Studying the effect of the combination of heating and water removal during

evaporative concentration on casein micelles would improve the understanding of the

behaviour of casein micelle during concentration and ultimately the properties of the

resulting products.

In this chapter, the effect of evaporative concentration on the hydration, composition

and size of casein micelles in fresh skim milk were examined for the first time. The

hydration and composition of casein micelles in concentrated milk was first investigated

by direct analysis of a series of concentrate samples. Additional experiments were

performed on samples re-diluted with water back to the original concentration to

examine the extent and rate of reversibility.

51

4.2 Materials and methods

To gain new knowledge into the alterations of casein micelles during evaporative

concentration a systematic approach had to be developed. A series of preparative and

analytical steps were performed for this purpose as outlined in Figure 4.1.

Figure 4.1 Analyses performed on evaporative concentrates

4.2.1 Skim milk and concentrated skim milk samples

Fresh skim milk was purchased from local supermarket with 33 g·L-1

protein, 1.5 g·L-1

total fat and 52 g·L-1

carbohydrate. All analyses were performed within two days of

purchase after first bringing the samples to room temperature and then allowing them to

equilibrate at this temperature for at least 1 hour. Skimmed milk samples were

concentrated at 50 °C in a rotary evaporator at 355±5 Torr (47.32±0.67 kPa) for

different times to acquire different concentrations of milk. Total solids content of the

samples was determined by oven drying 24 hours at 105 °C. Redilution of concentrated

samples back to the concentration of the original skimmed milk sample was done by

accurately replacing the mass lost though evaporation with distilled water.

52

4.2.2 Casein micelle hydration rate and mass balance

Accurately weighed samples of skimmed milk concentrates were added to pre-weighed

centrifuged tubes and ultracentrifuged at 35,000 rpm (75,940 g average) for 1 hour at

40 °C for concentrated samples and 20 °C for re-diluted samples (Ultra 90, 90 Ti rotor,

Beckman Instruments Inc., Palo Alto, CA). The supernatant was carefully removed and

the mass of the wet pellet determined. The pellet was dried at 105 °C for 24 hours in the

oven and the mass for the dry pellet recorded.

4.2.3 SDS-PAGE

Casein micelles were separated from the milk serum by ultracentrifugation (as described

in section 4.2.2). The protein content of the supernatants was analysed by SDS-PAGE

using a BioRad Criterion Cell electrophoresis unit (BioRad Laboratories, Richmond,

CA, USA). The SDS-PAGE was performed by diluting sample in 1 mM EDTA (pH 8)

buffer. First, 20 µl of diluted samples were mixed with 20 µl of BioRad Laemmli buffer

containing 5% beta mercaptoethanol and placed in a boiling water bath for 5 min. Next

aliquots (10 µl) of samples were loaded into 8-16% linear gradient precast Tris-HCl

criterion 18 well gels and run at 100 V for 130 min. Gels were stained with Biosafe

Coomassie Blue (BioRad, Laboratories, Richmond, CA, USA) and digitally scanned

and quantified using a BioRad Gel Dox XR + Imager (BioRad Laboratories, Richmond,

CA, USA).

4.2.4 Calcium measurement

Proteins remaining in the supernatants from ultracentrifuation were precipitated by

mixing 0.5 g supernatant with 5.5 g of 24% (w/w) trichloroacetic acid (TCA) and 4.5 g

distilled water. Precipitated protein was removed by vacuum filtration through

Whatman No. 1 filter paper with the filtrate collected in a pre-weighed flask. The

proteins on the filter paper were washed with 2 g of 12% (w/w) TCA solution and the

total mass of filtrate recorded. Filtrate samples (4 g) were then mixed with LaCl3

suppressant (2 g) and water (16 g). The calcium content was determined accompanied

with commercially available AAS calcium standards (Fluka, Sigma-Aldrich) by AAS at

422.7 nm using a Varian AA 140 (Agilent Technologies, Santa Clara, CA) with an air

acetylene flame.

53

4.2.5 Turbidity and viscosity measurements

Turbidity was measured by transmission of light (860 nm) through a 2 mm pathlength

quartz cuvette using Cary 3E UV-Visible spectrophotometer. The relative turbidity was

determined by dividing the turbidity of re-diluted milk concentrates by the turbidity of

the original skimmed milk sample. Since only alterations to the casein micelles were of

interest, variation resulting from differences in the fat content of different samples was

avoided by subtracting the turbidity of the fat globules. To measure the turbidity

contributed by the fat globules, samples were diluted 10 fold in 10 g·L-1

EDTA buffer

adjusted to pH 7. The relative turbidity was therefore calculated as follows:

(4.1)

Where: τsample and τskim are the turbidity of the re-diluted milk concentrate sample and

the original skim milk respectively,

τsample fat and τskim fat are the turbidity of the re-diluted milk concentrate sample

and the original skim milk diluted in EDTA multiplied by 10 to account for the dilution.

Viscosity data was obtained by capillary viscometry (type 531-03/0c, Schott AG),

performed in triplicate at 25 °C.

4.2.6 Particle size analysis

Hydrodynamic particle size analysis was performed by photon correlation spectroscopy

using a Malvern Autosizer 4700 PCS (Malvern instruments Ltd., Malvern,

Worcestershire, United Kingdom) with a 488 nm laser. Light was measured at

scattering angle of 90 ° and the temperature was maintained at 25 °C. Data presented

are the intensity weighted average particle size calculated by the method of cumulants.

Samples were diluted to approximately 1/10,000 of the concentration of the original

skim milk in a simplified simulated milk ultrafiltrate buffer (as described in section 3.4).

54

4.3 Results

4.3.1 Direct measurements of concentrated milk

Three different samples of skim milk were evaporated to different solids concentration

at 50 °C under vacuum. It took approximately 20, 35, 40 and 43 min to reach 14%, 25%,

35% and 40% TS, respectively. To prevent lactose crystallisation, samples of

concentrates were kept at 50 °C until ultracentrifugation and were maintained at 40 °C

during ultracentrifugation. Supernatants were carefully removed and an analysis of the

total mass of the pellet and the amount of solid material in the pellet was performed by

oven drying overnight (24 h). The hydration of the pellets (mass of water per mass of

total solids) was seen to decrease steadily from approximately 2.5 g/g for fresh

skimmed milk to about 1.2 g/g for skimmed milk concentrated to 42% (w/w) solids

(Figure 4.2A). A change in the mass fraction of total milk solids included in the pellets

as a function of concentration could not be observed within the limits of accuracy of the

data (Figure 4.2A). From this data the relative removal of water from the supernatant

and pellet fractions was determined. Removal of water from the supernatant accounts

for the majority of water removed and is approximately in line with the overall amount

of water removed from the milk (Figure 4.2B). Despite the scatter in the data, it is clear

that the removal of water from the pellet was considerably lower than that removed

from the supernatant across the entire range of concentrations. Water was therefore

removed from the serum in preference to removal from the micelles.

55

Figure 4.2 Hydration and water content of casein micelles during evaporation.

Hydration of the pellets (closed symbols) and mass fraction of total solids that are

pelleted (open symbols) during ultracentrifugation of concentrates kept at 40 °C as a

function of solids content (A). Percentage of original water in the supernatant (closed

symbols) and pellet (open symbols) fractions that remain after evaporative

concentration (B). Dashed line represents the overall percentage of water that is

removed as a function of solids concentration. Solid line is a guide for the eye. Different

symbols are the results from three separate fresh skim milk samples.

56

The concentration of calcium and casein in the supernatants from concentrates

ultracentrifuged at 40 °C were measured. This information was combined with the

known volume of supernatants so that the total amount of calcium and casein in the

supernatants could be determined. As the concentration of calcium increased during

evaporative concentration, the relative amount of total calcium in the supernatant

decreased as a function of water removal (Figure 4.3). At about 34% TS, less than 40%

of the serum calcium present in the fresh skim milk samples remained in the supernatant.

The relative amount of total casein in supernatants, as determined by SDS-PAGE

analysis, also decreased upon evaporative concentration. In contrast to the serum

calcium, the casein concentration appeared to decrease to below 40% of the original

immediately upon concentration to 12% solids and then remained between 40% and 50%

above 25% solids (Figure 4.3). The low concentration samples (12% and 16% solids)

were kept at 50 °C for an additional 1-1.5 h compared to the high concentration samples

(28% and 33% solids), which may explain the lower level of soluble casein in these

samples.

Figure 4.3 Total serum calcium and casein in the supernatant during evaporation.

Total amount of serum calcium () and casein () in the supernatant relative to the

supernatant of original skim milk. Error bars are the standard deviation of duplicate

SDS-PAGE analysis. Measurement error was negligible for the calcium data.

57

4.3.2 Measurements of re-diluted concentrated milk

The results above suggest that a number of important changes to the casein-mineral

system occur during evaporative concentration. To obtain a more complete

understanding of this behaviour, including insight into the reversibility, analyses were

preformed on concentrates rediluted to the solids concentration of the original skimmed

milk samples by addition of distilled water. The first of these analyses was an

examination of pellet hydration to understand whether or not the micelles can be fully

and rapidly rehydrated. The total amount of water and solids associated with the pellet

returned to 92% and 99%, respectively, of that in the original skim milk within the time

constraints of measurement (i.e. ca 1h) (Table 4.1). The micelle in the rediluted

concentrates were mostly but not completely rehydrated (ca 2.6 g water/g solids) relative to

those in the original milk (ca 2.8 g water/g solids).

Table 4.1 Rehydration of casein micelles upon redilution of concentrated skim milk.

Comparison of pelleted solids, pelleted water, and pellet hydration for ultracentrifuged

skim milk and a corresponding sample of concentrate (34% TS) rediluted to 8.7% TS.

Sample Pellet hydration (g

water/g solids)

Pelleted solids1

(g/100 g skim)

Pelleted water1

(g/ 100 g skim)

Original skim milk 2.78±0.025 3.01±0.005 8.37±0.09

34% Concentrate2 2.60±0.002 2.97±0.01 7.73±0.02

1 Mass of solids pelleted per 100 g of original skim milk used to produce concentrate.

2 rediluted to 8.7% and immediately ultracentrifuged

Error represents the standard deviation of triplicate measurements.

The size of casein micelles in the concentrated samples could not be measured directly

by conventional dynamic light scattering as they are too optically dense. Turbidity and

dynamic light scattering measurements were performed immediately upon redilution of

concentrate samples, giving information about the immediate reversibility of alteration

caused by concentration. The considerable amount of water removed from the micelles

(Figure 4.2) indicates the micelles must be in a contracted state in the concentrates. As

58

the micelles were almost fully rehydrated upon redilution (Table 4.1) a somewhat

similar micelle size would be expected. Measurements of casein micelle size were

performed with about 3 min of diluting the concentrates to about 1/10,000 of the

original skim milk concentration in simplified simulated ultrafiltrate buffer (described

in section 3.4). The average micelle size in rediluted samples initially decreased slightly

(ca 5%) as a function of evaporative concentration up to about 25% solids, above which

the size started to increase. The turbidity of samples diluted back to the original

concentration (8.7% TS) by addition of water were higher than the original milk and

showed a steady increase as a function of solids concentration (Figure 4.4).

Figure 4.4 Turbidity and average micelle size of rediluted evaporative concentrated milk.

Turbidity (open symbols) and average micelle size (closed symbols) of concentrated

skim milk samples diluted to the original solids concentration relative to the un-

concentrated skim milk samples. The different symbols represent data obtained from

different batches of skim milk. Error bars represent the standard deviation of at least

duplicate PCS measurements. Measurement error was negligible for the turbidity data.

Turbidity was corrected for the presence of fat globules.

59

Due to experimental constraints imposed by ultracentrifugation it was not possible to

acquire supernatant samples immediately upon redilution. Instead, supernatant samples

were obtained for a range of concentrate samples 2.5 h after redilution to original

concentration. The concentration of calcium and casein in the supernatants were

measured. After redilution for 2.5 h, the amount of calcium in the supernatant had

returned to its initial values for both the 12% and 20% samples and was very close for

the 45% samples (Figure 4.5). The amount of casein however, remained considerably

below the initial values for all samples.

Figure 4.5 Total serum calcium and casein in the supernatants of rediluted evaporative

concentrates. Total amount of serum calcium () and casein (CN) () in the

supernatants of rediluted concentrates relative to those of the original skim milk.

Concentrated samples were rediluted with distilled water back to 8.7% solids and left to

equilibrate for 2.5 h before ultracentrifugation. Error bars are the standard deviation of

duplicate SDS-PAGE analyses. Measurement error was negligible for the calcium data.

To further investigate post dilution reversibility, detailed kinetic studies were performed

on samples concentrated to ca 40% solids. The average particle size, turbidity, viscosity

60

and pH of a sample concentrated to 39% solids were measured as a function of time

after re-dilution to the original concentration. The average particle size was initially

higher than that of the original milk and dropped to close to that of the original milk

with the first hour after redilution (Figure 4.6A). The viscosity, however, remained

relatively constant and slightly below that of the original skim milk. The turbidity of

rediluted milk decreased particularly rapidly during the first 20 min or so and then

continued to drop more slowly, eventually dropping below the turbidity of the original

skim milk (Figure 4.6B). The turbidity presented here did not match the results in

Figure 4.4, owing to the fact that a correction for the presence of fat globules was not

applied. It is not entirely clear why the initial particle size of the 39% solid sample

examined in the kinetic study (Figure 4.6A) was higher than that previously observed

for similar samples (Figure 4.4). This could be due to a slight difference in experimental

procedure-the kinetic study sample was initially rediluted with water before dilution in

buffer for DLS measurement, whereas the other samples were immediately diluted in

DLS buffer containing minerals.

The pH initially increased in a similar manner to the decrease in turbidity, but instead

appeared to stabilise close to the original pH after about 2 h (Figure 4.6B). The amount

of soluble casein in the supernatants was determined for two samples concentrated to

similar solids content and shown to slowly increase from about 40% to about 50% of

that in supernatants of the original skim milk (Figure 4.6C).

61

Figure 4.6 Relative alterations of average micelle size, viscosity, pH, turbidity and

soluble caseins of rediluted evaporative concentrates.

Relative average micelle size () and viscosity () of skim milk rediluted to original

concentration (8.7 % solids w/w) from concentrates of 39% solids (w/w) as a function

of time after redilution (A). Relative alterations of turbidity () and pH () of skim

milk rediluted to original concentration (8.7 % solids w/w) from concentrates of 45%

+/- 1% solids (w/w) () as a function of time after redilution (B). Turbidity was not

corrected for fat globules. The concentration of casein in the supernatants of a 42% ()

and a 44% () concentrate relative to that of the original skim milk as a function of

time after re-dilution to 8.7% solids (C). Error bars are the standard deviation of

duplicate SDS-PAGE analyses.

62

4.4 Discussion

4.4.1 Hydration of casein micelles

Water in milk is found in two important stores, as inter-micellar water of the serum and

the intra-micellar water associated with the micelles. In the serum of native skim milk,

much of the water can be considered unstructured bulk water with only a portion of the

molecules associated with the lactose, whey proteins and various milk salts (Fox et al.,

1998a). The micellar water occupies the internal ‘channels’ (Dalgleish, 2011a) as well

as being associated with both the internal and external hydrophilic components,

including the CCP, internal casein, and hairy layer. While it has long been recognised

that water removal could cause shrinkage of micelles (Morr, 1975) direct quantification

of the extent of micelle dehydration as a function of evaporation does not appear to have

been performed. The fundamental effect of evaporative concentration of skim milk is

simply the removal of water. It is clear from these results (Figure 4.2) that both intra-

and inter-micellar water were removed and that water was removed preferentially from

the serum. It appears reasonable that the free water in the bulk serum will be more

mobile and available for evaporation than the water bound within micelles. This has the

effect that water within the micelles is only removed after much of the bulk water has

been evaporated. As a result of this, the inter-micelle distance must be less, and the

micelle size must be greater, than would be expected if water was removed equally from

within and outside the casein micelles. This observation should help explain the

rheological behaviour of concentrated milk systems.

The redilution results show that the micelles rapidly regained most of their original

water content upon dilution to the original concentration. There was a lower micelle

hydration of the rediluted milk samples, which is presumably due to a denser micelle

structure that includes a greater proportion of both CCP and casein (Figure 4.3). The

viscosity results confirm this interpretation as the rediluted samples show a lower

viscosity, indicative of a lower volume fraction, than the original skim milk even after

dilution for 5 h (Figure 4.6A). This shows that irreversible changes to the hydration and

structure of casein micelles occurred during evaporative concentration. The rapid

rehydration would suggest that the micelles behave somewhat like a sponge, quickly

absorbing water when it becomes available. This is in agreement with previous

observations in which fully dehydrated micelle in skim milk powder were indirectly

found to immediately rehydrate upon reconstitution (Martin et al., 2007). The

63

incomplete rehydration suggests that changes to the structure of the micelles must occur

to diminish the overall water holding capacity of the micelles. The fact that the

hydration data was obtained by ultracentrifuation suggests this change is not due solely

to a contraction of the hairy layer, since this layer would be compressed in the pellet.

Whether the decrease is associated with the water bound directly to the internal

components of the micelles or the water in the open internal spaces within the micelle is

not clear. It is possible that new protein associations are formed on water removal that

permanently collapse some of the open micelle structure as discussed in the sponge

model in Chapter 2.

4.4.2 Composition of casein micelles

In native skim milk only a small proportion of the casein is present in the serum. The

partitioning of casein between the micelles and the serum is highly temperature

dependent, with the amount of soluble casein decreasing with increasing temperature

(Ali et al., 1980; Creamer et al., 1977; Davies et al., 1983; Downey et al., 1970; Rose,

1968). Evaporative concentration invariably involves increasing the temperature of milk,

typically to 40-50 °C. It is therefore not surprising to have seen a decrease in the amount

of casein in the supernatants of concentrate samples (Figure 4.3). No direct attempt was

made in this chapter to separate the effects of heating and concentration and it is not

clear whether or not the increased temperature can entirely account for the observed

behaviour, in particular the irreversibility of the casein partitioning (Figure 4.5).

Irreversibility of protein partitioning was previously observed with reconstituted

skimmed milk powder (SMP) (Martin et al., 2007).

During evaporative concentration serum calcium will be driven from the serum to the

micelle via at least two mechanisms:

(1) The capacity of the mineral system to maintain serum calcium and phosphate

species will be reduced due to removal of water.

(2) The increased temperature lowers the solubility of calcium phosphate. The water

removal process clearly caused a reduction in the amount of serum calcium (Figure 4.3)

meaning an increase in the amount calcium associated with the micelles.

64

This has also been observed previously (Le Graët et al., 1982; Vujicic et al., 1966). It is

still unclear exactly how and where the additional calcium is deposited on the micelles.

It is not known for instance whether or not it associates directly with existing CCP

nanoclusters. In addition, as the mineral concentration in the serum rises due to

evaporation, the dynamics of the mineral equilibrium between CCP and the soluble

phase will be affected; however, information is incomplete and further investigation is

still required (Holt, de Kruif, Tuinier & Timmins, 2003; Nair, Alexander, Dalgleish &

Corredig, 2014). The increase in pH (Figure 4.6B) upon dilution is consistent with the

reversal of calcium back into the serum (Figure 4.5). The reversibility of the calcium

partitioning was seen for SMP, however the kinetics appeared to be much slower

(Martin et al., 2007).

Although it is evident that the contraction of micelles is almost immediately reversible

upon re-dilution, the reversibility of the calcium and caseins shows quite different

behaviours. The serum calcium returns to the serum completely over 1 h, while only a

small amount of the original soluble caseins return to the serum within 5 h. The slow

reversibility of the soluble casein may be due to increased associations that results from

both the contraction of the micelles due to water removal and the uptake of casein and

calcium resulting from the increased temperature.

4.4.3 Size of casein micelles

According to dynamic light scattering results on rediluted concentrates (Figure 4.4), the

micelle size appeared to decrease as a function of concentration up to about 25% TS and

then increase at higher concentrations. The reason for the decrease is likely to be an

overall contraction or shrinkage of the micelles due to the incomplete rehydration. This

is consistent with a lower viscosity as observed in this chapter (Figure 4.6A). The

increase in particle size at higher concentration, as measured by DLS, can be explained

by the presence of a small number of larger particles, perhaps formed from aggregation

of micelles. It has previously been observed that the size of the casein micelles in

concentrated milk can be affected by the holding time. One previous study looked at

particle size on diluted skim milk concentrates (45% TS) as a function of storage at

50 °C (Bienvenue et al., 2003). Samples immediately measured after concentration had

only one peak characteristic of casein micelles, whereas additional larger peaks (>500

65

nm) appeared after 6 h of storage, indicating aggregation of micelles. The increased

micelle size observed in the previous study on SMP (Martin et al., 2007) may have

resulted from the time kept under concentrated conditions. The concentrated samples in

that study were not able to be immediately analysed. Concentrates are often held for a

period of time before spray-drying, which could be the reason that the reconstituted

SMP micelles were larger compared to the results in this investigation here. Based on

these understandings, temperature effects on casein micelle were studied in the next

chapter.

The observed increase in turbidity (Figure 4.4) is most likely primarily due to an

increase in the micelle refractive index resulting from the increase in optically dense

CCP and casein. Any increase in particle size due to aggregation would also contribute

to the increase; however the overall decrease in volume fraction due to micelle

contraction would reduce it. The observed decrease in turbidity after redilution (Figure

4.6B) is consistent with the solubilisation of CCP, which would decrease the refractive

index. The eventual decrease of the turbidity below that of original skim milk (without a

continue increase in pH) would be consistent with a population of micelles that occupy

less total volume due to the micelles not being fully rehydrated. This could result in a

lower turbidity despite having a higher average size according to DLS results (Figure

4.6A) and an equivalent refractive index. The rapid initial decrease in turbidity appears

in part due to the initial decrease in micelle size (Figure 4.6A).

These results would suggest that the size of casein micelles is affected by two separate

mechanisms (shown in Figure 4.7). Firstly the micelles undergo contraction due to

water removal, which is not completely reversible upon redilution. This results in

micelles that must be considerably contracted in the concentrated state, and while

expanding upon rehydration, occupy a lower volume fraction than in fresh skim milk.

Secondly, at higher concentration micelles undergo some aggregation (or reformation

into larger micelles), that continues as a function of holding time at high concentration.

66

Figure 4.7 Casein micelle alterations during skim milk evaporative concentration and

re-dilution.

(Shading indicates the water content of the micelles, dark: less water, light: more water).

4.5 Conclusions

During evaporative concentration of skim milk, casein micelles were dehydrated; but

water was removed from the serum preferentially to removal from within the micelles.

The partitioning of both calcium and casein was shifted from the serum towards the

micellar phase. On reversal of the concentration process by re-dilution the micelles were

immediately rehydrated, although incompletely. The micelles remained somewhat

contracted, although the formation of some larger micelles was evident at higher solids

content. The alteration to the mineral balance and the presence of larger micelles were

less permanent, while loss of soluble casein was more permanent.

67

5 Temperature-dependent dynamics of bovine casein micelle in the

range 10-40 °C

(This Chapter has been published as Liu, D. Z., Weeks, M. G., Dunstan, D. E., &

Martin, G. J. O. (2013). Temperature-dependent dynamics of bovine casein micelles in

the range 10 – 40 °C. Food Chemistry, 141 (4), 4081-4086.)

5.1 Introduction

During the processing of milk, temperature is an important variable that affects a wide

range of physicochemical properties (Fox et al., 1998a). Whilst the native temperature

of bovine milk is 37 °C, it is usually stored cold (<10 °C) to control microbial growth.

The temperature can then be varied over a wide range during processing. Whilst heat

treatment of milk at temperatures above 70 °C can considerably alter the properties of

milk (Livney, Corredig & Dalgleish, 2003), changes in temperature below 50 °C also

have a significant effect on the various components of milk, including the casein.

Casein represents approximately 80% of the protein in milk and is predominantly

present in the form of large hydrated assemblies called casein micelles (CM) (Dalgleish,

2011a). CMs consist of four different species of casein proteins and calcium phosphate,

and are in dynamic equilibrium with the milk serum (Walstra, 1990). In particular,

calcium phosphate and casein are exchanged between the micelles and the serum, with

the partitioning of these components being influenced by temperature (Davies et al.,

1960; Rose, 1968).

The amount of calcium in the serum is known to decrease as a function of increasing

temperature, with calcium shifting from the serum into the micelles as CCP (Davies et

al., 1960; Rose et al., 1959). In a comprehensive study of the effect of temperature on

mineral balance in milk serum calcium was shown to decrease very rapidly (<2 min) as

a function of temperature on heating milk from 4 to 20-90 °C (Pouliot, Boulet & Paquin,

1989a). The reversibility on cooling of milk heated to 85 °C was considerably slower,

taking up to 60 min to reach 90-95% reversal; however, the reversibility of milk

warmed to 40 °C was not investigated (Pouliot et al., 1989b). Whilst the rate of

reversibility on cooling was attributed to the complex equilibrium between calcium

phosphate and citrate salts, it has also been shown that the rate of exchange of calcium

68

and phosphate between diffusible and colloidal phases is increased at higher

temperature (Zhang, Fujii & Aoki, 1996).

It is generally understood that casein dissociates from micelles at low temperature, and

that β-casein is the predominant protein released (Creamer et al., 1977; Downey et al.,

1970; Rose, 1968). Whilst the equilibrium between soluble and micellar casein is

known to be affected by temperature, the rate at which equilibrium is reached after

temperature change has not been properly investigated. In one study in which milk was

cooled from 37 to 0 or 5 °C, β-casein release was shown to occur over within about 60

min (Creamer et al., 1977). In contrast, the concentration of soluble casein was found to

continue to change at least 3 days after cooling milk from 20 °C to temperatures in the

range 4-15 °C (Ali et al., 1980). Casein released during storage at 4 °C has been shown

to be completely reversible after 18 h of incubation at 20 °C (Davies et al., 1983),

however the kinetics were not investigated.

In addition to affecting the partitioning of calcium and casein, the size and hydration of

casein micelles are also influenced by temperature. At high temperature (i.e. >70 °C)

attachment of denatured whey proteins and heat induced aggregation are known to

increase the hydrodynamic size of casein micelles (Anema et al., 2003; Tran Le, Saveyn,

Hoa & Van der Meeren, 2008). In the range 0-50 °C, casein micelle size is also affected

by temperature, but not due to interactions with whey protein. This temperature range is

particularly important for processing such as evaporative concentration and membrane

filtration of milk. Dynamic light scattering measurements performed at 6, 20, and 50 °C

showed the effect of temperature on micelle size to be different depending on the

dilution solvent used (milk ultrafiltrate, SMUF, or water) (Beliciu & Moraru, 2009). In

water or SMUF micelles were larger at increasing temperature. In UF permeate the

micelles were approximately the same size at 6 or 20 °C but considerably smaller at

50 °C. Whilst UF permeate should be closest to the native environment of the casein

micelles in undiluted milk, the permeates used in this study were not obtained at the

same temperature as the measurements and would therefore have an altered mineral

content (Beliciu et al., 2009). According to results obtained by differential

centrifugation, cooling of milk to 4 °C decreases the population of larger micelles and

increases the population of smaller micelles (Davies et al., 1983; Ono et al., 1990). This

effect was shown to be fully reversible upon re-warming to 20 °C after 18 h; however

no indication of the kinetics of this reversal was provided (Davies et al., 1983). It has

69

also been found that the apparent voluminosity of casein micelles decreases with

increasing temperature (Dewan, Bloomfield, Chudgar & Morr, 1973; Snoeren et al.,

1984; Walstra, 1979).

Detailed kinetic information on the effect of temperature on casein micelle has so far

been limited by difficulties in measuring changes to casein micelles occurring over

short time periods in native milk samples. In particular, direct real-time measurements

of soluble casein, serum calcium, micelle size and micelle hydration are not possible

using conventional methods. To obtain close to real-time information about changes to

casein micelles, pH and turbidity measurements were employed in this chapter. These

measurements have very short response times, are sensitive to small changes in casein

micelle properties and able to be applied in-situ on milk samples at native concentration.

As these methods give indirect information about changes that are occurring within the

skim milk system, the results need to be interpreted in the context of more

comprehensive information on the equilibrium relationship underlying the various

alterations as a function of temperature and an understanding of the physico-chemical

properties of the skim milk system. The pH of skim milk results from the mineral

system in the serum. Although complex, the predominant alteration effecting pH that

will result from changes in the temperature is the exchange of calcium between the CCP

in the casein micelles and the soluble calcium in the serum (Fox et al., 1998a):

2

4

2 HPOCa HCCP

In this way, pH can be used as an indirect indicator of the equilibration of the mineral

system, including the partitioning of calcium between the micelle and the serum. As

discussed in previous chapters, in skim milk the casein micelles are responsible for the

majority of the measured turbidity and therefore any variation in turbidity can be

attributed to changes in the casein micelles (Martin et al., 2007). In this chapter, a

comprehensive study of temperature dependent equilibrium changes to casein micelles

in skim milk is combined with real-time measurement of dynamics between 10 and

40 °C (as the upper limit for ultracentrifugation is 40 °C).

Whilst much is already known about the general effect of temperature on casein

micelles in bovine milk, most studies have examined only one selected aspect of the

casein system (e.g. casein partitioning or micelle size). In addition, information on the

cooling

heating

70

dynamics of the casein system in response to temperature changes is lacking. A detailed

comparison of kinetic response to heating and cooling within the low temperature range

has not been performed and highly time resolved information is yet to be obtained. In

this chapter, a comprehensive investigation was performed on the effect of temperature

on casein and calcium partitioning between the micelle and the serum as well as the size

and hydration of casein micelle. This study examines both the equilibrium and kinetic

aspects of calcium and casein alterations in response to temperature. By examining

whether there is temporal correlation between calcium and casein alterations in response

to temperature, a better understanding of the level of physical interconnection between

these two phenomena can be gained.


A systematic approach was required to get new information relating to the kinetics of

the temperature dependence of casein micelles. Detailed summary of the analyses

performed on fresh milk as a function of temperature is shown in Figure 5.1.

Figure 5.1 Analysis performed on fresh skim milk as a function of temperature

71

5.2.1 Skim milk samples

Pasteurised fresh skim milk containing 33 g·L-1

protein, 1.5 g·L-1


carbohydrate was purchased from a local supermarket and stored at ca 10 °C and

analysed within 2 days. For equilibrium studies, milk samples were held to the desired

temperature for at least one hour before commencing analysis.

5.2.2 Fraction of casein by centrifugation

Centrifugation of skim milk samples was performed using an Ultra 90 ultracentrifuge

fitted with 90 Ti rotor (Beckman Instruments Inc., Palo Alto, CA, USA). According to

Ono et al., (1990), to account for differences in sedimentation velocity resulting from

the dependence of viscosity on temperature, centrifugation times (shown in Table 5.1)

were adjusted based on the equations previously described in section 3.5. The casein

concentration in the supernatants was determined by densitometric analysis of SDS-

PAGE gels as described in section 4.2.3.

Table 5.1 Fractionation of definite sized micelles

Casein and Casein

Micelles maintained

in supernatant

Medium, Small

casein micelle,

Soluble1

Small casein

micelle, Soluble

Soluble

Centrifugation Force 5700 g 25000 g 75940 g

10 °C 78 min 78 min 78 min

20 °C 60 min 60 min 60 min

40 °C 39 min 39 min 39 min 1Large micelle is in the pellet

5.2.3 Casein micelle hydration and serum calcium measurement

Skim milk samples maintained at 10, 20, and 40 °C were centrifuged at 75,940 g for 78,

60, or 39 min respectively. The hydration of casein micelles was determined by

gravimetric analysis of supernatants and pellets as described in section 4.2.2, and the

calcium content of the supernatants determined by AAS as previously described in

section 4.2.4.

72

5.2.4 Determination of casein micelle volume fraction

Skim milk UF permeates were generated from skim milk equilibrated and maintained at

10, 20 and 40 °C±0.1 °C using Lab-scaleTM

TFF system (Millipore, Billerica, MA)

fitted with cutoff of 10 kDa regenerated cellulose membrane. The viscosity of skim

milk and the corresponding UF permeates were measured in triplicate at 10, 20 and

40 °C±0.1 °C by capillary viscometry (type 531-10, Schott AG). The volume fraction of

casein micelles was calculated on the basis of the relative viscosity of the skim milk and

UF permeates at each temperature according to Eiler’s equation as described in section

3.2.

5.2.5 Dynamic light scattering

Milk contains about 1.62 mg/kg riboflavin which fluoresces strongly on excitation by

light of wavelengths from 400 to 500 nm. This can affect the fluorescence and dynamic

light scattering (DLS) measurements in that wavelength range when milk ultrafiltration

permeates involved as riboflavin goes through the membrane during milk ultrafiltration.

Particle size analysis of the casein micelles was performed by photon correlation

spectroscopy of skim milk samples diluted 75 fold in UF permeates obtained at 10, 20

and 40 °C. The temperature during DLS measurements was maintained at 10, 20 and

40 °C respectively. Scattered light was measured at an angle of 90 ° using a Brookhaven

BI-9000AT autocorrelator with a BI-200M goniometer (Brookhaven Instruments

Corporation, Holtsville, NY) fitted with a 633 nm laser (to avoid the effect of

riboflavin). The intensity weighted average hydrodynamic particle size was determined

using the method of cumulants as previously described (Martin et al., 2007).

5.2.6 Turbidity and pH measurement

0.6 ml aliquot of skim milk sample was transferred into a 2 mm path length quartz

cuvette and the turbidity was measured as described in section 4.2.5. pH was measured

using Aqua-pH pH-mv-temperature meter (TPS Pty Ltd., Brisbane Australia) and a

temperature probe was fitted for automatic adjustments of temperatures involved in the

pH tests.

73

5.2.7 Temperature kinetic studies

For the kinetic studies, small samples (ca 30 ml) of skim milk were warmed to the

desired starting temperature and pre-equilibrated for at least 1 h, before being cooled or

further warmed in a thin walled plastic cylinder immersed in a water bath held at 10, 20

or 40 °C. Temperature and pH probes were immersed in the samples during

warming/cooling to allow simultaneous real-time measurements. To maximise

warming/cooling rate and the response time of the probes, samples were mixed using a

magnetic stirrer bar. For turbidity measurements, samples were quickly transferred to

the temperature equilibrated quartz cuvettes and immediately measured (<1 min).

5.3 Results and discussion

5.3.1 Micelle equilibrium as a function of temperature

Soluble calcium and pH both decreased as a function of increasing temperature between

10 to 40 °C (Figure 5.2A), in agreement with previous observations (Davies et al., 1960;

Pouliot et al., 1989a; Rose et al., 1959). This is consistent with the known tendency for

calcium to shift from the serum to the micelle as temperature is increased (Fox et al.,

1998a) and shows that pH can be used to indicate changes to the mineral balance, in

particular the partitioning of calcium between the micelle and the serum. Soluble casein

also decreased at increased temperature (Figure 5.2B), again in general agreement with

previous studies (Creamer et al., 1977; Downey et al., 1970; Rose, 1968). Although not

quantified, the predominant soluble casein observed in SDS-PAGE gels was expectedly

β-casein (shown in Figure 5.3).

74

Figure 5.2 Equilibrium state of skim milk as a function of temperature.

(A) Serum calcium (sCa) (,) relative to that of skim milk at 10 °C and pH (,).

(B) Turbidity (,) and soluble casein (sCN) (,) relative to that of skim milk at

10 °C. (C) Micelle hydration (,) and micelle volume fraction (,) relative to that

of skim milk at 10 °C, and average diameter (,). Error bars represent the standard

deviation of duplicate (serum calcium, pH, turbidity, soluble casein, hydration, and

volume fraction) or triplicate measurements (average diameter) of a single sample.

Square and triangles represent individual experiments performed on two different skim

milk samples.

75

Figure 5.3 SDS-PAGE image of soluble milk proteins in ultracentrifugation (75940 g)

supernatants at different temperatures.

Both the hydration of the casein micelles as determined by centrifugation, and the

voluminosity of casein micelles determined by viscometry, were lower at higher

temperature (Figure 5.2C). This is again consistent with previous observations (Dewan

et al., 1973; Snoeren et al., 1984; Walstra, 1979). The data suggest that micelles are

contracting due to dehydration resulting from increased temperature. The DLS

measurements show there was not a simple relationship between the size of the casein

micelles and temperature, with largest average diameter observed at 40 °C and the

lowest at 20 °C (Figure 5.2C). The data set obtained by Beliciu et al. (2009) also

showed a complex relationship between average micelle size and temperature, which

was dependent on the diluents used in the light scattering measurements. In their study

they did not use controlled permeates obtained at the measurement temperatures as they

were not specifically interested in determining changes to micelles in response to

temperature. In this chapter, temperature matched permeates were used to provide a

native environment for the micelles during these measurements, giving a better

indication of the effects of temperature on average micelle size. The effects of other

temperature related changes on the light scattering measurements were considered.

76

Whilst changes in the refractive index of the micelles and the serum had negligible

influence on the average size, the viscosity difference of the serum at the two

temperatures had to be directly accounted for. Whilst attempts were made, it was not

possible to obtain reliable and meaningful size distribution data from DLS

measurements of the highly polydisperse skim milk system. Consequently, only

intensity-weighted average diameter (Figure 5.2C) and polydispersity results are

reported here. The average micelle diameter was within a typical range of 170-200 nm

(Martin et al., 2007) across the temperatures tested, with the minimum size of 177 nm at

20 °C and the highest value of 200 nm at 40 °C. The polydispersity trended with the

average diameter, highest at 40 °C (0.149±0.008), lowest at 20 °C (0.124±0.017), with

an intermediate value at 10 °C (0.130±0.017). The intensity weighted average size value

gives an indication of relative changes in micelle size, but is strongly influenced by

larger particles and does not give information about changes throughout the broad size

distribution of casein micelles. To examine the effect of temperature on casein micelle

size in more detail, differential centrifugation was used to fractionate the micelle

population on the basis of sedimentation velocity (as discussed in section 3.5). The

amount of soluble casein decreased whilst the population of small casein micelles was

found to increase as temperature increased from 10 to 40 °C (Figure 5.4). This could be

explained by micellization of the soluble casein, which has been shown to be greater at

higher temperatures due to increased hydrophobic effect (Mikheeva et al., 2003). Less

simple trends in the populations of medium and large micelles were observed.

Nonetheless, the minimum amount of larger micelle material was obtained at 20 °C

which is compatible with average diameter results obtained by dynamic light scattering.

The combination of hydration, voluminosity, average diameter, and micelle

fractionation data suggests a relatively complex rearrangement of the casein micelles

occurs as a function of temperature. Despite the overall contraction of the casein

micelles indicated by the hydration and voluminosity results, the size of the larger

micelles appears to be greater at 40 °C than at 10 or 20 °C.

77

Figure 5.4 Relative amount of casein present as soluble casein, ‘small’ casein micelles

(CM), ‘medium’ CM, or ‘large’ CM in skim milk as a function of temperature as

determined by different centrifugation.

Error bars represent the standard deviation of duplicate measurements of samples from

duplicate experiments.

The turbidity of skim milk was found to increase as a function of increased temperature

(Figure 5.2B). The turbidity of skim milk is predominantly due to casein micelles, and

was not affected by microbial activity. Whilst the relationship between turbidity and the

physico-chemical properties of the casein micelle population is complex, a number of

observations can be made. Larger and more optically dense casein micelles scatter more

light. Therefore increases in the size and refractive index (RI) of the micelles will result

in an increase in turbidity assuming other parameters are constant. The observed

decreased in soluble calcium at higher temperatures is due to an increase in CCP which

increases the RI and hence turbidity. The dehydration and contraction of the micelles

will increase the RI, but will decrease the total volume fraction occupied by the casein

micelles which will have the opposite effect, making it difficult to predict the exact

effect on the turbidity. The effect of the complicated changes in casein micelle size

determined by dynamic light scattering and differential centrifugation on the turbidity

are also difficult to be accurately predicted.

78

Overall, it can be surmised that the pH of skim milk is indicative of alterations to the

mineral system, whilst the turbidity is a function of alterations to both the mineral and

micelle systems, including soluble casein, micelle voluminosity and size. The real-time

measurements of pH and turbidity can therefore be used to investigate the dynamics of

this system. These results can be interpreted on the basis of these equilibrium results

which showed that the soluble casein, soluble calcium and pH of skim milk all

decreased as a function of increasing temperature (Figure 5.2A and B) and that the

hydration and volume fraction of the casein micelles also decreased as a function of

increasing temperature (Figure 5.2C).

5.3.2 Micelle dynamics

A number of kinetic experiments were performed both warming and cooling milk. Two

examples sets of data are shown in Figure 5.5. Real-time measurements of the

temperature showed that the milk samples were heated or cooled to about 80% of the

total temperature difference with 5 min and to 95% within 15 min. On warming from 10

to 20 °C the relative pH increase tracked instantaneously with that of the temperature

increase, suggesting that the rate of pH change was instantaneous within the limits of

measurement. Similarly, the relative pH change on warming from 10 to 40 °C and

cooling from 40 or 20 to 10 °C also occurred within the time resolution of the

measurement (Figure 5.5).

The response of the milk turbidity to changes in temperature were somewhat different to

that of pH. On warming from 10 to 20 °C there was a slight lag (2-5 min) in the relative

increase in turbidity to that of temperature (Figure 5.5). However, on warming from 10

to 40 °C no lag was evident. This suggests that the kinetics of turbidity change are

temperature dependent, with the alterations that give rise to a portion of the turbidity

change occurring more rapidly at higher temperature. On cooling from 20 to 10 °C the

lag was even more pronounced, and on cooling from 40 to 10 °C the reduction in

turbidity was much slower, recovering only 50% of the turbidity change in 40 min,

whilst eventually returning to the original turbidity (i.e. the equilibrium turbidity at

10 °C) overnight. The rate of casein rearrangement may have been higher on warming

than cooling due to increased kinetics associated with the final temperature (20 or 40 °C

compared to 10 °C).

79

Figure 5.5 Dynamics of skim milk warmed and cooled between 10 and 20 °C (A), and

10 and 40 °C (B). Relative change in temperature ((), pH () and turbidity () as a

function of warming (left to right) and cooling (right to left) time. For clarity the data

presented are single measurements of one representative sample. Consistent results were

obtained from similar experiments performed with five other separate samples.

80

Since the pH response to temperature change was essentially instantaneous, any

contributions to the turbidity alterations resulting from the perturbations in the mineral

system such as partitioning of calcium between the micelle and the serum would also be

instantaneous. Therefore the observed lag in turbidity must be due to alterations to

casein such as the soluble casein, micelle size or hydration. It has previously been

shown that removal of calcium by EDTA addition results in the release of soluble

casein from the micelles (Griffin et al., 1988; Lin et al., 1972). The equilibrium results

presented here are compatible with this understanding, whilst the kinetic experiments

suggest that the rate of mineral re-equilibration is greater than that of protein release.

The mineral rearrangement consistently appeared to be very rapid whereas under some

circumstance (i.e. cooling from 40 to 10 °C) the protein rearrangement was much

slower (milk turbidity and soluble casein concentrations returned back to original states

after overnight storage at 10 °C). As the casein micelles become less hydrated and have

a denser structure (Trejo et al., 2011), migration and rearrangement of the protein

components would be expected to be slower than the transportation of calcium and

other mineral ions.

5.4 Conclusions

Casein micelles and the mineral distribution in skim milk are both dependent on

temperature in the range 10-40 °C. The dynamic response of the mineral system to

changes in temperature appears almost instantaneous where as re-equilibration of the

casein micelles is much slower, particular upon cooling. This temporal separation is

evidence that these two processes are somewhat independent and that it is possible to

induce changes in the properties of casein micelles through warming of the milk that are

not immediately reversed upon cooling. Consequently, for milk that has been re-cooled

after warming or heating, the length of time spent after cooling is likely to affect

subsequent processing operations, such as ultrafiltration, that are influenced by micelle

properties.

81

6 Alterations to the composition of casein micelles and retentate serum

during ultrafiltration of skim milk at 10 °C and 40 °C

(This Chapter has been published as Liu, D. Z., Weeks, M. G., Dunstan, D. E., &

Martin, G. J. O. (2014). Alterations to the composition of casein micelles and retentate

serum during ultrafiltration of skim milk at 10 °C and 40 °C. International Dairy

Journal, 35 (1), 63-69.)

6.1 Introduction

Ultrafiltration (UF) is a dairy processing unit used in the manufacture of cheese and

dairy ingredients and in milk standardization (Gésan-Guiziou, 2013). During UF, water,

soluble minerals and lactose are removed from milk system and consequently milk

proteins are concentrated in the retentates (Mistry et al., 2004). Temperature is an

important parameter in milk processing that affects a wide range of physicochemical

properties (Fox et al., 1998a; Liu, Weeks, Dunstan & Martin, 2013). The importance of

temperature on skim milk UF has been recognised since its inception, and a number of

early studies investigated the effect of temperature on permeation flux rates and the

microbial quality of retentates (Kapsimalis et al., 1981; St-Gelais, Haché & Gros-Louis,

1992). It was understood that low temperature can control microbial growth but results

in lower flux rates than UF performed at higher temperatures, regardless of the

membrane pore size. As a result of this trade-off and to avoid temperatures most suited

to microbial growth, UF of milk is performed either at or below 10 °C or between 40 to

50 °C (Gésan-Guiziou, 2013).

Casein and casein micelles, as discussed in previous sections, is an important

component in skim milk. During UF of skim milk, the casein micelles are known to be

responsible for processing efficiency (Bouzid et al., 2008; Rabiller-Baudry et al., 2008;

Rabiller-Baudry et al., 2005) and a better understanding of casein micelle behaviour

during UF would therefore improve the efficiency (David et al., 2008; Rabiller-Baudry

et al., 2005) and the quality of products. However, as mentioned in Chapter 2, only

limited research appears to have been performed to investigate the effect of UF on the

composition, size or structure of casein micelles (Singh, 2007).

82

Casein micelles are affected by temperature (Davies et al., 1983; Liu et al., 2013; Rose,

1968) and these effects remain to be understood in the context of UF processing of skim

milk. Of particular importance to UF processing is the exchange of calcium phosphate

and casein between the micelles and the serum that is directly influenced by temperature

(Davies et al., 1960; Liu et al., 2013; Rose, 1968). While the temperature of UF has

been shown to affect the bulk composition and properties of UF retentates and

permeates (McKenna, 2000; Pouliot et al., 1989a; Pouliot et al., 1989b; Renner & Abd

El-Salam, 1991; Rose et al., 1959; St-Gelais et al., 1992), a detailed study of the effects

of UF temperature on the composition of casein micelles and retentate serum has yet to

be performed. In addition, the effect of UF on the size and hydration of casein micelles

is still not clear, and the influence of temperature on these properties has not been

elucidated. The aim of this chapter was to investigate and compare the effect of UF

concentration at different processing temperatures on the hydration, composition and

size of casein micelles and the composition of the retentate serum. This information will

increase the understanding of the effect of temperature on the operational behaviour of

skim milk UF and the properties of the resulting retentates.


To get knowledge into the casein micelle alterations during UF, a systematic approach

had been developed. Detailed summary of the analyses performed on UF retentates and

permeates is shown in Figure 6.1.

83

Figure 6.1 Analyses performed on UF retentates and permeates

6.2.1 Skim milk samples

Pasteurised fresh skim milk containing 40 g·L-1

protein, 1.5 g·L-1


carbohydrate was purchased from a local supermarket and all experiments and analyses

were performed within two days and repeat experiments were performed on separate

batches of milk.

6.2.2 Ultrafiltration and pH measurements

Skim milk samples were concentrated to different concentrations (volume concentration

factor 2, 3 and 4) at 10 °C or 40 °C using a LabscaleTM

TTF system (Millipore, Billerica,

MA, USA) fitted with a 10 kDa cut-off polyethersulfone membrane at a constant

transmembrane pressure of 137.9 kPa. All skim milk samples were equilibrated at the

required temperature for 1 h before commencing UF. For experiments examining the

effects of altering the temperature during ultrafiltration, skim milk was initially

concentrated two fold at 40 or 10 °C, and then further concentrated to a concentration

factor of 3 (CF3) at 10 or 40 °C respectively (equilibration between these temperatures

occurred in less than 13 min).

pH of the original milk and UF retentate was measured using an Aqua-pH pH-mv-

temperature meter (TPS Pty Ltd., Brisbane, Australia) at UF processing temperature

(10 °C and 40 °C) and a temperature probe was fitted for automatic adjustments of

temperatures involved in the pH tests.

84

6.2.3 Casein micelle separation and hydration

Samples of skim milk and retentate were accurately weighed and added to pre-weighed

centrifuge tubes and ultracentrifuged at desired temperature as discussed in section 3.5.

The hydration was measured as described in section 4.2.2.

6.2.4 Protein and calcium quantification

The protein content of milk samples and supernatants obtained by ultracentrifugation

was analysed by SDS-PAGE as described in section 4.2.3. The concentration of the

caseins (α-, β- and κ-casein) was quantified densitometrically after scanning the stained

gels using BioRad Gel Dox XR+ Imager (BioRad Laboratories, Richmond, CA, USA).

Native-PAGE was used to investigate the content of proteins including individual native

whey proteins in the UF permeate and retentate. Permeates were diluted 4 times with

native loading buffer (Biorad, Laboratories, Richmond, CA, USA) and 10 µl of mixture

was loaded into 8-16% linear gradient precast Tris-HCl criterion 18 well gels (Biorad,

Laboratories, Richmond, CA, USA) before running at 100 V for 130 min. Supernatants

of the ultracentrifuged original skim milk and UF retentates were diluted 8 times with

native loading buffer and 10 µl, 5 µl, 3.3 µl and 2.5 µl of mixture was loaded into the

gels for original, CF2, CF3 and CF4 UF milk respectively. Gels were stained with

Biosafe Coomassie Blue (BioRad Laboratories, Richmond, CA, USA) before imaging

and densitometric quantification of the individual protein bands.

Measurement of the calcium concentration was performed by atomic absorption

spectroscopy as described in section 4.2.4.


Dynamic light scattering was used to determine the intensity-weighted average

hydrodynamic diameter of casein micelles in skim milk and UF concentrated samples.

Measurements were performed on samples diluted 75 fold in either UF permeates

obtained at 10 °C and 40 °C or simplified simulated milk ultrafiltrate (Martin et al.,

2007). The temperature during light scattering measurements was maintained at 10 °C

or 40 °C respectively. Measurements were performed as described in section 5.2.5.

85

6.2.6 Determination of lactose content

Prior to lactose determination, protein was removed from samples of milk or

resuspended pellets from ultracentrifugation using Carrez reagent following the

international standard protocol (ISO, 2002). Lactose concentration was then measured

using a Lactose Assay Kit (Abcam, Cambridge, UK) according to the manufacturer

instructions.


Skim milk samples were concentrated to different concentrations by UF at 10 °C and

40 °C respectively. 40 °C is at the upper limit of high temperature ultrafiltration, and

was selected as it was not possible to conduct these experiments beyond this

temperature. Native-PAGE was used to analyse the protein content in both permeate

and supernatants of ultracentrifuged UF retentates (ultracentrifugation was performed at

the UF processing temperature of 10 or 40 °C). As shown in Figure 6.2, no casein could

be observed in permeates at either 10 or 40 °C, with a small amount of α-lactalbumin

and trace bands of β-lactoglobulin evident. The total protein content of the permeate

accumulated up to CF4 was determined by total protein nitrogen measurement

(determined by external laboratory testing using standard method (DTS Food

Laboratories, Kensington, VIC, Australia)) to be 1.8 g·L-1

and 2.0 g·L-1

for the 10 °C

and 40 °C respectively. The relative amounts of both native α-lactalbumin and native β-

lactoglobulin did not change significantly in the supernatants of UF retentates at either

temperature (Figure 6.2 and Figure 6.3), indicating that minimal interaction between the

whey proteins and casein micelles resulted during UF.

86

Figure 6.2 Native-PAGE analysis of UF permeates and the supernatants of the

ultracentrifuged retentates (60 min ultracentrifugation).

Also according to Figure 6.2, β-casein was the predominant casein to be released from

the micelles into serum at 10 °C compared to 40 °C, with κ-casein and α-casein

relatively stable. This is consistent with previous observations (Creamer et al., 1977;

Davies et al., 1983; Downey et al., 1970; Liu et al., 2013; Rose, 1968).

87

Figure 6.3 Native-PAGE analysis of whey protein content in the serum of retentates

from ultrafiltered skim milk. Relative concentration of α-lactalbumin () and β-

lactoglobulin () in the serum of UF retentates at 10 °C (A) and 40 °C (B). Error bars

represent standard deviation of duplicate measurements of one single sample.

To investigate changes to the partitioning of milk components between the micelles and

the serum during UF, supernatants were carefully removed and the concentration of

soluble casein and calcium were measured. Consistent with previous observations in

this approximate temperature range (Creamer et al., 1977; Liu et al., 2013; Rose, 1968),

the proportion of soluble casein in the skim milk was higher at 10 °C than 40 °C (Figure

88

6.4A and 6.4B). Depending on the pH, the amount of soluble casein may decrease if the

temperature is increased beyond this range (Anema, 1998). Since all casein was retained

during UF (as shown in Figure 6.2), the total amount of casein in the retentates was the

same as that in the original milk samples (Figure 6.4A and B). The amount of soluble

casein in the retentates decreased slightly and the amount of micellar casein increased

correspondingly as a function of concentration at both temperatures (Figure 6.4A and B).

This indicates that some of the soluble casein shifted from the serum to the micelles

during UF. As permeate is removed during UF the total volume of serum decreases, so

although there is a shift of casein from the serum to the micelles, there is a large

increase in the concentration of soluble casein in the serum (Figure 6.4E and F). An

apparent increase in the amount of non-micellar casein has previously been observed,

and this was attributed to the release of casein from the micelles (McKenna, 2000).

However, the results here show that although the concentration of soluble casein

increased during UF, there was a net shift of casein from the serum to the micelles.

89

Figure 6.4 Alterations to the casein micelle and serum compositions of UF retentates as

a function of volumetric concentration factor (CF). UF at 10 °C (A, C, E) and 40 °C (B,

D, F). A and B: Amount of soluble casein (), micellar casein () and total casein ()

remaining in UF retentates and original unconcentrated skim milk. C and D: Amount of

serum calcium (), micellar calcium () and total calcium () remaining in UF

retentates and original unconcentrated skim milk. E and F: Relative concentration of

soluble casein () and serum calcium () present in the serum of UF retentates

relative to that of unconcentrated skim milk at 10 °C and the retentate pH (). Error

bars represent standard deviation of duplicate measurements of samples from triplicate

experiments.

0.0

0.5

1.0

1.5

2.0

2.5

3.0R

ete

nta

te c

asein

(g p

er

L o

f o

rig

inal m

ilk)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Rete

nta

te c

asein

(g p

er

L o

f o

rig

inal m

ilk)

0.0

0.2

0.4

0.6

0.8

1.0

Rete

nta

tecalc

ium

(g p

er

L o

f o

rig

inal

mil

k)

0.0

0.2

0.4

0.6

0.8

1.0

Rete

nta

te c

alc

ium

(g p

er

L o

f o

rig

inal m

ilk)

6.5

6.6

6.7

6.8

6.9

7.0

0.0

0.5

1.0

1.5

2.0

2.5

1 2 3 4

Rete

nta

te p

H

Rela

tive c

on

cen

trati

on

in

seru

m

Concentration Factor

6.5

6.6

6.7

6.8

6.9

7.0

0.0

0.5

1.0

1.5

2.0

2.5

1 2 3 4

Rete

nta

te p

H

Rela

tive c

on

cen

trati

on

in

seru

m

Concentration Factor

A B

FE

DC

90

The concentration of calcium in the original skim milk, the UF retentates, and in the

supernatants resulting from ultracentrifugation of the retentates, was measured by AAS.

The relative amount of micellar calcium was determined from the difference between

the total calcium in the retentate and the amount of calcium in the supernatants. After

concentration to CF2 the amount of micellar calcium was relatively unchanged for milk

ultrafiltered at both 10 and 40 °C (Figure 6.4C and D). A slightly higher percentage of

the calcium was removed on concentration to CF2 at 10 °C (14%) than at 40 °C (12%)

(Table 6.1). As little calcium had been removed from the micelles at either temperature

at CF2 (Figure 6.4 C and D), the calcium removed from the milk to this point originated

primarily from the serum. The differences in the rate of calcium removal could be

expected to result from the higher concentration of serum calcium at lower temperature

(Figure 6.4E and F), and a possible temperature dependence of the calcium rejection

coefficient. On further concentration to CF3 the behaviour of milk at different

temperatures diverged, with removal of calcium from the micelles evident at 10 °C, but

not 40 °C (Figure 6.4C and D). Correspondingly, an increase in the removal of calcium

from the retentate was evident at 10 °C due to release of micellar calcium, whereas

calcium removal was relatively low at 40 °C between CF2 and CF3 (Table 6.1). At

10 °C the removal of calcium from the micelles appeared to cease between CF3 and

CF4 (Figure 6.4C), as did the removal of calcium from the retentate (Table 6.1). At

40 °C, the calcium continued to remain with the micelles (Figure 6.4D) and the amount

of calcium removed from the retentate was low (Table 6.1). As shown in Figure 6.3E

and 6.3F the pH of both the retentates was reasonably stable during UF, with slight

variations reflective of mineral re-equilibration between the micelles and serum at the

different temperatures (Fox et al., 1998a).

The hydration of the casein micelles was determined by measuring the water content of

the pellets gravimetrically by 24 hours oven drying. The micelle hydration was seen to

increase from about 2.79 g water/g solids for fresh skim milk to approximately 3.16 g water/g

solids for milk concentrated four-fold at 10 °C, and from 1.99 g water/g solids to 2.23 g water/g

solids at 40 °C (Table 6.1). This behaviour is in contrast to concentration by evaporation,

during which the casein micelles were progressively dehydrated (Liu, Dunstan &

Martin, 2012). The marked difference in hydration at two different temperatures is

consistent with previous observations of casein micelles at different temperature

(Snoeren et al., 1984), and could be expected to affect of UF processing such as

91

resistance to flux caused by concentration polarization and gel layer formation. The

increase in micelle hydration could reflect an increase in micellar water, a decrease in

micellar solids, or a combination of the two. For the two major non aqueous micelle

components, micellar calcium was reduced during UF at 10 °C but not obviously at

40 °C (Figure 6.4C and 6.4D) and the micellar casein was increased at both

temperatures (Figure 6.4A and 6.4B). The partitioning of the other non aqueous micelle

components, lactose, was also investigated. While the total amount of lactose in the

retentate was reduced due to removal into the permeate, the amount of lactose

associated with the micelles remained low and relatively constant during UF (Table 6.1).

This suggests that the increase in micelle hydration is primarily reflective of an increase

in micellar water, which is consistent with previous observations of micelle swelling

during UF that became more prominent on subsequent diafiltration (McKenna, 2000).

Table 6.1 Micelle and mineral alterations during UF.

CF

Removal

of calcium1

Micelle

Hydration2

(g water/g solid)

Average effective diameter3 (nm)

Total lactose

in retentate4

(g/Loriginal milk)

Micellar lactose4

(g lactose/g wet pellet)

Buffer

Permeate

10 °C Original

2

3

4

N/A

14%

31%

31%

2.79 ± 0.10

2.96± 0.20

3.00 ± 0.10

3.16 ± 0.05

188.9±10.5

187.4±10.5

193.6±2.3

191.9±1.4

193.5±10.0

191.5±11.6

183.7±1.8

188.4±5.3

45.9 ± 0.6

n.d.

n.d.

12.5 ± 0.5

0.035 ± 0.003

n.d.

n.d.

0.043 ± 0.003

40 °C Original

2

3

4

N/A

12%

17%

19%

1.99 ± 0.04

1.97 ± 0.12

2.14 ± 0.08

2.23 ± 0.05

197.2±6.1

186.4±2.3

182.9±4.6

199.5±10.7

200.9±12.0

186.4±9.7

195.3±11.5

195.9±5.3

47.2 ± 0.6

n.d.

n.d.

13.2 ± 0.5

0.042 ± 0.001

n.d.

n.d.

0.039 ± 0.003

1 % of total calcium (serum + micellar) in the original milk that was removed from the

UF retentates i.e. lost to the UF permeates.

2 Errors represent the standard deviation of duplicate measurements of duplicate

experiments.

3 Errors represent the standard deviation of triplicate measurements of a single

experiment.

4Errors represent the standard deviation of quadruplicate measurements of a single

experiment.

92

The hydration of the micelles gives an indication of the relative compactness or

voluminosity of the micelles during UF, with the data indicating less hydrated, more

compact micelles were present at 40 °C than at 10 °C. The hydration of the micelles can

be considered a temporary property (Martin et al., 2007). To investigate more

permanent alterations to the average size of casein micelles, dynamic light scattering

measurements of fresh skim milk and UF retentate samples were preformed. As casein

micelles very rapidly re-hydrate, differences in the hydration of the micelles (Martin et

al., 2007) are not seen using this method (which requires dilution before measurement)

and only more permanent structural changes are observed. As shown in Table 6.1, only

minor variations in the average size were observed at 10 °C. At 40 °C there appeared to

be a slight decrease in average micelle size on concentration to CF 2 which was

reversed on further concentration to CF 4. The similar size of micelles in rediluted UF

concentrates as measured by DLS here is consistent with previous observations of

rehydrated MPC powder (Martin et al., 2010), and suggest permanent alterations to

casein micelle size caused by UF are minor. The slight increase in micelle size due to

increased hydration is temporary and only observable in UF retentates in their

concentrated state. Previous investigations of casein micelle size alterations during UF

processing have employed electron microscopy, which requires dehydrating and cross-

linking the micelles (McKenna, 2000; Srilaorkul et al., 1991) and may be affected by

the higher concentrations of soluble casein in the retentates that have been observed in

this chapter.

93

Figure 6.5 Alterations to skim milk after UF concentration to CF2 at 10 °C or 40 °C

followed by concentration to CF3 at 40 °C or 10 °C. The amount of calcium removed

from the retentates, the total amount of soluble casein in the retentates, and the

hydration of the casein micelles in the retentates are presented relative to the values for

retentates obtained at 10 °C. Error bars represent the standard deviation of duplicate

measurements of samples from duplicate experiments.

According to the results above, the major difference in the removal of calcium resulting

from UF performed at different temperatures occurred between CF2 and CF3 (Table 6.1,

Figure 6.4C and D). While the removal of calcium from the milk system was similar

when concentrated to CF2 by UF at both 10 °C and 40 °C, the processing time was

almost doubled at 10 °C. To further investigate the effects of temperature on processing

efficiency and calcium removal, skim milk samples were concentrated to CF2 at either

10 or 40 °C and then concentrated further to CF3 at 40 or 10 °C respectively. The

results show that the amount of calcium removed from the CF3 retentate obtained from

a two-step UF process at 40 °C then 10 °C was greater than that from concentrated at

40 °C, but lower than those at 10 °C. The reverse two-stage UF was also tested by

concentrating to CF2 at 10 °C followed by 40 °C UF to CF3. More calcium was

removed from the system than at 40 °C but less than the 40 °C to 10 °C two-stage UF

process. The redistribution of calcium between the micelles and serum has been shown

0.0

0.2

0.4

0.6

0.8

1.0

Ca removed Soluble Casein Hydration

Am

ou

nt

Rela

tive t

o 1

0 °

C

10 °C

40 → 10 °C

10 → 40 °C

40 °C

94

to be extremely rapid in response to temperature change (as discussed in Chapter 5),

which explains why intermediate levels of calcium removal can be obtained when the

temperature is changed during UF. Chapter 5 also indicates that as the effect of

temperature on the minerals in skim milk is effectively instantaneous; changes to the

casein were observed to occur more slowly, especially during cooling. Further analyses

were performed on samples obtained by the two-step UF processes to examine the

effects on the casein micelles. The amount of soluble casein in the retentates was

highest in order of UF performed at 10 °C, 40-10 °C, 10-40 °C, then 40 °C (Figure 6.5).

This is similar to the trend in calcium removed (Figure 6.5), indicating that the

temperature between CF2 and CF3 had a greater influence on the final level of soluble

casein than the temperature between CF1 and CF2. Although, as discussed in Chapter 6,

casein re-equilibrates less rapidly in response to temperature than calcium, little

difference in the behaviours of the two components was observed during the two-step

UF process, presumably because the processing time was sufficiently long. While the

calcium removal and the partitioning of casein between the micelles and serum were

intermediate to the two processing temperatures and dependent on the order in which

they performed, the micelle hydration in CF3 retentates (Figure 6.5) was only

dependent on the temperature of the final processing step (CF2 to CF3). This is

consistent with previous observations that micelle rehydration is a rapid process (Martin

et al., 2007) and the hydration state of micelles can be considered a temporary property.

These results show that the temperature of UF processing influence both permanent

alterations such as the removal of calcium, and temporary effects such as the hydration

of the micelles.

95

Figure 6.6 Casein micelle alterations during UF

6.4 Conclusions

The casein micelle alterations during UF at 10 and 40 °C were summarised in Figure

6.6. During UF concentration of skim milk the progressive removal of calcium was

affected by the partitioning of calcium between the micelles and the serum which was

influenced by processing temperature. Performing UF at different temperatures

therefore altered the final calcium content of the retentates. The composition of casein

micelles including the hydration, calcium and casein content were all altered to some

extent during UF and affected by the temperature of operation. These findings have

practical consequences relating to both filtration performance and the use of UF

retentates. The influence of temperature on casein micelle hydration and the partitioning

of micellar components will affect the retentate viscosity and the fouling behaviour

which determine the overall membrane flux. Understanding how the partitioning of

calcium and casein between the micelle and the serum are affected by temperature is

important for the use of UF retentates for MPC manufacture and cheese standardization.

96

7 Lactose effects on casein micelles: The regulator of micelle hydration

7.1 Introduction

Lactose is a dissacharide of β-1,4-linked glucose and galactose that is an important

component of milk. As early as in the 1920s, Whitaker et al. (1927) reported that lactose

is partly responsible for the density and viscosity of skim milk having a higher

temperature dependency than water. It has also been found that lactose is responsible for

around half of the osmotic pressure of milk (Jenness & Sloan, 1970) and there is a

negative correlation of lactose concentration with total salt osmolarity (Holt, 1993).

During milk secretion, lactose formation and casein phosphorylation both take place in

the Golgi region. Current theories suggest that lactose is the principal regulator of the

final ratio of water and caseins in milk (Farrell Jr et al., 2006; Jenness, 1974) and that

the concentration of casein and lactose have an inverse correlation during secretion

(Jenness et al., 1987). The presence of lactose in milk system alters the properties of the

serum and casein micelles (Farrell Jr et al., 2006). It has high efficiency of binding with

calcium (Abrams, Griffin & Davila, 2002; Charley & Saltman, 1963; Gaiani, Ehrhardt,

Scher, Hardy, Desobry & Banon, 2006) and also regulates the water content in and

around proteins (Farrell Jr et al., 2006). Mozersky, et al. (1991) reported that the overall

colloidal protein mass of reformed casein micelles decreased by 45 to 90%, as

calculated by sedimentation field flow fractionation, on addition of 300 mM lactose.

Decrease in pH, conductivity, water activity (Gao, van Leeuwen, Temminghoff, van

Valenberg, Eisner & van Boekel, 2010), casein micelle voluminosity (Dewan et al.,

1973) have also been observed to be dependent on the concentration of sugars in milk

(Mora-Gutierrez, Kumosinski & Farrell, 1997).

During dairy processing, the concentration of lactose can be altered significantly. While

it is clear that lactose concentration can have various effects on the physicochemical

properties of milk, there is limited information on the effects of post-secretion alteration

in lactose concentration on the properties and structure of casein micelles. As discussed

in the previous chapters, although both evaporation and ultrafiltration concentrate

proteins in milk system; the effect on micelle hydration is different. During evaporation,

the micelle hydration decreases as a function of concentration, whilst in the UF, the

hydration increases during processing. The reason for that might be because of the

changes of lactose concentration in the milk system: as evaporation removes only water;

97

while lactose passes through membrane during UF. However, detailed information

about lactose effect on casein micelles during milk processing has so far been limited.

Milk protein concentrate (MPC) is manufactured by removing minerals and lactose

from skim milk system by ultra- and dia-filtration. The retentate is further concentrated

by evaporation and then spray dried. It contains whey proteins and caseins in the same

proportions as the original milk, but with less lactose. Solubilised MPC can provide a

casein micelles suspension in solutions with an artificially low lactose concentration,

which extends the range over which the effects of lactose on casein micelles can be

studied. In this chapter, the lactose effects on casein micells were investigated in both

skim milk and MPC systems.


7.2.1 Skim milk and MPC solutions

Pasteurized fresh skim milk was purchased from local supermarket with a composition

as described in section 6.2.1. MPC 85 powder (MG Natra. Pro., Australia) was

resuspended in SMUF or distilled water respectively to the final concentration of 37.7

g·L-1

. MPC 85 solutions were stored at ca 10 °C overnight for full hydration. Milk

samples and MPC solutions were brought to the desired temperature and then allowed

to equilibrate for at least 1 h with different amount of lactose (AnalaR ®, England)

added.

7.2.2 Volume fraction and pH

The viscosity of different samples and the corresponding SMUF or water with different

concentration of lactose was measured as described in section 5.2.4. The volume

fraction of casein micelles was calculated as described in section 3.2 and 5.2.4. The pH

was measured as described in section 5.2.6.

7.2.3 Casein micelle hydration

MPC solutions and skim milk were ultracentrifuged at desired temperature and the

hydration of casein micelles was measured and calculated as described in section 4.2.2.

98


Dynamic light scattering was used to determine the average diameter of casein micelles

in different samples. Milk samples were diluted 75-fold in SMUF with corresponding

lactose concentration. The temperature during light scattering measurements was

maintained at 20 °C. Measurements were performed as described in section 5.2.5.

7.2.5 Casein and Calcium quantification

The soluble protein concentration in milk serum was analysed by SDS-PAGE as

described in section 4.2.3. Measurement of the calcium concentration was performed by

atomic absorption spectroscopy as described in section 4.2.4.

7.2.6 Determination of lactose content

The lactose concentration in milk samples and micelle pellets was measured as

described in section 6.2.6.


7.3.1 Lactose effects on casein micelles in MPC solutions

MPC 85 powder was dissolved in distilled water or SUMF and different amounts of

lactose were added to the solution to reach concentrations of 1.5 g·L-1

, 15 g·L-1

, 30 g·L-1

,

45 g·L-1

, 60 g·L-1

, 75 g·L-1

, 85 g·L-1

, 100 g·L-1

. The pH of the MPC85 solutions was

measured directly and the supernatants from ultracentrifugation were carefully removed

for serum calcium measurement. As shown in Figure 7.1A and C, both the pH and

serum calcium concentration decreased as a function of lactose concentration in MPC

85 solutions at 20 °C. This indicates that some of the serum calcium shifted from the

serum to the micelles at high lactose concentrations, which is consistent with previous

observations (Gao et al., 2010). As a result of the increasing lactose concentration in the

MPC system, the casein micelles were dehydrated and a gradual increase in the amount

of lactose associated with micelles was also observed (as shown in Figure 7.1 B and D

and Table 7.1). This is again consistent with the known tendency for the decrease of

micelle voluminosity when adding sugar (Dewan et al., 1973), which might be due to

99

micelle dehydration and contraction. In addition, during this re-arrangement, calcium-

lactose complexes might form (Charley et al., 1963) and the balance of calcium might

be shifted from the serum to the colloidal phase.

Figure 7.1 Pelletization of casein micelles and composition of the serum of MPC 85

disolved in water (A, B) and SMUF (C, D) as a function of lactose concentration.

A and C: Serum calcium (sCa) () relative to the amount in original solution without

adding extra lactose and the pH of the solution (). B and D: The amount of water ()

and solids () pelleted from ultracentrifugation. Error bars represent standard deviation

of duplicate measurements of samples from duplicate experiments.

100

Table 7.1 Micellar lactose and micelle hydration in MPC 85

Total lactose

(g·L-1

)

Micellar lactose 1


Micelle hydration 1

(g water/g solid)

MPC 85 in water

1.5 0.0020±0.0001 2.77±0.05

15 0.0045±0.0001 2.66±0.04

45 0.0242±0.0003 2.60±0.03

85 0.0396±0.0006 2.44±0.05

MPC 85 in SMUF

1.5 0.0017±0.0001 2.85±0.03

15 0.0056±0.0001 2.74±0.04

45 0.0255±0.0005 2.60±0.01

85 0.0341±0.0005 2.44±0.03

1 Errors represent the standard deviation of duplicate measurements of samples from


The turbidity was observed to be inversely proportional to the overall lactose

concentration (Figure 7.2). Casein micelles are the predominant contributor to milk

turbidity and the relationship between turbidity and the physico-chemical properties of

casein micelle is complex. Due to the increased amount of lactose and minerals

associated with micelles, the casein micelles are more optically dense and hence have an

increased refractive index relative to the serum and will scatter more light. On the other

hand, the dehydration-induced contraction of the micelles will decrease the total volume

fraction occupied by the casein micelles, which leads to the decrease in the scattering

volume and turbidity. Therefore, the exact relationship between the observed changes in

turbidity and changes to the casein micelles is difficult to accurately predict.

101

Figure 7.2 Relative turbidity of MPC 85 solutions as a function of lactose concentration.

(), MPC 85-SMUF; (), MPC 85-Water. Error bars represent standard deviation of

duplicate measurements of samples from duplicate experiments.

7.3.2 Lactose effects in skim milk

To further investigate the lactose effect on casein micelles, different amount of lactose

were dissolved into skim milk at room temperature to a final concentration of 46 g·L-1

,

60 g·L-1

, 73 g·L-1

, 85 g·L-1

, 100 g·L-1

. The concentration of soluble casein was

measured by SDS-PAGE and serum calcium concentration was measured by AAS. As

shown in Figure 7.3A, the pH was relatively stable and the serum calcium concentration

decreased slightly at high lactose concentrations. The concentration of the soluble

casein decreased about 7% when the lactose concentration was above 73 g·L-1

indicating that a minor shift of casein from serum to micelle phase occurred. Similar to

MPC85, skim milk showed a similar trend of decreasing micelle hydration and

increasing of micellar solids as a function of increasing lactose concentration (shown in

Figure 7.3B). According to the dynamic light scattering results the average micelle size

decreased as a function of lactose concentration. And the micelle voluminosity was also

shown to decrease when adding lactose (Figure 7.3C), which confirmed the micelle

contraction due to loss of micellar water. The increase of the ultracentrifugation pelleted

lactose (Table 7.2) suggested that casein micelles are able to accommodate extra lactose.

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

0 15 30 45 60 75 90 105

Re

lati

veTu

rbid

ity

Lactose concentration (g/L)

102

Table 7.2 Micellar lactose and micelle hydration in Skim milk

Total lactose

(g/L)

Micellar lactose 1


Micelle hydration 1

(g water/g solid)

46 0.0393±0.0019 2.33±0.01

60 0.0432±0.0022 2.31±0.01

85 0.0476±0.0025 2.14±0.03

100 0.0496±0.0031 2.07±0.02

1 Errors represent the standard deviation of duplicate measurements of samples from


103

Figure 7.3 Alterations of casein micelles and serum of skim milk as a function of

lactose concentration.

A: pH () of skim milk samples, serum calcium () and soluble casein () relative to

original milk without adding lactose. B: The amount of water () and solids ()

pelleted from ultracentrifugation. C: Average micelle size () and relative volume

fraction of casein micelles () as a function of lactose concentration. Error bars

represent standard deviation of duplicate measurements of samples from duplicate

experiments.

104

7.3.3 Lactose effects during milk processing

The hydration of casein micelles was determined by measuring the water content of the

ultracentrifugation pellets gravimetrically by overnight oven drying. As the data shown

in Table 7.1 and 7.2, the micelle hydration decreased as a function of lactose

concentration. This result confirmed that lactose can regulate the water content of casein

micelles in milk system. To further investigate this effect, skim milk samples with

different lactose concentration (46 g·L-1

, 60 g·L-1

, 85 g·L-1

and 100 g·L-1

for tests

performed at 10 °C, and 46 g·L-1

, 60 g·L-1

, 85 g·L-1

, 100 g·L-1

, 150 g·L-1

and 200 g·L-1

for tests performed at 40 °C) were stored at ca 10 °C for equilibration and then kept at

10 °C and 40 °C for at least 1 h before ultracentrifugation. The hydration of the casein

micelles as a function of lactose concentration at different temperature is summarized in

Figure 7.4 alongside other previously obtained micelle hydration data from evaporative

concentration on the basis of the lactose concentration. During evaporative

concentration, the lactose concentration increased and the casein micelles were

dehydrated as water was removed. In contrast, during ultrafiltration both lactose and

water are removed from the milk system and micelles remain fully hydrated (Table 6.1).

While UF and evaporative concentration have other factors that could influence micelle

hydration (chiefly altered protein concentration), the lactose addition experiments do

not. Also, the resuspension of MPC in SMUF and water can account for any deviation

in behaviour that could result from altered mineral content, in particular calcium and

phosphate. As a whole these results suggest that the lactose concentration is primary

major factor (along with temperature) that determines the hydration, and therefore the

volume faction occupied by the casein micelles; and their contribution to the bulk

viscosity. In addition, early study comparing the effect of lactose and sucrose on the

size of casein micelles in reconstituted bovine milk, the results indicated that the

presence of lactose decreased the apparent micellar protein mass in all samples while

the effects of sucrose were strongly dependent on sample preparation (Mozersky et al.,

1991). These results suggest that the effects of lactose on casein micelles are due to

different mechanisms to those resulting from the presence of sucrose; in particular

lactose may cause effects involving calcium chelation (Charley et al., 1963).

105

Figure 7.4 Micelle hydration alterations in response to lactose concentration.

7.4 Conclusions

Casein micelle are dehydrated and contracted on addition of lactose. The lactose content

of micelles increases as a function of the total lactose concentration in milk, and the

internal micellar lactose can be removed by diafiltration. These observations indicated

that lactose is able to exchange between serum and micelle phase. On addition of

lactose, a shift of calcium from the serum to the micelles was observed in MPC 85

solutions; whilst in skim milk the serum mineral concentration was relatively stable.

The concentration of soluble casein decreased slightly in skim milk as a function of

lactose concentration. Overall, this study has shown that the lactose concentration

influences the hydration and voluminosity of casein micelle as well as the viscosity of

the whole system.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 50 100 150 200 250 300

Mic

elle

hyd

rati

on

(g

wat

er/

g so

lid)

Lactose concentration (g/L)

MPC85 in water

MPC85 in SMUF

Skim milk 10 °C

Skim milk 20 °C

Skim milk 40 °C

Evaporation 50 °C

106

8 Conclusions and recommendations

8.1 Conclusions

The objectives of this thesis were to obtain a greater understanding of the casein micelle

alterations during processing, in particular during milk evaporative concentration and

ultrafiltration (UF). The key alterations that were uncovered as a result of this research

are summarised in Figure 8.1. Further, the response of casein micelles to temperature

and lactose concentration alterations was also explored. A brief overview is outlined

here.

Figure 8.1 A schematic overview of the effect of evaporative concentration and

ultrafiltration on casein micelles as revealed through this study

Evaporation inducted casein micelle alterations

Concentration of skim milk by evaporation is an important unit in a number of dairy

processes. Therefore, the effects of the combination of heat and water removal on casein

micelles during skim milk evaporative concentration were observed. The mass balance

and hydration results revealed that during the evaporation process, while micelles were

107

dehydrated, water was removed preferentially from the serum. The amount of soluble

casein and calcium in the serum decreased as a function of increasing solids content,

indicating a shift of these components to the micelles. Furthermore, the reversibility of

these alterations was examined by re-dilution of milk concentrates with water. It was

discovered that casein micelles were immediately rehydrated upon re-dilution. In

addition, serum calcium and pH returned to their original state over a number of hours;

however, only a small percentage of original soluble casein returned to the serum over

the 5 h period investigated. These results showed that casein micelles are significantly

affected by evaporative concentration and that the alterations are not completely and

rapidly reversible. This helps explain previous observations on reconstituted SMP

(Martin et al., 2007) as well as the rheological properties of concentrated milk (Vélez-

Ruiz et al., 1998), which is of high importance for milk processing.

Temperature induced casein micelle alterations

To gain additional insight into the casein micelle alteration during milk processing,

further studies were performed in temperature induced changes. In this study, a

comprehensive physico-chemical investigation of casein micelles in skim milk was

performed between 10 °C and 40 °C. When fully equilibrated, the amount of soluble

casein, serum calcium, the hydration and volume fraction of the casein micelles and the

pH of skim milk all decreased as a function of increasing temperature.

Real-time measurements of turbidity and pH were used to investigate the dynamics of

the system during warming and cooling of milk. Changes in pH are indicative of

changes to the mineral system and the turbidity is a measure of alterations to the casein

micelles. The pH and turbidity showed that alterations to both the casein micelles and

the mineral system occurred very rapidly on warming. However, while mineral re-

equilibration occurred very rapidly on cooling, changes to the casein micelle structure

(reflected by turbidity) continued after 40 min of measurement, returning to equilibrium

after 16 h equilibration.

These results indicate that casein micelle structure and the mineral system of milk were

both dependent on temperature in the range 10-40 °C. The dynamic response of the

mineral system to changes in temperature appeared almost instantaneous whereas

108

equilibration of caseins was considerably slower, particularly upon cooling. In addition,

the length of time spent on warming or cooling is likely to affect the subsequent

processing operations.

UF induced casein micelle alterations

UF is widely used in the dairy industry for recovering, fractionating and concentrating

proteins in milk process streams. Based on the information about the temperature effects,

alterations to the hydration, composition and size of casein micelles and the distribution

of calcium during membrane separation were investigated during UF at 10 °C and 40 °C

respectively. The observation indicated that the hydration of casein micelles increased

during UF process to a four-fold concentration due to the increase of micellar water.

More micellar calcium was removed from skim milk during UF at low temperature.

To further investigate the effects of temperature on processing efficiency and calcium

removal, skim milk samples were concentrated to CF2 at either 10 or 40 °C and then

concentrated further to CF3 at 40 or 10 °C respectively. The amount of calcium

removed from the retentates was highest in order of UF performed at 10 °C, 40-10 °C,

10-40 °C, then 40 °C, and the processing time used was shortest in order of UF

performed at 40 °C, 10-40 °C, 40-10 °C then 10 °C. The micelle hydration in CF3

retentates was only dependent on the temperature of the final processing step.

The understanding of the casein micelle alterations during UF has practical influence on

filtration operations, the composition and hydration of casein micelles determine the

viscosity and fouling behaviours of the UF retentates and ultimately the processing

efficiency. Furthermore, this helps improve the use of UF retentates for cheese and

MPC manufacture.

Lactose effects on casein micelle hydration

As discussed above, the removal of water during evaporative concentration of skim

milk was found to dehydrate casein micelles. Conversely, during milk UF, casein

micelles remained highly hydrated, although proteins were concentrated. The results

109

suggested that temperature and lactose concentration are the important factors affecting

hydration and volume fraction of casein micelles and hence the viscosity of the milk.

Casein micelle models

The casein micelle models discussed in Chapter 2 all present the micelle as a dynamic

entity. From the conclusions of evaporation and ultrafiltration of skim milk, it appears

that the sponge model, which describes the casein micelle as a compressible particle

(Bouchoux et al., 2010), can most readily accommodate the observed alterations to the

casein micelles, specifically the hydration, size and volume fraction changes. This is

also consistent with recent TEM results (Dalgleish, 2011b; Dalgleish et al., 2012;

McMahon et al., 2008). However, it fails to describe the underlying mechanisms behind

the shift of minerals and caseins between serum and micelle phase. On the other hand,

the hydrophobic interactions and CCP nanocluster links proposed in the dual-binding

model and nano-cluster model, are better able to explain the observed changes in the

partitioning of caseins and minerals, which was also confirmed by recent studies (de

Kruif & Huppertz, 2012a; Holt et al., 2013). Decreasing the temperature is known to

increase the solubility of calcium phosphate and decrease the strength of hydrophobic

interactions, which result in the shift of caseins and calcium mineral towards the serum.

Accordingly, a combination of these models may help in developing improved

understanding of the overall structure of casein micelles. Consequently, the casein

micelle can be described as a highly hydrated sponge-like particle with partly

compressible inner structure. The caseins inside associated with hydrophobic

interactions and other weak bonds as well as CCP links, respond to environmental

changes contemporaneously.

In summary, it is concluded from this work that the state of casein micelles and the

partitioning of caseins and calcium between serum and micelle phase are influenced

considerably during processing and temperature alteration. The conclusions resulting

from this thesis have important implications for improving processing efficiency and

products properties.

110

8.2 Recommendations for Future Research

While the work in this thesis has provided new insights into the behaviour of casein

micelles during processing, it also indicates areas of opportunity for further research.

Detailed investigation of internal structure of casein micelles

The focus of this thesis was on understanding alterations of casein micelles during

processing, and detailed information of the internal structure of casein micelles was not

obtained. Such investigation would develop a better understanding of the casein

micelles and their response to environmental changes. This could be done by

discovering the changes to the state and structural organisation of the caseins and CCP.

According to early studies, the micellar calcium falls into two categories, namely easily

exchanged and difficult to exchange (Wahlgren et al., 1990; Yamauchi et al., 1969;

Zhang et al., 1996). As discussed in this thesis, some micellar calcium was proved to be

difficult to remove during UF. A focused study examining the colloidal minerals would

facilitate the understanding of casein micelle internal structures. Based on previous

study of the equilibrium thermodynamic model of mineral-casein interactions (Holt,

2004) and recent analysis of CCP nanocluster distribution inside casein micelles (de

Kruif et al., 2012b), this study would concentrate on measuring and comparing the

amounts of major ionic species in different casein micelle systems, such as UF/DF

retentates, skim milk, MPC in different buffers at different pH. A further study

comparing the distribution of CCP nano-clusters in casein micelles in these different

milk system using small angle X-ray scattering would provide more insight into this.

This could be combined with cryo-TEM investigation of the internal structural changes

of the casein micelles in response to environmental and processing conditions.

Further study of casein micelles in UF polarisation and fouling layer

As mentioned above, casein is an important component of the concentration

polarization and the gel layer during UF. However, there is currently only limited

knowledge about casein micelle structure and UF fouling. To gain major insight into

this, comparison of the structure of the casein micelles in the bulk milk system and the

111

gel layer need to be done. The particle-particle interactions and effects of temperature

and minerals on casein micelles also need to be taken into account. Ultimately, this will

lead to better membrane performance and higher processing efficiency.

Diafiltration induced alterations and further study of lactose effects on casein micelles

There is also an opportunity to increase the understanding of diafiltration effects on

casein micelles in future study. With the combination of UF and DF, most of the CCP

and micellar lactose will be removed from casein micelles due to extensive addition of

water. While there is literature covering the effect of UF/DF effects on cheese

processing, concentrated milk fermentation and milk coagulation (Alvarez et al., 1998;

Caron, St-Gelais & Pouliot, 1997; Covacevich & Kosikowski, 1977; Ernstrom,

Sutherland & Jameson, 1980), information on the casein micelle structure and lactose

effects were not examined. A further understanding could ultimately lead to

optimisation of processing parameters and improvement of the functional properties of

the end products.

112

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Appendix: Physical properties of whole bovine milk (Sherbon, 1988;

Singh et al., 1997; Walstra et al., 1984)

Osmotic pressure

aw

Boiling point

Freezing point

Refractive index, nD20

Specific refractive index

Density (20 °C)

Specific gravity (20 °C)

Specific conductance

Ionic strength

Surface tension (20 °C)

Coefficient of viscosity

Thermal conductivity (2.9% fat)

Thermal diffusivity (15-20 °C)

Specific heat

pH (25 °C)

Titratable acidity

Coefficient of cubic expansion (273-333K)

Redox potential (25 °C, pH 6.6, in equilibrium with

air)

700 kPa

0.993

100.15 °C

-0.522 °C (approx.)

1.344-1.349

0.2075

1030 kg·m-3

1.0321

0.0050 ohm-1

·cm-1

0.08 M

52 N·m-1

2.127 mPa·s

0.559 W·m-1

·K-1

1.25×10-7

·m2·s

-1

3.931 kJ·kg-1

·K-1

6.6

1.3-2.0 meq OH- per 100 ml

(0.14-0.16% as lactic acid)

0.0008 m3·m

-3·K

-1

+0.25 to +0.35 V

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

LIU, ZHE

Title:

Fundamental study of physical and biochemical alterations to casein micelles during milk

evaporation and ultrafiltration

Date:

2013

Citation:

Liu, Z. (2013). Fundamental study of physical and biochemical alterations to casein micelles

during milk evaporation and ultrafiltration. PhD thesis, Department of Chemical and

Biomolecular Engineering, The University of Melbourne.

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Fundamental study of physical and biochemical alterations to casein micelles during milk

evaporation and ultrafiltration

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