Katholieke Universiteit Leuvenlib.ugent.be/fulltxt/RUG01/001/789/759/RUG01-001789759... · 2012. 3....

Katholieke

Universiteit

Leuven

FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME (IUPFOOD) MASTER OF SCIENCE IN FOOD TECHNOLOGY

Option Food Science and Technology

Academic year 2010-2011

Emulsifying Properties of Milk Fat Globule Fragment Prepared from

Butter Milk by Microfiltration

by Md. Asaduzzaman

Promotor : Prof. Dr. Ir. Koen Dewettinck

Tutor : Que Phan Thi Thanh

Master's dissertation submitted in partial fulfilment of the requirements for the degree of Master of Science in Food Technology

The author and promoter give the permission to consult and copy parts of this work for personal use

only. Any other use is under the limitations of copyrights laws, more specifically it is obligatory to

specify the source when using results from this Master’s Dissertation.

Gent, August 2011 The promoter The author ………………………………………. ………………………..

Prof. dr. ir. K. Dewettinck Md. Asaduzzaman

ii

ACKNOWLEDGMENTS

In the name of Allah, the most gracious, the most merciful, all praise is God Lord of all creation. I would sincerely like to thank all those who helped and inspired me to complete this dissertation.

It is my honour to express the most special thanks and gratitude to my promoter Prof. Dr. ir. Koen Dewettinck for providing me the invaluable opportunity to carry out the research and by giving continuous inspiration, suggestion, support and guidance during this research period. I am indebted to my supervisor Que Phan Thi Thanh for her wonderful guidance, support, intellectual guidance and company given to me to cope with everything throughout my research study.

I am also grateful to Mrs. Kathleen Anthierens and Ruth Van den Driessche for their friendly assistance and generous help on every occasion. Your cordial guidance highly motivated me to continue no matter how difficult things were, being far from my home country.

To all members and staff of the Laboratory of Food Technology and Engineering, I am grateful for their cooperation and warm friendship. Especially Eveline, Thein, Bart, Natalie, kim, Benny, Corine, and Bea for their invaluable technical assistance.

I also like to thanks prof. dr. ir. Bruno De Meulenaer and prf. dr. ir. Paul Van der Meeren for giving me the access to the equipment at their laboratories.

I am greatly indebted to VLIR for providing me the scholarship to pursue this master program, without which this study work would have not been possible and also wish to extend my sincere thanks to the VLIR staff for their cordial concern about the international student.

I will always treasure the happy moments with my Bangla community family and my circle of friends and to express my thanks for their initiative to make of students feel secured and ever blessed.. My stay in Belgium became more exciting thanks to Kamrul, Banti, Akter, Ifty, Shaown, Salatul, Wahid, Nipa, Neelanjona, Robiul and the great Simum.

To my fellow scholars Fahrizal, Puji, Joyce, Lily, Tarak, Thu, Bao, Biniam, Dian, Nathalia and Zahra, the fun times that we had, the tours we made and the crazy moments will always inspire me and I believe that friendship never ends……

I would like to express my special thanks to my beloved parents, sister, brother; they have given me all the necessary opportunities, and were there when needed. Tasmia, my beloved daughter, you're the very best that ever happened to me. I miss you a lot every day, every moment. Last but not least I am so indebted to my lovely wife who always shares with me love, happiness and sorrow.

Md. Asaduzzaman Ghent, August 2011

iii

TABLE OF CONTENTS ACKNOWLEDGMENTS ..................................................................................................................... ii

LIST OF TABLES ................................................................................................................................. v

LIST OF FIGURES .............................................................................................................................. vi

LIST OF ABBREVIATION ................................................................................................................ vii

LIST OF APPENDICES ..................................................................................................................... viii

ABSTRACT ............................................................................................................................................ x

CHAPTER I: INTRODUCTION ........................................................................................................ 1

CHAPTER II: LITERATURE REVIEW ........................................................................................... 3

2.1 Introduction of dairy by-products used in the emulsification process .......................................... 3

2.2 Milk fat globule membrane (MFGM) ........................................................................................... 3

2.2.1 Origin ..................................................................................................................................... 4

2.2.2 Structure ................................................................................................................................. 4

2.2.3 Composition of MFGM .......................................................................................................... 6

2.2.3.1 Lipids ............................................................................................................................... 7

2.2.3.2 Proteins ............................................................................................................................ 8

2.3 Separation and isolation of the MFGM materials ......................................................................... 9

2.4 Emulsifying properties of the MFGM .......................................................................................... 10

2.5 Dairy emulsion system ................................................................................................................ 12

2.5.1 Definition .............................................................................................................................. 12

2.5.2-Factors effect on stability of dairy emulsions ...................................................................... 13

2.5.2.1 Effect of proteins ........................................................................................................... 13

2.5.2.2 Effect of the MFGM ....................................................................................................... 14

2.5.2.3 Effect of homogenization .............................................................................................. 14

2.6 Whipping properties ................................................................................................................... 15

2.6.1 Whipped cream .................................................................................................................... 15

2.6.2 Characterization of whipped cream ..................................................................................... 15

CHAPTER III: MATERIALS AND METHODS ............................................................................................ 18

3.1 Materials and chemicals .............................................................................................................. 18

3.1.1 Materials .............................................................................................................................. 18

3.1.2 Chemicals ............................................................................................................................. 19

3.2 Compositional analysis of the experimental materials ............................................................... 19

3.3 Experiment 1: Characteristics of emulsions stabilized with milk fat globule membrane

fragments .......................................................................................................................................... 20

iv

3.3.1 Emulsion preparation ........................................................................................................... 20

3.3.2 Determination of emulsion properties ................................................................................ 21

3.3.2.1 Microscopic observation ............................................................................................... 21

3.3.2.2 Rheological characteristic ............................................................................................. 21

3.3.2.3 Particle size distribution measurement ........................................................................ 21

3.3.2.4 Creaming stability .......................................................................................................... 22

3.4 Experiment 2: whipping properties of the MFGM fragments .................................................... 22

3.4.1 Experimental procedure ....................................................................................................... 22

3.4.2 Determination of whipping properties ................................................................................ 23

3.4.2.1 Overrun ......................................................................................................................... 23

3.4.2.2 Serum loss ..................................................................................................................... 23

3.4.2.3 Firmness ........................................................................................................................ 23

3.5 Statistical analysis........................................................................................................................ 24

CHAPTER IV: RESULTS AND DISCUSSION ............................................................................................. 25

4.1 Composition of the experimental materials ............................................................................... 25

4.2 Creaming stability ........................................................................................................................ 28

4.3 Particle size distribution .............................................................................................................. 29

4.4 Microscopic observation ............................................................................................................. 34

4.5 Rheological characteristics .......................................................................................................... 35

4.6 Whipping properties of the recombined cream ......................................................................... 40

CHAPTER V: CONCLUSION .................................................................................................................... 43

FUTURE PERSPECTIVE ........................................................................................................................... 44

LIST OF REFERENCES ............................................................................................................................. 45

v

LIST OF TABLES

Table 1. Estimated average composition of the milk fat globule membrane… 6

Table 2. Protein components of the MFGM………………………………….. 8

Table 3. Composition of the formulation materials…………………………... 25

Table 4. Average Sauter mean diameter D3.2 of emulsions at different

homogenization pressure…………………………………………….

32

Table 5. Average surface-weighted mean diameter D3.2 of different

emulsions at 90/20 bar……………………………………………….

33

Table 6. Rheological parameters of emulsions homogenized at 90/20 bar….. 37

Table 7. Rheological parameters of emulsions prepared with different dairy

materials and homogenized at different pressures…………………..

39

Table 8. Whipping parameter of recombined cream containing BMP and

MFGM-BMP………………………………………………………...

40

vi

LIST OF FIGURES

Figure 1. Structure of the fat globule with detailed arrangement of the main

MFGM…………………………………………………………………

5

Figure 2. Schematic of the fluid mosaic membrane according to Singer and

Nicolson…………………………………………………………….......

5

Figure 3. SDS-PAGE of different dairy products…………………………........... 9

Figure 4. Protein profiling of different dairy materials by SDS-PAGE………….. 27

Figure 5. Creaming stability of emulsions prepared with different dairy materials

upon storage at 4°C……………………………………………………..

28

Figure 6a. Particle size distribution of different emulsions homogenized at

different pressures, after dilution in water and SDS…………………….

30

Figure 6b. Particle size distribution of different emulsions homogenized at

different pressures, after dilution in water and SDS…………………….

31

Figure 7. Particle size distribution of emulsions prepared with different material

at homogenization pressure 90/20 bar……………………....................

32

Figure 8. Microscopy images of emuslsions prepared with BMP, MFGM-BMP,

MFGM-BMW, SC and SMP at different homogenization pressures

(0/20, 90/20, and 210/20 bar)…………………………………………..

34

Figure 9. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM-

BMP and SC) at different homogenization pressures, after 1 day of

storage………………………………………………………………….

36

Figure 10. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM-

BMP and SC) homogenized at 90/20 bar……………………………….

37

Figure 11. Whipping properties of cream containing BMP and MFGM-BMP……. 40

vii

LIST OF ABBREVIATION

ADPH Adipophilin

ANOVA Analysis of variance

AOAC Association of Official Analytical Chemists

BPM Buttermilk powder

BSA Bovine serum albumin

BTN Butyrophilin

CD36 Cluster of differentiation 36

IEP Isoelectric point

MFGM Milk fat globule membrane

MFGM-BMP MFGM isolated from buttermilk

MFGM-BMW MFGM isolated from buttermilk whey

MUC 1 Mucin 1

O/W Oil-in-water

PAS 6/7 Periodic acid Schiff 6/7

SC Sodium-Caseinate

SDS Sodium dodecylsulfate

SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis

SMP Skim milk powder

XO Xanthine oxidase

viii

LIST OF APPENDICES

Appendix 1 Flow curve for the emulsions prepared with BMP, SMP, MFGM-

BMW, MFGM-BMP and SC at different homogenization pressures

Appendix Table 1 ANOVA on protein content of different dairy materials

Appendix Table 2 ANOVA on fat content of different dairy materials

Appendix Table 3 ANOVA on ash content of different dairy materials

Appendix Table 4 ANOVA on lactose content of different dairy materials

Appendix Table 5 ANOVA on polar lipids content of different dairy materials

Appendix Table 6 ANOVA on surface-weighted mean D3,2 of BMP after dilution in SDS.

Appendix Table 7 ANOVA on surface-weighted mean D3,2 of BMP after dilution in

water

Appendix Table 8 ANOVA on surface-weighted mean D3,2 of SMP after dilution in SDS

Appendix Table 9 ANOVA on surface-weighted mean D3,2 of SMP after dilution in water

Appendix Table 10 ANOVA on surface-weighted mean D3,2 of SC after dilution in SDS.

Appendix Table 11 ANOVA on surface-weighted mean D3,2 of SC after dilution in water

Appendix Table 12 ANOVA on surface-weighted mean D3,2 of MFGM-BMW after

dilution in SDS.

Appendix Table 13 ANOVA on surface-weighted mean D3,2 of MFGM-BMW after

dilution in water.

Appendix Table 14 ANOVA on surface-weighted mean D3,2 of MFGM-BMP after dilution

in SDS.

Appendix Table 15 ANOVA on surface-weighted mean D3,2 of MFGM-BMP after dilution

in water.

Appendix Table 16 ANOVA on surface-weighted mean D3,2 of different dairy materials at

90/20 bar after dilution in SDS

Appendix Table 17 ANOVA on surface-weighted mean D3,2 of different dairy materials at

90/20 bar after dilution in water

Appendix Table 18 ANOVA on consistency index (K) of emulsions prepared with BMP

after 1 day storage.

Appendix Table 19 ANOVA on consistency index (K) of emulsions prepared with BMP

after 8 days storage.

Appendix Table 20 ANOVA on consistency index (K) of emulsions prepared with SMP

after 1 day storage.

Appendix Table 21 ANOVA on consistency index (K) of emulsions prepared with SMP

after 8 days storage.

Appendix Table 22 ANOVA on consistency index (K) of emulsions prepared with SC after

1 day storage.

ix

Appendix Table 23 ANOVA on consistency index (K) of emulsions prepared with SC after

8 days storage.

Appendix Table 24 ANOVA on consistency index (K) of emulsions prepared with MFGM-

BMW after 1 day storage


BMW after 8 days storage


BMP after 1 day storage


BMP after 8 days storage

Appendix Table 28 ANOVA on consistency index (K) of emulsions prepared with different

dairy materials after 1 day storage

Appendix Table 29 ANOVA on consistency index (K) of emulsions prepared with different

dairy materials after 8 days storage

Appendix Table 30 ANOVA on Power law index (n) of emulsions prepared with BMP

after 1 day of storage.

Appendix Table 31 ANOVA on Power law index (n) of emulsions prepared with BMP

after 8 days of storage.

Appendix Table 32 ANOVA on Power law index (n) of emulsions prepared with SMP after

1 day of storage.

Appendix Table 33 ANOVA on Power law index (n) of emulsions prepared with SMP after

8 days of storage.

Appendix Table 34 ANOVA on Power law index (n) of emulsions prepared with SC after 1

day of storage.

Appendix Table 35 ANOVA on Power law index (n) of emulsions prepared with SC after 8

days of storage.

Appendix Table 36 ANOVA on Power law index (n) of emulsions prepared with MFGM-

BMW after 1 day of storage


BMW after 8 days of storage


BMP after 1 day of storage


BMP after 8 days of storage.

Appendix Table 40 ANOVA on Power law index (n) of emulsions prepared with different

dairy materials after 1 day of storage

Appendix Table 41 ANOVA on Power law index (n) of emulsions prepared with different

dairy materials after 8 days of storage

x

ABSTRACT

The milk fat globule membrane (MFGM) consists of specific membrane proteins and

phospholipids which possess nutritional and technological functionality. The MFGM

materials produced from buttermilk and buttermilk whey by microfiltration were used to

stabilize oil-in-water emulsions. The emulsifying properties of these materials were also

compared with BMP, SMP and sodium-caseinate. Whipping properties of recombined cream

enriched with MFGM-BMP was also investigated. The objective of this study was to

investigate the potentiality of MFGM materials as food emulsifier and how this potentiality

does influences the whipping properties of recombined dairy cream.

Emulsions containing MFGM-BMP showed significantly smaller particle size distribution

than MFGM-BMW, BMP and SMP. Microscopic observation was also in favor smaller

particle size distribution of MFGM-BMP. In addition MFGM-BMP showed good creaming

stability. Similar emulsions prepared with BMP, SMP, SC and MFGM-BMW showed

extensive flocculation with the only exception of MFGM-BMW for which a good stability to

creaming was found. Emulsions prepared with SC showed smaller particle size distribution

but a poor stability compared to the other emulsions. The Newtonian flow behavior was

observed for emulsions prepared with MFGM-BMP, whereas, shear thinning and thixotropic

behaviors were exhibited by the other materials. These research results indicated that a

selective concentration of MFGM isolated from buttermilk powder by microfiltration has the

potential for the development of ingredients that differ substantially from that of MFGM-

BMW and other dairy materials.

In addition to the conclusions described above, it can be concluded from an additional study

on the whipping properties of MFGM-BMP that recombined cream with MFGM-BMP has a

characteristic longer whipping time, higher overrun, similar firmness and a relatively higher

serum loss as compared to recombined cream with BMP. Therefore, more detailed work is

needed on the quantification and functionality of MFGM materials in the development of

microstructure and physical appearance of whipped cream.

1

CHAPTER I: INTRODUCTION

The fat globules in milk are surrounded by the MFGM, a true biological membrane, mainly

composed of polar lipids and unique membrane specific proteins, which have an interesting

nutritional functionality. The polar lipids are amphiphilic in nature; contain a hydrophobic tail

and a hydrophilic head group, which mainly contribute to the emulsifying properties of the

membrane. The MFGM materials can be used as emulsifiers or stabilizers, resulting in a

combined technological and nutritional functionality (Dewettinck et al., 2008)

Several side streams of the dairy industry such as buttermilk and buttermilk whey are wrongly

considered as inferior products. The aqueous phase derived as by-product from the butter

making process is known as buttermilk. It is rich in milk fat globule membrane (MFGM)

fractions and also contains all water soluble material present in milk such as lactose, casein

and whey protein. On the other hand, whey is the by-product of the cheese and casein

manufacturing process. It still contains some residual fat which consists of lipoprotein

particles, MFGM and small fat globules (Rombaut et al., 2007). It has been shown that dairy

side streams can be an interesting source of functional compounds such as the MFGM.

Although the MFGM concentration in whey is less than in buttermilk, the absence of casein

makes it easier to separate and makes it an interesting source of MFGM materials (Morin et

al., 2006).

In recent years there has been increasing interest in accumulating knowledge on the

composition and properties of the MFGM materials. The composition and properties of the

MFGM is completely different from that of milk or plasma. The MFGM prevents flocculation

and coalescence of milk fat globules as natural emulsifier and has a protective action against

enzymatic action on the fat globules. The properties of the MFGM largely depend on the type

of treatment of the milk during processing e.g. heating, cooling, homogenization, evaporation,

drying etc. (Evers, 2004) and the method of isolation and separation of the MFGM from raw

materials (Singh, 2006).

A number of studies have been conducted to investigate the potentiality of MFGM materials

in food and non- food applications. Lopez et al. (2007) and Michalski et al. (2007)

investigated the influence of MFGM enrichment on the microstructure of different cheeses by

using small fat globules; Thompson and Singh (2006) produced liposomes from the MFGM

using the microfluidization technique. Also several applications of MFGM materials, based

on their emulsifying properties (due to high polar lipid content), have been reported such as

2

their use as baking improver, their function as fat disperser and anti-staling agent in bakery

goods; for the prevention of spattering, browning and dispersion of sediment in margarine; for

the reduction of the viscosity and to prevent crystallization in chocolate and as wetting,

dispersion and stabilization agent in instant products (Vanhoutte et al., 2004; Szuhaj, 1983;

Vannieuwenhuyzen, 1981).

The side streams of dairy processing still have a lower economical value compared with the

mainstream products. Proper utilization of these sources to isolate the functional MFGM

material and then apply it in the development of new products may bring great economical

and technological success. Food processing industries have been emphasizing on the use of

MFGM materials as natural components, to improve the nutritional value and create specific

functionalities of food products. Although several workers (Roesch et al., 2004, Sodini et al.,

2006)) found the MFGM isolated from buttermilk to be a good emulsifier but opposite results

were also reported by others (Correding and Dalgleish, 1997; Wong and Kits, 2003).

However, this study focuses on the characterization of oil-in-water emulsions prepared with

MFGM materials produced by microfiltration from buttermilk and buttermilk whey. The

properties of these materials will also be compared with buttermilk powder (BMP), skim milk

powder (SMP) and sodium-caseinate. This comparative study will allow us to select MFGM

material with unique interfacial functionality.

Hence, the objective of this study is to investigate the potentiality of MFGM materials as food

emulsifier and how this potential does influence the whipping properties of recombined dairy

cream.

3

CHAPTER II: LITERATURE REVIEW

2.1 Introduction of dairy by-products used in the emulsification process

Many common dairy products are emulsions such as creams, whipped cream, ice cream,

yoghurt etc. Emulsifying properties of milk derived components influence the physical

characteristic of dairy emulsions. Skim milk powder, sweet buttermilk powder, butter-derived

aqueous phase, whey proteins, casein dispersions, phospholipids and purified milk fat globule

membrane (MFGM) suspensions have been used successfully to make emulsions (Elling et

al., 1996; Tomas et al., 1994; McCrae et al., 1999). Milk proteins are highly valued for their

emulsifying and emulsion stabilizing properties. Skim milk powder is rich in protein e.g.

caseins and whey proteins. Caseins from milk have high surface active properties and are

adsorbed readily at the O/W interface. Whey proteins are good emulsifying and foaming

agents. The lipid fractions of whey protein concentrate or whey protein isolate influence the

whipping properties of cream (Ennis and Mulvihill, 1998). Buttermilk is rich in MFGM

derived fractions. The use of buttermilk in food systems is closely related to its particular

composition in emulsifying components such as phospholipids which can act as emulsifier in

dairy emulsions. MFGM contains various proteins and phospholipids which are efficient

natural surface-active materials for emulsion formation. Hence, MFGM materials should be

considered as a potential emulsifying agent for certain foods and other emulsions (Kanno et

al., 1991)

2.2 Milk fat globule membrane (MFGM)

The dispersion of milk fat globules in milk is not a simple O/W emulsion. The fat globules are

surrounded by a complicated membrane, which cannot be considered as a simple

monomolecular film of surface active material. Instead, the membrane has several distinct

layers that are laid down during synthesis in the secretory cells of the mammary gland. This

membrane is well known as the milk fat globule membrane (MFGM). The membrane is

different from that of either milk or plasma and represents a unique biophysical system

(Singh, 2006). The MFGM acts as a natural emulsifying agent, protecting fat globules from

enzymatic action and preventing flocculation and coalescence of fat globules in milk

(McPherson & Kitchen, 1983; Walstra, 1995).

4

2.2.1 Origin

The membrane surrounding milk lipid globules is essentially a tripartite structure that

originates from the apical plasma membrane, the endoplasmic reticulum and possibly from

other intracellular compartments. The MFGM fraction originating from the apical membranes

has a typical bilayer appearance and is termed the primary membrane. The material derived

from the endoplasmic reticulum has the appearance of a monolayer of proteins and polar

lipids that covers the triacylglycerol-rich core lipids to the globules before secretions (Keenan

and Mather, 2006).

The triglycerols are synthesized in or on the surfaces of rough endoplasmic reticulum

membranes and accumulate in the form of micro-lipid droplets in the cytoplasm. These

intracellular droplets are covered by a diffuse interfacial layer, which consists of

phospholipids, glycosphingolipids, cholesterol and proteins. Micro-lipid droplets grow in

volume by fusion with each other to form cytoplasmic lipid droplets of various sizes, which

are then transported to the apical pole of the cell through the cytoplasm and are secreted from

the epithelial cell. During secretion the droplets are coated with the outer plasma membrane

from the cell. The composition of the fat globule membrane is similar to that of the apical

plasma membrane of secretory cells (Keenan & Dylewski, 1995; Keenan, 2001 and Heid &

Keenan, 2005). Apart from the MFGM, secretory cell fragments can also be secreted into the

lumen. They are rich in polar lipids, have a similar composition as the MFGM and comprise

only 4% of the total milk fat (Deeth, 1997; Keenan et al., 1999).

2.2.2 Structure

The MFGM is highly structurized and contains unique polar lipids and membrane specific

proteins. The natural MFGM consist of three distinct layers, viewed from the inside lipid core

to outwards, - a monolayer of proteins and polar lipids surrounding the intracellular fat

droplet, - an intermediate electron dense proteinaceous coat on the inner face of the bilayer

and finally, - a true bilayer membrane of polar lipids and protein. The entrained cytoplasmic

materials between the inner coat and outer bilayer membrane form the cytoplasmic crescents.

(Danthine et al., 2000; Evers, 2004; Michalaski et al., 2002). The MFGM originates from

several distinct layers with a total thickness varying from 10 to 20 nm (Wooding, 1971;

Walstra et al., 1999).

5

Figure 1. Structure of the fat globule with detailed arrangement of the main MFGM. A double layer of

polar lipids is placed on an inner monolayer of polar lipids. Membrane-specific proteins are distributed

along the membrane. ADPH is located in the inner polar lipid layer, XDH/XO is located in between both

layers. MUC1, BTN, CD36 and PASIII are located in the outer layer. PAS6/7 and PP3 are only loosely

attached at the outside of the MFGM. The choline-containing phospholipids, PC and SM, and the

glycolipids, cerebrosides and gangliosides, are largely located on the outside of the membrane, while PE,

PS and PI are mainly concentrated on the inner surface of the membrane. (cited from Dewettinck et al.,

2008)

The bilayer membrane of the MFGM is derived from the apical plasma membrane of the

secretory cell and the most widely accepted model for this type of membrane is the fluid

mosaic model. This suggests that the phospholipid bilayer serves as backbone of the

membrane, which exists in a fluid state. Peripheral membrane proteins are partially embedded

or loosely attached to the bilayer. Trans-membrane proteins extend through the lipid bilayer.

Carbohydrate moieties from glycolipids and glycoproteins are oriented outwards. Cholesterol

is present in the polar lipid bilayer (Dewettinck et al., 2008)

Figure 2. Schematic of the fluid mosaic membrane according to Singer and Nicolson (1972).

6

2.2.3 Composition of MFGM

The amount and composition of the MFGM varies considerably depending on the fat globule

size and the fat content of milk, which is in turn, is influenced by several others factors such

as type of feed, breed, age, health and stage of lactation of cows (Keenan, 2001; Keenan &

Dylewski, 1995). The major components of the MFGM comprise membrane specific proteins

(mainly glycoproteins), phosphor- and sphingolipids. Protein and phospholipids together

account for over 90% of the membrane dry weight but the relative proportions of lipids and

proteins may vary widely (Singh, 2006). The average composition of the MFGM is given in

the table 1.

Table 1. Estimated average composition of the milk fat globule membrane (Walstra et al.,

2006)

Component mg/100gfat globules g/100g MFGM dry matter Protein 1800 70 Phospholipids 650 25 Cerebrosides 80 3 Cholesterol 40 2 Monoglycerides +a ? Water + - Carotenoids + Vit. A 0.04 0.0 Fe 0.3 0.0 Cu 0.01 0.0 Total � 2570 100 a+; present, but quantity unknown

The composition of the milk fat globule membrane also changes when subjected to different

processing treatment such as cooling, agitation, heating and aging. (Evers, 2004). Kirst,

(1996) divided the factors that affect the composition of the MFGM into 3 groups, viz.

physiological, chemical/enzymatic and physical/mechanical. Physical/mechanical factors are

related to milk handling during and after milking. Pre-factory milk handling includes air

inclusion, pumping and stirring of milk, changes in temperature and changes in time. Milk

handling at the factory involves aging, agitation, heat treatment, separation, homogenization

and changes in water content (McPherson and Kitchen, 1983). Sometimes the presence of air

is wanted (e.g. butter making process) but the mixing of air or gas with milk or cream

significantly reduces the stability of the fat globules. When milk fat globules and air come

into contact with each other the milk fat globule membrane is disrupted. Consequently the

7

MFGM materials spread over the air/milk plasma interface and are released through the milk

plasma when the air bubbles collapse (van Boekel and Walstra, 1989).

Large fat globules are more susceptible to shear stress than the smaller ones. Wiking et al.,

(2003) reported that the resistance of the fat globules membrane against coalescence during

agitation is determined by the size of the fat globule, the fat content, the milk temperature and

the shear rate. Ye et al., (2002) showed that, when fat globules are heated in absence of serum

proteins, there is a tendency to form a complex between butyrophilin and xanthine

oxidoreductase by disulphide bonds at temperature as low as 60°C. At higher temperature

(80°C) a significant amount of serum proteins, particularly β-lactoglobulin, interacts with the

MFGM (Lee and Sherbon, 2002). Homogenization of the milk decreases the fat globule size

and increases the milk fat surface area; consequently new materials from the milk serum

(predominantly casein) come to cover the free surface and change the composition of the

membrane (Darling and Butcher, 1978).

2.2.3.1 Lipids

Milk fat globule membranes consist mainly of polar lipids and a negligible amount of neutral

lipids (triglycerides, diglycerides, monoglycerides, cholesterol and its esters). High melting

triglycerides are the major fraction of the neutral lipids in the MFGM (Wooding & Kemp,

1975). However these triglycerides appear to originate from contamination by the core of the

fat globules during the isolation process of the membrane (Walstra, 1985). Hence, the method

of isolation has great influence on the triglyceride content of the MFGM. The MFGM

associated triglycerides contain higher proportions of long chain saturated fatty acids than that

of the globule core fat. Cholesterol amounts to 90% of the sterols content of the MFGM

(Jensen and Newberg 1995)

In bovine milk, about 60% of the phospholipids are associated with the milk fat globules and

the rest is located in the membrane material of skim milk (Patton and Keenan, 1975). The

major polar lipid fraction present in the MFGM consists of pholphatidylcholine (PC), 35%;

phosphatidylethanolamine (PE), 30%; sphingomyelin (SM), 25%; phosphatidylinositol (PI),

5%; phosphatidylserine (PS), 3%. glucosylceramide (GluCer), lactosylceramide (LacCer).

Gangliosides (Gang) are present in trace amounts (Danthine et al., 2000; Deeth, 1997)

The MFGM phospholipids contain high levels of long chain (> C14) fatty acids; short and

medium chain (C4 -C14 ) fatty acids are virtually absent. Especially, PE is highly unsaturated

followed by PI and PS. PC is rather saturated as compared to other glycerophospholipids.

8

Sphingomyelin (SM) is very uncommon in its fatty acid pattern and consists of around 70-

97% of saturated FA and of a high proportion (40-50%) of fatty acids having a chain length of

C20 or more. These two physical features contribute to less fluidity and maintaining rigidity of

the MFGM (Bitman and Wood, 1990).

2.2.3.2 Proteins

Depending on the isolation method, the protein content of the MFGM varies from 25-60%

(Singh, 2006) and the type of proteins largely depends on the origin or source of the milk. It is

well accepted that the MFGM protein fractions represent only 1-4% of the total milk proteins

(Park, 2009). The major MFGM proteins are xanthine oxidase, butyrophilin (BTN),

adidophilin (ADPH) and periodic acid Schiff (PAS) 6/7; the minors proteins are polymeric

immunoglobulin receptor protein, apolipoprotein E, apolipoprotein A1, 71 kg/mol heat-shock

cognate protein, clusterin, lactoperoxidase, immunoglobulin heavy chain and eptidylprolyl

isomerase A, actin, fatty acid binding protein, cluster of differentiation 26 and mucin (Fong et

al., 2007). Furthermore parts of the proteose peptone fraction like proteose peptone 3 (PP3)

originate from the MFGM (Campagna et al., 2001).

Table 2. Protein components of the MFGM (cited from Singh, 2006).

Proteins Molecular weight (g/mol) Mucin I (MUC 1) 160,000 – 200,000 Xanthine oxidase 150,000 Pass III 95,000 – 100,000 CD36 or PAS IV 76,000 – 78,000 Butyrophilin 67,000 Adipophilin (ADPH) 52,000 PAS 6/7 48,000 – 52,000 Fatty acid binding protein (FABP) 13,000 BRCA 1 210,000

The protein composition is highly dependent on the isolation method and the analytical

procedure. Moreover, all proteins are not equally connected with the MFGM. Some are

integral proteins, some are peripheral proteins and others are only loosely attached. Upon

separation by SDS-PAGE the MFGM material is resolved into 7-8 major bands. However,

several minor proteins are still unidentified (Dewettinck, at al., 2008).

9

Figure 3. SDS-PAGE of different dairy products. Names of MFGM proteins are given at the right, the

other proteins are named at the left. Separation is performed on gradient (4–12%) polyacrylamide gels

with the Xcell Surelock system. Visualization was done with SilverXpress silver stain. On each lane, 250

ng of protein was loaded: (1) raw milk; (2) skimmed milk; (3) acid buttermilk; (4) butter serum, aqueous

fraction obtained from churning of cream; (5) acid buttermilk whey, soluble fraction obtained from

acidification of sweet-cream buttermilk; (6) acid buttermilk quark, coagulated fraction obtained from the

acidification of the sweet-cream buttermilk; (7) MFGM-isolate; and (8) mark 12 molecular weight

standard (Rombaut, 2006). (Cited from Dewettinck, 2008)

2.3 Separation and isolation of the MFGM materials

Buttermilk is considered to be a suitable source of the MFGM for commercial-scale

production, mainly because of its low cost and relatively high content of MFGM components.

Buttermilk is a side stream of the butter making process and has limited commercial value.

Food processing industries have been emphasizing on the utilization of natural components to

improve the nutritional value and create specific functionalities of food products (Innocente et

al., 1997). Many attempts have been made to separate and isolate MFGM material from

commercial buttermilk. The similarity in size of casein micelles and MFGM components is

the first major consideration taken into account to achieve an effective fractionation of

buttermilk (Sachdeva and Buchhiem, 1997). Micro- and ultrafiltration of buttermilk depends

on the filtration conditions such pore size and type of membrane material, temperature, pH,

and the type of buttermilk (Morin et al., 2004; Rombaut et al., 2007).

Correding et al. (2003) reported that increasing the number of diafiltration from 2 to 6 reduces

casein contamination in the retentate from 30% to 6% but increasing the diafiltration number

has the disadvantage of loss of MFGM material (Rombaut et al 2006). Correding et al. (2003)

10

use citrate to dissociate the casein micelles of buttermilk; the MFGM material is then

recovered by high speed centrifugation. In later studies they observed that microfiltration of

citrate treated buttermilk through a membrane of 0.1 µm nominal pore size is more suitable

for isolating MFGM materials than high speed centrifugation. Rombaut et al. (2006) reported

that the addition of sodium citrate dissociates not only the casein micelles but also the MFGM

fragments and high citrate concentrations may result in loss of polar lipids (64% recovery)

during filtration due to blocking and fouling of the filter membrane with MFGM particles.

Whey contains 0.4 to 0.5% of residual fat which is the main source of small fat globules,

lipoprotein particles and milk fat globule membrane fragments (Rombaut et al., 2007).

Isolation of the MFGM from whey (whey buttermilk, acid buttermilk whey) by filtration is

favorable because whey is free of caseins. Absence of casein facilitates to obtain concentrated

MFGM material by filtration, although the MFGM concentration of whey is limited compared

with other dairy fractions such as buttermilk or butter serum. Morin et al. (2006) reported that

whey buttermilk is favorable for MFGM isolation by filtration due to absence of casein and

the expected transmission of MFGM proteins through the membrane was lower when using

whey buttermilk compared to regular buttermilk.

2.4 Emulsifying properties of the MFGM

MFGM materials are found in quite significant amounts in different dairy products especially

in cream, butter, buttermilk, cheese and cheese whey. The unique functionality of MFGM

materials have led to research and developing technologies for isolation, separation and use in

different food emulsions (Singh, 2006). The MFGM enriched dairy products are often

preferred for their emulsifying properties and capacity to improve flavour and texture

especially for reduced fat food products (Ward et al., 2006). Lopez et al. (2007) have

discussed the influence of the MFGM on the microstructure and flavour development of

Emmental cheese.

Milk fat globule membranes are composed of mainly polar lipids and negligible amounts of

neutral lipids. Polar lipids are amphiphilic molecules which contain a hydrophobic tail and a

hydrophilic head group. This unique feature largely contributes to the emulsifying properties

of the membrane. MFGM materials are considered to be an efficient and natural emulsifying

agent due its amphiphilic nature and original function in stabilizing fat globules in whole

milk. Corredig and Dalgleish (1998a) prepared a model soy oil-in-water emulsion with

MFGM materials from fresh raw cream and found that the newly formed oil droplets covered

11

by the MFGM material, behave differently from emulsions stabilized by other milk proteins

i.e. no displacement occurs on the addition of small molecular weight surfactant (Tweens and

Triton X-100) and the droplets are not affected by the presence of proteins such as an addition

of β-lactoglobulin or caseins. This is due to fact that strong interaction may exist at the

interface and phospholipids component of the MFGM may lower the interfacial tension

(Corredig & Dalgleish, 1998c). MFGM materials isolated from industrial buttermilk are poor

emulsifiers of soy oil-in-water emulsions as compared to those isolated from fresh cream

(Corredig & Dalgleish, 1997). This is related to the intensity of membrane protein

denaturation and association with β-lactoglobulin during heat treatment and the churning

process following the manufacturing of buttermilk.

Type of raw material, pretreatment and method of separation have a significant effect on the

composition of MFGM isolates and consequently on their emulsification properties. In

reconstituted milk fat emulsion (20-80 mg MFGM/g fat), it was observed that MFGM can

stabilize 25 times its mass of milk fat. Droplets size decreases linearly with increasing

membrane concentration which is comparable to those of homogenized milk. The surface

protein coverage also increases at acid pH with the increase of MFGM concentration in the

emulsion; by using > 80mg MFGM material/g fat it is possible to prepare stable milk fat

globules that have similar stability as the natural milk fat globules (Kanno, 1989; Kanno, et

al., 1991). However, Wong & Kitts (2003); Corredig & Dalgleish (1997) found that

commercial buttermilk has inferior emulsifying and stabilizing capacity than non-fat dried

milk. This may be due to the fact that heat treatment (80°C for 2.5-20 min.) and churning

have an influence on the behavior of the membrane and could have caused excessive

denaturation of membrane proteins which influences the association of whey protein with

MFGM (Houlihan et al., 1992).

Sodini et al. (2006) studied the compositional and functional properties of sweet, sour and

whey buttermilk and reported that sweet and cultured buttermilk exhibit lower emulsifying

properties and higher viscosity and lower pH whereas the functional properties of whey

buttermilk were independent of pH. The emulsifying properties of those three types of

buttermilk are better than milk and whey but have lower foaming capacity. However, among

the above listed buttermilk samples, whey buttermilk was found to have the highest

emulsifying properties and the lowest foaming capacity. Possible reasons for this could be the

higher ratio of phospholipids to protein in whey buttermilk compared with sweet or cultured

buttermilk (Sodini et al., 2006). Innocente et al. (1997) studied the change in surface

12

properties of the soluble fraction of the MFGM (SFMFGM) at different temperatures (4-

40°C) and observed that during preparation of emulsions temperature affects their functional

properties. A temperature of even as low as 65°C results in loss of emulsifying properties of

the membrane fraction. The stability of oil-in-water emulsion prepared with MFGM material

depends on the heat treatment of the cream (Corredig & Dalgleish, 1998b).

Roesch et at. (2004) studied the emulsifying properties of the MFGM fraction obtained by

microfiltration of commercial buttermilk and buttermilk concentrate (BMC). An emulsion

prepared with 10% soybean oil and with >0.25% MFGM isolate (60% w/w proteins) from

buttermilk, showed good stability against creaming and small particle size distribution

increased with MFGM concentration, whereas a similar emulsion prepared with BMC showed

extensive flocculation. These findings are in disagreement with the previous work by

Corredig and Dalgleish (1997). These authors observed that the MFGM isolate is a poor

emulsifier compared to a whole isolate of buttermilk (containing whey proteins, caseins, and

protein of MFGM). Emulsions with 10% soybean oil needed much higher percentage (>8%)

of MFGM isolate to produce a droplet size distribution similar to that found for the emulsions

prepared with 1-2% (w/v) whole isolate.

From the different emulsification studies it can be deduced that the source, the intensity and

frequency of the heat treatment, the type of fractionation procedure as well as the preparation

condition results in major differences in the efficiency of MFGM material in emulsion

preparation. The solubility of MFGM materials is influenced by the degree of denaturation of

the MFGM proteins, the intensity of complex formation between MFGM proteins and the

lipid fractions and the association of whey proteins with the MFGM caused by the heat

treatment (Corredig and Dalgleish, 1998b). Hence, one of the recommendations could be that

MFGM isolate should hydrate completely before its use in emulsification.

2.5 Dairy emulsion system

2.5.1 Definition

Milk is a well known natural oil-in-water emulsion. The fat droplets in milk are surrounded

and stabilized by their own membrane, the bilayer membrane of the MFGM. Dairy cream, the

fat rich fraction of whole milk, is also an oil-in-water emulsion in which fat globules are

dispersed in a continuous aqueous phase. According to Dewettinck and Fredrick (2009), the

inner layer of this surrounding membrane, existing of globular protein and phospholipids, lies

on a layer of high melting glycerides and has no enzymatic activity. The outer layer has

13

enzymatic activity and determines agglutination, adsorption phenomena and the stability of

the fat globule. The fat globules range in diameter from approximately 0.1 to 20 µm with a

mean value of 3.5 µm.

2.5.2-Factors effect on stability of dairy emulsions

The stability of milk fat emulsions is related to the physical and chemical characteristic of

milk and dairy products. Thermodynamically, emulsions are not stable. Stability is a time

dependent kinetic phenomenon and is largely dependent on the size distribution of the

globules (Dewettinck and Fredrick 2009). Fox and McSweeney, (1998) reported that

emulsion stability strongly depends on the integrity of the MFGM. Lipid emulsions are

inherently unstable systems due to the difference in density and the interfacial tension

between the lipid and the aqueous phases.

2.5.2.1 Effect of proteins

Milk proteins are well known for their emulsifying and emulsion stabilizing properties.

Proteins play a major role in the stabilization of the fat globules against partial coalescence

through adsorption on the interface which results in electrostatic repulsion and increased

thickness and viscoelasticity of the interfacial layer (Dalgleish, 2006; Goff, 1997). During

emulsification, milk proteins become rapidly adsorbed on the O/W interface as individual

molecules or in the form of aggregates (Walstra and Smulders, 1997). The newly formed

layer results in steric stabilization and protects finely dispersed droplets against recoalescence.

It provides long term stability towards creaming and flocculation during subsequent storage

(Dalgleish 1997).

The type of proteins affects the degree of stabilization, for example caseins from milk are

highly surface active and adsorb readily the whey proteins. In practice casein stabilized

emulsions have fine, small and stable droplets (Graham and Philips 1979). The protein-fat

ratio in emulsions also influences the degree of stabilization. At a lower ratio the amount of

proteins is not enough to cover the overall O/W interface and as a result not all fat globules

are stabilized by proteins. During emulsification these uncovered fat globules may aggregate

and form clusters (Fredrick et al., 2010). Van Camp et al., (1996) observed the effect of the

fat-protein ratio on emulsions stabilized by whey protein concentrate and sodium caseinate

and reported that an increase in the fat-protein ratio resulted in a decreased whipping time,

overrun and increased firmness and stability of the whipped emulsion.

14

In milk based emulsions, whey proteins adsorb on the interface and also act as bridging

material in the homogenization induced flocculation of fat droplets. Heat denaturation after

homogenization retards desorption of whey proteins and inhibit protein loss on washing

(Dickinson, 1986). Shimizu et al., (1981) reported that the amount of whey proteins adsorbed

on the fat globules is dependent on pH during emulsification and the gross adsorption features

are as follows: at lower pH greater adsorption of α-lactalbumin and at IEP greater adsorption

of serum albumin.

2.5.2.2 Effect of the MFGM

The milk fat globule membrane is a natural surface active material used in emulsion

preparation. The MFGM is closely involved in creaming and agglutination of milk and those

processes are highly affected by the treatment of dairy products such as cooling, heating and

homogenization (McPherson and Kitchen, 1983). The stability and acceptability of dairy

products are determined by the response of MFGM materials to these treatments (Houlihan et

al., 1992).

Mechanical treatments during processing of milk cause damage of the native MFGM and

reduce the fat globule size and increase the interfacial area. The MFGM can no longer entirely

cover the newly formed fat globule, and as a result some caseins micelles and whey proteins

adsorb onto the surface. Homogenization induces such a high shear force that the fat globules

are disrupted and their average diameter falls below 1 µm. The new droplets are almost

entirely composed of milk proteins. Since the interfacial and electrostatic properties are

changed, the damage of the fat globule membrane can significantly affect the stability and

properties of dairy products (Walstra, 1994). Kanno et al., (1991) concluded that the

adsorption of protein on the milk fat surface is affected by pH, the MFGM concentration and

the milk fat content and that the MFGM could be a potential emulsifier for certain food and

other emulsions.

2.5.2.3 Effect of homogenization

Homogenization is the most common method to improve the stability of a dairy and food

emulsion. It results in smaller particle sizes with more uniform size distributions, hence

limiting the rate of phase separation (Biasutti et al., 2010). Droplet size distribution is perhaps

the most important factor in determining the emulsion properties like consistency, rheology,

stability, colour and taste (Stang et al., 2001). During homogenization proteins are absorbed

from the continuous phase into the newly created fat globule membrane. The composition and

15

the structure of this absorbed layer influence the stability and the rheological properties of

emulsion (Dickinson, 1998).

Phipps (1975) and Mulder & Walstra (1974) reported that, for homogenization pressures

between 0.25 – 40.5 MPa, the average fat globule diameter (d) of emulsions decreases with

emulsification pressure (P) in a relation d~P(-0.6). Robin et al. (1992) used a microfluidizer to

prepare butteroil-in-water emulsions and observed that the average size of the particles

decreased with an increase in pressure and reached a minimum value at around 60 MPa.

Homogenization could be a critical factor in the network formation process. San Martin-

Gonzalez et al. (2009) reported that an emulsion containing 30% oil and 2-3.5% casein

showed a gel like structure when homogenized at pressures between 20 to 100 MPa.

The main effect of homogenization is the reduction in droplet size and consequently in the

increase of the O/W surface area. The increase in homogenization pressure reduces the

droplet size of the emulsions and thus improves the shelf life of the products by reducing the

creaming rate. High pressure homogenization resulted in the increase of the surface activity of

emulsifying molecules; it may improve the efficiency of the product e.g. coating ability or

penetration action (Flouryu et at., 2000)

2.6 Whipping properties

2.6.1 Whipped cream

The fat rich fraction of whole milk is known as cream. Dairy cream can be mixed with air and

the volume of the resulting colloid becomes roughly double of the original cream. Thus, the

whipped cream can be defined as a foamed dairy product, consisting of a dispersion of gas

bubbles, surrounded by partially coalesced fat at the air-serum interface and supported by a

high viscosity serum phase (Smith et al., 2000). According to Hotrun et al. (2005) the

whipping process consists of three stages: 1) introduction of air bubbles and simultaneous

protein adsorption into their surfaces, 2) accumulation and adsorption of fat globules onto the

surfaces and 3) formation of a fat globule aggregate network. The whipping of cream

introduces air bubbles in the structure and fat globules become partially coalesced and form a

network which stabilizes the incorporated air.

2.6.2 Characterization of whipped cream

Whipped creams are thermodynamically unstable. Destabilization of whipped cream takes

place over time and is undesired. It is difficult to control these physical properties and they

16

affect the characteristics of whipped cream such as flavour, appearance and mouth feeling

(Walstra et al., 2006). Serum loss, Ostwald ripening and bubble coalescence are the three

principle mechanism of instability of whipped cream. All of these mechanisms occur

simultaneously and result in complete phase separation: aqueous, fat and the air phase

(Fredrick et al., 2010). Ostwald ripening is the disappearance of small air cells, resulting in

the formation of bigger air cells due to the difference in Laplace pressure. It largely depends

on the solubility of the gaseous phase into the serum phase. On the other hand bubble

coalescence is the merging of two neighboring air bubbles.

Consumers and dairy industries expect whipping cream to have certain desirable qualities

such as taste, shelf-life and whipping characteristics. The dairy industries express whipping

characteristics in terms of shorter whipping time, high overrun, good firmness and high

stability of whipped product (Bruhn and Bruhn, 1988). Whipping time is the time needed to

whip cream until it reaches maximum volume. Depending on the rate of partial coalescence,

the whipping time is changed and faster partial coalescence rate results in a shorter whipping

time (Hotrun et al., 2005). Serum loss is the amount of aqueous phase that is released from the

whipped cream after standing for a period of time. It is one of the indications of whipped

cream instability. Serum loss should be as minimum as possible. Overrun is defined as the

volume of air incorporated into the cream structure relative to the volume of the cream. For

traditional dairy whipped cream the desirable overrun is around 100-120% (Graf and Muller,

1965). Firmness is the expression of structural rigidity of the whipped cream. The texture,

stability and whipping properties of whipped cream depend on several factors. Whipping of

cream is in fact a destabilization mechanism whereby milk fat globules partially coalesce so

that the interfacial layer is not being too strong. A control destabilization or partial

coalescence of the emulsion is needed during further processing to develop an internal

structure of agglomerated fat which alters the texture and physical appearance of the product.

When the milk fat is homogenized in the presence of proteins e.g. caseins and whey proteins,

the resulting emulsion become too stable. When small molecular weight surfactants replace

protein on the interface, due their high affinity to the surface and their capability to reduce

surface tension, weaker spot on interface and partial coalescence are promoted (Goff, 1997).

MFGM materials, isolated from native milk fat globules, have good surface active properties.

It was observed that the reduction of interfacial tension of the surface covered by the MFGM

is similar to that covered by caseins (Chazelas et al., 1995). The MFGM can be used as an

emulsifier in reconstituted milk fat emulsions; early work was done by Kanno et al. (1991)

17

whereby they emulsified anhydrous milk fat (AMF) with MFGM fragments and investigated

the emulsifying properties (emulsion capability, foam and emulsion stability, and

whippability) of the MFGM fractions. They concluded that the amount of protein adsorbed

onto the surface of the milk fat globules is affected by the pH, the concentration of the

MFGM and the fat content. When emulsions are prepared with MFGM materials (derived

from untreated cream) the newly formed oil droplets covered by the MFGM material behave

differently from the emulsions stabilized by other milk proteins: no displacement occurs on

the addition of small molecular weight surfactant. Also the addition of milk proteins (β-

lactoglobulin or caseins) after emulsification does not seem to have remarkable affect on the

composition of the interface (Corredig and Dalgleish 1998c).

18

CHAPTER III: MATERIALS AND METHODS

3.1 Materials and chemicals

3.1.1 Materials

Buttermilk powder was purchased from Friesland-Campina (Lummen, Belgium); Buttermilk

whey was obtained from a local dairy company (Bϋllinger Butterei Bϋllingen, Belgium).

Skim milk powder was purchased from Hochdorf Swiss Milk AG (Hochdorf, Switzerland),

sodium caseinate from Acros-Organic (Geel, Belgium), , Lummen, Belgium), soy oil from a

local supermarket and milk fat from Friesland Campina. Deionized water from Millipore

ultrapure water purification system (Millipore SA, Malsheim, France) was used to

reconstitute the buttermilk powder.

Buttermilk powder was reconstituted in deionized water with 4% buttermilk solids. Trisodium

citrate, 1%, with > 99% purity was also added for dissociating the casein micelles into casein

species which are small enough to permeate through the membrane during the filtration

process (Roesch et al 2004, Rombaut et al 2006). After complete dissolution of the powder,

the pH was obtained at 7.6 and the solution was allowed to stand overnight at 4°C to ensure

complete hydration. Buttermilk whey was also stored at 4°C and before microfiltration; the

pH was adjusted to 7.5 by adding (1N) KOH (Rombaut et al 2007).

Isolation of the MFGM materials

The cross-flow microfiltration (MF) method with four steps continuous diafiltration was used

to isolate the MFGM materials from reconstituted buttermilk and buttermilk whey. The

microfiltration unit consist of a Millipore frame with Pellicon® 2 cassette filter

(PVGVPPC05), a hydrophilic PVDF membrane Durapore® with a pore size of 0.22 µm and a

membrane surface of 0.5 m2 (Screen type C). The feed-pump was a compressed air-operated

diaphragm pump (Chemicor series of Almatec, Kamp-lintfort, Germany). The feed flow rate

was adjusted at 200 L/h and the permeate flow rate was adjusted with a Millipore peristaltic

pump at 15 L/h. The microfiltration process was carried out at 40-45 °C. The trans-membrane

pressure was 0.35-0.55 bar. The dry matter (DM) content of the final MFGM material was

about 9% and the material was stored at < -20°C for further analysis and emulsion

preparation.

19

3.1.2 Chemicals

Chemicals for analysis were obtained from Chem-Lab (Zedelgem, Belgium) and Sigma-

Aldrich (Steinheim, Germany). For the analysis of phospholipids, high-performance-liquid-

chromatography (HPLC)-grade dichloromethane, supra-gradient methanol and HPLC grade

water were obtained from Biosolve (Valkenswaard, Netherland)

3.2 Compositional analysis of the experimental materials

The dry matter content of the samples was determined by measuring the loss of weight after

drying the samples to a constant weight at 105°C (Williams, 1984). The total protein content

of the samples was determined by the Kjeldhal method (Egan et al, 1981) using 6.38 as

conversion factor and the total fat content by gravimetric determination using the Röse-

Gottlieb method (IDF, 1987). In case of the MFGM samples (lower in mineral content) 3 mL

10% (w/v) CaCl2 solution was added before carrying out the extraction process. CaCl2 was

added to increase the ionic strength of the solution (MFGM) which then improves the phase

separation process during fat extraction (Le at al., 2010). Total ash content was determined by

heating and igniting the samples in an electric muffle furnace at 550°C (Williams, 1984) and

the lactose content by subtraction methods. All chemical analyses were measured in duplicate

with two replications.

Phospholipids were extracted using a mixed solution of chloroform: methanol (2:1),

chloroform: methanol (20:1), HPLC chloroform: methanol (88:12) and 10% (w/v) CaCl2

solution and the extracted material was stored in the freezer for further analysis. Samples were

analyzed using the HPLC method developed by Le et al., (2011). The Shimadzu HPLC

System (Tokyo, Japan) consisting of a controller (CBM-20A), an online degasser (DGU-

20A5), a solvent delivery module (LC-20AT), an autosampler (SIL-20AT) and a column oven

(CTO-20AC), was connected to an evaporative light-scattering (ELSD) detector (Alltech-

3300, Alltech Associates Inc., Lokeren, Belgium). The separation column was 150 x 3.0 mm

Prevail silica 3 µm, connected behind a 7.5 x 3.0 mm pre-column, and made of the same

material, with 5µm packing particle size. The column oven and sample chamber of the

autosampler was set at 40°C and 20°C respectively. Two solvent lines were used: line-A

contained dichloromethane and line-D contained methanol and acetic acid/triethylamine

buffer solution (with a ratio of 500:21 (v/v) and at pH 4.5). The ELSD parameters were set at

65°C for the tube temperature, 2.1 L/min for the nebulizer gas (N2) and 1 L/min for the

acquisition gain. The injection volume was 10 µL and each extract was injected twice. The

20

mobile phase pumping was in linear gradient program with volume ratios of A and D being as

follows: 96:4 at start to 88:12 at t=4 min. and to 6:94 at t=12 min. and back to 96:4 at t= 17

min. respectively. The pumping was then maintained at this condition until t=22.5 min. before

a new injection started. Total mobile phase flow rate was 0.5 mL/min.

The buffer solution was prepared by adding 7.16 mL acetic acid (From Acros-Organics,

Belgium) and 8.0 mL triethylamine (From Sigma-Aldrich, Belgium, ) to 117.8 mL HPLC

grade water (From Biosolve, Netherland).

Protein profiling by SDS-PAGE

In order to study the protein profiles of the experimental samples, reducing SDS-PAGE

(Sodium dodecyl sulfate polyacrylamide gel electrophoresis) was used. The separation is

based on the mobility of protein in the acrylamide matrix according to their molecular weight.

The separation system, all reagents and the precast gels were obtained from Invitrogen™

(Carlsbad, USA). The sample preparation and reduction was according to method developed

by Le et al., (2009). Approximately 16 µg of total protein was loaded on each well of a

precast gel (NuPAGE® NOVEX 4-12% Bis-Tris gel, 1.0 mm x 17 wells) and the same

amount of Mark 12 solution was also added as molecular weight (MW) standard.

Electrophoresis was performed in 50 min. at 200 V and 125 mA/gel.

After separation, the gel was washed 3 times (for 5 min.) with deionized water and stained for

one hour with Simply Blue™ Safe Stain (Coomassie blue).. After de-staining the wet gel was

scanned at 400 dpi using a high-resolution transmission scanner (UMAX Powerlook III,

Taipei, Taiwan) and was analyzed by Imagemaster Totallab Software (GE Healthcare,

Diegem, Belgium). Different types of protein were identified by comparing their MW with

the standard and named according to Mather, (2000).

3.3 Experiment 1: Characteristics of emulsions stabilized with milk fat globule membrane fragments

3.3.1 Emulsion preparation

Emulsions were prepared for buttermilk powder (BMP), skim milk powder (SMP), sodium

caseinate, milk fat globule membrane from buttermilk powder (MFGM-BMP) and milk fat

globule membrane from buttermilk whey (MFGM-BMW). Suspensions in deionized water

were made by dispersing each of the materials, using a magnetic stirrer. Samples were stored

overnight at 4°C for hydration. The final protein content in emulsions was standardized at

21

2.3g/100g. Emulsions were then prepared by adding soy oil at a concentration of 35% and the

pH was adjusted at 7.0. Samples were then warmed at 50°C and pre-homogenized at 13000

rpm for 2 minutes with a Ultra-Turrax (IKA®- Werke, Germany). The emulsions were then

passed through a two steps lab scale high pressure homogenizer (APV Systems, Albertslund,

Denmark) at 50°C. For each sample five different homogenization pressures (0/20, 30/20,

90/20, 150/20 and 210/20 bar) were applied to make five different emulsions. The emulsions

were stored in a refrigerator at 4°C for further analysis.

3.3.2 Determination of emulsion properties

3.3.2.1 Microscopic observation

Microphotographs of the different emulsions were obtained with an optical microscope

(Diaplan, Van Hopplynus Instrument, Germany) equipped with a Nikon Coolpix 4500 digital

camera. Samples were first diluted 10x and a sample drop was carefully placed on a glass

slide, covered with cover slip and observed with a plan fluor objective (10x magnifications).

Although the resolution was not sufficient to distinguish individual droplet, the method gave a

good approximation of the qualitative difference in the flocculation structures of the

emulsions.

3.3.2.2 Rheological characteristic

The rheological characteristics of the samples were measured at 1st and 8th days of storage

using a TA instruments AR2000 controlled-stress rheometer, equipped with a conical

concentric cylinder geometry (28 mm diameter) and a cup (30 mm diameter). For the

characterization of the flow curves, emulsion samples were mixed gently and passed through

a syringe to ensure homogeneity of the sample. After this step 20 g of each emulsion sample

was transferred into the cup of the rheometer for testing.

Flow curves were measured at a shear rate between 0.1 to 100 s-1 (31 measuring points) and at

a temperature of 20°C. The experimental data were fitted to the power law equation.

��

Where K is the consistency index and ‘n’ the flow behavior index. For Newtonian fluid, n= 1

and for non-Newtonian fluid n is far from 1.

3.3.2.3 Particle size distribution measurement

The particle size distribution of the emulsions was determined with a long bench Malvern

Mastersizer S (Malvern Instruments, Malvern, UK) with a MS17 automated sample

22

dispersion unit. The polydisperse analysis of the samples dissolved in water was performed

using a 300RF lens and the standard presentation code (1.5295, 0.01, 1.3300 for the relative

particle refractive index, particle absorption and dispersant refractive index respectively).

Measurement of the emulsions was carried out by dispersing the samples both in water and in

1% SDS (Sodium Dodecyl Sulfate) solution. SDS solution facilitates the separation of

flocculated fat particles. The Sauter mean diameter (D3.2) and volume-surface average particle

diameter (D4.3) were measured. The samples were measured twice and average values were

taken.

3.3.2.4 Creaming stability

The creaming of emulsion was measured by placing the samples in graduated tubes and stored

quiescently at 4°C for 8 days. Emulsion sample of 10 ml was placed in graduated tubes. The

volume of serum layer (Vs) formed at the bottom of the tubes was recorded and expressed as a

percentage of the total volume of emulsion (Vt) in the tube.

��

��

� 100

3.4 Experiment 2: whipping properties of the MFGM fragments

3.4.1 Experimental procedure

Materials used are milk fat, buttermilk powder and milk fat globule membrane from

buttermilk powder. The milk fat was added to the water phase of reconstituted buttermilk

(6.71%) and the MFGM-BMP (3.31%) in order to obtain 2.3% protein in the final emulsion.

The pH was adjusted to 7.0, heated at 60°C and mixed using the Ultra-Turrax at 13000 rpm

for 2 minutes. The mixtures were then homogenized using a two steps high pressure

homogenizer at 30/20 bars at 55°C and subsequently cooled to 5°C. The recombined creams

were then stored in refrigerator for 1 day at 5°C.

The recombined cream was whipped using a Hobart mixer with ‘D’ wire whip agitator at

medium speed (about 250 rpm). In order to avoid heating of the cream, the bowl and the

agitator were stored in the refrigerator at 5°C. Cream temperature was recorded before and

after whipping to make sure that the temperature was within 5°C. Approximately 1 liter cream

was whipped in each whipping experiment. Whipping continued until visually acceptable

whipped cream is obtained (i.e. when the cream does not flow around the wire) and whipping

time was recorded.

23

3.4.2 Determination of whipping properties

3.4.2.1 Overrun

To measure the overrun of the whipped cream, first an empty plastic cup was weighed, filled

fully with un-whipped cream and weighed again to know the mass of the un-whipped cream.

After filling the cup was tapped gently on the table to remove air bobbles and the surface was

flattened by scraping off with a knife. The same cup was used to measure the weight of the

whipped cream following the same procedure. The measurements of overrun were performed

6 times and the average values were used for comparison. The overrun of whipped creams

was calculated using the following equation:

% �� ! �"

�"

� 100

Where M1= Mass of the un-whipped cream with a certain volume

M2= Mass of the whipped cream with the same volume

3.4.2.2 Serum loss

The serum loss measurement was performed at 20°C for 1 hour and at 5°C for 24 hours by

putting whipped cream into a funnel that was placed on top of a flask. The empty flask was

weighed first and then approximately 30g of whipped cream was put into the funnel. The

liquid that leaked from the whipped cream and dripped onto the flask through the funnel was

weighed and the percentage serum loss was calculated using the following equation:

% �� #$��

�"

� 100

Where M1= Mass of the liquid that comes out from whipped cream after storage.

M2= Initial mass of the whipped cream.

3.4.2.3 Firmness

The compression force was performed using a TA 500 texture analyzer (Lloyd Instrument,

Bognor Regis, West Sussex, UK) to determine the firmness of the whipped cream. A

cylindrical probe with 500N load was used for measurement. The probe penetrates into the

sample to a depth of 20 mm at a rate of 1 mm/s with 0.2 N trigger values. The firmness of the

whipped cream samples was measured after 1 hour and 24 hours of storage at 5°C. The

sample cups were wrapped with aluminium foil to prevent drying during storage in the

refrigerator.

24

3.5 Statistical analysis.

All statistical analyses were performed using S-Plus® 8.0 package for Windows (Tibco

Software Inc., Palo Alto, CA, USA). Results of rheological and physical characteristics of the

emulsions were statistically processed using analysis of variance (one-way ANOVA) and

multiple comparisons of means were adjudged by the Tukey test when a significant difference

was observed. Two sample-T tests were performed to compare the whipping properties of

recombined creams. All the tests were performed at 5% level of significance.

25

CHAPTER IV: RESULTS AND DISCUSSION

4.1 Composition of the experimental materials The composition of different dairy materials (BMP, SMP, MFGM-BMP, MFGM-BMW and

SC) is shown in table 3. The result of proximate analysis indicated that the composition of the

different experimental materials is not the same at 5% level of significance.

Table 3. Composition of the formulation materials

Materials

Dry matter

(%)

Composition on dry matter (g/100g)

Total protein

Total lipid ash lactose Polar lipids

MFGM-BMP 8.61b ± 0.17 69.61c ± 1.31 23.80c ± 0.61 3.25a ± 0.18 3.33a ± 1.81 9.30c ± 0.31

MFGM- BMW

7.34a±0.07 22.88a ± 0.10 39.17d ± 0.64 28.34c ± 0.22 9.61b ± 0.69 12.14d± 0.19

BMP 95.77d±0 .03 34.25b ± 0.14 8.37b ± 0.30 7.13b ± 0.05 50.25c ± 0.20 3.27b ± 0.13

SMP 95.68d ±0.11 34.39b ± 0.02 1.93a ± 0.15 7.14b ± 0.01 56.53d ± 0.16 0.16a ± 0.01

SC 92.46c± 0.10 96.85d ± 0.01 _ 2.25a ± 0.03 _ _

Values are mean ± standard deviation of proximate composition of different samples expressed in percentage. Means having different superscripts are significantly different at overall 5% level of significance. Reported data are means for duplicate batches - Not analyzed for composition

Result of multiple comparisons of the means showed that the mean total protein and ash

content of MFGM-BMP, MFGM-BMW and SC are significantly (p<0.05) different from that

of BMP and SMP. No significant (p>0.05) difference in mean total protein and ash content

were found between BMP and SMP. Significant (p<0.05) differences in mean total lipids,

lactose and polar lipids content were observed among different dairy materials. MFGM-BMW

revealed the lowest total protein (22.88 g/100g) content but the highest total lipids (39.17

g/100g), ash (28.34 g/100g) and polar lipids (12.14 g/100g) content among dairy materials.

The low protein content is due to the fact that large amount of proteins mainly casein were

already coagulated and precipitated during acid coagulation of butter milk and high ash

content might be due to the addition of CaCl2 as process additives during the coagulation

process of buttermilk cheese. On the contrary, the MFGM-BMP contained 1.6 times lower

fat, 8.7 times lower ash, 2.8 times lower lactose and 1.3 times lower polar lipids but 3 times

more protein than MFGM-BMW. Similar results on the composition of MFGM-BMP and

MFGM-BMW also reported by Le et al., (2011). The relatively higher protein content in

MFGM-BMP could imply that some fractions of casein may contaminate the final product

26

due the similarity in size of casein micelles and MFGM fragments (Sachdeva and Buchheim,

1997).

When the composition of BMP was compared with that of MFGM-BMP, it was found that

MFGM-BMP concentration of polar lipids increased by a factor of 2.67 (table 3). Total

protein and lipids level also increased 2 and 2.8 times respectively in MFGM fraction.

Whereas, the lactose and ash content was found to decrease by a factor of 17 and 2.1

respectively in MFGM-BMP. These results are also in agreement with the data reported by Le

et al., (2011). The lower lactose and ash content in MFGM-BMP was expected because

during microfiltration the majority of lactose and minerals were drained out through permeate.

On the contrary, damaged fat globule membrane fractions rich in polar lipids were isolated as

retentate which contributed in the higher total lipids and polar lipids content of MFGM-BMP.

The polar lipids fraction comprises 40-50% of total lipids of MFGM materials (Singh, 2006).

Skim milk powder showed the lowest total lipids (1.93 g/100g) and polar lipids (0.16 g/100g)

and the highest lactose (56.53 g/100g) content as compared to other materials. The lowest

total lipids content in SMP is due the fact of cream separation from raw milk. However, BMP

presented 4.3 times higher total lipids and 20 times more polar lipids then SMP. The result on

the composition of BMP and SMP were comparable with the result of Elling et al., 1996;

Trachoo (2003) and Remeuf et al., (2003). Sodium caseinate has the highest total protein

content (96.46) among the materials but does not contain lipids, lactose and polar lipids.

Protein profiling by SDS-PAGE

The SDS-PAGE profile of different dairy materials used in this experiment is shown in Figure

4. The SDS-PAGE profile is used for qualitative interpretation of the composition of different

materials. The major MFGM proteins are MUC1, XO, PAS III, CD36, BTN, PAS 6/7 and

ADPH (Singh, 2006; Berglund et al., 1996). From the figure 4 it is observed that, in addition

to standard proteins the MFGM-BMP (lane-4) contained considerable amount of non-MFGM

protein such as caseins and whey proteins e.g. β-lactoglobulin and α-lactalbumin. XO and

BTN was found to 1.2 and 1.4 times higher ratio in MFGM-BMP than BMP. Corredig and

Dalgleish, 1998b, Ye et al., 2004 reported that heat treatment during buttermilk powder

production may induce the interaction between β-lactoglobulin, α-lactalbumin, κ-casein and

MFGM proteins which could be the reason for the presence of casein and whey proteins in the

MFGM fraction. Moreover, the casein micelles are roughly similar in size as compared to the

MFGM fraction, which also makes it possible that some of the caseins micelles might be

collected with the retentate during the isolation of MFGM from buttermilk powder.

Figure 4. SDS-PAGE of different dairy materials. (1) Skimmed milk powder; (2) Buttermilk Sodium caseinate; (4) MFGM isolated from buttermilk powder; (5) MFGM isolated from buttermilk whey and (6) mark 12 molecular weight standard. Separation was performed on gradient (4xanthine oxidase; CD36=cluster of differentiation 36; BSA= bovine serum albumin; BTN=butyrophilin; PAS 6/7= periodic acid Schiff 6/7; ADPH= adipophilin.

The major MFGM proteins such as

MFGM-BMW (lane-5). But BSA

distinguished. MFGM-BMW contained a 1.3 and 1.6 times higher ratio of

lactalbumin than MFGM-BMP but the amount of

similar. High molecular weight caseins

Similar observations were also reported by Rombaut et al., (2007). Because, during the

buttermilk cheese making process most of the caseins are separated out by

buttermilk whey is virtually free of caseins

almost similar protein profile with the exception of PAS 6/7 and ADPH

in SMP. But SMP showed a 2 times higher ratio of casein.

the data published by Le et al., (2011)

form of casein. As it was expected

(lane-3).

27

fraction, which also makes it possible that some of the caseins micelles might be


PAGE of different dairy materials. (1) Skimmed milk powder; (2) Buttermilk m caseinate; (4) MFGM isolated from buttermilk powder; (5) MFGM isolated from buttermilk

whey and (6) mark 12 molecular weight standard. The load on each lane contained 16Separation was performed on gradient (4-12%) polyacrylamide gels with Xcell Surelock system. XO=

ine oxidase; CD36=cluster of differentiation 36; BSA= bovine serum albumin; BTN=butyrophilin; PAS 6/7= periodic acid Schiff 6/7; ADPH= adipophilin.

he major MFGM proteins such as XO, Lactoferrin, CD36 and BTN were not observed in

5). But BSA, β-lactoglobulin and α-lactalbumin

BMW contained a 1.3 and 1.6 times higher ratio of

BMP but the amount of β-lactoglobulin was found to b

High molecular weight caseins (> 32 kg/mol) are also not found in MFGM


buttermilk cheese making process most of the caseins are separated out by

buttermilk whey is virtually free of caseins. SMP (lane-1) and BMP

almost similar protein profile with the exception of PAS 6/7 and ADPH

But SMP showed a 2 times higher ratio of casein. The results were in agreement with

Le et al., (2011). Sodium caseinate (SC) is a commercially purified

As it was expected, only the casein fractions were clearly distinguished in SC

fraction, which also makes it possible that some of the caseins micelles might be


PAGE of different dairy materials. (1) Skimmed milk powder; (2) Buttermilk powder; (3) m caseinate; (4) MFGM isolated from buttermilk powder; (5) MFGM isolated from buttermilk

The load on each lane contained 16µg total protein. crylamide gels with Xcell Surelock system. XO=

ine oxidase; CD36=cluster of differentiation 36; BSA= bovine serum albumin; BTN=butyrophilin;

BTN were not observed in

lactalbumin were clearly

BMW contained a 1.3 and 1.6 times higher ratio of BSA and α-

was found to be almost

also not found in MFGM-BMW.


buttermilk cheese making process most of the caseins are separated out by coagulation,

BMP (lane-2) showed an

almost similar protein profile with the exception of PAS 6/7 and ADPH which was not found

s were in agreement with

. Sodium caseinate (SC) is a commercially purified

only the casein fractions were clearly distinguished in SC

28

4.2 Creaming stability Emulsions prepared with different dairy materials (BMP, SMP, SC, MFGM-BMP and

MFGM-BMW) were studied for their stability. Figure 5 illustrate the creaming behavior of

different emulsions.

Figure 5. Creaming stability of emulsions prepared with different dairy materials upon storage at 4°C Emulsions prepared with BMP and SMP showed creaming behavior when lower

homogenization pressure is applied but showed better stability only at higher homogenization

pressure (>90/20 bar). SMP and BMP contain equal amounts of protein (table 3). The SDS-

PAGE analysis also confirms the unique pattern of casein and whey protein distribution (see

section 4.1). The casein and whey protein content of those materials play an important role in

stabilizing the emulsion. High pressure homogenization results in further unfolding of the

globular proteins and the exposure of hydrophobic sites. This influences the attachment of

proteins at the interface, reduces interfacial tension and stabilizes the emulsion (Roesch and

Corredig, 2003; San Martin-Gonzalez et al., 2009)

0

10

20

30

40

50

0 2 4 6 8 10

Se

pa

rati

on

(%

)

Storage time (day)

BMP

0

10

20

30

40

50

0 2 4 6 8 10S

ep

ara

ion

(%

)Storage (d)

SMP0/20 bar30/20 bar90/20 bar150/20 bar210/20 bar

0

10

20

30

40

50

0 2 4 6 8 10

Se

pa

rati

on

(%

)

Storage time (day)

SC

0

10

20

30

40

50

0 2 4 6 8 10

Se

pa

rati

on

(%

)

Storage time (day)

MFGM-BMP0/20

30/20

90/20

150/20

210/20

29

On the other hand emulsions prepared with MFGM-BMW showed good stability (no

separation) irrespective of homogenization pressure. This could be due the highest polar lipid

content of MFGM-BMW (Elling and Duncan, 1996). Emulsions prepared with MFGM-BMP

also showed good stability at homogenization pressure higher than 30/20 bar. The better

creaming stability of these emulsions can be attributed to the presence of more native milk fat

membrane constituents, such as phospholipids materials (Scott et al., 2003). In general the

higher homogenization pressure was found to be more efficient in yielding stable emulsions

(Elling and Duncan, 1996). From a colloidal point of view, it is well known that the reduction

of surface tension is an important factor in the formation and stabilization of emulsions.

Although high homogenization pressure increases the surface area, hydrophobic proteins and

a higher content of polar lipids of the MFGM materials facilitate emulsion stability (Kanno et

al., 1991).

Emulsions prepared with sodium caseinate showed greater instability irrespective of the

homogenization pressure. Polar lipids are absent in sodium caseinate. The lower stability

against creaming may be caused by flocculation of droplets or adsorption of proteins in an

aggregated form because aggregated proteins are less effective emulsifiers (Roesch et al

2004). Dalgleish, (1997) reported that during homogenization casein micelles are disrupted

and adsorb at the interface either as a whole or in fragments form. The presence of large

amounts of unabsorbed proteins causes phase separation due to depletion flocculation

(Dickinson and Golding, 1997).

4.3 Particle size distribution The particle size distribution of the emulsions prepared with different dairy materials (BMP,

SMP, MFGM-BMP, MFGM-BMW and SC) and homogenized at different pressures (0/20,

30/20, 90/20, 150/20 and 210/20 bar) are shown in figure 6a and 6b. These results show that

the average particle size of emulsions prepared with different materials decreases significantly

(p<0.05) with the increase in homogenization pressure (table 4). The results are in agreement

with the data published by Mulder & Walstra, (1974), Phipps, (1975) and Robin, et al.,

(1992). Emulsions prepared with SC at higher homogenization pressure (>150/20 bar )

showed a bimodal distribution with one population of about 0.5µm droplet diameter and the

second larger group with an average droplet size of about 1.5-2.0 µm. The presence of a

bimodal distribution is undesirable because it cases aggregation and flocculation of particles

and subsequently makes the emulsion unstable.

30

A B

Figure 6a. Particle size distribution of different emulsions homogenized at different pressures, after dilution in water and SDS. Lane-A after dilution in water and Lane-B after dilution in SDS.

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

BMP

0/20 bar30/20 bar

90/20 bar

150/20 bar210/20 bar

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

BMP

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

SMP

0/20 bar30/20 bar90/20 bar150/20 bar210/20 bar

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

SMP

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

MFGM-BMW


0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

MFGM-BMW

31

A B

Figure 6b. Particle size distribution of different emulsions homogenized at different pressures, after dilution in water and SDS. Lane-A after dilution in water and Lane-B after dilution in SDS. The bimodal distribution of particles can be due to clustering by bridging flocculation of fat

droplets. In case of bridging flocculation the surface of newly formed particles share a

common protein at the interface. The result of particle size distribution was also confirmed by

what we found in microscopic observation (figure 8). On the other hand a monomodal

distribution was found for emulsions prepared with BMP, SMP and MFGM-BMW. But, the

average particle diameters were found to be higher than that of MFGM-BMP and sodium

caseinate (table 4).

All emulsions after incubation with SDS showed a lower particle diameter as compared to

those after dilution in water (table 4). Dilution with SDS caused disruption of particle

aggregates and displacement of protein by a surfactant molecule. When measuring the particle

size distribution in the presence of SDS, it was possible to determine the size distribution of

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

MFGM-BMP


0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

MFGM-BMP

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

SC

0/20 bar

30/20 bar

90/20 bar

150/20 bar

210/20 bar

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

SC

32

Table 4. Average Sauter mean diameter D3.2 of emulsions at different homogenization pressure

Pressure

(bar)

D3.2 (µm) after dilution in water at different pressure

BMP SMP MFGM-BMW MFGM-BMP SC

0/20 8.33a ±0.67 6.44c ±0.42 5.16a±0.26 3.74e±0.16 4.07d±0.46

30/20 7.67a ±1.97 4.58a±1.23 5.59b±0.54 2.66d±0.12 2.69c±0.08

90/20 6.36a±0.83 4.63a±0.93 6.28c±0.23 1.89c±0.11 2.00b±0.19

150/20 6.96a ±1.27 6.14bc±1.24 6.27c±0.44 0.76b±0.06 0.60a±0.03

210/20 7.48a ±1.97 5.38bc ±1.37 5.23a±1.11 0.57a±0.07 0.40a±0.04

D3.2 (µm) after dilution in SDS at different pressure

0/20 3.79e±0.25 4.34e±0.47 3.47d±0.42 3.53e±0.06 3.91e±0.47

30/20 2.68d±0.12 2.93d±0.05 2.61c±0.08 2.63d±0.06 2.64d±0.06

90/20 2.14c±0.09 2.22c±0.09 2.11b±0.18 1.92c±0.07 1.98c±0.15

150/20 1.58b±0.03 1.65b±0.25 0.93a±0.20 0.76b±0.05 0.63b±0.09

210/20 1.11a±0.12 1.12a±0.30 0.88a±0.12 0.62a±0.09 0.39a±0.03

Data are expressed as sample means ± Standard deviation Means having different superscripts (by column) are significantly different at overall 5% level of significance.

the individual fat droplet and not of the aggregated or flocculated ones. The droplet size

distribution of emulsions prepared with SMP, BMP and MFGM-BMW diluted with water

were found to be quite different from those diluted with 1% SDS (figure 6a, 6b and table 4),

indicating the presence of flocculated oil droplets in the emulsions. In all cases the droplet

size was smaller when incubated with SDS, indicating the fact that the oil surface was

partially covered and therefore bridging between droplets in close proximity occurred.

Figure 7. Particle size distribution of emulsions prepared with different material at homogenization pressure 90/20 bar. A) After dilution in water. B) After dilution in SDS.

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

ABMP

MFGM-BMP

MFGM-BMW

S C

SMP

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100 1000

Vo

lum

e (

%)

Diameter (µm)

B

33

The effect of different materials on droplet size distribution is shown in figure 7. After

dilution in water, the emulsions prepared with MFGM-BMP and SC have a smaller particle

size compared to those of BMP, SMP and MFGM-BMW.

Table 5. Average surface-weighted mean diameter D3.2 of different emulsions at 90/20 bar

Materials D3.2 (µm) after dilution in water D 3.2 (µm) after dilution in SDS BMP 6.36a ±0.83 2.14a ±0.09 SMP 4.63b ±0.93 2.22a ±0.09 MFGM-BMW 6.28a ±0.23 2.11a ±0.18 MFGM-BMP 1.89c ±0.11 1.92b ±0.07 SC 2.00c ±0.19 1.98b ±0.15 Data are expressed as sample means ± Standard deviation Means having different superscripts (by column) are significantly different at overall 5% level of significance.

The observation was confirmed by the results of the average Sauter mean diameter (D3.2) of

different materials (table 5). The emulsions prepared with MFGM-BMP and SC were

characterized by a significantly lower (p<0.05) Sauter mean diameter as compared to BMP,

SMP and MFGM-BMW. The creaming stability of SC was found to be quite lower than that

of MFGM-BMP (see section 4.2).

It can also be noted that, even at higher homogenization pressure (> 90/20 bar), the emulsions

with BMP, SMP and MFGM-BMW showed larger value of the particle size distribution, in

opposition with what was found in case of MFGM-BMP and SC (table 4). In the case of

BMP and SMP a lack of surface active material could be a reason for the larger particle sizes.

Toams et al. (1994) also reported that an inadequate amount of proteins could cause some

aggregation of the fat droplets. On the other hand the chemical composition analysis showed

that MFGM-BMW contains a higher level of polar lipids (table 3). Instead of a higher polar

lipids content of MFGM-BMW, the larger diameter in water indicates that some other factors

are interacting with the absorption process at the surface. This could be due to the higher ash

content (minerals e.g., Ca) of MFGM-BMW which might have influence on the aggregation

process. Because Ca+2 is a bivalent cation it can bind two MFGM particles so at a higher pH

interaction between Ca+2 and MFGM fragments is favored which results in the formation of

MFGM aggregates (Rombaut and Dewettinck, 2007). Among the materials, MFGM-BMP

showed the smallest droplet size both in water and SDS solution. The higher phospholipids

content of MFGM materials might be the cause of better emulsifying properties (Sodini, et al.,

2006).

34

4.4 Microscopic observation Materials 0/20 bar 90/20 bar 210/20 bar

BMP

SMP

MFGMBMW

MFGMBMP

SC Figure 8. Microscopy images of emuslsions prepared with BMP, MFGM-BMP, MFGM-BMW, SC and SMP at different homogenization pressures (0/20, 90/20, and 210/20 bar).

35

Microstructural observations of emulsions prepared at different homogenization pressures are

depicted in figure 8. Observation of emulsion microstructure under optical microscope

enables to determine the variation in appearance and distribution of flocs between emulsions

prepared with different materials and homogenization pressures. Microscopic examination

showed that emulsions made with BMP, SMP and MFGM-BMW contain large aggregates

and the presence of aggregates increased with the homogenization pressure, pointing to an

extensive flocculation which is in agreement with the data on the particle size distribution.

This is probably due to the presence of a limited number of surface active materials other than

proteins since an increase in homogenization pressure results in a reduction in droplet size and

an increase in surface area. Whereas, emulsions prepared with MFGM-BMP and SC showed

individual oil droplets and no sign of flocculation at higher homogenization pressure.

Among the different materials, the best emulsions were obtained with MFGM-BMP. No

flocculation was observed for emulsions prepared at homogenization pressure even at 90/20

bar. Corredig and Dalgleish (1998a) found that the newly formed oil droplets covered by the

MFGM material, behave differently from emulsions stabilized by other milk proteins i.e. no

displacement occurs on the addition of small molecular weight surfactant and the droplets are

not affected by the presence of proteins such as an addition of β-lactoglobulin or caseins. This

is due to fact that strong interaction may exist at the interface and that the phospholipids of the

MFGM may lower the interfacial tension; consequently the particles remain individually

dispersed in the emulsion. Roesch et at. (2004) also found that good stability against creaming

and small particle size distribution of emulsion was obtained with an increase in the MFGM

concentration, whereas a similar emulsion prepared with BMP showed extensive flocculation.

Our microscopic analysis confirmed these results as described in the section on particle size

distribution. In general, larger flocs were shown in BMP, SMP and MFGM-BMW emulsions.

4.5 Rheological characteristics The emulsions prepared with different materials (BMP, SMP, MFGM-BMW and SC) at

different homogenization pressure showed non-Newtonian shear thinning behavior after 1 day

storage as shown in figure 9 (See also Appendix 1). Whereas, emulsions prepared with

MFGM-BMP (regardless of the homogenization pressure) showed a Newtonian flow behavior

and a very low shear stress against shear rate. BMP, SMP and MFGM-BMW showed a higher

initial shear stress which then increased gradually with the shear rate.

36

Figure 9. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM-BMP and SC) at different homogenization pressures, after 1 day of storage. The higher initial shear stress was related to an increased homogenization pressure. The

emulsions prepared with SMP showed a higher shear stress then followed by BMP, MFGM-

BMW and SC. However, it can also be noted that although SC showed a non-Newtonian flow

behavior, the shear stress against the shear rate was very low as compared to SMP, BMP and

MFGM-BMW (figure 9).

0

50

100

150

200

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

BMP0/20 bar30/20 bar

90/20 bar150/20 bar

210/20 bar

0

50

100

150

200

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

SMP0/20 bar

30/20 bar

90/20 bar

150/20 bar

210/20 bar

0

50

100

150

200

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

MFGM-BMW


0

1

2

3

4

5

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

MFGM-BMP


0

1

2

3

4

5

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

S-C


37

Figure 10. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM-BMP and SC) homogenized at 90/20 bar. A: after 1 day storage B: after 8 days of storage.

As shown in table 6 the consistency index (K) of SMP was significantly (p<0.05) higher than

that of others materials. On the other hand a significantly (p<0.05) lower consistency index

was observed for emulsion with SC. However, no significant difference (p<0.05) in the

consistency index was observed among the emulsions with BMP, MFGM-BMW and MFGM-

BMP. Furthermore, Power law index (n) of the different emulsions was also found to be

significantly different after a day of storage.

Table 6. Rheological parameters of emulsions homogenized at 90/20 bar

Materials /samples

Parameter after 1 day of storage Parameter after 8 days of storage Consistency

index K (Pa.sn )

Power law index

n

Consistency index

K (Pa.sn )

Power law index

n BMP 3.78bc±1.62 0.54b±0.10 11.48c±1.10 0.39c±0.01 SMP 5.04c±0.50 0.49b±0.07 5.54b±0.27 0.48b±0.02 MFGM -BMW 3.80bc±0.76 0.44bc±0.01 5.64b±0.45 0.35c±0.05 MFGM- BMP 2.20b±0.02 0.79a±0.01 0.02a±0.00 0.83a±0.01 SC 0.32a±0.02 0.36c±0.01 0.32a±0.03 0.35c±0.00

Data are expressed as sample means ± Standard deviation Means having different superscripts (by column) are significantly different at overall 5% level of significance. The Power law index of emulsions prepared with BMP, SMP, MFGM-BMW and SC was

significantly (p<0.05) lower than that of MFGM-BMP. Meanwhile, no significant difference

in the Power law index was observed among the emulsions with BMP, SMP and MFGM-

BMP. Emulsions that were stored for 8 days also showed shear thinning behavior (figure 10).

No significant difference (p>0.05) in the consistency index was observed between samples of

SMP and MFGM-BMW and also MFGM-BMP and SC. However, the consistency index of

0

10

20

30

40

50

60

70

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

A

BMPSMPS-CMFGM-BMP

MFGM-BMW

0

10

20

30

40

50

60

70

0 20 40 60 80 100 120

Sh

ea

r st

ress

(P

a)

Shear rate (1/s)

B BMPSMPS-CMFGM-BMPMFGM-BMW

38

the BMP samples was found to be significantly (p<0.05) higher than that of the other

materials. In the case of the Power law index, no significant difference (p>0.05) was observed

among the samples of BMP, MFGM-BMW and SC, whereas the MFGM-BMP sample

showed a significantly higher Power law index as compared to that of the other materials. The

consistency index and the Power law index for emulsions prepared at different

homogenization pressures are shown in table 7.

With regard to the consistency index and the Power law index under both storage conditions

(after 1 day of storage and 8 days of storage) for different emulsions, a similar trend was

observed. Emulsions with MFGM-BMP showed a lower consistency index and a higher

Power law index indicating a Newtonian type flow behavior. Whereas, the opposite results

were found in the case of emulsions prepared with BMP, SMP and MFGM-BMW. The

consistency index (K) in the Power law model describes the lowering in viscosity as the shear

rate increases, which correlates to the shear thinning property. An increased consistency index

and a decreased Power law index are indicative for a thinner consistency. The higher shear

stress for the samples prepared with SMP, BMP and MFGM-BMW indicated the apparently

viscous emulsion as compared to that of MFGM-BMP. The shear stress increased with the

homogenization pressure. These results are in agreement with the observation of Prentice,

(1992) and Kanno et al., (1991). Emulsions with SMP were apparently more viscous as

compared to others. Similar results also were reported by Scott et al., (2003), in which cream

containing SMP and low-melt butter oil was found to be more viscous than that of sweet

buttermilk and butter-derived aqueous phase. Among the materials, MFGM-BMP derived

samples showed very low shear stress and fluid like flow behavior (index n is close to 1). The

reason for this could be the higher polar lipids content of MFGM-BMP. Scott, (1999) also

reported that a higher phospholipids and unsaturated fatty acid content of buttermilk and

butter-derived aqueous phase may have contributed to an increased fluidity of the emulsions.

39

Table 7. Rheological parameters of emulsions prepared with different dairy materials and homogenized at different pressures.

Pressur

e

(bar)

Consistency index (K) and Power law index (n) after 1 day of storage


K n K n K n K n K n

0/20 0.13a±0.01 0.854a±0.01 0.06a±0.02 0.92a±0.04 0.32a±0.03 0.67a±0.03 2.10a±0.07 0.74a±0.03 0.07a±0.00 0.59a±0.00

30/20 0.75ab±0.62 0.75a±0.14 0.11a±0.05 0.96a±0.04 1.33a±0.11 0.54b±0.01 2.05a±0.01 0.72a±0.00 0.21b±0.00 0.37b±0.00

90/20 3.78b±1.62 0.54b±0.10 5.04b±0.50 0.49b±0.07 3.80b±0.76 0.44c±0.01 2.20a±0.02 0.79b±0.01 0.32c±0.02 0.36b±0.01

150/20 13.90c±2.74 0.40bc±0.03 21.31c±1.41 0.38c±0.01 9.44c±1.20 0.35d±0.02 2.39b±0.04 0.87c±0.01 0.42d±0.01 0.38b±0.01

210/20 20.37d±0.77 0.35c±0.01 27.03d±3.82 0.38c±0.02 10.69c±1.00 0.30d±0.01 2.43b±0.06 0.89c±0.02 0.48e±0.00 0.38b±0.00

Consistency index (K) and Power law index (n) after 8 days of storage


K n K n K n K n K n

0/20 0.07a±0.00 0.92a±0.00 0.11a±0.01 0.91a±0.00 1.09a±0.19 0.42ab±0.02 0.02a±0.00 0.78a±0.02 0.07a±0.00 0.56a±0.01

30/20 0.83a±0.74 0.76a±0.14 0.12a±0.04 0.91a±0.06 1.43a±0.52 0.51b±0.07 0.02a±0.00 0.78a±0.01 0.21b±0.01 0.37b±0.01

90/20 11.48b±1.10 0.39b±0.10 5.55b±0.27 0.48b±0.02 5.64b±0.43 0.35a±0.05 0.02b±0.00 0.83a±0.01 0.32c±0.03 0.35bc±0.00

150/20 11.99b±7.86 0.44b±0.09 27.35c±3.20 0.33c±0.01 8.01c±0.40 0.34a±0.02 0.01b±0.00 0.89b±0.00 0.47d±0.01 0.32c±0.00

210/20 14.40b±3.21 0.43b±0.05 30.02c±0.92 0.35c±0.01 7.05c±0.68 0.32a±0.01 0.02b±0.00 0.90b±0.02 0.45d±0.06 0.34bc±0.00

Data are expressed as sample means ± Standard deviation Means having different superscripts (by column) are significantly different at overall 5% level of significance.

40

4.6 Whipping properties of the recombined cream

The effect of MFGM-BMP on the whipping properties of cream compared to buttermilk

powder is shown in Figure 11.

Figure 11. Whipping properties of cream containing BMP and MFGM-BMP.

In the presence of MFGM-BMP, the whipping time and overrun increased as compared to the

cream with BMP. The increase in whipping time and overrun was significantly (p< 0.05)

higher than that of cream with BMP. The variation may be an indication of differences in

structure and composition of the surface layer of the fat droplets.

Table 8. Whipping parameter of recombined cream containing BMP and MFGM-BMP

Materials Whipping time (s)

Overrun (%)

Serum loss (%) Firmness (N) 1h at 20°C 24h at 5°C 1h at 5°C 24h at 5°C

BMP 798.0a ±11.4 117.7a±6.5 1.92a±0.23 0.0a±0.00 1.69a±0.12 1.67a±0.09 MFGM- BMP 1064.6b±33.4 132.6b±3.6 7.67b±0.37 0.37b±0.06 1.64a±0.07 1.71a±0.07

Values are mean ± standard deviation from duplicate batches Means having different superscripts (by column) are significantly different at overall 5% level of significance.

0

200

400

600

800

1000

1200

BMP MFGM BMP

Wh

ipp

ing

tim

e (

s)

Type of cream

0

20

40

60

80

100

120

140

160

BMP MFGM BMP

Ov

err

un

(%

)

Type of cream

0

1

2

3

4

5

6

7

8

9

1h at 20° C 24h at 5° C

Se

rum

loss

(%

)

Storage time

BMP MFGM BMP

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

1h at 5°C 24h at 5°C

Fir

mn

ess

(N

)

Storage time

BMP MFGM BMP

41

Cream with MFGM-BMP gave a significantly (p<0.05) higher serum loss as compared to that

of BMP both after 1 h at 20°C and 24 h at 5°C of storage. It should be noted here that BMP

did not show any serum loss after storage for 24 h at 5°C. With regards to the firmness there

was no significant (p>0.05) difference between cream with BMP and MFGM-BMP both after

1h and 24h.

In the production of whipped cream a controlled destabilization or partial coalescence of the

emulsion is needed to develop an internal structure of agglomerated fat globules which alters

the texture and physical appearance of the product (Goff, 1997). When the milk fat is

homogenized in the presence of BMP, the caseins and whey proteins of this material may

readily be absorbed and makes a relatively thick interfacial layer around the oil droplets. The

resulting emulsions become stable and inhibit partial coalescence. But the MFGM-BMP

contain a considerable amount of polar lipids (Table 3) which may influence the partial

coalescence and the development of a agglomerated fat structure in whipped cream. This was

reflected by a higher percentage of overrun of MFGM-BMP cream (table 8). On the other

hand MFGM-BMP showed a higher percentage of serum loss than BMP cream. This can be

linked to the functional properties of proteins that are adsorbed at the interface and the

rheological properties of the aqueous phase. Dickinson, (1992) reported that foam stability is

greatly affected by the rheological properties of the continuous phase and the viscoelastic

properties of the interfacial layer. In whipped cream the partially aggregated fat droplets are

evenly distributed around the air-serum interface and provide the stability and firmness of the

foam (Noda and Shiinoki, 1986). The milk proteins with good rheological properties form a

stable interfacial film that stabilizes the air-serum and fat-serum interfaces (Prentice, 1992).

The MFGM-BMP contains lower milk proteins e.g. casein and whey proteins (see section

4.1), which have very high water holding capacity. This could be the reason for the higher

serum loss in cream with MFGM-BMP. On the other hand BMP contained comparatively a

higher amount of milk proteins thus whipped cream with BMP could be expected to give

lower serum loss after a storage time of 1 h at 20°C. Van Lent et al., (2008) also observed no

fresh serum loss for creams reformulated with SMP.

The results show that MFGM-BMP cream has a significantly higher whipping time, overrun

and serum loss when compared with that of BMP. The higher overrun means that more air

was incorporated during whipping and that a less dense structure was formed. But in contrast,

the firmness of MFGM-BMP cream was found to be similar to that of BMP. The higher

42

serum loss of MFGM-BMP cream might be due to bubble coalescence and Ostwald ripening.

However, further investigations might be needed to confirm this hypothesis using for example

the microscopy technique. From this experiment it appears that the stability of recombined

cream rich in MFGM materials needs to be improved whilst the other properties are quite

appreciable. Therefore more detailed work is needed on the quantification and function of

MFGM materials in the development of microstructure and physical appearance of whipped

cream.

43

CHAPTER V: CONCLUSION

The emulsifying properties of MFGM fragments isolated from buttermilk and buttermilk

whey by microfiltration were evaluated. The properties of these materials were also compared

with those of BMP, SMP and SC (sodium-caseinate).

Emulsions containing MFGM-BMP showed smaller particle size distribution than MFGM-

BMW, BMP and SMP. Microscopic observation was also in favor smaller particle size

distribution of MFGM-BMP. In addition MFGM-BMP showed good creaming stability.

Similar emulsions prepared with BMP, SMP, SC and MFGM-BMW showed differences with

regard to extensive flocculation with the only exception however, that MFGM-BMW showed

a good stability to creaming. The smallest particle size distribution but the highest creaming

behavior was found in case of SC.

Newtonian flow behavior was observed for emulsions prepared with MFGM-BMP at all

homogenization pressure, whereas, at higher homogenization pressure (>30/20 bar),

emulsions containing BMP, SMP, SC and MFGM-BMW showed shear thinning and

thixotropic behavior with higher consistency index and lower power law index values. But the

opposite was observed at lower homogenization pressure. However, the creaming stability

was found to be very low in such a case.

Our data indicate that a selective concentration of MFGM isolated from buttermilk powder by

microfiltration has superior emulsifying properties than those of MFGM-BMW and other

dairy materials. However the MFGM-BMW fractions used in the present study are highly

contaminated with minerals (as shown by the higher ash content) and it is expected that the

differences in functionality found in our study will only be amplified when the fractions are

isolated from less contaminated buttermilk whey.

In addition to the conclusions described above, it can be concluded from additional

investigations on the functionality of MFGM-BMP in whipping cream that the recombined

creams with MFGM-BMP have longer whipping time, higher overrun, similar firmness and

relatively higher serum loss than those of recombined cream with BMP. From this

investigation it is evidenced that the stability of recombined cream rich in MFGM materials

needs to be improved whilst the other properties are quite appreciable. Therefore, more

detailed work is needed on the quantification and functionality of MFGM materials in the

development of microstructure and physical appearance of whipped cream.

44

FUTURE PERSPECTIVE

This research is a small part of the attempt to understand the emulsifying and whipping

properties of MFGM fragments. The author wished to recommend the following points for

future investigation:

1. Functional properties of MFGM fragments from different sources should be

investigated

2. Further investigation on the components of protein and fat contents of MFGM

materials should be conducted especially on casein and polar lipids.

3. The effect of different MFGM fragments on partial coalescence and whipping

properties of dairy recombined cream should be conducted.

45

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54

Appendix 1

A B

Figure 1a. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM-BMP and SC) at different homogenization pressures and day of storage. Lane A= after 1 day of storage; Lane B= after 8 day of storage.

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A B

Figure 1b. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM-BMP and SC) at different homogenization pressures and day of storage. Lane A= after 1 day of storage; Lane B= after 8 day of storage.

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56

Appendix table 1. ANOVA on protein content of different dairy materials Short Output: Call: aov(formula = Protein ~ Materials, data = composition, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 7587.675 1896.919 5449.278 2.787247e-009 Residuals 5 1.741 0.348 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-BMP-MFGM -35.400 0.59 -37.70 -33.00 **** BMP-BMW-MFGM 11.400 0.59 9.01 13.70 **** BMP-SC -62.600 0.59 -65.00 -60.20 **** BMP-SMP -0.139 0.59 -2.51 2.23 BMP-MFGM-BMW-MFGM 46.700 0.59 44.40 49.10 **** BMP-MFGM-SC -27.200 0.59 -29.60 -24.90 **** BMP-MFGM-SMP 35.200 0.59 32.90 37.60 **** BMW-MFGM-SC -74.000 0.59 -76.30 -71.60 **** BMW-MFGM-SMP -11.500 0.59 -13.90 -9.15 **** SC-SMP 62.500 0.59 60.10 64.80 ****

Appendix table 2. ANOVA on fat content of different dairy materials Short Output: Call: aov(formula = Fat ~ Materials, data = fat, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 3 1664.769 554.9231 2490.149 5.368906e-007 Residuals 4 0.891 0.2228 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-BMP-MFGM -15.40 0.472 -17.40 -13.50 **** BMP-BMW-MFGM -30.80 0.472 -32.70 -28.90 **** BMP-SMP 6.44 0.472 4.51 8.36 **** BMP-MFGM-BMW-MFGM -15.40 0.472 -17.30 -13.50 **** BMP-MFGM-SMP 21.90 0.472 19.90 23.80 **** BMW-MFGM-SMP 37.20 0.472 35.30 39.20 ****

Appendix table 3. ANOVA on ash content of different dairy materials Short Output: Call: aov(formula = Ash ~ Materials, data = composition, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 915.2216 228.8054 746.1276 3.999205e-007 Residuals 5 1.5333 0.3067 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-BMP-MFGM 3.8800 0.554 1.66 6.10 ****

57

BMP-BMW-MFGM -21.2000 0.554 -23.40 -19.00 **** BMP-SC 4.8800 0.554 2.66 7.10 **** BMP-SMP -0.0137 0.554 -2.24 2.21 BMP-MFGM-BMW-MFGM -25.1000 0.554 -27.30 -22.90 **** BMP-MFGM-SC 0.9970 0.554 -1.22 3.22 BMP-MFGM-SMP -3.8900 0.554 -6.11 -1.67 **** BMW-MFGM-SC 26.1000 0.554 23.90 28.30 **** BMW-MFGM-SMP 21.2000 0.554 19.00 23.40 **** SC-SMP -4.8900 0.554 -7.11 -2.67 ****

Appendix table 4. ANOVA on lactose content of different dairy materials Short Output: Call: aov(formula = Lactose ~ Materials, data = fat, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 3 4481.206 1493.735 1560.042 1.366912e-006 Residuals 4 3.830 0.957 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-BMP-MFGM 46.90 0.979 42.9 50.9 **** BMP-BMW-MFGM 40.60 0.979 36.7 44.6 **** BMP-SMP -6.28 0.979 -10.3 -2.3 **** BMP-MFGM-BMW-MFGM -6.28 0.979 -10.3 -2.3 **** BMP-MFGM-SMP -53.20 0.979 -57.2 -49.2 **** BMW-MFGM-SMP -46.90 0.979 -50.9 -42.9 ****

Appendix table 5. ANOVA on polar lipids content of different dairy materials Short Output: Call: aov(formula = PLs ~ Materials, data = PLs, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 3 360.0438 120.0146 3329.073 0 Residuals 12 0.4326 0.0361 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-BMP-MFGM -6.03 0.134 -6.42 -5.63 **** BMP-BMW-MFGM -8.87 0.134 -9.27 -8.47 **** BMP-SMP 3.11 0.134 2.72 3.51 **** BMP-MFGM-BMW-MFGM -2.85 0.134 -3.24 -2.45 **** BMP-MFGM-SMP 9.14 0.134 8.74 9.54 **** BMW-MFGM-SMP 12.00 0.134 11.60 12.40 ****

Appendix table 6. ANOVA on surface-weighted mean D3,2 of BMP after dilution in SDS. Short Output: Call: aov(formula = D32.SDS ~ pressure, data = BMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) pressure 4 17.34798 4.336995 732.271 0 Residuals 15 0.08884 0.005923 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 2.210 0.0544 2.040 2.380 **** 0/20-210/20 2.690 0.0544 2.520 2.850 ****

58

0/20-30/20 1.110 0.0544 0.942 1.280 **** 0/20-90/20 1.650 0.0544 1.490 1.820 **** 150/20-210/20 0.474 0.0544 0.306 0.642 **** 150/20-30/20 -1.100 0.0544 -1.270 -0.935 **** 150/20-90/20 -0.559 0.0544 -0.727 -0.391 **** 210/20-30/20 -1.580 0.0544 -1.750 -1.410 **** 210/20-90/20 -1.030 0.0544 -1.200 -0.865 **** 30/20-90/20 0.544 0.0544 0.376 0.712 ****

Appendix table 7. ANOVA on surface-weighted mean D3,2 of BMP after dilution in water. Short Output: Call: aov(formula = D32.water ~ pressure, data = BMP, qr = T, na.action = na.exclude ) Df Sum of Sq Mean Sq F Value Pr(F) pressure 4 8.91268 2.228171 2.221679 0.1156595 Residuals 15 15.04383 1.002922 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 1.370 0.708 -0.813 3.56 0/20-210/20 0.850 0.708 -1.340 3.04 0/20-30/20 0.666 0.708 -1.520 2.85 0/20-90/20 1.980 0.708 -0.208 4.17 150/20-210/20 -0.524 0.708 -2.710 1.66 150/20-30/20 -0.708 0.708 -2.890 1.48 150/20-90/20 0.605 0.708 -1.580 2.79 210/20-30/20 -0.184 0.708 -2.370 2.00 210/20-90/20 1.130 0.708 -1.060 3.32 30/20-90/20 1.310 0.708 -0.874 3.50

Appendix table 8. ANOVA on surface-weighted mean D3,2 of SMP after dilution in SDS. Short Output: Call: aov(formula = D32.SDS ~ Pressure, data = SMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 24.99836 6.249589 2501.203 0 Residuals 15 0.03748 0.002499 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 2.680 0.0353 2.570 2.790 **** 0/20-210/20 3.220 0.0353 3.110 3.330 **** 0/20-30/20 1.410 0.0353 1.300 1.520 **** 0/20-90/20 2.120 0.0353 2.010 2.230 **** 150/20-210/20 0.536 0.0353 0.427 0.646 **** 150/20-30/20 -1.270 0.0353 -1.380 -1.160 **** 150/20-90/20 -0.562 0.0353 -0.671 -0.453 **** 210/20-30/20 -1.810 0.0353 -1.920 -1.700 **** 210/20-90/20 -1.100 0.0353 -1.210 -0.989 **** 30/20-90/20 0.710 0.0353 0.601 0.820 ****

Appendix table 9. ANOVA on surface-weighted mean D3,2 of SMP after dilution in water. Short Output: Call: aov(formula = D32.water ~ Pressure, data = SMP, qr = T, na.action = na.exclude

59

) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 11.5986 2.89965 12.88274 0.00009573989 Residuals 15 3.3762 0.22508 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.308 0.335 -0.728 1.340 0/20-210/20 1.070 0.335 0.031 2.100 **** 0/20-30/20 1.860 0.335 0.829 2.900 **** 0/20-90/20 1.820 0.335 0.782 2.850 **** 150/20-210/20 0.759 0.335 -0.277 1.790 150/20-30/20 1.560 0.335 0.521 2.590 **** 150/20-90/20 1.510 0.335 0.474 2.550 **** 210/20-30/20 0.798 0.335 -0.238 1.830 210/20-90/20 0.751 0.335 -0.285 1.790 30/20-90/20 -0.047 0.335 -1.080 0.989

Appendix table 10. ANOVA on surface-weighted mean D3,2 of SC after dilution in SDS. Short Output: Call: aov(formula = D32.SDS ~ Pressure, data = SC, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 33.94439 8.486098 1197.248 0 Residuals 15 0.10632 0.007088 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 3.280 0.0595 3.1000 3.470 **** 0/20-210/20 3.520 0.0595 3.3300 3.700 **** 0/20-30/20 1.270 0.0595 1.0900 1.450 **** 0/20-90/20 1.930 0.0595 1.7400 2.110 **** 150/20-210/20 0.234 0.0595 0.0498 0.417 **** 150/20-30/20 -2.010 0.0595 -2.2000 -1.830 **** 150/20-90/20 -1.360 0.0595 -1.5400 -1.170 **** 210/20-30/20 -2.250 0.0595 -2.4300 -2.060 **** 210/20-90/20 -1.590 0.0595 -1.7700 -1.410 **** 30/20-90/20 0.658 0.0595 0.4740 0.842 ****

Appendix table 11. ANOVA on surface-weighted mean D3,2 of SC after dilution in water Short Output: Call: aov(formula = D32.water ~ Pressure, data = SC, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 37.10103 9.275258 631.0362 1.110223e-016 Residuals 15 0.22048 0.014698 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 3.470 0.0857 3.2100 3.740 **** 0/20-210/20 3.670 0.0857 3.4100 3.940 **** 0/20-30/20 1.380 0.0857 1.1200 1.650 **** 0/20-90/20 2.080 0.0857 1.8100 2.340 **** 150/20-210/20 0.197 0.0857 -0.0676 0.462 150/20-30/20 -2.090 0.0857 -2.3500 -1.820 **** 150/20-90/20 -1.390 0.0857 -1.6600 -1.130 ****

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210/20-30/20 -2.290 0.0857 -2.5500 -2.020 **** 210/20-90/20 -1.590 0.0857 -1.8600 -1.330 **** 30/20-90/20 0.695 0.0857 0.4300 0.960 ****

Appendix table 12. ANOVA on surface-weighted mean D3,2 of MFGM-BMW after dilution in SDS. Short Output: Call: aov(formula = D32.SDS ~ Pressure, data = MFGMBMW, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 19.83393 4.958482 668.3334 1.110223e-016 Residuals 15 0.11129 0.007419 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 2.5500 0.0609 2.360 2.730 **** 0/20-210/20 2.6000 0.0609 2.410 2.780 **** 0/20-30/20 0.8670 0.0609 0.679 1.060 **** 0/20-90/20 1.3700 0.0609 1.180 1.550 **** 150/20-210/20 0.0493 0.0609 -0.139 0.237 150/20-30/20 -1.6800 0.0609 -1.870 -1.490 **** 150/20-90/20 -1.1800 0.0609 -1.370 -0.991 **** 210/20-30/20 -1.7300 0.0609 -1.920 -1.540 **** 210/20-90/20 -1.2300 0.0609 -1.420 -1.040 **** 30/20-90/20 0.5000 0.0609 0.312 0.688 ****

Appendix table 13. ANOVA on surface-weighted mean D3,2 of MFGM-BMW after dilution in water. Short Output: Call: aov(formula = D32.water ~ Pressure, data = MFGMBMW, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 4.705772 1.176443 77.24187 8.002534e-010 Residuals 15 0.228460 0.015231 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -1.11000 0.0873 -1.380 -0.8420 **** 0/20-210/20 -0.07840 0.0873 -0.348 0.1910 0/20-30/20 -0.43700 0.0873 -0.707 -0.1680 **** 0/20-90/20 -1.12000 0.0873 -1.390 -0.8500 **** 150/20-210/20 1.03000 0.0873 0.763 1.3000 **** 150/20-30/20 0.67400 0.0873 0.404 0.9430 **** 150/20-90/20 -0.00802 0.0873 -0.277 0.2610 210/20-30/20 -0.35900 0.0873 -0.628 -0.0895 **** 210/20-90/20 -1.04000 0.0873 -1.310 -0.7710 **** 30/20-90/20 -0.68200 0.0873 -0.951 -0.4120 ****

Appendix table 14. ANOVA on surface-weighted mean D3,2 of MFGM-BMP after dilution in SDS. Short Output: Call: aov(formula = D32.SDS ~ Pressure, data = MFGMBMP, qr = T, na.action = na.exclude)

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Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 24.50037 6.125094 2569.194 0 Residuals 15 0.03576 0.002384 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 2.770 0.0345 2.660 2.870 **** 0/20-210/20 2.910 0.0345 2.800 3.020 **** 0/20-30/20 0.900 0.0345 0.793 1.010 **** 0/20-90/20 1.610 0.0345 1.500 1.710 **** 150/20-210/20 0.143 0.0345 0.036 0.249 **** 150/20-30/20 -1.870 0.0345 -1.970 -1.760 **** 150/20-90/20 -1.160 0.0345 -1.270 -1.060 **** 210/20-30/20 -2.010 0.0345 -2.120 -1.900 **** 210/20-90/20 -1.310 0.0345 -1.410 -1.200 **** 30/20-90/20 0.705 0.0345 0.599 0.812 ****

Appendix table 15. ANOVA on surface-weighted mean D3,2 of MFGM-BMP after dilution in water. Short Output: Call: aov(formula = D32.water ~ Pressure, data = MFGMBMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 28.05144 7.012859 6478.864 0 Residuals 15 0.01624 0.001082 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 2.97 0.0233 2.900 3.050 **** 0/20-210/20 3.16 0.0233 3.090 3.240 **** 0/20-30/20 1.07 0.0233 1.000 1.150 **** 0/20-90/20 1.84 0.0233 1.770 1.920 **** 150/20-210/20 0.19 0.0233 0.118 0.262 **** 150/20-30/20 -1.90 0.0233 -1.970 -1.830 **** 150/20-90/20 -1.13 0.0233 -1.200 -1.060 **** 210/20-30/20 -2.09 0.0233 -2.160 -2.020 **** 210/20-90/20 -1.32 0.0233 -1.390 -1.250 **** 30/20-90/20 0.77 0.0233 0.698 0.842 ****

Appendix table 16. ANOVA on surface-weighted mean D3,2 of different dairy materials at 90/20 bar after dilution in SDS. Short Output: Call: aov(formula = D32.SDS ~ Materials, data = pressure90, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 0.2284500 0.05711251 14.57357 0.00004737906 Residuals 15 0.0587837 0.00391891 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-MFGM-BMP 0.2140 0.0443 0.07760 0.3510 **** BMP-MFGM-BMW 0.0267 0.0443 -0.11000 0.1630 BMP-SC 0.1580 0.0443 0.02170 0.2950 **** BMP-SMP -0.0778 0.0443 -0.21500 0.0589 MFGM-BMP-MFGM-BMW -0.1880 0.0443 -0.32400 -0.0509 ****

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MFGM-BMP-SC -0.0559 0.0443 -0.19300 0.0808 MFGM-BMP-SMP -0.2920 0.0443 -0.42900 -0.1550 **** MFGM-BMW-SC 0.1320 0.0443 -0.00493 0.2680 MFGM-BMW-SMP -0.1040 0.0443 -0.24100 0.0322 SC-SMP -0.2360 0.0443 -0.37300 -0.0996 ****

Appendix table 17. ANOVA on surface-weighted mean D3,2 of different dairy materials at 90/20 bar after dilution in water. Short Output: Call: aov(formula = D32.water ~ Materials, data = pressure90, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 77.24445 19.31111 235.6218 2.430278e-013 Residuals 15 1.22937 0.08196 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-MFGM-BMP 4.4600 0.202 3.840 5.090 **** BMP-MFGM-BMW 0.0797 0.202 -0.545 0.705 BMP-SC 4.3600 0.202 3.730 4.990 **** BMP-SMP 1.7300 0.202 1.100 2.350 **** MFGM-BMP-MFGM-BMW -4.3800 0.202 -5.010 -3.760 **** MFGM-BMP-SC -0.1020 0.202 -0.727 0.523 MFGM-BMP-SMP -2.7300 0.202 -3.360 -2.110 **** MFGM-BMW-SC 4.2800 0.202 3.660 4.910 **** MFGM-BMW-SMP 1.6500 0.202 1.020 2.270 **** SC-SMP -2.6300 0.202 -3.260 -2.010 ****

Appendix table 18. ANOVA on consistency index (K) of emulsions prepared with BMP after 1 day storage. Short Output: Call: aov(formula = K1 ~ Pressure, data = BMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 1279.911 319.9777 143.229 9.340084e-012 Residuals 15 33.510 2.2340 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -13.800 1.06 -17.00 -10.500 **** 0/20-210/20 -20.200 1.06 -23.50 -17.000 **** 0/20-30/20 -0.619 1.06 -3.88 2.640 0/20-90/20 -3.660 1.06 -6.92 -0.393 **** 150/20-210/20 -6.470 1.06 -9.74 -3.210 **** 150/20-30/20 13.200 1.06 9.89 16.400 **** 150/20-90/20 10.100 1.06 6.85 13.400 **** 210/20-30/20 19.600 1.06 16.40 22.900 **** 210/20-90/20 16.600 1.06 13.30 19.900 **** 30/20-90/20 -3.040 1.06 -6.30 0.226

Appendix table 19. ANOVA on consistency index (K) of emulsions prepared with BMP after 8 days storage.

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Short Output: Call: aov(formula = K8 ~ Pressure, data = BMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 731.4966 182.8742 12.357 0.0001208303 Residuals 15 221.9885 14.7992 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -11.900 2.72 -20.30 -3.51 **** 0/20-210/20 -14.300 2.72 -22.70 -5.92 **** 0/20-30/20 -0.758 2.72 -9.16 7.64 0/20-90/20 -11.400 2.72 -19.80 -3.01 **** 150/20-210/20 -2.410 2.72 -10.80 5.99 150/20-30/20 11.200 2.72 2.76 19.60 **** 150/20-90/20 0.504 2.72 -7.90 8.90 210/20-30/20 13.600 2.72 5.17 22.00 **** 210/20-90/20 2.920 2.72 -5.48 11.30 30/20-90/20 -10.700 2.72 -19.10 -2.25 ****

Appendix table 20. ANOVA on consistency index (K) of emulsions prepared with SMP after 1 day storage. Short Output: Call: aov(formula = K1 ~ Pressure, data = SMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 2546.235 636.5588 188.5254 1.252221e-012 Residuals 15 50.648 3.3765 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -21.2000 1.3 -25.30 -17.200 **** 0/20-210/20 -27.0000 1.3 -31.00 -23.000 **** 0/20-30/20 -0.0487 1.3 -4.06 3.960 0/20-90/20 -4.9800 1.3 -8.99 -0.969 **** 150/20-210/20 -5.7200 1.3 -9.74 -1.710 **** 150/20-30/20 21.2000 1.3 17.20 25.200 **** 150/20-90/20 16.3000 1.3 12.30 20.300 **** 210/20-30/20 26.9000 1.3 22.90 30.900 **** 210/20-90/20 22.0000 1.3 18.00 26.000 **** 30/20-90/20 -4.9300 1.3 -8.94 -0.920 ****

Appendix table 21. ANOVA on consistency index (K) of emulsions prepared with SMP after 8 days storage. Short Output: Call: aov(formula = K8 ~ Pressure, data = SMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 3530.176 882.5441 394.2367 5.440093e-015 Residuals 15 33.579 2.2386 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -27.20000 1.06 -30.50 -24.000 **** 0/20-210/20 -29.90000 1.06 -33.20 -26.600 ****

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0/20-30/20 -0.00664 1.06 -3.27 3.260 0/20-90/20 -5.44000 1.06 -8.70 -2.170 **** 150/20-210/20 -2.67000 1.06 -5.94 0.596 150/20-30/20 27.20000 1.06 24.00 30.500 **** 150/20-90/20 21.80000 1.06 18.50 25.100 **** 210/20-30/20 29.90000 1.06 26.60 33.200 **** 210/20-90/20 24.50000 1.06 21.20 27.700 **** 30/20-90/20 -5.43000 1.06 -8.70 -2.160 ****

Appendix table 22. ANOVA on consistency index (K) of emulsions prepared with SC after 1 day storage. Short Output: Call: aov(formula = K1 ~ Pressure, data = SC, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.4297256 0.1074314 759.8813 0 Residuals 15 0.0021207 0.0001414 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -0.3490 0.00841 -0.3750 -0.323 **** 0/20-210/20 -0.4090 0.00841 -0.4350 -0.383 **** 0/20-30/20 -0.1400 0.00841 -0.1660 -0.114 **** 0/20-90/20 -0.2500 0.00841 -0.2750 -0.224 **** 150/20-210/20 -0.0600 0.00841 -0.0859 -0.034 **** 150/20-30/20 0.2090 0.00841 0.1830 0.235 **** 150/20-90/20 0.0991 0.00841 0.0731 0.125 **** 210/20-30/20 0.2690 0.00841 0.2430 0.295 **** 210/20-90/20 0.1590 0.00841 0.1330 0.185 **** 30/20-90/20 -0.1100 0.00841 -0.1360 -0.084 ****

Appendix table 23. ANOVA on consistency index (K) of emulsions prepared with SC after 8 days storage. Short Output: Call: aov(formula = K8 ~ Pressure, data = SC, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.4556919 0.1139230 86.88023 3.45902e-010 Residuals 15 0.0196690 0.0013113 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -0.4010 0.0256 -0.4800 -0.3220 **** 0/20-210/20 -0.3780 0.0256 -0.4570 -0.2990 **** 0/20-30/20 -0.1330 0.0256 -0.2120 -0.0537 **** 0/20-90/20 -0.2500 0.0256 -0.3300 -0.1710 **** 150/20-210/20 0.0232 0.0256 -0.0558 0.1020 150/20-30/20 0.2680 0.0256 0.1890 0.3470 **** 150/20-90/20 0.1510 0.0256 0.0716 0.2300 **** 210/20-30/20 0.2450 0.0256 0.1660 0.3240 **** 210/20-90/20 0.1270 0.0256 0.0484 0.2070 **** 30/20-90/20 -0.1180 0.0256 -0.1970 -0.0386 ****

Appendix table 24. ANOVA on consistency index (K) of emulsions prepared with MFGM-BMW after 1 day storage. Short Output: Call: aov(formula = K1 ~ Pressure, data = MFBMW, qr = T, na.action = na.exclude)

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Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 355.6925 88.92313 145.108 8.493761e-012 Residuals 15 9.1921 0.61281 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -9.12 0.554 -10.80 -7.420 **** 0/20-210/20 -10.40 0.554 -12.10 -8.660 **** 0/20-30/20 -1.01 0.554 -2.72 0.701 0/20-90/20 -3.48 0.554 -5.19 -1.770 **** 150/20-210/20 -1.25 0.554 -2.96 0.460 150/20-30/20 8.12 0.554 6.41 9.830 **** 150/20-90/20 5.64 0.554 3.93 7.350 **** 210/20-30/20 9.37 0.554 7.66 11.100 **** 210/20-90/20 6.89 0.554 5.18 8.600 **** 30/20-90/20 -2.47 0.554 -4.18 -0.765 ****

Appendix table 25. ANOVA on consistency index (K) of emulsions prepared with MFGM-BMW after 8 days storage. Short Output: Call: aov(formula = K8 ~ Pressure, data = MFBMW, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 164.1822 41.04556 177.6823 1.933342e-012 Residuals 15 3.4651 0.23101 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -6.910 0.34 -7.9600 -5.860 **** 0/20-210/20 -5.950 0.34 -7.0000 -4.900 **** 0/20-30/20 -0.333 0.34 -1.3800 0.717 0/20-90/20 -4.550 0.34 -5.6000 -3.500 **** 150/20-210/20 0.960 0.34 -0.0892 2.010 150/20-30/20 6.580 0.34 5.5300 7.630 **** 150/20-90/20 2.370 0.34 1.3200 3.420 **** 210/20-30/20 5.620 0.34 4.5700 6.670 **** 210/20-90/20 1.410 0.34 0.3590 2.460 **** 30/20-90/20 -4.210 0.34 -5.2600 -3.160 ****

Appendix table 26. ANOVA on consistency index (K) of emulsions prepared with MFGM-BMP after 1 day storage. Short Output: Call: aov(formula = K1 ~ Pressure, data = MFBMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.4532008 0.1133002 45.67111 3.163834e-008 Residuals 15 0.0372118 0.0024808 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -0.2850 0.0352 -0.3930 -0.17600 **** 0/20-210/20 -0.3270 0.0352 -0.4360 -0.21900 **** 0/20-30/20 0.0505 0.0352 -0.0583 0.15900

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0/20-90/20 -0.0988 0.0352 -0.2080 0.00999 150/20-210/20 -0.0429 0.0352 -0.1520 0.06590 150/20-30/20 0.3350 0.0352 0.2260 0.44400 **** 150/20-90/20 0.1860 0.0352 0.0770 0.29500 **** 210/20-30/20 0.3780 0.0352 0.2690 0.48700 **** 210/20-90/20 0.2290 0.0352 0.1200 0.33700 **** 30/20-90/20 -0.1490 0.0352 -0.2580 -0.04050 ****

Appendix table 27. ANOVA on consistency index (K) of emulsions prepared with MFGM-BMP after 8 days storage. Short Output: Call: aov(formula = K8 ~ Pressure, data = MFBMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.0001172830 0.00002932075 16.68584 0.00002137958 Residuals 15 0.0000263584 0.00000175722 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.006210 0.000937 0.003320 0.00910 **** 0/20-210/20 0.004520 0.000937 0.001630 0.00742 **** 0/20-30/20 0.000229 0.000937 -0.002660 0.00312 0/20-90/20 0.003360 0.000937 0.000461 0.00625 **** 150/20-210/20 -0.001690 0.000937 -0.004580 0.00121 150/20-30/20 -0.005980 0.000937 -0.008870 -0.00309 **** 150/20-90/20 -0.002850 0.000937 -0.005750 0.00004 210/20-30/20 -0.004290 0.000937 -0.007190 -0.00140 **** 210/20-90/20 -0.001170 0.000937 -0.004060 0.00173 30/20-90/20 0.003130 0.000937 0.000231 0.00602 ****

Appendix table 28. ANOVA on consistency index (K) of emulsions prepared with different dairy materials after 1 day storage. Short Output: Call: aov(formula = K1 ~ Materials, data = Pres.90.20, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 53.10636 13.27659 18.96393 9.864719e-006 Residuals 15 10.50145 0.70010 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-MFGM-BMP 1.5800 0.592 -0.243 3.410 BMP-MFGM-BMW -0.0197 0.592 -1.850 1.810 BMP-SC 3.4700 0.592 1.640 5.290 **** BMP-SMP -1.2600 0.592 -3.090 0.565 MFGM-BMP-MFGM-BMW -1.6000 0.592 -3.430 0.224 MFGM-BMP-SC 1.8800 0.592 0.056 3.710 **** MFGM-BMP-SMP -2.8500 0.592 -4.670 -1.020 **** MFGM-BMW-SC 3.4900 0.592 1.660 5.310 **** MFGM-BMW-SMP -1.2400 0.592 -3.070 0.585 SC-SMP -4.7300 0.592 -6.560 -2.900 ****

Appendix table 29. ANOVA on consistency index (K) of emulsions prepared with different dairy materials after 8 days storage. Short Output: Call: aov(formula = K8 ~ Materials, data = Pres.90.20, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F)

67

Materials 4 354.5583 88.63958 296.8134 4.418688e-014 Residuals 15 4.4796 0.29864 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-MFGM-BMP 11.5000 0.386 10.30 12.700 **** BMP-MFGM-BMW 5.8400 0.386 4.65 7.030 **** BMP-SC 11.2000 0.386 9.97 12.400 **** BMP-SMP 5.9400 0.386 4.74 7.130 **** MFGM-BMP-MFGM-BMW -5.6200 0.386 -6.82 -4.430 **** MFGM-BMP-SC -0.3080 0.386 -1.50 0.885 MFGM-BMP-SMP -5.5300 0.386 -6.72 -4.340 **** MFGM-BMW-SC 5.3200 0.386 4.12 6.510 **** MFGM-BMW-SMP 0.0949 0.386 -1.10 1.290 SC-SMP -5.2200 0.386 -6.42 -4.030 ****

Appendix table 30. ANOVA on Power law index (n) of emulsions prepared with BMP after 1 day of storage. Short Output: Call: aov(formula = n1 ~ Pressure, data = BMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.7469806 0.1867451 27.13987 1.027608e-006 Residuals 15 0.1032126 0.0068808 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.4490 0.0587 0.2680 0.63000 **** 0/20-210/20 0.4940 0.0587 0.3130 0.67600 **** 0/20-30/20 0.0973 0.0587 -0.0838 0.27800 0/20-90/20 0.3090 0.0587 0.1280 0.49000 **** 150/20-210/20 0.0452 0.0587 -0.1360 0.22600 150/20-30/20 -0.3520 0.0587 -0.5330 -0.17100 **** 150/20-90/20 -0.1410 0.0587 -0.3220 0.04060 210/20-30/20 -0.3970 0.0587 -0.5780 -0.21600 **** 210/20-90/20 -0.1860 0.0587 -0.3670 -0.00455 **** 30/20-90/20 0.2110 0.0587 0.0304 0.39300 ****

Appendix table 31. ANOVA on Power law index (n) of emulsions prepared with BMP after 8 days of storage. Short Output: Call: aov(formula = n8 ~ Pressure, data = BMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.8930863 0.2232716 31.92352 3.546373e-007 Residuals 15 0.1049093 0.0069940 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.4750 0.0591 0.2920 0.657 **** 0/20-210/20 0.4870 0.0591 0.3050 0.670 **** 0/20-30/20 0.1570 0.0591 -0.0261 0.339 0/20-90/20 0.5270 0.0591 0.3440 0.709 **** 150/20-210/20 0.0128 0.0591 -0.1700 0.195 150/20-30/20 -0.3180 0.0591 -0.5010 -0.135 **** 150/20-90/20 0.0520 0.0591 -0.1310 0.235 210/20-30/20 -0.3310 0.0591 -0.5130 -0.148 **** 210/20-90/20 0.0391 0.0591 -0.1430 0.222 30/20-90/20 0.3700 0.0591 0.1870 0.553 ****

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Appendix table 32. ANOVA on Power law index (n) of emulsions prepared with SMP after 1 day of storage. Short Output: Call: aov(formula = n1 ~ Pressure, data = SMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 1.359284 0.3398209 163.2342 3.596679e-012 Residuals 15 0.031227 0.0020818 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.546000 0.0323 0.4460 0.6450 **** 0/20-210/20 0.547000 0.0323 0.4470 0.6460 **** 0/20-30/20 -0.032600 0.0323 -0.1320 0.0671 0/20-90/20 0.435000 0.0323 0.3350 0.5340 **** 150/20-210/20 0.000969 0.0323 -0.0987 0.1010 150/20-30/20 -0.578000 0.0323 -0.6780 -0.4790 **** 150/20-90/20 -0.111000 0.0323 -0.2100 -0.0112 **** 210/20-30/20 -0.579000 0.0323 -0.6790 -0.4790 **** 210/20-90/20 -0.112000 0.0323 -0.2110 -0.0122 **** 30/20-90/20 0.467000 0.0323 0.3680 0.5670 ****

Appendix table 33. ANOVA on Power law index (n) of emulsions prepared with SMP after 8 days of storage. Short Output: Call: aov(formula = n8 ~ Pressure, data = SMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 1.362591 0.3406477 329.571 2.031708e-014 Residuals 15 0.015504 0.0010336 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.57500 0.0227 0.5050 0.6450 **** 0/20-210/20 0.55900 0.0227 0.4880 0.6290 **** 0/20-30/20 -0.00361 0.0227 -0.0738 0.0666 0/20-90/20 0.42900 0.0227 0.3590 0.4990 **** 150/20-210/20 -0.01640 0.0227 -0.0866 0.0538 150/20-30/20 -0.57900 0.0227 -0.6490 -0.5080 **** 150/20-90/20 -0.14600 0.0227 -0.2170 -0.0762 **** 210/20-30/20 -0.56200 0.0227 -0.6330 -0.4920 **** 210/20-90/20 -0.13000 0.0227 -0.2000 -0.0598 **** 30/20-90/20 0.43200 0.0227 0.3620 0.5030 ****

Appendix table 35. ANOVA on Power law index (n) of emulsions prepared with SC after 8 days of storage. Short Output: Call: aov(formula = n8 ~ Pressure, data = SC, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.1590173 0.03975433 219.0389 4.158895e-013 Residuals 15 0.0027224 0.00018149 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound

69

0/20-150/20 0.2420 0.00953 0.21300 0.2720000 **** 0/20-210/20 0.2240 0.00953 0.19500 0.2540000 **** 0/20-30/20 0.1950 0.00953 0.16500 0.2240000 **** 0/20-90/20 0.2180 0.00953 0.18800 0.2470000 **** 150/20-210/20 -0.0181 0.00953 -0.04750 0.0113000 150/20-30/20 -0.0475 0.00953 -0.07690 -0.0181000 **** 150/20-90/20 -0.0247 0.00953 -0.05410 0.0047100 210/20-30/20 -0.0294 0.00953 -0.05880 0.0000244 210/20-90/20 -0.0066 0.00953 -0.03600 0.0228000 30/20-90/20 0.0228 0.00953 -0.00662 0.0522000

Appendix table 36. ANOVA on Power law index (n) of emulsions prepared with MFGM-BMW after 1 day of storage. Short Output: Call: aov(formula = n1 ~ Pressure, data = MFBMW, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.3577031 0.08942578 197.8303 8.791856e-013 Residuals 15 0.0067805 0.00045203 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.3240 0.015 0.27800 0.3700 **** 0/20-210/20 0.3670 0.015 0.32100 0.4140 **** 0/20-30/20 0.1250 0.015 0.07890 0.1720 **** 0/20-90/20 0.2290 0.015 0.18300 0.2750 **** 150/20-210/20 0.0435 0.015 -0.00292 0.0899 150/20-30/20 -0.1990 0.015 -0.24500 -0.1520 **** 150/20-90/20 -0.0949 0.015 -0.14100 -0.0485 **** 210/20-30/20 -0.2420 0.015 -0.28900 -0.1960 **** 210/20-90/20 -0.1380 0.015 -0.18500 -0.0920 **** 30/20-90/20 0.1040 0.015 0.05730 0.1500 ****

Appendix table 37. ANOVA on Power law index (n) of emulsions prepared with MFGM-BMW after 8 days of storage. Short Output: Call: aov(formula = n8 ~ Pressure, data = MFBMW, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.1001287 0.02503218 12.05774 0.0001383987 Residuals 15 0.0311404 0.00207603 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 0.07910 0.0322 -0.02040 0.17900 0/20-210/20 0.09770 0.0322 -0.00177 0.19700 0/20-30/20 -0.09410 0.0322 -0.19400 0.00539 0/20-90/20 0.07170 0.0322 -0.02780 0.17100 150/20-210/20 0.01860 0.0322 -0.08090 0.11800 150/20-30/20 -0.17300 0.0322 -0.27300 -0.07370 **** 150/20-90/20 -0.00737 0.0322 -0.10700 0.09210 210/20-30/20 -0.19200 0.0322 -0.29100 -0.09230 **** 210/20-90/20 -0.02600 0.0322 -0.12500 0.07350 30/20-90/20 0.16600 0.0322 0.06630 0.26500 ****

Appendix table 38. ANOVA on Power law index (n) of emulsions prepared with MFGM-

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BMP after 1 day of storage. Short Output: Call: aov(formula = n1 ~ Pressure, data = MFBMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.09036893 0.02259223 45.90161 3.056343e-008 Residuals 15 0.00738282 0.00049219 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -0.1270 0.0157 -0.1760 -0.07890 **** 0/20-210/20 -0.1450 0.0157 -0.1940 -0.09660 **** 0/20-30/20 0.0239 0.0157 -0.0246 0.07230 0/20-90/20 -0.0464 0.0157 -0.0948 0.00206 150/20-210/20 -0.0177 0.0157 -0.0662 0.03070 150/20-30/20 0.1510 0.0157 0.1030 0.20000 **** 150/20-90/20 0.0810 0.0157 0.0325 0.12900 **** 210/20-30/20 0.1690 0.0157 0.1210 0.21700 **** 210/20-90/20 0.0987 0.0157 0.0503 0.14700 **** 30/20-90/20 -0.0702 0.0157 -0.1190 -0.02180 ****

Appendix table 39. ANOVA on Power law index (n) of emulsions prepared with MFGM-BMP after 8 days of storage. Short Output: Call: aov(formula = n8 ~ Pressure, data = MFBMP, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Pressure 4 0.05787187 0.01446797 45.16975 3.412766e-008 Residuals 15 0.00480453 0.00032030 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound 0/20-150/20 -0.114000 0.0127 -0.1540 -0.07540 **** 0/20-210/20 -0.125000 0.0127 -0.1640 -0.08610 **** 0/20-30/20 -0.000459 0.0127 -0.0395 0.03860 0/20-90/20 -0.048600 0.0127 -0.0877 -0.00956 **** 150/20-210/20 -0.010800 0.0127 -0.0498 0.02830 150/20-30/20 0.114000 0.0127 0.0749 0.15300 **** 150/20-90/20 0.065800 0.0127 0.0267 0.10500 **** 210/20-30/20 0.125000 0.0127 0.0857 0.16400 **** 210/20-90/20 0.076600 0.0127 0.0375 0.11600 **** 30/20-90/20 -0.048200 0.0127 -0.0873 -0.00910 ****

Appendix table 40. ANOVA on Power law index (n) of emulsions prepared with different dairy materials after 1 day of storage. Short Output: Call: aov(formula = n1 ~ Materials, data = Pres.90.20, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 0.4227895 0.1056974 31.10222 4.21311e-007 Residuals 15 0.0509758 0.0033984 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-MFGM-BMP -0.2510 0.0412 -0.3780 -0.12300 **** BMP-MFGM-BMW 0.0975 0.0412 -0.0298 0.22500

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BMP-SC 0.1790 0.0412 0.0518 0.30600 **** BMP-SMP 0.0493 0.0412 -0.0780 0.17700 MFGM-BMP-MFGM-BMW 0.3480 0.0412 0.2210 0.47500 **** MFGM-BMP-SC 0.4300 0.0412 0.3020 0.55700 **** MFGM-BMP-SMP 0.3000 0.0412 0.1730 0.42700 **** MFGM-BMW-SC 0.0817 0.0412 -0.0456 0.20900 MFGM-BMW-SMP -0.0482 0.0412 -0.1750 0.07910 SC-SMP -0.1300 0.0412 -0.2570 -0.00253 ****

Appendix table 41. ANOVA on Power law index (n) of emulsions prepared with different dairy materials after 8 days of storage. Short Output: Call: aov(formula = n8 ~ Materials, data = Pres.90.20, qr = T, na.action = na.exclude) Df Sum of Sq Mean Sq F Value Pr(F) Materials 4 0.6517178 0.1629294 176.2103 2.054801e-012 Residuals 15 0.0138695 0.0009246 95 % simultaneous confidence intervals for specified linear combinations, by the Tukey method Estimate Std.Error Lower Bound Upper Bound BMP-MFGM-BMP -0.43700 0.0215 -0.5030 -0.3700 **** BMP-MFGM-BMW 0.04030 0.0215 -0.0261 0.1070 BMP-SC 0.04300 0.0215 -0.0234 0.1090 BMP-SMP -0.08900 0.0215 -0.1550 -0.0226 **** MFGM-BMP-MFGM-BMW 0.47700 0.0215 0.4110 0.5430 **** MFGM-BMP-SC 0.48000 0.0215 0.4130 0.5460 **** MFGM-BMP-SMP 0.34800 0.0215 0.2810 0.4140 **** MFGM-BMW-SC 0.00274 0.0215 -0.0637 0.0691 MFGM-BMW-SMP -0.12900 0.0215 -0.1960 -0.0628 **** SC-SMP -0.13200 0.0215 -0.1980 -0.0656 ****

Katholieke Universiteit Leuvenlib.ugent.be/fulltxt/RUG01/001/789/759/RUG01-001789759... · 2012. 3....

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