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UNIVERSITY OF COPENHAGEN FACULTY OF SCIENCE Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia Ahrné and Denise Felix da Silva Submitted on: March 5, 2018

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Page 1: Master Thesis - ku...Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia

U N I V E R S I T Y O F C O P E N H A G E N

F A C U L T Y O F S C I E N C E

Master Thesis

Sissel Therese Brøkner Kavli (shl704)

Effect of varying feed dry matter content on

powder properties of micellar casein powder

Supervisor: Lilia Ahrné and Denise Felix da Silva

Submitted on: March 5, 2018

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Name of University: University of Copenhagen

Name of Faculty: Faculty of Science

Name of department: Department of Food Science

Name of Section: Ingredient and Dairy Technology

Location: Rolighedsvej 26,

2000 Frederiksberg,

Denmark

Thesis: Master thesis in Food Science and Technology

Author: Sissel Therese Brøkner Kavli

Student ID: shl704

Title: Effect of varying feed dry matter content on powder rehydration ability

of dried micellar casein powders

Supervisors: Lilia Ahrné and Denise Felix da Silva

Submitted: March 5, 2018

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Preface

The present work is a master thesis, completing the two-year MSc programme in Food Science and

Technology, with a specialisation in Dairy Science and Technology, at University of Copenhagen,

Denmark. The target group of this work is fellow students and everyone that holds an interest in

micellar casein isolate, drying technology and rehydration ability.

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Abstract

Micellar casein isolate (MCI) is a relatively new casein-based powder that is useful in food

production due to its functional properties, such as water holding capacity, gelling- and foaming

ability. A problem associated with casein-based powders is their poor rehydration ability. Powder

rehydration ability is known to be highly influenced by physico-chemical aspects of the powder,

such as composition, particle morphology and microstructure, which in turn are influenced by the

drying process. Thus in this work, the effect of varying feed dry matter content on the rehydration

ability of freeze- and spray dried powders were investigated.

Commercial MCI powder was rehydrated and used as feed material for further drying at dry matter

contents (DM) of 12%, 15% and 18%. The resulting freeze- and spray dried powders showed clear

differences in powder properties dependent on feed dry matter content at drying and the drying

technology applied. Increased feed DM decreased feed pH, the particle size distribution, the

viscosity and the rheological properties. Spray dried powders showed spherical particles while

freeze dried powders had a flaky shape, with significant differences in structure and particle size

distribution, The lowest moisture content was obtained for the freeze dried powder at 18% DM. The

powders rehydration ability was evaluated according to wettability, dispersibility and final

solubility. In comparison to the starting commercial powder, it was found that wettability and

dispersibility were improved in powders produced with lower DM content (12%). Additionally,

spray dried powders performed better than freeze dried. Final solubility was improved for both

drying methods compared to the solubility of the commercial powder. However, only spray dried

powders were observed to fully solubilise to a particle size distribution with almost no measured

values in the size class area larger than 1 μm.

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Acknowledgement

I would like to express my gratitude to my supervisor, Associate Professor, Lilia Ahrné for her

enthusiastic approach and dedication to this work and additionally for her knowledgeable guidance

and participation throughout.

Furthermore, to PhD Denise Felix Da Silva I owe my thanks for introduction to equipment used in

powder manufacture and sample analyses, and for her help and guidance during use. Additionally, a

thank to Associate Professor Jens Christian Sørensen for introduction to the Büchi lab-scale spray

dryer and for his supervision and answers to all questions.

Lastly, I am grateful for the countless hours of babysitting provided by my family and for the

support given by my boyfriend Sebastian.

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Table of Contents

Preface ................................................................................................................................................. II

Abstract .............................................................................................................................................. III

Acknowledgement ............................................................................................................................. IV

Abbreviations .................................................................................................................................. VIII

List of Figures .................................................................................................................................... IX

List of Tables ...................................................................................................................................... X

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

1.1 Objectives of Thesis ................................................................................................................... 3

2. Literature review .............................................................................................................................. 4

2.1 Principal components of MCI .................................................................................................... 4

2.1.1 Lactose ................................................................................................................................ 5

2.1.2 Proteins................................................................................................................................ 5

2.1.3 Minerals .............................................................................................................................. 7

2.1.4 The Casein Micelle Complex .............................................................................................. 8

2.2 Industrial Production of MCI Powder ........................................................................................ 9

2.2.1 Membrane filtration .......................................................................................................... 11

2.2.2 Spray Drying ..................................................................................................................... 12

2.2.3 Freeze drying..................................................................................................................... 15

2.3 The Rehydration Process ......................................................................................................... 15

2.3.1 Wetting and Swelling ........................................................................................................ 17

2.3.2 Sinking .............................................................................................................................. 18

2.3.3 Dispersion and Dissolution ............................................................................................... 18

3. Materials and Methods ................................................................................................................... 20

3.1 Preparation of MCI feeds ......................................................................................................... 20

3.2 MCI powder production by freeze- and spray drying .............................................................. 21

3.2.1 Spray drying ...................................................................................................................... 22

3.2.2 Freeze drying..................................................................................................................... 22

3.3 MCI feed characterisation ........................................................................................................ 23

3.3.1 Feed pH ............................................................................................................................. 23

3.3.2 Feed particle size distribution ........................................................................................... 23

3.3.3 Feed rheological behaviour ............................................................................................... 23

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3.4 MCI Powder characterisation ................................................................................................... 24

3.4.1 Powder pH......................................................................................................................... 24

3.4.2 Powder particle size distribution ....................................................................................... 24

3.4.3 Powder moisture content ................................................................................................... 24

3.4.4 Powder water activity........................................................................................................ 25

3.4.5 Powder colour ................................................................................................................... 25

3.4.6 Powder microstructure ...................................................................................................... 26

3.5 Characterisation of MCI powders rehydration ability ............................................................. 26

3.5.1 Powder wettability ............................................................................................................ 26

3.5.2 Powder dispersibility......................................................................................................... 27

3.5.3 Powders final solubility .................................................................................................... 27

3.6 Statistical data analysis ............................................................................................................ 27

4. Results and discussion ................................................................................................................... 28

4.1 Properties of MCI feed concentrates at different dry matter contents ..................................... 28

4.1.1 pH of MCI feed concentrates ............................................................................................ 28

4.1.2 Particle size ....................................................................................................................... 30

4.1.3 Rheological behaviour of the MCI feed concentrates ....................................................... 32

4.2 Properties of MCI powders obtained by freeze- and spray drying .......................................... 34

4.2.1 pH of the powders ............................................................................................................. 34

4.2.2 Particle size distribution .................................................................................................... 35

4.2.3 Moisture content and water activity in powders ............................................................... 38

4.2.4 Colour evaluation of the powders ..................................................................................... 40

4.2.5 Microstructure ................................................................................................................... 42

4.3 Rehydration ability ................................................................................................................... 45

4.3.1 Wettability of powders ...................................................................................................... 45

4.3.2 Dispersibility of powders .................................................................................................. 46

4.3.3 Final solubility of freeze- and spray dried powders .......................................................... 50

4.4 Multivariate data analysis ........................................................................................................ 55

5. Main conclusions ........................................................................................................................... 58

6. Future perspectives ........................................................................................................................ 60

7. References ...................................................................................................................................... 61

Appendix I.......................................................................................................................................... 74

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Appendix II ........................................................................................................................................ 75

Appendix III ....................................................................................................................................... 76

Appendix IV ....................................................................................................................................... 77

Appendix V ........................................................................................................................................ 78

Appendix VI ....................................................................................................................................... 79

Appendix VII ..................................................................................................................................... 80

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Abbreviations

α-la α-lactalbumin

αs1-cn αs1-casein

αs2-cn αs2-casein

β-cn β-casien

β-lg β-lactoglobulin

κ-cn κ-casein

AA Amino Acids

CCP Colloidal calcium phosphate

DF Diafiltration

LiCl Lithium chloride

MC Micellar casein

MCI Micellar casein isolate

MF Microfiltration

MPC Milk protein concentrate

MW Molecular weight

PCA Principal component analysis

PSD Particle size distribution

pI Isolectric point

RI Refractive index

SEM Scanning Electron Microscopy

SMP Skim milk powder

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List of Figures

Figure 1: Cross section of a casein micelle ........................................................................................................................ 8

Figure 2: Modifications of the casein micelle and the subsequent dissociation or association of numerous components

upon different environmental changes .................................................................................................................... 10

Figure 3: A simplified and schematic spray drying system ............................................................................................. 13

Figure 4: The rehydration process profile for powder particles ....................................................................................... 16

Figure 5: Experimental overview of the present study .................................................................................................... 20

Figure 6: Experimental setup for the drying process ....................................................................................................... 21

Figure 7: Size class distribution overview for feed concentrates ..................................................................................... 31

Figure 8: Feed concentrate flow curves ........................................................................................................................... 33

Figure 9: Powder pH measurements ................................................................................................................................ 35

Figure 10: Particle size distribution overview of bulk powder ........................................................................................ 37

Figure 11: Moisture content and water activity ............................................................................................................... 38

Figure 12: Colour evaluation displayed as ∆E and browning index (BI) ........................................................................ 41

Figure 13: Overview of SEM images .............................................................................................................................. 44

Figure 14: Wetting behaviour .......................................................................................................................................... 45

Figure 15: Dispersibility plotted as D (50) values over time ........................................................................................... 47

Figure 16: Overview of pH of rehydrated freeze- and spray dried powders .................................................................... 51

Figure 17: Particle size class distribution overview for rehydrated powder concentrates ............................................... 52

Figure 18: Flow curves obtained for rehydrated MCI powder solutions. ........................................................................ 53

Figure 19: Principal component analysis (PCA) .............................................................................................................. 56

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List of Tables

Table 1: Average chemical composition of bovine milk, skim milk powder and MCI ..................................................... 4

Table 2: Specifications of the dominant genetic variants of the proteins found in bovine milk ........................................ 7

Table 3: Technical specifications for the commercial micellar casein isolate, Promilk 852B. ........................................ 21

Table 4: Settings used during lab-scale Büchi spray drying, for feed concentrates ......................................................... 22

Table 5: pH, power law derived parameters (n and K) and particle size distribution parameters for feed concentrates .. 29

Table 6: Particle size distribution parameters for bulk powder. ....................................................................................... 36

Table 7: Drying efficiency for the freeze- and spray drying process. .............................................................................. 39

Table 8: Overview of microstructure characteristics........................................................................................................ 42

Table 9: Power law derived parameters (n and K) and particle size distribution parameters for rehydrated freeze- and

spray dried powders ................................................................................................................................................ 51

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1. Introduction

Drying is one of the major food processing operations for the preservation of perishable food

products. Powder production involves the stabilization of product components by removal of water.

There are several drying techniques available for powder production where spray drying is one of

the most commonly used methods, e.g. in the dairy industry for the production of dairy-based

powders (King, Downton and Flores-Luna, 1982; Schuck, 2013; Burgain et al., 2017). The use of

freeze-drying has in the last years become more widespread, in the preservation of heat-sensitive

food components in powder product.

For decades, the production of dairy-based powders has been a developing industry as the

consecutively emerging of new technologies provides opportunities within powder manufacture, i.e.

the progress within membrane filtration technology. With membrane technology, it is possible to

fractionate and concentrate e.g. milk proteins (Gelfand, 1996). Milk protein powders are systems

that differ in compositional proportions of lactose, fat, minerals and proteins but also in production

methods. Today, whey protein powders are used in a wide range of products, whereas the use of

casein-based powders is still emerging (Meena et al., 2017). The increasingly interest for casein-

based powders are found in their high heat stability, oppositely to whey proteins that are more

unstable and easily denature upon thermal treatment (Walstra and Jennes, 1984). There exist a

number of casein-based powders, where their way of manufacture influence the structure of the

caseins present and thus the powder properties (Crowley et al., 2016). Sodium caseinate (NaCas)

and calcium caseinate (CaCas) are produced by acid precipitation and resuspension with sodium- or

calcium hydroxide, respectively. Milk protein concentrate (MPC) is the concentration of serum

proteins and caseins by membrane filtration, whereas micellar casein (MCI) is the concentration of

mainly caseins (Felix da Silva et al., 2017). In MPC and MCI, caseins are present as casein micelles

compared to caseinates where the single caseins are precipitated out of the micelle (Beliciu, Sauer

and Moraru, 2012). Casein isolated by membrane filtration is thus not contaminated with any

additives that, e.g. can affect their flavour profile. Furthermore, the casein micelle structure remains

unaltered, similar to that of bovine milk (Walstra, Wouters and Geurts, 2006).

Casein-based powders have a wide range of applications, where their functional properties account

foaming, emulsifying, gel-forming, thickening, water-binding, and nutritional properties, that deals

with the complexity of food systems (Parkash, 1968; Kinsella, 1984; Mora-Gutierrez, Farrell and

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Kumosinski, 1995; Mimouni et al., 2009; Oliver, 2011; O’Mahony and Fox, 2013; Ji et al., 2016;

Nasser et al., 2017). Powders rehydration ability is an important functional property, as prolonged

or incomplete rehydration negatively affects industrial food manufacture. For industrial application

most powders are dissolved beforehand, and a slow rehydration process can prolong the production

or results in quality deficiencies, e.g. through the formation of lumps with non-hydrated powder

areas for powders with poor wetting or dispersion abilities (King, 1966; Kinsella, 1984; Schokker et

al., 2011; Crowley et al., 2015; Ji et al., 2016; Nasser et al., 2017). Furthermore, a complete

rehydration is a prerequisite for an effective expression of the underlying protein functionality

(Bouvier et al., 2013).

Some difficulties are present in the rehydration of casein-based powders, which results in prolonged

rehydration times or incomplete rehydration, especially exhibited at low temperatures and within

the time frames for food manufacture (Mistry and Pulgar, 1996). The prolonged rehydration is

probably a result of the formation of inter-linked casein micelles at the powder particle surface and

a low lactose content resulting in a slow dispersion of powder particles and likewise a slow particle

surface erosion (Richard et al., 2013). Feed composition, the drying process, and drying- and

storage conditions are the main parameters influencing the properties of the powder. By

understanding the influence of feed characteristics and processing methods on powder rehydration

ability, it is possible to tailor powder ingredients by varying different conditions. For example, the

variation in feed dry matter content (Kinsella, 1984; Singh, 2009; Gaiani et al., 2010; Schokker et

al., 2011; McSweeney and Fox, 2013; Schuck, 2013; Burgain et al., 2017).

Additionally, an important aspect of powder production is the reduction of energy-to-product ratio.

The amount of energy required for spray drying is considerably higher for that of evaporation

(Schuck, le Floch-Fouere and Jeantet, 2013), thus an increase in feed dry matter content for spray

drying represents a strong industrial opportunity in reducing the overall energy costs in powder

production. However, the subsequent effects of feed concentration on the physico-chemical

properties and powder functionality must be understood to achieve a successful rehydration process.

Several studies have investigated the impact from drying on the surface composition of powders

(Fäldt and Bergenståhl, 1994, 1996; Gaiani, Ehrhardt, et al., 2006; Kim, Chen and Pearce, 2009;

Wu et al., 2014; Xiao et al., 2016) and the influence of storage conditions (Haque et al., 2010; Gazi

and Huppertz, 2015; Nasser, Moreau, et al., 2017) related to powder rehydration ability, but an

investigation on the influence from varying MC feed dry matter content is still lacking.

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1.1 Objectives of Thesis

It is hypothesized that the characteristics of feed and conditions for processing influence the

functional properties of the final micellar casein powder product and the primary objective of the

present master thesis project is thus to investigate the effect of varying feed dry matter content on

properties of micellar casein powders dried by spray drying and freeze drying. The work is focused

on studying the influence of dry matter content on pH, particle size distribution, rheological

behaviour, colour, microstructure and their effects on powders rehydration ability, regarding

wettability, dispersibility, and final solubility. Thus the specific objectives of the present master

thesis are

i. To investigate the effect of varying dry matter content of feed (12%, 15% and 18%) prior

drying on feed properties.

ii. To compare the properties of freeze- and spray dried powders.

iii. To evaluate rehydration ability of MCI powder and how it is influenced by dry matter

content and drying technology.

Due to the present limited production of liquid micellar casein isolate the project is based on drying

of a rehydrated commercial micellar casein powder, which hold a known composition.

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2. Literature review

The functional properties of protein powders serve great advantages within food production. As

protein powders are increasingly used as ingredients in food and beverage manufacture it is

necessary to understand how new protein ingredients like micellar casein isolate (MCI) powder,

behave when in contact with water and which factors that influence the behaviour for proper

powder application and optimal protein functionality.

To get a better understanding of the drying behaviour and the mechanisms behind rehydration of

MCI powder and the challenges coupled with this, the principal components of milk will in the

following be outlined as well as the manufacture of casein powder and lastly, the rehydration

process will be defined.

2.1 Principal components of MCI

The composition of milk is responsible for its properties and application in further product

manufacture. Bovine milk consists of the components protein, fat, lactose, mineral and salt in

colloidal suspension (Walstra and Jennes, 1984). In Table 1, the average chemical composition of

bovine milk, skim milk powder (SMP) and MCI are outlined to show their diversity and the

possibilities that lie behind product fractionation and -manipulation as bovine milk is used as the

raw material in the production of all the listed powders despite their variability. From Table 1 it can

be seen that MCI powder has a high content of protein and a low content of lactose (Dahbi et al.,

2010) whereas the composition of SMP is different with a high lactose content and a lower protein

content (Ryder et al., 2018).

Table 1 Average chemical composition of bovine milk, skim milk powder and MCI. Adapted from Walstra and Jennes (1984),

Dahbi et al. (2010) and Ryder et al. (2018).

Technical specification Average quantity [% w/w]

Bovine milk SMP MCI*

Protein 3.3 35 81

- Casein 2.6 27.7 74.5

- Serum proteins 0.7 7 6.5

Lactose 4.6 53 4

Minerals 0.7 7.5 8.5

Fat 3.9 1 1.5

Water 87.3 3.5 5

*MCI is the commercial powder Promilk® 852 B.

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In the following sections, the major principal components of MCI, i.e. proteins, minerals and

lactose will be described to give an understanding of their composition and properties in milk. Milk

fat will not be included, as the content of MCI powder is so low that it can be disregarded (Table 1).

2.1.1 Lactose

Even though lactose represents a minor part of micellar casein powder (Table 1) its structural

properties have a significant role in the structure of the powder and are related to its rehydration

ability.

Lactose. The principal carbohydrate of bovine milk is lactose. It is a reducing disaccharide

consisting of the two monosaccharides; D-galactose and D-glucose joined in a β-1,4-glycosidic

linkage and is present as a colloidal solution (Walstra and Jennes, 1984). The structure of the

carbohydrate has great importance for its physico-chemical properties. The disaccharide exists

partially as an open-chain form with an aldehyde group that can form a hemiacetal and thus a ring

structure. The hemiacetal can exist as two isomers, α or β which is interchanged through

mutarotation. α-lactose crystallizes as a monohydrate, while β-lactose forms anhydrous crystals. α-

hydrates are hard and non-hygroscopic. When lactose in solution undergoes rapid drying, the

resulting structure remains in an amorphous state, mainly due to the insufficient time for

crystallisation to occur. The amorphous structure is hygroscopic by nature thus its presence at

powder particle surface improves rehydration (Bhandari and Howes, 1999).

2.1.2 Proteins

Bovine milk consists of different proteins. The two main protein groups are categorized into milk

proteins and whey or serum proteins. They differ in many aspects (Walstra and Jenness, 1984)

where some of them that are relevant for the behaviour during powder production and during

powder rehydration will be covered in this section.

Caseins. The dominating proteins of bovine milk are caseins. They represent ~80% of the total

protein content (Creamer, Richardson and Parry, 1981; Griffin and Anderson, 1983; Dalgleish and

Corredig, 2012) and were originally defined as the protein part that precipitates by acidification of

milk to its isoelectric point (pI) of pH 4.6 (20 ⁰C) where they are insoluble (Jenness et al., 1956).

Caseins are divided into four different polypeptides classified according to their primary amino acid

sequences (~150 to 200 residues), denoted αS1-, αS2-, β- and κ-casein with molar ratios of

approximately 11:3:10:4, respectively. They differ in many aspects such as numbers of

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phosphoseryl groups, the presence of cystine, glycosylation etc. which influence the behaviour of

caseins (Jenness et al., 1956; Griffin and Anderson, 1983; Walstra and Jenness 1984). Each

polypeptide fraction can be further subdivided into different genetic variants where some

specifications for the most common variants are given in Table 2.

All caseins are phosphoproteins that contain a number of phosphate groups. The phosphate groups

are esterified to serine residues through posttranslational phosphorylation. As αS- and β-cn contain a

high number of phosphate groups and thereby are phosphorylated to a high degree, they are highly

anionic and have a strong tendency for metal ion binding, mainly Ca2+

. Oppositely, k-cn only

contains one phosphorylation and is thus not sensitive to calcium (Bockian, Stewart and Tappel,

1956; Swaisgood, 2003). κ-cn is the only casein that is glycosylated, leaving its C-terminal polar

and hydrophilic. The N-terminal (1 to 105) is hydrophobic and possess two intramolecular

disulphide bonds (Broyard and Gaucheron, 2015).

Caseins are very heat stable and can hardly be denatured as they have little secondary and no

tertiary structure. This is due to the high levels of proline residues in their amino acid sequences,

which hinder the formation of α-helix and β-sheet structures. E.g. caseins can withstand heat

treatment at 100 ⁰C for 24 hours or 140 ⁰C for 20 to 25 minutes without coagulating in milk at

physiological pH (6.7) (Walstra and Jennes, 1984). The lack of a stable secondary and tertiary

structure and the resulting random coil polypeptide structure with a high degree of molecular

flexibility exposes most of the hydrophobic residues of the caseins resulting in their high surface

hydrophobicity, where β-cn is found to be most hydrophobic. This is why caseins have strong

tendencies for their association, which typically is via hydrophobic bonding (Swaisgood, 2003).

Serum proteins. The remaining protein part of bovine milk is serum proteins. They represent ∼20%

of the total protein content and are, contrarily to caseins, soluble at pH 4.6. The most abundant

proteins are β-lactoglobulin (β-lg) and α-lactalbumin (α-la). They comprise ∼50% and ∼20%,

respectively, of the total serum proteins found in bovine milk. Minor serum proteins include bovine

serum albumin (BSA), proteose peptones, lactoferrin, and immunoglobulins Serum proteins are

globular proteins and the heat stability is typical for that, where unfolding of structure and total

denaturation is obtained by heating at 90 ⁰C for 10 minutes (Kinsella, 1984; Walstra and Jennes,

1984; Swaisgood, 2003; Oliver, 2011).

Oppositely to caseins, serum proteins are rich in cysteine (Cys) residues (thiol groups, R-SH) and

disulphide bonds (S-S). β-lg contains five Cys residues, of which four are linked by (S-S) bonds as

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Cystine, leaving one highly reactive Cys open for thiol-disulphide interchange. The thiol group is

exposed following thermal denaturation and commonly reacts with the intermolecular disulphide

bonds of κ-cn (King, 1965; Livney and Dalgleish, 2004).

Table 2 Specifications of the dominant genetic variants of the proteins found in bovine milk. Adapted from Walstra and Jennes

(1984), Swaisgood (1992), Swaisgood (2003) and Fox and McSweeney (2003).

Properties

Genetic variant

αS1-cn

(B)

αS2-cn

(A)

β-cn

(A)

κ-cn

(A)

β-lg

(B)

α-la

(B)

MW [kDa]1 23.6 25.2 24.0 19.0 18.44 14.2

AA residues2 199 207 209 169 162 123

Phosphoserine 8 11 5 1 0 0

Phosphorylation

sites

Ser46, Ser48, Ser64,

Ser66-68, Ser75,

Ser115

Ser8-10, Ser16, Ser56-58,

Ser61, Ser129, Ser131,

Ser43

Ser15, Ser17-19,

Ser35

Ser149

- -

Cysteine3 0 2 0 2 5 8

S-S 0 4 0 2 2 4

Hydrophobicity 25 23 29 22 29 28

Proline 17 10 35 20 8 2

Secondary structure Low Low Low Low High High 1MW = Molecular weight 2AA = Amino acid 3Number of thiol groups

4Usually present as a dimer at room temperature with a molecular weight of ~36 kD.

A summary of some specifications for the dominant genetic variants of caseins and serum proteins

can be seen in Table 2.

2.1.3 Minerals

The minerals of milk contain cations (calcium, magnesium, sodium and potassium) and anions

(inorganic phosphate, citrate and chloride) (Gaucheron, 2004).

Calcium Phosphate. Sodium and potassium are soluble and present almost entirely as ions

dissolved in the serum phase. Calcium and phosphate are, however, much less soluble and exist

partly in a dissolved and partly in an insoluble form (Walstra and Jenness, 1984). Most calcium and

phosphate are bound covalently to casein and are present as colloidal calcium phosphate where the

involvement participates in neutralizing the proteins phosphoseryl residues and bridging the caseins

(Horne, 2017). The distribution and equilibrium state of calcium phosphate is affected by changes

in environmental conditions, such as pH or temperature. The quantity of calcium phosphate

associated with caseins increase with heating and decreases with decreasing pH (Walstra and

Jennes, 1984).

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2.1.4 The Casein Micelle Complex

Even though the molecular weight of casein molecules is relatively low, around 95% of the caseins

form aggregates that are present in a combination with calcium phosphate (colloidal calcium

phosphate, CCP). Collectively, they form the casein micelle. It is a roughly spherical (Walstra and

Jennes, 1984) complex where the protein fraction represents approximately 93% and the remainder

being CCP (Noble and Waugh, 1965; De Kruif et al., 2012). The casein micelle is initially formed

by polymerisation where hydrophobic regions of the caseins associate through clustering and where

the phosphopeptides are linked into calcium phosphate clusters (Noble and Waugh, 1965; Waugh

and Talbott, 1971; Griffin and Anderson, 1983).

Figure 1 Cross section of a casein micelle. α- and β-cn (orange) are attached to calcium phosphate (grey spheres). Some β-cn (blue)

are bound hydrophobically to other caseins. κ-cn is divided into para-κ-cn (green) and caseinomacropeptides (black). Not drawn to

scale and exaggerated for clarity. Adapted from Dalgleish and Corredig (2012).

The internal structure of the casein micelle has been tried elucidated in several models, but till date,

there are still disagreements on the exact structure (Bouchoux et al., 2010). The most commonly

referred models are the sub-micellar model and the nanocluster model. In the sub-micellar model, it

is suggested that the micelle is composed of clustered submicelles (∼106 Da and 10 to 15 nm in

diameter) that are held together by CCP, giving a micelle with an open porous structure (Morr,

1967). In the nanocluster model, CCP is randomly distributed within the casein micelle surrounded

by casein (Holt, 1992). Despite the different models, there is consensus on some aspects. A

common perception of the casein micelle structure is that at physiological pH of milk the casein

micelle has a net negative charge and is sterically and electrostatically stabilized from aggregation

by κ-cn situated on the micelle surface with the hydrophobic caseins αS- and β-cn located in the

interior of the micelle. k-cn is found with its hydrophilic C-terminal protruding 5 nm to 10 nm from

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the micelle surface into the surrounding solvent providing colloidal stability as interactions of

hydrophobic regions is prevented. The protruding κ-cn is commonly referred to as a hairy layer or a

polymer/polyelectrolyte brush (Noble and Waugh, 1965; Waugh and Talbott, 1971; Griffin and

Anderson, 1983; De Kruif and Zhulina, 1996; Holt and Horne, 1996; Dalgleish, 1998). The

illustration of the casein micelle suggested by Dalgleish and Corredig (2012) illustrated in Figure 1,

have included the main casein micelle structural agreements. From Figure 1 it can be seen that αs-

and some β-casein (orange) are attached to calcium phosphate clusters (grey spheres) and k-cn

(green and black) is found on the outer layer of the micelle. Some of the β-cn are bound by

hydrophobic interactions and can dissociate from the micelles porous structure upon cooling (Fox

and Brodkorb, 2008).The polyelectrolyte layer does not interfere with the thiol-disulphide

interactions between k-cn and serum proteins as the serum proteins can penetrate the layer and form

disulphide bonds with the inner part of the κ-cn (Livney and Dalgleish, 2004).

Casein micelles are structurally stable systems but on environmental changes, i.e. acidification or

temperature change the stabilization is altered. At physiological pH of bovine milk the serum phase

is supersaturated in calcium phosphate. The casein micelle is in dynamic equilibrium with the

serum phase where the exchange of calcium, phosphate and water can occur upon environmental

changes (Walstra and Jennes, 1984). The distribution of calcium phosphate between the serum- and

colloid phase depends on the pH, ionic strength, calcium activity, and temperature which affect the

solubility of calcium phosphate (Schuck, le Floch-Fouere and Jeantet, 2013). Acidification to pI of

casein micelles (pH 4.6) results in the reduction of steric repulsion between the casein micelles and

the subsequent consequence is a collapse of the polymer brush which results in aggregation of the

colloidal system (De Kruif and Zhulina, 1996). Following a temperature drop the solubility of CCP

is increased. CCP is solubilized into the serum phase and the stability of the casein micelle is

impaired (Gaucheron, 2004). The more calcium that is removed, the greater the dissociation of

single caseins from the casein micelle (Xu et al., 2016).

2.2 Industrial Production of MCI Powder

The major component of MCI powder is casein. As casein represents a minor part of bovine milk, it

is necessary to isolate caseins prior to powder production. Beforehand, bovine milk is skimmed and

pasteurized so fat is present in a small quantity and bacteria inactivated (Crowley et al., 2015).

Traditionally, caseins have been isolated through methods that build on the concepts of precipitating

the caseins out of the micelle, i.e. rennet and acid precipitation (Holt and Horne, 1996; Dalgleish

and Corredig, 2012; O’Mahony and Fox, 2013).

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By applying membrane technologies instead of precipitation it is possible to isolate casein kept at

its micellar state, containing colloidal calcium phosphate (Burgain et al., 2016). The composition

and physical state of bovine milk components make membrane fractionation a suitable method,

hence whey proteins, lactose, and part of the minerals are present in colloidal solution; casein in

colloidal suspension; and fat globules in emulsion (King, 1966; Walstra and Jenness, 1984;

Gelfand, 1996). Following membrane filtration, the concentrate is spray dried into powder by

removal of water. Several environmental changes influence the structure of the casein micelle that

can be destroyed to a greater or lesser extent by dissociation of single caseins and minerals from the

micelle (Gaucheron, 2004). An overview of the physico-chemical changes occurring upon a number

of environmental changes is illustrated in Figure 2.

Figure 2 Modifications of the casein micelle and the subsequent dissociation or association of numerous components upon different

environmental changes, such as cooling, acidification and heat treatment. Adapted from Gaucheron (2004).

Largely, cooling release β-cn and CCP; heating denatures serum proteins which associate with the

casein micelle, κ-cn is released and the solubility of CCP is decreased and, acidification release

single caseins and CCP is solubilized (Gaucheron, 2004; Dalgleish and Corredig, 2012).

In the following, the principles behind the technologies that are used in MCI powder production

will be described and their contribution to product formation related to physico-chemical changes

occurring during powder production elucidated.

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2.2.1 Membrane filtration

The different components of milk can be separated into fractions in various ways depending on the

wanted outcome (Walstra, Wouters and Geurts, 2006; Schuck, 2013). Membrane processing is a

molecular separation technique that separate components based on their molecular size and is useful

in milk protein fractionation. It enables the separation of milk into permeate and retentate. The

retentate part is retained by the membrane whereas the permeate part is permitted through the

membrane pores (Walstra and Jennes, 1984; Bylund, 1995).

In the production of MCI, the first production step is microfiltration (MF) of pasteurized skim milk.

MF is a crossflow membrane filtration method where the feed flows over a membrane under

pressure, retaining the solids (Pierre et al., 1992). It is referred to as a tangential filtration method

because the liquid is circulated tangentially to the membrane in order to limit fouling development

(Schuck, le Floch-Fouere, and Jeantet, 2013). The pressure-driven process is commonly used in the

production of MCI with a pore size of 0.1 μm and retains components with molecular weights

larger than ~100 kDa. Casein micelles vary in size from 0.01 to 0.3 μm while the majority have an

approximate average micelle size varying from 0.13μm to 0.16 μm and are larger in molecular

weights compared to most serum proteins (~10 kDa) (Mulder and Walstra, 1974; Walstra and

Jennes, 1984; Swaisgood, 2003) (Table 2). The outcome of the MF process of skim milk is a

retentate, consisting of micellar casein, and a permeate, consisting of the majority of whey proteins.

However, aggregates of whey protein will be retained by the membrane and thus found in the

retentate as well (Rosenberg, 1995; Broyard and Gaucheron, 2015).

When using membrane applications in combination, more advanced approaches arise. Further

treatment of MF retentate with diafiltration (DF) improves the quality of the recovered components

as it dilutes soluble components, such as lactose and minerals from the MF retentate. In DF the

concentrate is diluted with water and the filtration proceeds until the wanted solute removal is

obtained. It is possible to purify the retentate up to 90% protein on a total solids basis (Pierre et al.,

1992; Rosenberg, 1995; Claire Gaiani et al., 2007).

Albeit membrane filtration is intended for keeping casein at its micellar state, containing CCP, it is

notable that the physico-chemical properties of the casein micelle are altered and that the outcome

of the filtration is casein in association colloids that closely resemble the micellar one. One

important change is the removal of CCP that is specially altered by the DF step (Gaucheron, 2004;

Ferrer, Alexander, and Corredig, 2014). The degree of CCP removal is influenced by the

temperature and pressure at which filtration is performed, where more CCP is removed with

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decreasing temperature and increasing pressure (200 MPa to 300 MPa) (Orlien, Boserup and Olsen,

2010; Schuck, le Floch-Fouere and Jeantet, 2013; Liu et al., 2014).

Despite the physico-chemical changes that follow membrane processing the technology is of good

use in food production as it provides the possibility of accomplishing both the fractionation and

concentration of components in liquid systems without phase changes and still remains desirable

physical and chemical characteristics.

2.2.2 Spray Drying

There is a variety of different processing methods available for drying. One of the most commonly

used methods for powder production is spray drying (Parkash, 1968; Schuck, 2013). Spray drying is

a drying technique that dehydrates the incoming feed by removal of water and retaining of solids in

a dry state by evaporation (Bhandari and Howes, 1999; Schuck, 2009). Depending on the type of

powder to produce, there are different drying setups to use (King, 1965). In the following, a

simplified setup is described as divided into the concentration of feed material and the actual

drying, starting from the liquid feed enter the machinery and finishing at the collection of dry

particles.

2.2.2.1 Initial concentration of feed

For industrial applications, the feed for spray drying undergoes an initial concentration process

where it is evaporated under pressure to a dry matter content of approximately 20% to 25% for

protein isolates (Schuck, le Floch-Fouere and Jeantet, 2013). For evaporation, the concentrate is

held near its boiling point (Skanderby et al., 2009) and vacuum is applied to prevent heat damage as

the temperature can be reduced. This is much used in the industry as the removal of water by

evaporation requires less energy compared to that of drying (Bylund, 1995). However, the process

is not uncomplicated as several physicochemical properties of the feed are changed. The most

apparent one is the change in rheological behaviour as the viscosity increases and changes the

solution into a shear-thinning liquid. Caseins aggregate via hydrophobic bonding as a consequence

of the tightly packed system (King, 1965). The salt equilibria and conformation of proteins also

change. Liquid milk is saturated with calcium phosphate. The solubility change as a consequence of

heating, where the resulting influence is a heat-induced shift in mineral equilibria towards the

colloidal phase. This reduces the ionic calcium (Ca2+

) and decreases its activity. However, during

evaporation, the activity of Ca2+

is increased as the volume decrease upon concentration which

increases the level of Ca2+

and decreases pH. Furthermore, the tendency for protein association

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increase promoting casein micelle aggregation which results in an increase in micellar size

(Walstra, 1984; Oldfield, Taylor and Singh, 2005; Singh, 2009). During concentration, the

temperature exceeds the denaturation temperature of serum proteins (65 ⁰C to 75 ⁰C (Schuck, le

Floch-Fouere and Jeantet, 2013)) and the complex between denatured serum proteins and κ-cn is

established via thiol-disulphide interchange (Livney and Dalgleish, 2004). It is mainly in the

concentration stage that denaturation occurs compared to the remaining spray drying process

(Bhandari, 2013).

2.2.2.2 Drying of feed

For drying, the now concentrated feed is taken up by the system and transported to the drying

tower. The drying tower can be separated into three stages consisting of atomizing of concentrate,

evaporation of concentrate and collection of dry particles (Bylund, 1995). Mainly atomization and

evaporation will be described in the following.

There are many configurations of the drying tower used in spray drying and the simplified

arrangement shown in Figure 3 is limited to the introduction of liquid feed into the heating chamber

and subsequent water evaporation.

Figure 3 A simplified and schematic spray drying system, where the feed is introduced into the drying chamber for atomization from

the top and dry particles are collected from the bottom where it is transferred to a cyclone. Adapted from Singh and Heldman (2013).

Atomizing. The atomizer is a spray ball device which serves to disperse the large feed droplets into

finely dispersed ones. The consequence is enlargement of the droplets specific surface area and is

done for a more effective and rapid drying process with minimal heat damage (Schuck, 2009). The

most common atomizers for the dairy industry are pressure nozzles and rotary atomizers (spinning

discs). Pressure nozzles force the feed through a small opening at high pressures (up to 30 MPa)

whereas the rotating atomizer is based on centrifugal energy. It consists of a spinning disc where the

incoming feed is distributed on the disc surface by centrifugal forces and discharged at high rotation

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speeds, e.g. 100 m*s-1

at the edge of the disc (King, 1965; Walstra, Wouters and Geurts, 2006). It

has the ability to handle highly viscous concentrates and crystalized products and is commonly used

for most conventional milk products (Westergaard, 2011). The degree of atomization mainly

depends on feed material properties, such as viscosity where viscous solutions result in the

atomization of larger droplets (Schuck, 2009). The ideal atomizing process would be a spray of

droplets of equal sizes that thereby exhibit same drying time. In practice, this has still not been a

possible development, hence the spray dried powder particles vary in size and drying history

(Westergaard, 2011).

Evaporation. Following atomization, the evaporation initiates. The powder is formed by atomising

the concentrated milk into a stream of hot air (180 ⁰C to 220 ⁰C) (King, 1965; Skanderby et al.,

2009). The high surface-to-mass-ratio that is obtained in the atomization process increases the

evaporation rate of the droplet as the thermal efficiency is improved due to an increase in surface

area available for contact and exchange with the surrounding hot air (Schuck, 2009; Westergaard,

2011).

In the evaporation process, there are two main mechanisms responsible for the dehydration; the heat

and mass transfer. Heat is transferred from the air to the droplet due to temperature differences

between the droplet surface and the hot air. Additionally, water is transferred from the droplet to the

air due to the difference in partial pressure of water vapour. Thus air is used both as the heater and

as the carrier gas for removal of water (Schuck, 2009; Singh and Heldman, 2013). The evaporation

is initiated from the droplet surface and is maintained throughout the drying process by the

migration of capillary water from the interior of the particle towards the particle surface. During

evaporation, the solute surface concentration increase and a skin is formed. It simultaneously leads

to the migration of milk components towards the surface to replace the aqueous phase (Fäldt and

Bergenståhl, 1996; Nijdam and Langrish, 2006). However, as evaporation progress, a concentration

gradient across the radius of the particle is established. This gradient causes the solutes to diffuse

inwards to the centre of the droplet. As components exhibit different diffusivities the composition

of the surface will be greatly influenced by this (Woo and Bhandari, 2013). Heat and mass transfer

of a droplet can be expressed mathematically as in Equation 1 and 2, respectively (Singh and

Heldman, 2013; Woo and Bhandari, 2013). It is seen that the proportions of the droplet, its

composition, heat transfer and conductivity etc. all play a significant role in the drying process.

𝑑𝑇

𝑑𝑡= ℎ𝐴(𝑇𝑎𝑖𝑟 − 𝑇𝑑𝑟𝑜𝑝𝑙𝑒𝑡) −

𝑑𝑚

𝑑𝑡∆𝐻𝑒𝑣𝑎𝑝 (1)

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𝑑𝑚

𝑑𝑡= ℎ𝑚𝐴(𝜌𝑑𝑟𝑜𝑝𝑙𝑒𝑡 𝑠𝑢𝑟𝑓𝑎𝑐𝑒 − 𝜌𝑎𝑖𝑟) (2)

where, dT/dt is the rate of temperature change [K*s−1

], dm/dt is the drying rate [kg*s−1

], A is the

droplet surface area [m2], (Tair-Tdroplet) is the temperature difference [K], ∆Hevap is the latent heat of

evaporation [kJ*kg−1

], h is the convective heat transfer coefficient [W*m−2*

K−1

], hm is the

convective mass transfer coefficient [m*s−1

] and ρdropletsurface – ρair is the vapour concentration

difference [kg*m−3

].

When evaporation has finished and the powder particles are formed, they are ready to leave the

spray dryer. Some particles are too small in size and are separated in a cyclone before the finished

bulk powder can be collected (Walstra, Wouters and Geurts, 2006).

2.2.3 Freeze drying

Freeze drying is the evaporation of water under vacuum. It can be divided into the two stages where

the product is frozen, and the product is dried by direct sublimation of the ice under reduced

pressure (Bhandari and Howes, 1999). The heat and mass transfer during freeze-drying occur from

two product stages that is as ice or as dry product. When the product is frozen, the heat transfer will

be rapid. As the freeze-drying initiates and the product dry its heat transfer decrease due to low

thermal conductivity and the diffusion of water vapour is low due to the slow diffusion rate under

vacuum (Singh and Heldman, 2013). While spray drying is evaporation from droplets, freeze-

drying is the evaporation from a static surface where the product volume is maintained but the

structure becomes more porous. The main advantage of the freeze drying process is the low

temperature at which it is exceeded preventing thermal degradation (King, 1965; Liapis &

Marchello, 1984). However, the freezing results in an irreversible disintegration of the casein

micelle structure when subjected to high pressure, such as 100 MPa for 1 hour. Furthermore, the

temperature decrease weakens the hydrophobic interactions of the casein micelle and part of β-cn

dissociate from the micelle and a smaller part of the calcium phosphate dissolves, resulting in a

decrease of micelle size (Walstra, 1984). Even though freeze drying is a gentle powder forming

process resulting in high-quality products it is not widely used due to a high energy demand

(Schuck, le Floch-Fouere and Jeantet, 2013).

2.3 The Rehydration Process

After powder production has finished, the obtained powder product facilitate a number of powder

functionalities. One powder functionality, that is of great importance for the application of powder

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is the powder particles association with water, i.e. its rehydration ability. The rehydration process

consists of a combination of the stages wetting, swelling, sinking, dispersing and dissolution, as

seen in Figure 4. Some stages, such as wetting/swelling and dispersion/dissolution overlap or

happen concurrently and are difficult to completely differentiate, as rehydration is a dynamic

process (Crowley et al., 2016).

Figure 4 The rehydration process profile for powder particles, divided into the stages 1) Wetting, 2) Swelling, 3) Sinking, 4)

Dispersion and 5) Dissolution. The profile is highly schematic for clarity and some stages may overlap or happen concurrently.

Adapted from Crowley et al. (2016).

The rehydration behaviour is affected by several factors, such as composition of raw material,

rehydration conditions (e.g. stirring rate, temperature (Jeantet et al., 2009)), processing methods and

the resulting structural influences that follow (e.g. presence of pores, agglomeration) etc. (Parkash,

1968; Kinsella, 1984). As the powder particle surface is the first part that comes into contact and

interacts with water, the rehydration is additionally affected by powder surface composition and it

has been found that most rehydration steps are directly influenced by this to a higher or lower

degree (Fiildt and Bergenstahl, 1996; Kim, Chen and Pearce, 2002). It is crucial for its application

that the rehydration of commercial powder is rapid, especially for further use in industrial

applications but also for consumer convenience. Problems associated with rehydration vary from

dairy power to dairy powder. For example, for whey protein powder the limiting step is wetting,

whereas for casein-based powders it has been found to be dispersion (C. Gaiani et al., 2007). If the

rehydration is incomplete the resulting consequence is quality deficiencies. Depending on the

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limiting rehydration step it could be lump formation with non-hydrated regions or lack of protein

expression which alter product quality (King, 1966).

The knowledge necessary for understanding the powder behaviour during rehydration will be

outlined in the following sections where each section represents one or several rehydration steps.

2.3.1 Wetting and Swelling

When bulk powder is placed onto the water surface the first rehydration step is wetting. As wetting

and swelling happen concurrently, these are both described in the following.

Wetting. The first stage of rehydration, wetting, is defined as the absorption of water following bulk

powders initial contact with the water surface. When the bulk powder overcomes the surface

tension it sinks into solution where powder disappears from the water surface. Poor wettability is

seen in powders that fail to wet sufficiently and remain floating on the surface, whereas powders

with good wettability sink readily upon surface contact (King, 1966; Parkash, 1968).

Wetting is directly dependent on the contact angle (θ) between powder- and water surface and

hence particle size, as large particles form small contact angles. A contact angle less than 90 ⁰

represents a successful particle wetting. The contact angle is typically found from Youngs equation

listed in Equation 3 (Crowley et al., 2016).

𝛾𝑆𝐺 = 𝛾𝐿𝑆 + 𝛾𝐿𝐺 ∙ 𝑐𝑜𝑠𝜃 (3)

Where γSG is the interfacial tensions between the solid and gaseous phase, γLS is the liquid and solid

phase and γLG is the liquid and gaseous phase.

Wetting time can vary from a few seconds to minutes, depending on the characteristics of the

presented powder where several parameters affect the wetting behaviour either by improving or

prolonging the process. Factors that improve wetting decrease the contact angle and vice versa. The

hydrophobicity or hydrophilicity of the components present at the powder surface is of major

importance, where the presence of lipids will decrease wettability and the presence of lactose will

increase wettability (Kim, Chen and Pearce, 2002; Havea, 2006). Furthermore, agglomeration of

particles improves powder wetting behaviour as the capillary network of the agglomerated powder

particles induce inwards water diffusion where the interstitial air is replaced by water. Further, the

increase in particle size that follows agglomeration has a larger effect on the surface tension

compared to non-agglomerated powders which improve wetting behaviour (Bockian, Stewart and

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Tappel, 1956). If the powder particles absorb water too quickly it has been found that there is an

establishment of a partly wetted-partly dry surface barrier film between the powder and water

impeding further wetting (Skanderby et al., 2009).

Swelling. Swelling occurs due to water uptake and is the increase in particle size as a consequence

hereof. Swelling has been found to occur in powders high in casein; however, for powders high in

serum proteins, the same tendency has not been clearly distinguished (C. Gaiani et al., 2007; Gaiani

et al., 2006).

2.3.2 Sinking

After bulk powders contact with the water surface and its subsequent initial water uptake, the

powder is ready to sink into the solution.

Sinking. The swelled particles sink into the solution by the release of occluded air entrapped inside

the bulk powder and its replacement by water. As water replaces the remaining air, the particles

become denser and hence increasingly sink through the solution (Vos et al., 2016). Powder

sinkability can limit dispersion and solubility at higher solids concentrations due to an increased

viscosity where sinking is prevented (Schober and Fitzpatrick, 2005). However, in today’s

industrial powder application, where it is common that tanks etc. are equipped with agitation

(Westergaard, 2011) the sinkability can be neglected. It becomes drastically abbreviated so that the

dispersion phase of the rehydration process is initialised more or less directly following the

wetting/swelling stage.

2.3.3 Dispersion and Dissolution

Following wetting, swelling, and sinking of powder into the water, particles are ready to be

dispersed into the solution and further for total disruption of particle structure and subsequent

dissolution. Dispersion and dissolution overlap in mechanism and both will be described in the

following.

Dispersion and Dissolution. Dispersion is the separation of bulk powder into agglomerates, the

dispersion of agglomerates into primary powder particles and further, the erosion of primary

particles and the subsequent dissolution of particle components, in water (Walstra and Jennes, 1984;

Kinsella, 1984). It has been suggested that high protein concentrate powders contain two groups of

dissolving components during erosion of primary particles; (i) fast dissolving i.e. lactose, serum

proteins and minerals (dispersion) and (ii) slow dissolving consisting primarily of casein and

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colloidal calcium phosphate (dissolution) (Bockian, Stewart and Tappel, 1956; Arnaud Mimouni et

al., 2010). As particles are penetrated by water the release of ions are promoted, seen as an increase

in conductivity (Uluko et al., 2016). For casein-based powders the slow dissolving components is

mainly due to the appearance of a surface skin layer of inter-linked casein micelles present at the

particle surface preventing successful dissolution of particles as the hydrophobic bonds between the

casein micelles have to be overcome, making the physical state of casein a critical factor limiting

the rate of dispersion and dissolution (King, 1966; Parkash, 1968; A. Mimouni et al., 2010).

In the preceding section an introduction to the principal components of MCI, production of MCI

and the rehydration process have been elucidated. In the evaluation of the principal components of

MCI, some different aspects important for the interaction of milk components, either as the casein

micelle complex or molecular interactions occurring upon environmental changes has been

elucidated. For example, the structure of proteins, including their differences in phosphorylation,

hydrophobicity, sensitivity to and influence from minerals, i.e. calcium and its effect on the

colloidal stability of milk or the stability of the casein micelle complex.

In the evaluation of MCI production, a simplified production process for the manufacture of

micellar casein powder has been described according to initial fractionation by membrane

technology, the concentration of feed prior to drying and the drying itself by the technologies spray

drying and freeze drying. As well as the influence of each process step on some physicochemical

aspects of the feed and powder particles has been outlined.

Lastly, the rehydration process and its phases have been evaluated, i.e. wetting, swelling, sinking,

dispersion, and dissolution where the behaviour of bulk powder, primary powder particles and the

erosion of particle structure has been described in relation to its phase.

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3. Materials and Methods

This study was based on rehydration of a commercial powder, that was afterwards spray or freeze

dried into six different powders at three concentrations. Several analyses were used to characterize

feed and powder properties and further, the rehydration ability of the powders. An overview of the

experimental setup is displayed in Figure 5.

Figure 5 Experimental overview of the present study, divided into overall steps consisting of feed characterisation, powder

production, powder characterisation and characterisation of powders rehydration ability.

3.1 Preparation of MCI feeds

The raw material was the commercially produced powder Promilk® 852 B. It was provided by

Ingredia (Bagsværd, Denmark) produced 21st of September 2017. The product bag (25 kg) was

stored at room temperature in a dark and dry place throughout the study. The physico-chemical

specifications are listed in Table 3.

For feed preparation, commercial MCI powder was rehydrated. Water (25 ± 1 ⁰C) was added to a

large container, wherein sodium azide (0.05% w/w) was dissolved to avoid microbial spoilage

during feed preparation (De Kruif and Zhulina, 1996; Huppertz and De Kruif, 2007). The

commercial MCI powder was added to the liquid solution until solid contents of 12%, 15% and

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18% (w/w) and manually stirred for approximately five minutes until no non-hydrated areas were

visible. The feed concentrates were transferred to glass bottles holding a magnet, sealed with a lid

and left to rehydrate for 48 hours at ~25 ± 1 ⁰C on agitation, in accordance with the studies by

Gaiani et al. (2010) and Sadek et al. (2014).

Table 3 Technical specifications for the commercial micellar casein isolate, Promilk 852B, displaying its composition.

Technical specification

Average quantity [% w/w]

MCI*

Protein 81%

- Casein 74.5%

- Serum proteins 6.5%

Lactose 4%

Minerals 8.5%

Fat 1.5%

Water 5%

3.2 MCI powder production by freeze- and spray drying

The powder was produced by two different processing methods; spray drying and freeze drying.

The setup for both is seen in Figure 6.

Figure 6 Experimental setup for the drying process for static freeze drying (left) and lab scale spray drying (right).

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3.2.1 Spray drying

Spray dried powders were prepared with a Büchi Mini Spray Dryer B-290, which is a one-stage lab

scale spray dryer that can dry approximately 200 ml sample per hour.

The settings used can be seen in Table 4 (Hogan et al., 2001; Nasser, Moreau, et al., 2017).

Table 4 Settings used during lab-scale Büchi spray drying, for feed concentrates at varying dry matter content (12%, 15% and 18%)

and the resulting Toutlet.

Sample

concentration

Tinlet [⁰C] Toutlet [⁰C] Pump speed [%] Aspirator [%] Nozzle

cleaner Q-flow

180 - 9 99 2 45

12% 180 66 11 99 2 45

15% 180 66 11 99 2 45

18% 180 68 11-14 99 2 45

As the outlet temperature is influenced by the settings used and thereby also controlled by these,

pump speed was either increased or decreased where an increased feed uptake decreased Toutlet and

vice versa. The spray drying was done by placing the feed tube in the sample container. All

parameters, but pump speed, were kept constant throughout the drying. Toutlet was generally kept

constant at 66 ⁰C. However, for samples with a DM content of 18% Toutlet increased rapidly and

was generally at 68 ⁰C (Table 4). However, this was controlled by an increase in pump speed.

During spray drying, it was noticed that for the 18% sample there was a rapid accumulation of

burned residues on the spraying nozzle. When finished, the spray dried powder was transferred to a

glass bottle, sealed with a lid and stored at 5 ⁰C until use.

3.2.2 Freeze drying

Freeze dried powders were prepared with an Edwards static freeze dryer, from Buch & Holm A/S.

It holds 30 petri dishes (~40 ml per dish) per drying cycle (72 hours). The feed was transferred to

the petri dishes and frozen at -80 ± 1 ⁰C for a minimum time of 24 hours and a maximum of 72

hours, to ensure complete and uniform freezing. The samples that were frozen for 72 hours were

held at -80⁰C in accordance with Baldwin and Truong (2007), to avoid an increase in insolubility.

The petri dishes were placed in the freeze drier without lids directly from the freezer. Temperature

and vacuum were activated and samples stayed in the freeze dryer for 72 hours. The powders of

each freeze-dried petri dish were manually grinded with a pestle and a mortar, for 2 minutes (~1.5

round per second) per plate. Additionally, the powders were standardized using a ceramic sieve

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(pore size of 1 mm) for removal of large non-grinded particles in accordance with the study by

Seville, Kellaway and Birchall (2002). The finished powder from all petri dishes was mixed and

transferred to a glass bottle, sealed with a lid and stored at 5 ± 1 ⁰C until use.

3.3 MCI feed characterisation

The feed was characterised according to pH, particle size distribution, and rheological behaviour.

3.3.1 Feed pH

pH was measured with a pH-meter holding a penetration electrode (Testo 205, Testo, Lenzkrich,

Germany). Prior to measurements, the pH-meter was calibrated using buffers of pH 4.0, pH 7.0 and

10.0. Measurements were performed by directly measuring in the feed solutions. Results obtained

were based on an average of six measurements.

3.3.2 Feed particle size distribution

The particle size distributions of feed concentrates were measured with the Malvern Mastersizer

(Malvern instruments Ltd. Worcestershire, UK) equipped with the Malvern Mastersizer 3000

attachment. Prior to measurements, settings for the shape of particle, the refractive index of sample,

the refractive index of dispersant and the absorption were set to non-spherical, 1.57 (Griffin and

Griffin, 1985), 1.33 and 0.01 respectively (Gaiani et al., 2005). Prior to each measurements a

cleaning cycle was run, system initialized and lastly background measured. The measurements were

performed by adding samples dropwise into the dispersion unit to reach the ideal obscuration range

of 8. Measurements were conducted at 2000 rpm. Results obtained consisted of the particle size

distribution parameters D (3;2), D (4;3), D (10), D (50) and D (90). Results for all concentrations

were based on an average of three measurements for two solutions per feed concentrate (2 x 3).

3.3.3 Feed rheological behaviour

The rheological behaviour of feed concentrates was determined by flow curves (shear stress and

viscosity versus shear rate) was obtained using a HAAKE RheoStress 600 (Thermo Fisher

Scientific Inc., Karlsruhe, Germany) rheometer containing a stainless steel bob-cup geometry with a

rotating cone (d = 40 mm). Approximately 40 mL of feed concentrate was placed into the cup and

submitted to an up-down shear rate sweep from 0 to 300 s-1

with a 4 mm gap at room temperature

(20 ± 1 °C). The flow curves were fitted to the Power Law model (Equation 4) by nonlinear

regression using Origin Pro 9.1 (OriginLab Corporation, Northampton, MA 01060 USA). Results

were based on an average of three replicates.

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𝜎 = 𝐾γ̇𝑛 (4)

Where σ is shear stress [Pa], γ is shear rate [s-1

], K is the consistency coefficient [Pa.sn] and n is the

flow behavior index

3.4 MCI Powder characterisation

MCI powders were characterised according to pH, particle size distribution, moisture content and

water activity, colour and microstructure.

3.4.1 Powder pH

The powder pH was measured as described in section ‘3.3.1 Feed pH’, for all powders at t = 20

minutes following water immersion.

3.4.2 Powder particle size distribution

Powder particle size distribution was measured as described in section ‘3.3.2 Feed particle size

distribution’ but with a dry feeder attachment, Malvern Mastersizer Aero S.

The settings for optical model presentation for particles dispersed in air were set at a pressure of 2

bars and a feed rate during measurements of 61%. Approximately 1 to 2 g of powder was loaded

directly into the dispersion unit that consisted of a sieve and a small ball for breakage of powder

lumps. At start of the analysis, the sample intake was activated and the powder was shaken through

the sieve to a transport bond and transferred to the measuring area of the machinery. When

measurements had finished, the machinery was stopped and cleaned. Between measurements of

powders with same DM content, the equipment was brushed clean in the feeding area whereas the

equipment was dismantled for proper cleaning of tubes, between measurements of powders with

varying DM content. Results for all concentrations were based on an average of three

measurements.

3.4.3 Powder moisture content

Moisture content was determined in accordance with Ardö (2014) and Schokker et al. (2011) by

oven drying. It is a gravimetric method that consists of drying samples overnight (16 to 18 hours)

under atmospheric pressure at a temperature of 102 ± 1 ⁰C until constant weight where all moisture

is assumed evaporated. Approximately 5 g clean pumice was added to beakers and dried in the oven

at 100 ± 1 ⁰C for 1 hour until constant weight to ensure a dry environment. Approximately 1 to 2 g

of sample was placed in the beaker and dried overnight. The moisture content was calculated

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according to Equation 5. Results for all concentrations were based on an average of three performed

measurements.

𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 100 − (𝑚2−𝑚0

𝑚1∙ 100%) (5)

Where m0 is the mass of the beaker [g], m1 is the mass of the powder placed in the beaker for drying

[g] and m2 is the mass of the beaker with dried powder sample [g].

3.4.4 Powder water activity

Water activity (aw) was measured using an Aqua Lab 3 TE (Decagon Devices, Inc., USA) at

temperature 20 ± 1 ⁰C. Plastic containers were filled half with powder and left in the machinery

until measurement had finished. Beforehand, a measurement on a hydrated lithium chloride (LiCl)

solution with known aw of 0.11 was measured to ensure the equipment was calibrated correctly and

measured precisely. Results for all concentrations were based on an average of six samples.

3.4.5 Powder colour

Colour was measured with a Chroma Meter CR-400 (Konica Minolta Business Technologies, Inc.,

Tokyo, Japan) and was evaluated according to the colour coordinates L* (black = 0, white = 100),

a* (red = +, green = -) and b* (yellow = +, blue= -). ∆E and browning index (BI) was calculated

from Equation 6 and 7 (Nasser, Moreau, et al., 2017).

∆𝐸 = √𝐿2 + 𝑎2 + 𝑏2 (6)

𝐵𝐼 =100∙[(

𝑎+1.75∙𝐿

5.647∙𝐿+𝑎−3.012∙𝑏)−0.31]

0.17 (7)

The colorimeter was standardized using a white calibration plate. The powder was placed in a small

circular container and the colorimeter held towards the surface of the powder. Six determinations

were performed for each sample and results were expressed as average values. Between each

replicate, the powder was disrupted and moved around in the container for variation. The

colorimeter was thoroughly cleaned between measurements on powders with varying dry matter

content.

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3.4.6 Powder microstructure

The microstructure of MCI powders was evaluated using scanning electron microscopy (SEM).

SEM was used for imaging of powders at all concentrations, including the commercially produced

powder. SEM consists of two steps divided into pre-treatment of samples and scanning of samples.

Pre-treatment. The powder was distributed onto stubs with double-sided adhesive carbon tabs, in a

thin layer and placed with round shaped tweezers in a sample stage in a vacuum chamber. The

chamber was sealed, the cylinder with gas opened and vacuum decreased. The sample stage holds a

quantity of 6 samples per coating and coat by rotating the samples under a spray of palladium for 20

seconds. After coating, samples were transferred to a holder and ready for scanning.

Scanning. The scanning was carried out using a FEI Quanta 200 scanning electron microscope (FEI

Company, Hillsboro, USA) equipped with a back-scattered electron detector. The SEM system was

vented with nitrogen gas and samples were loaded into the sample holder, with round shaped

tweezers. SEM door was closed, the pump was activated and the system went into high-vacuum

mode. When a sample of interest had been located, the electron beam was started and imaging

performed at appropriate magnifications.

3.5 Characterisation of MCI powders rehydration ability

Powders rehydration ability was evaluated by wettability, dispersibility, and final solubility.

3.5.1 Powder wettability

Dynamic wettability measurements were based on a modified Washburn method. One to two g

sample was added to a plastic tube with an open bottom (d = 15 mm). Filter paper (d = 125 mm)

was gauzed to the tube to hold the powder in place and the tube was fixed just above the water

surface (20 ± 2.0 ⁰C). After 10 minutes, the samples were removed and weighed to determine the

wettability in form of g water taken up per gram of powder. Equation 8 is modified from Selomulya

and Fang (2013). Results for all concentrations were based on an average of six measurements.

𝐻𝑦𝑑𝑟𝑎𝑡𝑖𝑜𝑛 =𝑚𝑡𝑢𝑏𝑒+𝑝𝑜𝑤𝑑𝑒𝑟+𝑤𝑎𝑡𝑒𝑟−𝑚𝑡𝑢𝑏𝑒+𝑝𝑜𝑤𝑑𝑒𝑟

𝑚𝑡𝑢𝑏𝑒+𝑝𝑜𝑤𝑑𝑒𝑟−𝑚𝑡𝑢𝑏𝑒 [𝑔 𝑤𝑎𝑡𝑒𝑟 𝑝𝑒𝑟 𝑔 𝑝𝑜𝑤𝑑𝑒𝑟] (8)

Where mtube is the mass of the tube used [g], mtube+powder is the mass of tube with loaded powder [g]

and mtube+powder+water is the mass of tubes with powder after wetting time [g]

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3.5.2 Powder dispersibility

Dispersibility was determined by the change seen for the particle size distribution parameter D (50)

over time. The settings can be seen in section ‘3.4.2 Powder particle size distribution’. Each powder

was hydrated in demineralised water (25 ⁰C) to fit a solution of 5% (w/w), placed on a heating pad

set to a temperature of 25 ⁰C with agitation and measured at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h,

and 48h.

3.5.3 Powders final solubility

Final solubility was evaluated by measuring pH, particle size distribution, and rheological

behaviour.

3.5.3.1 Rehydrated powders pH

pH was measured in accordance with section ‘3.3.1 Feed pH’.

3.5.3.2 Rehydrated powders particle size distribution

Particles size distributions were measured in accordance with section ‘3.3.2 Feed particle size

distribution’.

3.5.3.3 Rehydrated powders rheological behaviour

Rheological behaviour of rehydrated powders were measured in accordance with section ‘3.3.3

Feed rheological behaviour’.

3.6 Statistical data analysis

The statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc,

California, USA) based on three or six replicates. Means of samples and their standard errors were

used to determine the variation between replicates and to display in plots. One-way ANOVA was

used to compare the population variation at a significance level of p < 0.05. Further, ANOVA was

complimented with Tukey’s post-test with corrections for multiple comparisons for comparison of

differences between groups and differences denoted with different letters.

Multivariate data analysis was conducted with principal component analysis (PCA). All

computations were made using MATLAB R2017A (The MathWorks, Natick, MA). PCA models

were developed using PLS Toolbox version 8.2.1 (Eigenvector Research, Inc. Wenatchee, WA).

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4. Results and discussion

In the present section, the results obtained from the conducted experiments have been presented and

discussed. The section is divided into subsections on feed solution (rehydrated commercial powder),

dry powder (freeze- and spray dried) and rehydrated powder solutions (freeze- and spray dried)

where i.e. pH, particle size, rheological behaviour, moisture content and water activity, colour,

microstructure, and rehydration ability concerning wettability, dispersibility, and final solubility

were investigated.

4.1 Properties of MCI feed concentrates at different dry matter contents

To characterise the feed used in the manufacture of freeze- and spray dried powders, properties such

as pH, particle size, and rheological behaviour was evaluated for rehydrated (48 hours) concentrates

at dry matter contents of 12%, 15% and 18% (w/w). As the feeds constituted a commercially

produced powder it is important to consider the physico-chemical changes that might have occurred

during the production of the commercial powder. These changes to the casein micelle may not be

readily reversed during rehydration (Martin, Williams and Dunstan, 2007). Several factors influence

pH, particle size and rheological behaviour of feed, such as time and temperature of heating, pH of

the solution prior to heating, levels of soluble calcium and phosphate and dry matter content (Singh

et al, 1995). Unfortunately, these parameters are unknown for the production process of the

commercial powder and estimations based on the feed values analysed in this project aims mainly at

comparing the effects of dry matter content on physical properties of the feed. It is known that

membrane filtration; concentration by evaporation and spray drying are commonly a part of the

commercial production of MCI powders. Regardless of the specificities for the environmental

factors, it can be assumed that some shifts in mineral equilibria have occurred as well as serum

protein denaturation as a consequence of the commercial drying process.

4.1.1 pH of MCI feed concentrates

The pH values of the feeds prepared from the commercial powder are presented in Table 5 for

concentrates with DM content of 12%, 15% and 18%. It can be seen that pH varied slightly between

feed concentrations where pH decreased (6.87 ± 0.009 to 6.69 ± 0.005) with increasing DM content.

However, feeds with DM 15% and DM 18% were not significantly different (p > 0.05).

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Considering the commercial processing, the application of diafiltration dilutes the soluble

components of the serum phase, creating a mineral imbalance. As the mineral concentration

becomes increasingly higher in the serum phase, the ratio between the serum phase and the colloidal

phase in increased. The resulting influence is an increase in pH value (Broyard and Gaucheron,

2015). The mineral imbalance affects the coherence of the casein micelle which results in loss of

CCP as the equilibrium is shifted towards the soluble phase and pH is decreased (Li and Corredig,

2014). The casein micelle becomes less negatively charged with decreased pH and vice versa. As

found by Le Graët and Gaucheron (1999) there are lower levels of CCP associated with the casein

micelle at lower pH. Same pH tendency was found by Li and Corredig (2014) and Gonzalez-Jordan

et al. (2015). Thus, the increase in feed DM content caused a decrease in pH that may later lead to

less negatively charged caseins and lower levels of CCP associated with the casein micelle. This

may have an effect on the rheology of the feed as discussed later. The degree of CCP removal from

the casein micelle may also have been influenced by the production process of the commercial

powder. The temperature and pressure at which filtration has been performed influence the amount

of CCP removed from the casein micelle where more CCP is removed at decreasing temperature

and increasing pressure (Schuck, le Floch-Fouere and Jeantet, 2013; Liu et al., 2014). However,

Ferrer, Alexander and Corredig (2014) found that diafiltration did not result in a significant

decrease of CCP.

Table 5 pH, power law derived parameters (n and K) and particle size distribution parameters for feed concentrates for (t = 48 hours)

displayed at DM contents of 12%, 15% and 18% (w/w).

Feed Parameters Feed Concentrate

12% 15%2 18%

pH 6.87 ± 0.009a 6.67 ± 0.059b 6.69 ± 0.0047b

n 0.92 ± 0.010a 0.73 ± 0.013b 0.50 ± 0.003c

K 0.014 ± 0.001a 0.14 ± 0.019b 2.45 ± 0.119c

D (3;2) [μm] 0.081 ± 0.001a 0.091 ± 0.002a 0.094 ± 0.001a

D (4;3) [μm] 6.168 ± 0.329a 9.795 ± 6.704a 12.65 ± 2.914a

D (10) [μm] 0.033 ± 0.001a 0.038 ± 0.0002a 0.038 ± 0.0002a

D (50) [μm] 0.130 ± 0.002a 0.147 ± 0.006a 0.154 ± 0.003a

D (90) [μm] 16.933 ± 1.81a 29.125 ± 17.39b 49.30 ± 12.716c 1Particle size distribution (PSD) parameters: D (3;2), D (4;3), D (10), D (50), and D (90) representing the mean surface area, the

volume mean diameter, and particle sizes in the 10%, 50% and 90% quantiles of the distribution. 2Values for feed 15% DM had large std errors and variations in results that could resemble contamination of equipment. Therefore,

measurements for PSD is only based on 4 replicates compared to 12% and 18% that are based on six replicates (a-e) Values for a given parameter within each row for all powders that are significantly different from each other (p < 0.05) based on

two-way ANOVA.

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The filtration process is followed by heat treatments (evaporation and spray drying).The

temperature increase reduces the solubility of calcium phosphate which shifts the mineral equilibria

towards the colloidal phase; whereafter, solubilised calcium phosphate associate with the casein

micelle (Walstra and Jennes, 1984; Holt and Horne, 1996; Oldfield, Taylor and Singh, 2005;

Dalgleish and Corredig, 2012). However, as the concentration process progress and the serum phase

become increasingly concentrated, the mineral equilibrium is shifted anew. The serum phase

becomes supersaturated with calcium and the formation of tricalcium phosphate with a concomitant

release of protons occur, accompanied by a decrease in pH (Walstra and Jennes, 1984; Dalgleish

and Corredig, 2012; Schuck, le Floch-Fouere and Jeantet, 2013).

The feed pH values (Table 5) indicate that the commercial powder may not have been largely

affected by the commercial processing, as pH values are similar to that of bovine milk. If the heat

treatment is exhibited at lower temperatures than 95 ⁰C for a few minutes, the modifications in

mineral equilibrium are considered reversible (Gaucheron, 2004). It is thus hypothesised that the pH

difference noticed between feed concentrations were likely due to the decreased inter-particle

distance between the casein micelles that changed the ionic strength of the solution where pH

decreased as a consequence of the solubilisation of CCP (Nieuwenhuijse, Timmermans and

Walstra, 1988; Le Graët and Gaucheron, 1999; Schuck, le Floch-Fouere and Jeantet, 2013). Despite

the findings from Beliciu, Sauer and Moraru (2012) on dilution of micellar casein with RO water,

where the pH differences seen at different concentrations are attributed to the pH of the diluent

(7.65), the pH influence from demineralised water in this study is evaluated to have no influence on

the pH measurements.

4.1.2 Particle size

An overview of feed particle size distribution (PSD) parameters can be seen in Table 5 for

concentrates with DM content of 12%, 15% and 18%. It can be seen that the mean particle size

diameter, D (4;3), increased with increasing DM content. However, particle sizes obtained at for

different feed concentrations are not significantly different (p > 0.05). Although, considering the

differentiation of D (90), representing 90% of the powder particles, all DM concentrations are

significantly different from each other (p < 0.05). Additionally, the associated size class

distributions are presented in Figure 7. The overall size class distribution had similar volume

densities at all concentrations where they shared the same bimodal distribution. In the size class

area of ~100 μm there was, however, an increase in concentrate with 18% DM content, but

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differentiation between DM 15% and DM 12% is difficult based on the size class distribution

overview.

The particle size distribution of the feeds may have been influenced by interactions that occurred

during the commercial MCI powder production process. It is expected that the commercial

microfiltration process has included the use of membranes with a pore size of 0.1 μm retaining

casein micelles and removing components larger than 0.1 μm. At native conditions, the casein

micelles are well separated so that inter-particle interactions are weak and do not interfere with the

structure of the micelles (Bouchoux et al., 2010). However, upon membrane filtration,

concentration by evaporation and spray drying it is possible that the casein micelle rearrange. The

rearrangement might change the voluminocity where it either decreases, i.e. due to structural

collapse or increase, i.e. due to the association of denatured serum proteins to the casein micelle

surface and aggregation of casein micelles as a response to the stress exerted at any concentration or

filtration process (Bouchoux et al., 2010). The size class area visible from 0.01 μm to ~1 μm was

thus considered to be a mixture of rearranged casein micelles and single caseins.

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2

4

6

S iz e C la s s e s [µ m ]

Vo

lum

e d

en

sis

ty [

%] 1 2 %

1 5 %

1 8 %

Figure 7 Size class distribution overview for feed concentrates (t = 48 hours), displayed at dry matter contents of 12%, 15% and

18%. Values are an average of six replicates, however for feed concentrate with 15% dry matter the size class distribution is based on

four replicates only, due to high standard errors assumed to be from equipment contamination.

The processing may furthermore have resulted in a shift in mineral equilibria and denaturation of

serum proteins (α-la and β-lg) and their subsequent association with the casein micelle by

disulphide bonding (Mulder and Walstra, 1974). The degree of association is highly influenced by

pH of the solution. Approximately 80% of the denatured serum proteins (α-la and β-lg) are

associated with the casein micelle at pH 6.5 as surface-bound aggregates. Increasing pH to 6.7

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maintains approximately 80% of serum proteins in the serum phase where κ-cn dissociates from the

casein micelle for serum protein interaction in the form of serum phase aggregates. Upon further pH

increase to pH 6.9, all serum proteins are found in the serum phase (Nieuwenhuijse, Timmermans

and Walstra, 1988; Anema and Klostermeyer, 1997; Anema, Lowe and Li, 2004; Del Angel and

Dalgleish, 2006; Anema, 2007). According to feed pH measurements (Table 5), the mean particle

size decreased with increasing pH. At lower pH, the negative charge of the casein micelle is

decreased (Dalgleish and Corredig, 2012). Beliciu, Sauer and Moraru (2012) used the zeta-potential

as an indicator for colloidal stability for casein micelles and found that micelles at higher pH had

less propensity for aggregation as the repulsive forces at decreased pH is reduced, leading to casein

micelle aggregation. Furthermore, Sauer and Moraru (2012) found that casein, i.e. mainly κ-cn and

β-cn, dissociation increased with increasing pH. The measured volume density in the size class area

higher than 0.3 μm (Figure 7) is assumed to be aggregated micelles or association of serum proteins

(that equals ~6% of the proteins present in the commercial MCI powder, Table 3) to the surface of

the casein micelle. Likewise, the observed increase in particle size for feed concentrates with

increasing DM content and decreasing pH values, is likely a combination of increased aggregation

of casein micelles, an increased association of serum proteins to the casein micelles and less single

casein dissociation from the casein micelle.

4.1.3 Rheological behaviour of the MCI feed concentrates

The rheological behaviour of rehydrated feed was evaluated according to viscosity and shear stress

as a function of shear rate. Flow curves are displayed at 12%, 15% and 18% (w/w) and presented in

Figure 8. It can be seen that shear stress and viscosity increased with increasing DM content for

feed solutions. The replicated measurements for shear stress and viscosity had good reproducibility

with low standard error values. To characterise the rheological behaviour of the feed concentrates,

the consistency coefficient (K) and the flow behaviour index (n) were estimated from the Power

Law model and are shown in Table 5. All measured samples showed non-Newtonian and shear-

thinning behaviour (n < 1), where the apparent viscosity decreased at increasing shear rate. The

value n decreased with increased casein concentration, which equals a more pronounced shear-

thinning effect at higher DM content (0.92 ± 0.01 to 0.50 ± 0.003). The consistency index, K,

increased with DM content. Additionally, both n and K were found to be significantly different at

all concentrations (p < 0.05).

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0 1 0 0 2 0 0 3 0 0

0

1 0

2 0

3 0

4 0

5 0

S h e a r ra te [s-1

]

Sh

ea

r s

tre

ss

[P

a]

1 2 %

1 5 %

1 8 %

0 1 0 0 2 0 0 3 0 0

0 .0 0 1

0 .0 1

0 .1

1

1 0

S h e a r ra te [s-1

]

Ap

pa

ra

nt

vis

co

sit

y [

Pa

*s

]

1 2 %

1 5 %

1 8 %

Figure 8 Feed flow curves (t = 48 hours) displayed as shear stress (top) and apparent viscosity (bottom) as a function of shear rate (0

to 300 s-1) at dry matter contents of 12%, 15% and 18%.

The interactions between colloidal particles contribute to the rheology of solutions (Hristov et al.,

2016). Skim milk behaves as a Newtonian fluid, where the viscosity is independent of shear rate

(Karlsson et al., 2005). However, upon concentration, the rheological behaviour is changed.

Bouchoux et al. (2009) investigated MCI solutions at different concentrations and found that in

concentrates with up to 10% DM content, solutions behave as Newtonian liquids. Dahbi et al.

(2010) found it to be at a concentration less than 8%. Furthermore, increasing DM content up to

40% DM shifts the solutions into non-Newtonian liquids. At more than 40% DM content the

solutions behave as elastic gels (Bouchoux et al., 2009). All feed solutions evaluated in this study

contained a fairly high DM content (12%, 15% and 18%) where the interactions between casein

micelles were increased due to a tightly packed environment with little distance between the

micelles (De Kruif, 1997; Bouchoux et al., 2010). The more frequently the casein micelles interact

the higher the viscosity of the solution due to casein micelle aggregation (De Kruif, 1997;

Bienvenue, Jimenez-Flores and Singh, 2003). The increased viscosity with increased DM content is

a known feature shared by all colloidal suspensions (Dahbi et al., 2010) and the viscosity-to-DM

ratio for micellar casein solutions are confirmed by several authors (Beliciu, Sauer and Moraru,

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34

2012; Beliciu and Moraru, 2011; Gaiani, Scher, et al., 2006). The high viscosity of casein

dominated solutions is attributed to the open structure of casein micelles which equals a high

specific volume contributing to the formation of highly viscous solutions as a consequence hereof

(O’Mahony and Fox, 2013). Following powder introduction to water, the solutions became highly

viscous with a gel-like appearance (data not shown). This was pronounced for solutions with DM

content of 15% and 18%., the latter, appeared almost solid-like. As the solutions were left to

rehydrate over time the viscosity decreased and the solutions began to flow.

Any factors that alter the aggregation state of casein micelles, such as pH and heat treatment, affect

the viscosity (Singh et al, 1995). Considering feed pH and feed mean particle size distributions at all

concentrations (Table 5) the increase in viscosity with increasing DM content was probably highly

influenced by the increased particle size at decreased pH and a higher tendency for aggregation

(Schuck, le Floch-Fouere and Jeantet, 2013).

4.2 Properties of MCI powders obtained by freeze- and spray drying

Feed solutions with dry matter content of 12%, 15% and 18% (w/w) were dried into six lab scale

produced powders by freeze drying (FD 12%, FD 15% and FD 18%) and spray drying (SD 12%,

SD 15%, SD 18%), as previously described. The powder characteristics were evaluated according

to its properties, i.e. pH, particle size distribution, moisture content, colour and microstructure. For

some analyses, the powder properties are compared to those of the commercial powder that was

used to produce the concentrate feed, as a reference.

4.2.1 pH of the powders

An overview of pH for powders rehydrated for 20 minutes is presented in Figure 9, at 12%, 15%

and 18% DM content. There were clear tendencies for pH to decrease with increasing DM content

for both freeze- and spray dried powders where the differences were found statistically significant

for all samples at all concentrations (p < 0.05).

The pH differences at different DM concentrations for freeze- and spray dried powders were

equivalent to the differences seen in the evaluation of pH of feed concentrates. It was estimated for

feed pH that the pH values between feed concentrates with varying DM content might differed due

to increased ionic strength with increasing DM content which leads to a decrease in pH

(Nieuwenhuijse, Timmermans and Walstra, 1988; Le Graët and Gaucheron, 1999; Schuck, le Floch-

Fouere and Jeantet, 2013). This hypothesis was also found applicable for measurements on powder

pH.

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FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

6 .5

6 .6

6 .7

6 .8

6 .9

7 .0

7 .1

P o w d e r T y p e

pH

a

b

c

d

e

f

Figure 9 Powder pH measurements (t = 20 m) at concentrations of 12%, 15% and 18% DM content for freeze- and spray dried

powders.

Furthermore, the pH differences between freeze- and spray dried powders were likely a

consequence of a shift in the mineral equilibrium due to a changed solubility of CCP at varying

temperatures influencing pH (Gaucheron, 2004). Freeze dried powders had a lower pH compared to

that of spray dried powders at same concentrations (FD 12% versus. SD 12%, FD 15% versus SD

15%, FD 18% versus SD 18%,) as the mineral equilibria may have been shifted towards the serum

phase for freeze dried powders and towards the colloidal phase for spray dried powders. By

comparing the powder pH values to feed pH values the powder pH for freeze-dried powders was

more similar to that of feed, indicating that the mineral equilibria was influenced to a higher degree

by the elevated temperature during spray drying compared to the decreased temperature during

freeze drying.

4.2.2 Particle size distribution

An overview of particle size distribution (PSD) parameters is seen in Table 6 for powders freeze-

and spray dried at 12%, 15% and 18% (w/w). It can be seen that the mean particle size diameter, D

(4;3), increased with increasing DM content for both freeze- and spray dried powders which were

found to be significantly influenced by DM content for freeze dried particles (p < 0.05) but not for

spray dried particles (p > 0.05). It should further be noted that there were found high standard errors

for the mean particle diameters obtained for freeze dried powders at all concentrations. This was

attributed to the manual grinding applied in the formation of freeze dried blocks into powder.

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36

Additionally, the associated size class distributions are presented in Figure 10. It can be seen that

the volume density at different size classes did not vary much for same powder types but was highly

influenced by processing methods (freeze-, spray dried or commercial). Furthermore, all powders

shared a monomodal distribution. However, the distributional peaks were situated at different size

classes as can be seen for the mean particle size diameter listed in Table 6. It should be noted that

there was a clear differentiation between particle size classes for freeze dried powders at all

concentrations (FD 12% < FD 15% < FD 18%) (Figure 10 A) but, a differentiation for spray dried

powders at DM 12% and DM 15% is more difficult (Figure 10 B). However, SD 18% is slightly

shifted towards a higher size class distribution.

Table 6 Particle size distribution parameters for bulk powder prepared with different drying technologies; freeze drying (12%, 15%

and 18%), spray drying (12%, 15% and 18%) and commercial. Spray dried powders were produced using a laboratory-scale spray

dryer (Büchi B-290) and freeze dried powders by static freeze drying (Edwards).

PSD

Parameter1

Freeze dried Spray dried Commercial

12% 15% 18% 12% 15% 18%

D(3;2) [μm] 62.55 ± 0.46a 77.25 ± 0.21b 88.0 ± 2.00c 3.46 ± 0.08d 3.77 ± 0.21d 5.63 ± 0.09d 33.0 ± 4.54e

D(4;3) [μm] 207.5 ± 1.25a 257.5 ± 4.92b 288.0 ± 6.60c 7.21 ± 0.20d 7.72 ± 0.69d 11.50 ± 0.25d 70.67 ± 0.82e

D (10) [μm] 31.50 ± 0.41a 42.80 ± 0.19b 49.60 ± 1.23b 2.05 ± 0.01c 2.09 ± 0.05c 2.60 ± 0.03c 18.53 ± 1.79d

D (50) [μm] 158.0 ± 1.63a 191.5 ± 0.47b 222.0 ± 5.89c 5.72 ± 0.02d 6.01 ± 0.28d 8.19 ± 0.24d 57.67 ± 0.47e

D (90) [μm] 456.5 ±2.94a 541.0 ± 6.94b 611.0 ± 16.44c 14.53 ± 0.66d 15.70 ± 1.2d 23.1 ± 0.62d 139.7 ± 2.05e

1Particle size distribution (PSD) parameters: D (3;2), D (4;3), D (10), D (50), and D (90) representing the mean surface area, the

volume mean diameter, and particle sizes in the 10%, 50% and 90% quantiles of the distribution. (a-d) Values for a given parameter within each row for all powders that are significantly different from each other (p < 0.05) based on

two-way ANOVA.

In spray drying, it is well established that the initial size of droplets at time of drying has a major

influence on the final powder particle size (Schuck, le Floch-Fouere and Jeantet, 2013). The initial

droplet size depends mainly on feed properties, such as viscosity (Schuck, 2009; Liu, Dunstan and

Martin, 2012) and operating settings such as atomization, feed flow rate and type of nozzle (Jeantet

et al., 2009; Kemp et al., 2015). In the evaluation of feed properties, it was found that viscosity

increased with increasing DM content. This was directly related to the increased powder particle

size diameter with increasing DM content, where the atomization process is expected to have

resulted in larger droplets and thereby larger powder particles with increasing viscosity and

implicitly DM content. Likewise, the decreased distance between casein micelles with increasing

DM content also promotes aggregation of casein micelles leading to an additional increase in size

(Schokker et al., 2011). According to operating settings used during the spray drying, feed flow rate

was determined by the pump speed for feed intake. Pump speed was kept constant during spray

drying of feed concentrates with DM content of 12% and 15% (11%, corresponding to 110 ml per

Page 48: Master Thesis - ku...Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia

37

hour) but was varied during spray drying of feed concentrate with a DM content of 18% (11% to

14%, corresponding to 110 ml to 140 ml per hour). The pneumatic nozzle, that the Büchi B-290 was

equipped with, atomize feed by high air velocity that is independent of liquid flow and the increased

feed uptake might have contributed to the increased particle size found for SD 18% (Westergaard,

2011).

A

0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

F D 1 2 %

F D 1 5 %

F D 1 8 %

0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

S D 1 2 %

S D 1 5 %

S D 1 8 %

0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

C om

B

C

Figure 10 Particle size distribution overview of bulk powder, A: freeze dried powders (DM 12%, DM 15% and DM 18%), B: spray

dried powders (DM 12%, DM 15% and DM 18%), and C: commercial powder.

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38

As the spray drying equipment has a high influence on the resulting particle size it is complicated to

evaluate particle size as a consequence of DM content in accordance with other authors work as the

production method and drying settings are highly variable.

4.2.3 Moisture content and water activity in powders

The water content of powders is an important characteristic and was in this work characterised

according to moisture content and water activity.

Moisture content. The moisture content of all powders was evaluated by calculating the mass loss

following evaporation to constant weight by oven heating. An overview of the results is presented

in Figure 11, where each bar represents a powder type (FD 12%, FD 15%, FD 18%, SD 12%, SD

15%, SD 18% and Commercial). As the drying time was similar for all powders it was expected

that moisture content would decrease with increasing dry matter content, as seen in Figure 11 for

both freeze dried (5.4% to 8.7%) and spray dried (6.3% to 6.9) powders. Although, the tendency

was more pronounced for freeze dried powders, where the influence of DM content on moisture

content is statistically significant (p < 0.05). For spray dried powders the influence from DM

content was not found statistically significant (p > 0.05) between SD 12% and SD 15%, and

between SD 12% and SD 18%. However, SD 15% and SD 18% are different (p < 0.05).

FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

Co

mm

erc

ial

0

2

4

6

8

1 0

P o w d e r T y p e

Mo

istu

re

[%

]

a

b b

c

d de e

FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

Co

mm

erc

ial

0 .0 0

0 .0 5

0 .1 0

0 .1 5

0 .2 0

0 .2 5

P o w d e r T y p e

aw

a

be

c

be

f

g

Figure 11 Moisture content (left) and water activity (right) for all powder types. Freeze- and spray dried powders were measured at

DM content of 12%, 15% and 18%.

The surface area influence the evaporation of water from the drying droplet where an increased

surface area improves the evaporation due to a greater thermal efficiency and thereby a more

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39

successful drying (Schuck, 2009). Considering the mean particle size (D (4;3), Table 6) it was

found that powder particle size increased with increasing DM content and likewise exhibit a larger

surface area at increased DM. This fact may explain the higher moisture content present in spray

dried particles dried at 12% DM content. The low differences in moisture content for spray dried

powders were probably a result of their small sizes at all concentrations and thereby low variability

because the total volume is small. The increased difference for freeze dried powders might be due

to a higher variation in particle size. It may also be associated with the bound water in casein

micelles (Huppertz et al., 2017). Naturally, there is a high amount of water associated with the

casein micelle (4 g H2O per g of protein) (Broyard and Gaucheron, 2015) whereas roughly 0.25 g

remains bound per g dry casein (Mulder and Walstra, 1974). A portion of the associated water

appears to be non-freezable (Walstra and Jennes, 1984), which has been estimated to be ~0.5 g

water per g dry matter for micellar casein isolate by Huppertz et al. (2017).

To evaluate the efficiency of the drying process it is possible to establish a ratio between the

moisture content present in the finished powder and the water content of the feed. It can be seen

from Table 7 that the drying efficiency was constant for the spray drying process but varied for the

freeze drying process, where the efficiency increase with DM content. This is probably associated

with the lower water content present from the beginning of the drying process at higher

concentrations. It should be noted that in the production of spray dried powder at 18% DM content,

the feed intake was slightly higher (pump speed of 14%) compared to SD 12% and SD 15%, where

it was 11% (Table 4). However, it seems that the differences in pump speed may be too low to

significantly interfere with the drying efficiency.

Table 7 Drying efficiency for the freeze- and spray drying process for powders at DM content of 12%, 15% and 18%.

Processing method Dry matter content (w/w)

12% 15% 18%

FD 90.5% 91.4% 93.3%

SD 92.2% 92.2% 92.2%

Water activity. The availability of water in powder is evaluated by measuring its water activity, aw.

It is used as a measure for how easy a powder can interact with water, either during rehydration

(wanted) or during storage (unwanted). An overview of aw for all powder types are displayed in

Figure 11. It can be seen that the aw decreased with increasing DM content for both freeze- and

spray dried powders and that it was possible to significantly differentiate the aw between powders

that vary in DM content for both drying technologies (p < 0.05) but a significant differentiation

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40

between processing methods is not strict as aw of FD 15% was similar to SD 12% and SD 15% (p >

0.05). The aw for freeze dried powders had a high variance between powders dried at different

concentrations, where the highest aw was found for DM 12%. The aw for spray dried powders did

not vary much between concentrations, although a similar tendency for a highest aw at a dry matter

concentration of 12% was observed. The same tendency was found for moisture content, which is

probably an indication of that aw of dried milk products is largely correlated with its moisture

content (Schuck, 2009). This is easily detected in Figure 11. However, the differences found in

moisture content for FD powders were more pronounced for aw. Differences in aw are due mostly to

the state of proteins and lactose (Schuck, 2009). The commercial powder had the highest aw

compared to all powders. A high initial aw (0.11 < aw < 0.90) have been reported to have a positive

effect on rehydration behaviour where disruption of particle structure is improved (Gianfrancesco et

al., 2011). However, a high aw can have problematic consequences for powder storage as absorption

of environmental moisture can produce liquid bridges evolving into solid bridges (Hardy, Scheer

and S, 2002).

It should further be noted that moisture content values for the commercial powder were slightly

higher than expected (7% vs 5%, as given by the supplier). Considering the high aw of the

commercial powder it is likely related to moisture uptake from the surrounding environment.

4.2.4 Colour evaluation of the powders

The measured colour coordinates (a*, b* and L) were calculated into measures representing

browning (BI) and the colour difference (∆E). The findings are presented in Figure 12, where

images of all powders can be seen in the top of the BI graph. ∆E values did not vary much for the

different powder types and were located within the range from 90 to 96 for freeze dried powders, 97

to 100 for spray dried powders and 98 to 100 for the commercial powder. Based on the statistics it

was possible to significantly differentiate the freeze dried powders from the spray dried and

commercial powder, but not to differentiate between spray dried and commercial powders. In the

analysis of BI it was possible to see a clear differentiation between powders produced with varying

processing methods, where all were found significantly different (p < 0.05). However, the statistical

differentiation between powders dried with varying DM content was not possible for either BI or

∆E (p > 0.05) and it seems that there are no significant tendencies related to DM content and colour

development.

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FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

Co

mm

erc

ial

0

5

1 0

1 5

P o w d e r T y p e

BI

a a a

b

b b

c

FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

Co

mm

erc

ial

0

5 0

1 0 0

P o w d e r T y p e

E

ab a bc c c c

Figure 12 Colour evaluation displayed as ∆E and browning index (BI) for freeze- and spray dried powders at all concentrations, and

the commercial powders. ∆E and BI are based on the colour coordinates a*, b* and L. Images are added on top of the BI bar plot for

the visualisation of powder appearance.

Freeze dried powders at all concentrations had the highest BI values and, as seen on the top images

in Figure 12, visibly appear darker compared to spray dried powders and the commercial powder.

Subsequently, the commercial powder appeared darker than the spray dried powders at all

concentrations. The colour perception might be attributed to the structure and size of the powder

particles that influence the way light is reflected. As found by Felix da Silva et al. (2018) the

lightness of particles increased with decreasing particle size, where small particles appeared to have

a lighter colour compared to large particles. Considering the size difference of powder particles

(Table 6) and processing method, the darkest powder is freeze dried powder which also has the

highest mean particle diameter (207.5 ± 1.25, 257.5 ± 4.92 and 288.0 ± 6.60) and oppositely with

spray dried powders (7.21 ± 0.20, 7.72 ± 0.69 and 11.50 ± 0.25). The commercial powder is

situated in between freeze- and spray dried powders when comparing both BI and mean particle

diameter (70.67 ± 0.82). It is commonly suggested that browning of products are influenced by the

non-enzymatic browning reaction that occurs between lactose and lysin groups, denoted the

Maillard reaction. Maillard is influenced by temperature and time. A temperature increase results in

an increased reaction rate and subsequent a darker powder appearance with time (Walstra and

Jennes, 1984). Nasser et al. (2017) investigated micellar casein powders and found that the

browning was plausibly related to Maillard even considering the low lactose content (~1% to

~2.5%). As the lactose content of the commercial powder used for this project was 4% (Table 3) it

is not possible to reject that the Maillard reaction has occurred. However as BI for freeze dried

Page 53: Master Thesis - ku...Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia

42

powders that has exhibited no thermal impact was found to be higher compared to BI of spray dried

and commercial powders, it is unlikely that the browning difference visible for powders

manufactured with different processing methods is explained by non-enzymatic browning, even

considering that it likely has occurred in the production of the commercial powder and thus to some

degree are present in the feed concentrates. Nonetheless, the visible colour difference in spray dried

powders could be explained by the increased lactose content present at time of drying in feed

concentrates with increasing DM concentrations. Although, the overall colour perception is likely

more attributed to the structure and size of particles rather than the occurrence of Maillard.

4.2.5 Microstructure

The microstructure of all powders was evaluated by Scanning Electron Microscopy (SEM), to

assess morphological differences. An overview of powder particle appearances is listed in Table 8.

Single particles are shown at different magnifications to show their structural appearance. FD

powders are shown at magnification 500, SD at magnification 2000, and powder at magnification

1000. Furthermore, different criteria are listed such as appearance, the presence of dents, the

apparent size of particles, the degree of shrinkage (- equals no shrinkage, + equals some shrinkage

and ++ equals a high degree of shrinkage), particle surface appearance and agglomeration. As

single particles are not representable for the majority of particles, SEM images of larger areas are

displayed in Figure 13. The images are divided by powder type (FD 12%, FD 15%, FD 18%, SD

12%, SD 15%, SD 18% and Commercial). Freeze dried powders are displayed at magnification 80,

250 and 500, spray dried at 1000, 2000 and 4000 and commercial at 500, 100 and 2000.

Table 8 Overview of microstructure characteristics for freeze dried and spray dried powders at concentrations DM 12%, DM 15%

and DM 18% and the commercial powder product. Images of single particles are obtained at different magnifications with focus on

the appearance of single powder particles.

Criteria FD SD

Commercial 12% 15% 18% 12% 15% 18%

Magnification 500 500 500 2000 2000 2000 1000

Example of

particles

Appearance Flakes Flakes Flakes Spherical Spherical Spherical Spherical

Dents - - - +2 +2 +2 +

Size of

particles Large Large Large

Small and

very small

Small and

very small

Small and

very small

Large and

small

Shrinkage - - - ++ ++ ++ +

Surface Smooth

with fissures

Smooth

with fissures

Smooth with

fissures Smooth Smooth Smooth Smooth

Agglomeratio - - - + + + + 1’-‘ denotes no and ‘+’ denotes yes. 2 Spontaneous agglomeration due to electrostatic particles

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A

B

C

D

E

F

Page 55: Master Thesis - ku...Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia

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G

Figure 13 Overview of SEM images obtained for A: FD 12%, B: FD 15%, C: FD 18%, D: SD 12%, E: SD 15%, F: SD 18%, G:

Commercial. The magnification used for each images is noted in left corner. FD powders are displayed at magnification 80, 250 and

500. SD powders are displayed at 1000, 2000 and 4000. Commercial powder is displayed at 500, 100 and 2000.

As particle structure is strongly affected by the drying technique applied (Schuck, 2009) the freeze-

and spray dried powders were expected to exhibit very different microstructure. The freeze dried

particles were flaky due to the grinding method applied and had a smooth surface. However, the

structure was highly porous as freeze drying is sublimation of ice under pressure maintaining the

structure present when the specimen is frozen. The spray dried particles were roughly spherical and

had a smooth surface with dents. It has been confirmed by several authors that the appearance of

spray dried droplets containing a high protein content (~80%) and low lactose content (~4%)

resulted in these characteristics (Mistry and Pulgar, 1996; Arnaud Mimouni et al., 2010; Burgain et

al., 2016). As SEM images were visualised at high resolutions with clear images at nanoscale, i.e.

magnification of 2000 times for Büchi spray dried powders, it was concluded that the smooth

surface represents the true surface structure of the powder particles. The smooth surface has been

attributed to compaction and shrinkage of protein material, i.e. casein micelles (Tamime, 2007).

Chew et al. (2014) investigated the structure of dried single MPC droplets at DM 15% (w/w). It was

found that an increase in DM content of MPC droplets led to reduced shrinkage and increased rate

of temperature rise during drying. Particles with higher dry matter contents may form a crust in a

much shorter drying period, and consequently maintain their spherical shape (Wu et al., 2014). This

finding has been difficult to confirm for the spray dried powders at varying DM content as there are

large variations within same powder type of both appearances of particles and size (Figure 13). The

large difference in particle appearance may be a consequence of the ineffective atomization

obtained at 11% to 14% pump speed (Kemp et al., 2015). However, by comparing spray dried

powders with the structure of the commercial powder, which is expected to have been dried at

higher concentrations than possible at lab-scale, it could be confirmed that the commercial powder

particle appeared less shrunk.

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4.3 Rehydration ability

The rehydration capacity was measured in terms of wetting, dispersibility and final solubility of

powders. Wetting was evaluated by measuring the amount of water taken up by a controlled amount

of powder for a specified period of time. Dispersibility of powder particles was investigated based

on the decrease in particle size of the PSD parameter D (50) upon rehydration of a solution (5%

w/w) during 48 hours. Changes in the rheological behaviour of feed and rehydrated powders were

used to evaluate the final solubility, which was further evaluated according to pH and particle size

distribution.

4.3.1 Wettability of powders

In the process of capillary rise wetting, water penetrates into the structure by capillary force (Ji, J.

Fitzpatrick, et al., 2016). The wettability was analysed as a measure of g water taken up per g

powder. Results are presented in Figure 14. Spray dried powders at all concentrations exhibited a

higher wettability compared to freeze dried and commercial powders. Spray dried powders had

similar wettability at different concentrations and were thus not found significantly different (p >

0.05). Likewise, freeze dried powders shared similar wettability at all concentrations and were

furthermore found similar to the wettability obtained for the commercial powder (p > 0.05).

However, it was possible to distinguish spray dried powders from freeze dried powders and the

commercial powder (p < 0.05), although the wettability of FD 15%, SD 12% and SD 18% powders

were similar.

FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

Co

mm

erc

ial

0 .0

0 .5

1 .0

1 .5

2 .0

P o w d e r T y p e

We

ttin

g [

g H

2O

/g p

ow

de

r]

a

a c

a

b cb

b c

a

Figure 14 Wetting behaviour measured as g water taken up per gram of powder for freeze-and spray dried powders at all

concentrations and for the commercial powder.

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The possible differentiation between freeze- and spray dried powders may be related to the volume

mean diameter, D (4;3), in which smaller particles were found for spray dried powders compared to

both commercial and freeze dried particles (Table 6). These results indicated improved wettability

for small single particles in comparison to large agglomerated (commercial) or flaky (freeze dried)

particles. This is not in agreement with previous studies where it has been found that an

agglomerated structure has a positive effect on wettability as agglomerated particles form large

pores with a porous structure and the subsequent increase in particle size form small contact angles

between the powder surface and the penetrating water. Water can more easily penetrate into a

porous structure thus agglomeration improve water transfer (Freudig, Hogekamp and Schubert,

1999). This hypothesis was confirmed by Ji, John Fitzpatrick, et al. (2016) who found that an

agglomerated structure drastically increased the water penetration for micellar casein when

comparing agglomerated and non-agglomerated micellar casein powder. However, as seen in Figure

14, the commercial powder takes up the lowest amount of water compared to all powders.

Wettability is the ability of powder particles to overcome surface tension at the powder-liquid

interface (Selomulya and Fang, 2013). Surface composition of particles is an important aspect for

powder wettability, as the particle surface is the first particle part to interact with the water. The

presence of hydrophobic components, such as lipids or proteins, at the particle surface exhibit

particles with poor wetting properties (Havea, 2006) whereas the presence of hygroscopic

components, such as lactose, exhibit particles with good wetting properties (Fäldt and Bergenståhl,

1994). The high content of hydrophobic components of MCI powders was seen as the formation of

non-hydrated powder regions divided by a powder barrier at the powder-water interface where

further water penetration was prohibited. Gaiani et al. (2011) found that the optimal particle size for

a rapid wetting process was approximately 210 μm, for micellar casein. This is similar to the mean

particle diameter for powders freeze dried at a DM content of 12% (207.5 ± 1.25, Table 6).

However, FD 12% exhibited far lower wettability measures compared to spray dried powders at all

concentrations. This is likely an indication of the flaky structure of the freeze dried particles

complicating a comparison with others data as the capillary rise wetting might be highly affected by

the particle structure (Ji, J. Fitzpatrick, et al., 2016).

4.3.2 Dispersibility of powders

The dispersibility process of powder particles can be expressed by the PSD parameter D (50) as a

function of time (t = 0, 15 m, 30 m, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h) (Mimouni et al., 2009). An

overview of the dispersibility is displayed in Figure 15, for freeze dried (DM 12%, DM 15% and

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DM 18%), spray dried (DM 12%, DM 15% and DM 18%) and commercial powder, measured at a

concentration of 5% (w/w) during rehydration.

0 0 .2 5 0 .5 1 2 4 8 2 4 4 8

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

t [h r s ]

D(5

0)

[m

]F D 1 2 %

F D 1 5 %

F D 1 8 %

0 0 .2 5 0 .5 1 2 4 8 2 4 4 8

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

t [h r s ]

D(5

0)

[m

]

S D 1 2 %

S D 1 5 %

S D 1 8 %

0 0 .2 5 0 .5 0 1 2 4 8 2 4 4 8

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

t [h r s ]

D(5

0)

[m

]

C o m m e rc ia l

Figure 15 Dispersibility plotted as D (50) values over time, for freeze dried (12%, 15% and 18%), spray dried (12%, 15% and 18%)

and commercial powder. The timeline is displayed in hours, however time points are not plotted with true distances.

Overall, it was found that SD 12% had the fastest dispersion compared to all powders (4 hours),

followed by SD 15%, SD 18% and FD 12% (8 hours) and lastly, FD 15%, FD 18% and commercial

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(24 hours). This indicates that decreased DM content at drying improved faster dispersion and that

spray dried powders disperse faster compared to freeze dried powders. As the size of bulk powder

particles exhibited high variability between powder types (Table 6) the dispersibility profiles at t =

0 are notably different. Freeze dried particles were large in size compared to the remaining powders

which are expected to be the main reason for the extremely large differences in D (50) at t = 0

(~158 μm to ~222 μm versus ~5.7 μm to ~8 μm).

During dispersion agglomerated particles are dispersed into primary particles from where casein

micelles are released by water erosion followed by complete dissolution (Ji, John Fitzpatrick, et al.,

2016). The appearance of particle structure is important for its dispersibility. Agglomeration of

micellar casein powders had no beneficial effect on rehydration but it rather prolonged the

dispersion as dissolving of solid bridges between agglomerated particles became an additional

dispersion mechanism (Ji, John Fitzpatrick, et al., 2016). Ji, John Fitzpatrick, et al. (2016) measured

dispersibility of MCI powder and found that micellar casein powder (non-agglomerated) dispersed

fully in 20 minutes reaching a D (50) value at nanoscale. This is far from the findings listed in this

study. However, Baldwin (2010) found that a total solubilisation of MCI powders took 8 hours or

longer.

As the evaluation of dispersibility was only based on size class distribution measured for 50% of the

particles present in the solution it does not necessarily illustrate the full context of particle size

distributions. From Appendix I to VII the particle size distribution curves can be seen at t = 0, 15

m, 30 m, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h for all powders at all concentrations. It is observed that the

solutions still exhibit a bimodal distribution at rehydration time of 48 hours even though it seems

that the powder particles are all fully solubilised with a D (50) value near 0 μm at t = 24 hours

(Figure 15). For spray dried powders the dispersibility was similar at all concentrations until t = 2

hours where SD 12% decreased and were fully dispersed at t = 4 hours. For SD 15% and SD 18%,

the tendency was different. Both powders increased in D (50) at t = 4 hours. This is reckoned to be

swelling of particles where water is taken up. The swelling was likewise seen for the commercial

powder at t = 8 hours. As the swelling step was absent for freeze dried powders it indicates that

swelling is either absent for powder flakes or that swelling of flakes was not visible at the time

interval used in this study. At t = 24 hours all powders were fully dispersed. Agglomerated powder

was expected to swell faster than non-agglomerated powder as water easier penetrates into the

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agglomerated structure (Ji et al., 2015). However, this was not found in the agglomerated

commercial powder in this project where swelling occurred at t = 8 hours.

Dispersion of particles is further influenced by its surface composition. Lactose is a fast dissolving

monosaccharide and its presence facilitate rehydration by favouring water penetration towards the

core of the powder particle and thereby accelerating dissolution of particle structure and the release

of components from the particle surface (Richard et al., 2013; Gaiani et al., 2010). However, the

low content of lactose of MCI (4%, Table 3) was expected not to contribute to the rehydration as

hydrophobic components (proteins) are the main powder constituents. Furthermore, Gianfrancesco

et al. (2011) found that the presence of lactose had no significant effect on dispersion of micellar

casein powder as there are no interactions between lactose and casein micelles.

Caseins are prompt to adsorb to the liquid-air interface during drying. Arnaud Mimouni et al.

(2010) investigated rehydration of MPC powders and found that the interactions between casein

micelles resulted in a gel-like structure that has been found to consist of inter-linked casein

micelles. The inter-linkage might be associated with the low lactose content where a continuous

matrix cannot be established and where interactions of casein micelles are thus facilitated (Gazi and

Huppertz, 2015). The skin formation inhibits the transfer of water through the particle surface

resulting in poor rehydration ability (Sadek et al., 2014) but also retains the release of individual

micelles into the serum phase (Ji, John Fitzpatrick, et al., 2016). Ji, John Fitzpatrick, et al. (2016)

used SEM imaging to visualise the difference in particle appearance during rehydration where

erosion of the interlinked casein micelle surfaces as water was allowed transport was clearly visible.

Prior water immersion the particle surfaces were smooth but with rehydration time surface

roughness increased due to solubilisation of components. It has further been found that single casein

act as structure breakers that control the establishment of inter-linked casein micelle on the particle

surface (Schokker et al., 2011). Sauer and Moraru (2012) found an increased amount of single

caseins present in the serum phase of MCI powders with increasing pH. Considering the powder

pH values (Figure 9) the improved dispersibility for spray dried powders (t = 4 to 8 hours)

compared to freeze dried powders (t = 8 to 24 hours) might be a consequence of the pH difference

where spray dried powders had a higher pH compared to freeze dried powders and plausibly a

higher amount of single casein present in the serum phase. The same tendency was found for

powders varying in DM content, where DM 12% exhibited an improved dispersibility where

powder pH was likewise higher. The increase in single casein results in reduced adsorption of

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casein micelles at the air-liquid interface during spray drying, as the smaller single casein will

adsorb preferentially at the interface rather than the larger casein micelles (Schokker et al., 2011).

It has furthermore been established that an increased protein content prolongs dispersibility as a

consequence of aggregation (Schokker et al., 2011). This could be confirmed for powders dried at a

DM content of 18% which had lowest moisture content for both processing methods (and thereby

highest protein content in dry powder) and a longer dispersion time compared to powders dried at a

DM content of 12%. However, it is necessary to conduct more measurements between t = 4 hours

and t = 8 hours to be able to fully conclude on this. A faster dispersion has furthermore been

coupled with increased aw for micellar casein powder (Gianfrancesco et al., 2011). Considering this,

the dispersion of commercial powder (aw = 0.22) and freeze dried at a DM content of 12% (aw =

0.18) should show faster dispersion compared to the remaining powders. However, this was not

found from the dispersibility data in Figure 15, where the fastest dispersed powder is SD 12% (t = 4

hours) which had a aw of 0.12.Though, FD 12% did have low particle sizes at t = 4 hours. It is

suspected that the high aw for the commercial powder is overruled by the agglomerated structure.

Or, that it has resulted in an uptake of moisture from the environment where liquid bridges could

have evolved into solid bridges, prolonging the dispersion process (Hardy, Scheer and S, 2002).

4.3.3 Final solubility of freeze- and spray dried powders

Final solubility was evaluated by pH, particle size distribution and rheological behaviour of

rehydrated powders (t = 48 hours) to concentrations of 12%, 15% and 18% (w/w) by comparing the

findings with the results obtained for feed concentrates (rehydrated commercial powder).

An overview of pH measurements is presented in Figure 16. Overall, it was found that pH

decreased with increasing DM content for both freeze- and spray dried powders. Further, the

rehydrated MCI powder solutions produced by different processing methods had similar pH values

at same concentrations where they are similar (p > 0.05) except at 12% where they are different (p <

0.05). By comparing these pH values with powder pH (Figure 9) it was seen that the difference

between pH of freeze- and spray dried powders were initially larger but during 48 hours of

rehydration pH approach that of feed concentrates (Table 5), however, slightly higher. This

tendency indicated that the changes occurring during freeze- and spray drying were to some extent

reversible over time, where equilibria between powders with similar DM content may be obtained.

As stated by Martin, Williams and Dunstan (2007), during rehydration the native equilibrium

between colloidal calcium and serum calcium is slowly re-established, confirming the hypothesis.

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FD

12%

FD

15%

FD

18%

SD

12%

SD

15%

SD

18%

6 .5

6 .6

6 .7

6 .8

6 .9

7 .0

7 .1

P o w d e r T y p e

pH

a

bd

c

b

d

c

Figure 16 Overview of pH of rehydrated freeze- and spray dried powders (t = 48 hrs) with DM contents of 12%, 15% and 18%.

An overview of particle size distribution (PSD) parameters are listed in Table 9 and size class

distributions displayed in Figure 17, for freeze- and spray dried powders at all concentrations. It

was observed that the mean particle diameter, D (4;3), remained to increase with increasing DM

content and that particle sizes were larger in the rehydrated solutions for freeze dried powders

compared to spray dried powders, as previously observed in both feed and dry powder data. It was

furthermore established from the size class distribution that there was a higher volume density

present at size classes varying from 5 μm to 100 μm for freeze dried powders compared to the

volume density present for spray dried powders within the same size class area.

Table 9 Power law derived parameters (n and K) and particle size distribution parameters for rehydrated freeze- and spray dried

powders at DM contents of 12%, 15% and 18%.

PSD

Parameter1

Freeze dried Spray dried

12% 15% 18% 12% 15% 18%

n 0.998 ± 0.01a 0.908 ± 0.01bd 0.722 ± 0.02c 0.978 ± 0.00a 0.891 ± 0.02d 0.742 ± 0.00c

K 0.010 ± 0.00a 0.044 ± 0.00a 0.264 ± 0.63b 0.017 ± 0.00a 0.061 ± 0.01a 0.328 ± 0.02b

D(3;2) [μm] 0.07 ± 0.00a 0.08 ± 0.00a 0.08 ± 0.001a 0.07 ± 0.00a 0.07 ± 0.00a 0.07 ± 0.00a

D(4;3) [μm] 2.43 ± 0.125a 2.85 ± 0.235a 4.14 ± 0.276b 0.53 ± 0.123c 0.94 ± 0.121cd 1.41 ± 0.19d

D (10) [μm] 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a

D (50) [μm] 0.12 ± 0.00a 0.12 ± 0.001a 0.13 ± 0.002a 0.11 ± 0.00a 0.11 ±0.00a 0.11 ± 0.00a

D (90) [μm] 0.51 ± 0.011a 2.06 ± 1.41b 6.16 ± 1.45c 0.34 ± 0.004a 0.36 ± 0.007 a 0.37 ± 0.005a

1Particle size distribution (PSD) parameters: D (3;2), D (4;3), D (10), D (50), and D (90) representing the mean surface area, the

volume mean diameter, and particle sizes in the 10%, 50% and 90% quantiles of the distribution. (a-e) Values for a given parameter within each row for all powders that are significantly different from each other (p < 0.05) based on

two-way ANOVA.

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0 .0 1 0 .1 1 1 0 1 0 0

0

2

4

6

8

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

F D 1 2 %

F D 1 5 %

F D 1 8 %

0 .0 1 0 .1 1 1 0 1 0 0

0

2

4

6

8

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

S D 1 2 %

S D 1 5 %

S D 1 8 %

Figure 17 Particle size class distribution overview for rehydrated powder concentrates at DM contents of 12%, 15% and 18%

measured at t = 48 hours.

The rheological behaviour of rehydrated powders was evaluated according to viscosity and shear

stress as a function of shear rate. Flow curves of MCI solutions are displayed at 12%, 15% and 18%

(w/w) in Figure 18. Furthermore, power law derived parameters, n and K, are listed in Table 9. It

can be seen that the rheological behaviour exhibited similar rheological tendencies as found for feed

concentrates (Table 5), where viscosity and shear-thinning behaviour increased with DM content

for both rehydrated freeze- and spray dried powders. However, measurements for rehydrated freeze-

and spray dried powders were equivalently related whereas only minor differences were visible in

their flow profiles. By comparing with the feed flow profiles (Figure 8) it was observed that the

apparent viscosity of rehydrated powders was decreased by the extra drying process, where they

became less shear-thinning.

A

0 1 0 0 2 0 0 3 0 0

0

1 0

2 0

3 0

4 0

5 0

S h e a r ra te [s-1

]

Sh

ea

r s

tre

ss

[P

a]

F D 1 2 %

F D 1 5 %

F D 1 8 %

0 1 0 0 2 0 0 3 0 0

0

5

1 0

1 5

2 0

2 5

S h e a r ra te [s-1

]

Sh

ea

r s

tre

ss

[P

a]

S D 1 2 %

S D 1 5 %

S D 1 8 %

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B

0 1 0 0 2 0 0 3 0 0

0 .0 0 1

0 .0 1

0 .1

1

1 0

S h e a r ra te [s-1

]

Ap

pa

ra

nt

vis

co

sit

y [

Pa

*s

]

F D 1 2 %

F D 1 5 %

F D 1 8 %

0 1 0 0 2 0 0 3 0 0

0 .0 0 1

0 .0 1

0 .1

1

1 0

S h e a r ra te [s-1

]

Ap

pa

ra

nt

vis

co

sit

y [

Pa

*s

]

S D 1 2 %

S D 1 5 %

S D 1 8 %

Figure 18 Flow curves obtained for rehydrated (t = 48 hours) MCI powder solutions displayed as A: shear stress by shear rate and B:

apparent viscosity by shear rate for freeze dried (left) and spray dried (right) powders.

The dispersion and dissolution of powder particle components happen concurrently and are

therefore not completely distinguished in the literature. Final solubility is though an important

functional property of casein-based powders and a prerequisite for proper protein expression. For a

proper solubility overview, it is necessary to briefly summarise the main protein-related

mechanisms that occur as a consequence of powder processing which might influence

solubilisation. During heat treatment, α-la and β-lg denature and attach to the casein micelle by

bonding to κ-cn (McKenna et al., 1999). Furthermore, there is the formation of serum phase

aggregates of κ-cn and β-lg (Havea, 2006), the formation of inter-linked casein micelles on the

particle surface (A. Mimouni et al., 2010) and the dissociation of single caseins from the casein

micelle (Sauer and Moraru, 2012) which are influenced by environmental factors, such as pH.

McKenna et al. (1999) found the insoluble part to be related to casein micelle attachment to fat

globules. However, as the fat content of micellar casein isolate, used in this study is 1.5%, which is

not significant. Anema et al. (2006) and Fang et al. (2012) found that the insoluble material is

related to caseins rather than whey proteins and that the structure of the insoluble part must be

porous for serum proteins diffusion. Havea (2006) did not consider the serum phase protein

aggregates of κ-cn and β-lg to play an important role in the formation of insoluble material. Gazi

and Huppertz (2015) correlated the micellar and non-micellar fractions of caseins and suggested

that it was the casein micelles that became insoluble. It has further been suggested that the problem

associated with powders high in casein is the slow release of components from the particle surface

prolonging the rehydration process (McKenna et al., 1999; Havea, 2006; Gazi and Huppertz, 2015)

rather than to the actual formation of an insoluble material (A. Mimouni et al., 2010; Arnaud

Mimouni et al., 2010; Udabage et al., 2012). This phenomenon related to inter-linkage of casein

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micelles on the particle surface was discussed for dispersion of particles in the latter rehydration

section. However, inter-linked casein micelles explained as the cause of insoluble development is

questionable as prolonged rehydration times eventually lead to full solubility of casein-based

powders (Arnaud Mimouni et al., 2010; Schokker et al., 2011; Liu, Dunstan and Martin, 2012). As

stated by A. Mimouni et al. (2010) the inter-linked layer of casein micelles present on the particle

surface has large enough pores between the micelles to enable water diffusion into the core of

powder particles for diffusion of non-micellar solutes initiating and maintaining the dispersion and

solubilisation.

For the size class distribution displayed in Figure 17 for rehydrated powders, it can be seen that the

volume density found in the size class area between 5 μm to 100 μm is of importance, as a size

classes smaller than 1 μm have been regarded as fully solubilised powder. from the differences

exhibited between the article size distributions of rehydrated freeze- and spray dried powders, it can

be noticed that the total solubilisation was improved for spray dried powders that started

rehydration at higher pH values compared freeze dried powders, where a higher CCP content was

expected. CCP has been found to be involved in maintaining the structure of the casein micelle and

its solubilisation is accompanied by the release of single caseins (Anema and Klostermeyer, 1997;

Meena et al., 2017). However, it seems that for freeze- and spray dried powders, where the latter

contain a higher amount of CCP, the solubility was improved. There are several suggestions on

methods for calcium removal to improve solubility of powders, such as found in caseinates.

Although, these methods might be more associated with a faster re-equilibration of the mineral

balance during powder rehydration (Martin, Williams and Dunstan, 2007).

The re-equilibrium that occurred over time was greatly visible. The viscosity at t = 20 min (analysis

not performed) was highly viscous with a gel-like appearance. As the solution rehydrated the

solutions became more flowable. The pH changes that likewise occurred over time for the same

solutions (t = 20 min versus t = 48 hours) were probably due to the retained mineral balance. At t =

20 the solution was still divided into powder and water. As particles solubilized it seemed that the

mineral equilibrium re-established as pH decreased for spray dried powders, where the excepted

heat-induced increased CCP content solubilised, which lead to a decrease in pH.

pH of spray dried powders was elevated at all concentrations compared to that of freeze dried

powders. Compared to the particle size of rehydrated powders (Table 9), freeze dried powders had

larger mean particle diameters and a larger volume density situated in the size class area of 5 μm to

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100 μm, compared to spray dried powders. This might be correlated to the increased powder pH of

spray dried powders, where there is a higher release of single caseins and lower degree of casein

micelle aggregation promoting dispersion and solubilisation. However, pH differences between

rehydrated freeze- and spray dried powders might be too small to have an influence. Although, the

decreased solubility of freeze dried powders might be affected by a prolonged re-equilibration of

minerals. Udabage et al. (2012) found that the solubility of powder immersed in skim milk was

highly improved compared to that in water. The improved solubility was attributed to the higher

mineral salt content in milk, which promoted the re-equilibration of mineral equilibria, providing a

driving force for the release of the casein from the dispersed powder particle.

Several suggestions have been made for the improvement of rehydration of casein-based powders.

Jeantet et al. (2009) found that environmental changes, i.e. increasing temperature and/or agitation

resulted in decreased rehydration time. Schokker et al. (2011) suggest the use of physical processes

that improve the dissociation of casein micelles, i.e. high-pressure processing. Furthermore, storage

conditions have been evaluated as important factors influencing the solubility, where prolonged

rehydration is a common effect of improper storage. Gazi and Huppertz (2015) found that inter-

linkage of casein micelles increased with increasing storage temperature and –time. As confirmed

by A. Mimouni et al. (2010) and Anema et al. (2006).

4.4 Multivariate data analysis Principal component analysis provided an overview of the correlations found between the analysed

parameters in this study. Mean centering and scaling were used as pre-processing and no outliers

were removed from the data. Scores (left) and loading (right) plots are presented in Figure 19,

where dry matter content is denoted a colour and processing method a shape.

Figure 19 A represents all data obtained for freeze- and spray dried powders, only excluding

dispersibility data (feed pH, powder pH, rehydrate pH, feed particle diameter, dry particle diameter,

rehydrated particle diameter, colour coordinates a, b and L, moisture content, water activity, feed

viscosity, rehydrated viscosity and wettability) and, B is limited to represent the analyses that were

also conducted for the commercial powder (dry particle diameter, colour coordinates a, b and L,

moisture content, water activity and wettability). The first two principal components (PC1 and PC2)

explained 84.1% and 83.4% of the total variability for the listed plots in Figure 19 A and Figure 19

B, respectively.

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A

B

Figure 19 A: Principal component analysis (PCA) displaying the main variance in all analyses but dispersibility data for powders

spray dried at 12%, 15% and 18% dry matter content and freeze dried at 12%, 15% or 18% dry matter content, B: PCA plot (scores

and loadings) for analyses conducted on colour coordinates, dry powders mean diameter (D(4;3)), Dry surface area, wettability,

moisture content and aw for powders spray dried at 12%, 15% and 18% dry matter content and freeze dried at 12%, 15% or 18% dry

matter content and the commercial MCI powder.

It can be seen that there was a clear differentiation between powders that varied in dry matter

content (12%, 15% and 18%) at production and between powders produced by freeze- or spray

drying (Figure 19 A). When only the analyses conducted for the commercial powder was included it

was possible to correlate the properties of the commercial powder with the properties of the freeze

dried powders (Figure 19 B). It should be noted, that the scores for the commercial powder were

located in the direction towards decreased dry matter content. This was attributed to the similar

moisture contents of FD 12% and the commercial powder, where the loading moisture content and

aw pulls the commercial powder scores towards this direction. Furthermore, the division of scores

for freeze dried powders was correlated to the dry mean particle size diameter. As freeze dried

powders had a large variation in dry mean particle size diameter (Table 6), this is expected to be the

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main parameter contributing to its division visible in Figure 19 B.

Feed properties (fourth quadrant) increasingly influence samples towards the direction of increased

DM content where measurements from powders produced at 18% DM content is pulled towards the

feed properties direction. Wetting is oppositely correlated to mean particle diameter of dry powder

particles, which were confirmed by the wettability data where wettability increased with decreased

mean particle diameter. It is further observed that wetting is located in the same area as scores for

spray dried powders, pulling the spray dried data points into the direction of the wetting loading.

Spray dried powders were found to have increased wettability (Figure 14). Solubility is correlated

with the mean particle diameter of dry powder particles where the large particles of freeze dried

powders solubilised less than spray dried powders (Figure 17).

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5. Main conclusions

The dry matter content of the feed and the drying technique has an effect on the rehydration ability

of freeze- and spray dried powders. This work showed that it was possible to produce MCI powders

with significant differences in powder properties and powder rehydration ability, by varying feed

dry matter content and drying technique. Since a commercial MCI powder was used as raw material

it was possible to compare the properties of the powders obtained by freeze- and spray drying with

the commercial powders.

Feed dry matter content changed the inter-particle distance between the casein micelles where

increased dry matter resulted in a more tightly packed environment. At increased dry matter, pH

was decreased; a larger mean particle size distribution and an increased viscosity with a higher

degree of shear-thinning behaviour were observed.

The subsequent drying process, freeze- or spray drying, was found to be of great importance for the

morphology and properties of the powder, i.e. pH, mean particle size diameter, microstructure,

colour and moisture content highly varied for MCI powders produced by freeze- or spray drying.

The variations were partly attributed to the structural development of the particles that occurred as a

result of the drying method, such as the formation of spherical particles in spray dried powders and

sublimated flakes in freeze dried powders, and partly to the thermal differences and water loss

mechanisms exhibited between the two methods. For the range of dry matter contents evaluated

(12%, 15% and 18%) it was furthermore observed, that powder properties were interrelated with

feed properties, e.g. increased feed viscosity at increased dry matter content resulted in larger

powder particles with darker colour during spray drying, while the increase of dry matter reduced

significantly the water content of powders by freeze drying.

In the evaluation of rehydration ability of MCI powders wettability, dispersibility and final

solubility were characterised. Wetting was found to be improved for small spray dried powder

particles rather than for freeze dried flaky powder particles and commercial agglomerated particles.

Compared with the commercial powder, dispersibility was found to be improved for spray dried

powders and freeze dried powders, which were related to powder properties, such as pH, particle

size and morphology. MCI powders dried at a dry matter content of 12% for both freeze- and spray

dried powders showed an expressive improvement of dispersibility compared with the other dry

matter contents. The dispersibility of the commercial powder was found longer compared to freeze-

and spray dried powders, probably as a consequence of the agglomerated structure or high aw that

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might have formed solid bridges. Further studies are required to fully understand the behaviour of

powder particles. Final solubility was improved for both freeze- and spray dried powders compared

to the commercial powder. Spray dried powders were found largely solubilised (PSD < 5 μm)

whereas freeze dried powders contained particles in the size class area from 5 μm to 100 μm. This

was evaluated to possibly be related to a prolonged mineral re-equilibration for freeze dried

powders that exhibited lower pH values compared to that of spray dried powders. It was further

established that some heat-induced changes were reversible. This was primarily concluded based on

the pH equilibrium established in rehydrated powders (t = 48 hours) compared to pH of powders (t

= 20 min).

The selection of methods used in this work allowed evaluation of the effect of formulation and

processing on rehydration properties of powders, however, other methods to evaluate internal- and

surface structure of the particles are needed to better understand MCI particle formation during

drying and its consequences for rehydration ability.

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6. Future perspectives

The present work found correlations associated with feed dry matter content and its physico-

chemical effects on rehydration behaviour of powders. The findings and experiences gained

throughout have governed thoughts on ideas for future work.

As this study was based on rehydration of a commercially produced powder, it is necessary to

define the physico-chemical differences that were related to the setup for this work rather than

differences associated with the commercial drying process, and a comparison of analyses conducted

on freshly produced liquid micellar casein isolate is needed.

Further, it is necessary to evaluate the dispersibility at additional time points, in the time area

between four to twenty-four hours to obtain more detailed knowledge on the dispersibility

differences observed in this study.

Additionally, since pH values were found to be different at t = 20 minutes compared to t = 48 hours,

it is suggested to conduct zeta-potential analyses to characterize the electrical charge of the casein

micelles and the colloidal stability of the solutions. This may contribute to a better understanding of

the differences exhibited between the solutions and how they re-equilibrate upon rehydration

Few mechanisms of insolubility development have been proposed for casein-based powders and a

full elucidation of the phenomena of insolubility is required.

Some work has superficially investigated alternative dissolution media for the rehydration of

casein-based powders, such as skim milk. However, the underlying mechanism and factors that

enhance or prolong the rehydration are not fully understood and more work is needed in this area.

Some work has been dedicated to investigating the prediction of insolubility development in casein-

based powders. However, the different drying history exhibited between powder particles in spray

dried powders complicate the development of a precise model for insolubility prediction and more

work is needed.

Investigation on the rehydration-induced changes in the refractive index for casein micelles is also

needed. It has been found that the solubilisation of CCP possibly influence this, where the refractive

index decrease as CCP is dissolved.

Overall, the development of precise analyses that e.g. are valid for measurement of bimodal particle

size distributions is also imperative.

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Appendix I

Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for freeze dried powder dried

at 12% dry matter content and measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

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Appendix II Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for freeze dried powder dried

at 15% dry matter content and measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

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Appendix III Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for freeze dried powder dried

at 18% dry matter content and measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

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Appendix IV Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for spray dried powder dried

at 12% dry matter content and measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

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Appendix V Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for spray dried powder dried

at 15% dry matter content and measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t= 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

Page 90: Master Thesis - ku...Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia

79

Appendix VI Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for spray dried powder dried

at 18% dry matter content and measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

1 0

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

Page 91: Master Thesis - ku...Master Thesis Sissel Therese Brøkner Kavli (shl704) Effect of varying feed dry matter content on powder properties of micellar casein powder Supervisor: Lilia

80

Appendix VII Overview of PSD curves at t = 0, 15m, 30m, 1h, 2h, 4h, 8h, 24h, 48h for the commercial powder

measured at a 5% solution (w/w).

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 0

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 5 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 3 0 m

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 1 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 2 4 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2

4

6

8

t = 4 8 h

S iz e C la s s e s [µ m ]

Vo

lum

e D

en

sit

y [

%]