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Katholieke Universiteit Leuven FACULTY OF BIOSCIENCE ENGINEERING INTERUNIVERSITY PROGRAMME (IUPFOOD) MASTER OF SCIENCE IN
Katholieke Universiteit Leuven FACULTY OF BIOSCIENCE ENGINEERING INTERUNIVERSITY PROGRAMME (IUPFOOD) MASTER OF SCIENCE IN

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

Formulation and characterization aspects of low fat whipping cream by Water/Oil/Water technology

by Lien Vermeir

Promotor : Prof. dr. ir. Paul Van der Meeren

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

The author and promoters 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 thesis.

Gent, June 2011

The promotor

the author

Prof. dr. ir. Paul Van der Meeren

Lien Vermeir

Acknowledgements

I would like to thank my promotor, supervisor and mentor, Prof. dr. ir. Paul Van der Meeren, whose guidance, insights and stimulating suggestions helped me in all the time of research. Deep gratitude is expressed to the members of the department of Applied Physical Chemistry:

Eric, Marios, Maryam, Paolo, Quenten, Saskia and Zhu. Lastly, I offer my regards to all of those who supported me in any respect during the completion of my thesis: members of the lab of FTE, the staff of IUPFOOD in Gent and Leuven and last but not least, my family.

Table of contents

Table of contents

Abstract

1

Chapter 1: Literature review

2

1.1 Water-in-oil-in-water emulsions

2

1.1.1 Water-in-oil-in-water technology

2

1.1.2 Preparation of a w/o/w-emulsion

3

1.1.3 Encapsulation efficiency

4

1.1.4 Instability of w/o/w-emulsions

6

1.1.4.1 Thermodynamic instability of a w/o/w-emulsion

6

1.1.4.2 Water transport between the w 1 and w 2 -phase

9

1.1.4.3 Gravitational instability of a w/o/w-emulsion

11

1.1.5 Desired characteristics of a whippable w/o/w-cream

12

1.2 Traditional dairy whipping cream

12

1.3 Changes during whipping of cream

13

1.3.1 Three stages during whipping of cream

13

1.3.2 Foam stabilization by partial coalescence

15

1.3.2.1 Determination of partial coalescence

17

1.3.2.2 Distinction between partial coalescence and complete coalescence

17

1.3.3 Alternatives to partial coalescence

18

1.4 Mimicing whipping cream

19

1.5 Quality characteristics of whipped cream

21

1.5.1 Overrun

22

1.5.2 Whipping time

23

1.5.3 Textural analysis

24

1.5.4 Physical stability

24

1.5.4.1 Coarsening of foam

24

1.5.4.2 Drainage

25

1.6 Factors determining functional properties of whipping cream

26

1.6.1 Temperature

26

1.6.1.1 Tempering

27

1.6.1.2 Heat treatment

28

1.6.2 Fat content

28

1.6.3 Homogenization

28

Table of contents

Chapter 2: Materials and methods

31

2.1 Commercial butters

31

2.1.1 Preparation of butter samples

31

2.1.2 Analysis of butter samples

32

2.2 Water-in-oil emulsions

32

2.2.2 Materials needed for the preparation of w/o-emulsions

32

2.2.3 Composition of the w/o-emulsions

33

2.2.4 Preparation of the w/o-emulsions

34

2.3 Quantitative particle size analysis of water in w/o-emulsions

36

2.3.1 Pulsed field gradient-Nuclear Magnetic Resonance

36

2.3.2 The pfg-NMR experiment

37

2.3.2.1

Calibration procedure

39

2.3.3 Restricted diffusion in w/o emulsions

39

2.3.3.1 Data procesing by the Droplet Size application

41

2.3.3.2 Data processing by Excel

42

2.3.3.3 Data processing by Matlab

46

2.3.4 Statistical methods

48

2.3.5 Important issues during pfg-NMR analysis

49

2.3.5.1 Temperature of the emulsion during pgf-NMR analysis

49

2.3.5.2 Assumption of a lognormal size distribution

50

2.3.5.3 Advantages of pfg-NMR analysis for determination of water droplet size

50

2.4 Qualitative particle size analysis of water in w/o-emulsions

51

2.4.1 Fluorescence microscopy

51

2.4.2 Fluorescence

52

2.4.3 EosinY

53

2.4.4 Fluorimetric analysis of w/o emulsions

54

2.4.4.1 Fluorimeter

54

2.4.4.2 Determination of a suitable concentration of eosinY

54

2.4.4.3 Determination of maximum excitation and emission wavelength

55

2.4.5 Confocal laser scanning microscopy

58

2.5 Water-in-oil-in-water emulsions

59

2.5.1 Composition of the w/o/w-emulsions

59

2.5.2 Preparation of the w/o/w-emulsions

59

2.5.2.1

Method A

59

Table of contents

2.5.2.3 Method C

61

2.5.2.4 Method D

64

2.6 Quantitative analysis of the enclosed water volume and yield of water-in-oil-in-water

64

emulsions

2.6.1 The CPMG-experiment

64

2.6.2 Determination of the enclosed water volume

70

2.6.3 Determination of the yield of a double emulsion

71

2.6.4 Statistical methods

72

2.7 Fat globule analysis by laser diffraction

72

2.8 Profilometric analysis of double emulsions

72

2.9 Whipping of a commercial dairy cream and w/o/w-emulsions

73

2.9.1 Commercial dairy cream

73

2.9.2 W/o/w emulsions

73

2.9.2.1 First attempt to prepare whippable double emulsions

73

2.9.2.2 Second attempt to prepare whippable double emulsions

73

2.9.2.3 Third attempt to prepare whippable double emulsions

74

2.9.2.4 Fourth attempt to prepare whippable double emulsions

74

2.9.3 Determination of the whipping time

74

2.9.4 Determination of the overrun of a whipped emulsion

74

2.9.5 Physical destabilization (drainage) of whipped emulsions

75

Chapter 3: Results and discussion

76

3.1 Optimization of wayer droplet size analysis

76

3.1.1 Temperature experiment by using a thermocouple

76

3.1.2 Repeatability experiment

78

3.2 Quantitative particle size analysis of water droplets in commercial butters

80

3.3 Quantitative particle size analysis of water droplets in w/o-emulsions

85

3.3.1 Analysis of emulsions with composition H0/1, H0.5/1, M0/1 and P0/1

85

3.3.2 Analysis of emulsions with composition H0/1. H0.5/1, M0/1, P0/1, P0.5/1 and

88

P0.5/2

3.3.2.1

Statistical analysis of emulsions H0/1, H0.5/1, P0/1, M0/1, P0.5/1 and

89

P0.5/2

3.3.2.2

Difference between different data processing methods

91

3.3.2.3

Influence of the type of fat on the mean water droplet size

91

Table of contents

3.3.3 Elevation of the water fraction of emulsions based on Hozol

93

3.3.4 Effect of the decrease of the homogenization pressure of the Microfluidizer M110S on the water droplet size in a w/o emulsion

96

3.3.5 Analysis of emulsions with the hydrophilic surfactant whey protein isolate

98

3.4 Qualitative particle size analysis of water droplets in w/o-emulsions by fluorescence microscopy

99

3.4.1 Preliminary investigation of eosinY-solutions by fluorimetry

99

3.4.2 Determination of the maximum excitation and emission wavelength of eosinY (5µg/mL) in water

99

3.4.3 Determination of the maximum excitation and emission wavelength of eosinY (5µg/mL) in an aqueous phosphate buffer (pH6.7)

101

3.4.4 Determination of the maximum excitation and emission wavelength of eosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v)

102

3.4.5 Determination of the maximum excitation and emission wavelength of eosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v) and sodium caseinate (1.25%,w/v)

103

3.4.6 Selection of the filter block in the fluorescence microscope

104

3.4.7 Fluorescence microscopic images of water droplets in a w/o-emulsion

105

3.4.8 Imaging of water droplets in water in oil emulsions by confocal laser scanning microscopy

106

3.5 Determination of the enclosed water volume and yield of w/o/w-emulsions

107

3.5.1 Method optimization

107

3.5.1.1 Optimization of the preparation method

107

3.5.1.2 Finding an appropriate concentration of MnCl 2

110

3.5.1.3 Investigation of the permeability of the oil phase by a temperature experiment

110

 

3.5.1.3.1 Analysis of the area under the curve at different temperatures

113

3.5.1.3.2 Analysis of the signal amplitude at different temperatures

116

3.5.1.3.3 Analysis of the relaxation time at different temperatures

117

3.5.1.4 Variation of the duration of mixing of the double emulsions

121

3.5.1.5 Effect of the reduction of the duration of mixing with an Ultraturrax S25-

122

 

10G

3.5.1.6 Effect of quick cooling on the percentage of enclosed water volume

123

3.5.2 Method application

124

3.5.2.1

Variation of the composition of the fat phase

124

Table of contents

yield of double emulsions made by Method B

 

3.5.2.1.2 Effect of variation of the fat phase on the T 2 -distribution of double emulsion made by Method B

125

3.5.2.1.3 Effect of the fat composition on the enclosed water volume and yield of double emulsions made by Method C

126

3.5.2.1.4 Effect of the variation of the fat phase on the T 2 -distribution of double emulsion made by Method C

128

3.5.2.1.5 Effect of the fat composition on the enclosed water volume and

129

yield of double emulsions made by Method D

 

3.5.2.2

Effect of the concentration of the hydrophilic emulsifier in the external

130

 

water phase on the enclosed water volume

3.6 Fat globule analysis by a Malvern Mastersizer S

130

3.7 Visualization of double emulsions by light microscopy

131

3.8 Research on the thickness of the separated cream layer of double emulsions

131

3.8.1 Determination of the thickness of the separated creamy layer with a ruler

131

3.8.2 Profilometric analysis of double emulsions

132

3.9 Whipping of a commercial dairy cream

136

3.9.1 Whipping time of a whipped commercial dairy cream

136

3.9.2 Overrun of a whipped commercial dairy cream

136

3.9.3 Physical destabilization (drainage) of a whipped commercial dairy cream

136

3.10

Whipping of w/o/w-emulsions

136

3.10.1 First attempt

 

136

3.10.2 Second attempt

137

3.10.3 Third attempt

138

3.10.4 Fourth attempt

138

General conclusions

139

List of references

141

Abstract

Abstract

A proper foundation is established with the prospect of manufacturing a food grade

recombined whipping cream by water/oil/water technology. The w/o/w technology can offer the opportunity to produce a cream in which the oil droplets are filled with water droplets and hence a low-energy-dense cream can be created.

In this thesis three main parts can be distinguished. In the first section, as a water-in-oil-

in-water emulsion requires an appropriate water-in-oil emulsion, research was performed on w/o-emulsions. The composition of an emulsion is determined by the type of fat, the emulsifier and the water content. The water droplet size was affected by the type of fat, the emulsifier concentration and the driving air pressure during preparation. Visualization of water droplets might be possible by light microscopy and the use of the fluorescent dye eosinY. In the second section, the manufacturing of w/o/w-emulsions is optimized by various ways of manufacture and characterized by T 2 -analysis, laser diffraction and profilometry. A yield of about 42 to 62% could be obtained if the double emulsion was made by intensive and less intensive homogenization during the first step of the preparation of double emulsions,

respectively. Moreover the homogenization intensity during the second step needed to be sufficiently small. The duration of mixing in the second step and the concentration of sodium caseinate in the external water phase affected the yield. In a third section, whipping of w/o/w- creams was attempted. Although by the use of xanthan gum in the external water phase a gravitational stable cream was obtained, the desired textural change during whipping was not observed.

In the whole process, due attention is paid to proper characterization techniques, with

special focus on low-resolution NMR as a non-invasive and non-destructive method. This technique not only enabled water droplet analysis in the primary w/o-emulsion (based on diffusometry), but also allowed the discrimination between internal and external water in the multiple emulsion (based on T 2 -analysis) and determination of the thickness of the separated cream layer and its water content in double emulsions (based on profilometry).

Overall, it was shown that multiple emulsions with about 30wt% of dispersed phase could be obtained with only 20wt% of fat using food-grade emulsifiers (i.e. PGPR and sodium caseinate). In order to ensure good whipping properties, further research will be needed. Hereby, the composition of the interfacial layers, as well as fat crystallization will be important issues.

Chapter 1 Literature review

Chapter 1

Literature review

1.1 Water-in-oil-in-water emulsions

1.1.1 Water-in-oil-in-water technology

A w/o/w emulsion, which is a double emulsion, consists of two non-miscible liquids, water and oil. Small water droplets are dispersed in larger oil globules, which are themselves dispersed in an aqueous continuous phase. Three potential benefits are the possibility of lowering the fat content, entrapping (and releasing) therapeutic, nutritional or odourous compounds in the internal water droplets and separation of incompatible substances (Márquez and Wagner, 2010). In this thesis, emphasis lies on the potential of w/o/w-emulsions to reduce the fat content. The accelerated increase in the incidence of cardiovascular diseases at worldwide level has led consumers demand healthier low-fat food that reduces or helps to maintain triglyceride blood levels (Lobota-Calleros et al., 2009). Most double emulsions are very polydispersed. The range of the size of double emulsion droplets can be quite extensive: the size of multiple droplets can be between 2-5 µm and 15- 50µm, containing respectively a few and 50-100 water droplets. In this regard, three classes can be distinguished: type A, B and C (Figure 1.1), which differ from each other in the amount of water droplets entrapped in the multiple droplet (Garti and Bisperink, 1998).

in the multiple droplet (Garti and Bisperink, 1998). Figure 1.1 Three different types of entrapment of

Figure 1.1 Three different types of entrapment of water droplets in the multiple droplet (Garti and Bisperink, 1998).

Chapter 1 Literature review

1.1.2 Preparation of a w/o/w-emulsion

Multiple emulsions can be produced in a one-step or a two-step process. With regard to food applications, a two-step process (Figure 1.2) is more common and consists of a first emulsification step at higher shear forces than the second step, resulting in a w 1 /o-emulsion and w 1 /o/w 2 -emulsion, respectively. The second emulsification step is a critical step. A too high homogenization pressure results in swelling and rupture of the internal water droplets (w 1 ) and coalescence of the w 1 -droplets with the outer water phase (w 2 ) (Hindmarsch et al., 2005). A lower homogenization pressure might result in more coalescence in virtue of too large multiple emulsion droplets. Besides the homogenization pressure, the amount of homogenization cycles and the temperature are important. High temperatures and a high amount of homogenization cycles, assuming sufficient emulsifier, result in smaller multiple droplets, which might compromise the encapsulation of water (Lindenstruth and Müller, 2004).

the encapsulation of water (Lindenstruth and Müller, 2004). Figure 1.2: Scheme of the preparation of a

Figure 1.2: Scheme of the preparation of a w/o/w- emulsion (Remon & Vervaet, 2003)

Mostly, the mixing of the w 1 /o-emulsion and the w 2 -phase is done at a lower temperature than the preparation of the w 1 /o-emulsion. Water-in-oil emulsions are made at 50 to 70°C, whereas w/o/w-emulsions are prepared at room temperature (Min et al., 2010; Lutz et al., 2009) or in an ice bath (Frasch-Melnik et al., 2010b). O’Regan and Mulvihill (2010) prepared the w/o/w- emulsions at 60°C.

Chapter 1 Literature review

Emulsification can be performed by homogenization, membrane emulsification and micro- channel emulsification.

Evaluation of the type of double emulsion can be done by dilution with water. Water will not be miscible with an o/w/o-emulsion, whereas the reverse is true for an w/o/w-emulsion. Alternatively, a water-soluble compound, e.g. methylene blue, can be added to the double emulsion, followed by visual evaluation of the miscibility and/or microscopical investigation. Methylene blue in an w/o/w-emulsion makes the sample turn blue, whereas in an o/w/o- emulsion, this won’t have a profound effect on the color (Tirnaksiz and Kalsin, 2005). An overview of different compositions of double emulsions in literature is given in Table 1.1.

1.1.3 Encapsulation efficiency

The encapsulation efficiency is determined by the conditions during emulsification (e.g. homogenization pressure and temperature) and the factors affecting the release of internal water. Regarding the latter, shear action or the presence of a concentration gradient influences the encapsulation of the w 1 -phase. Depending on the direction of the osmotic gradient swelling or shrinkage of the internal water droplets can occur (Lutz et al., 2009). The yield of encapsulation after emulsification can be determined by the entrapment of a marker compound in the w 1 -phase and its detection in the w 2 -phase. An overview of markers and their detection is given in Table 1.2. For example, the entrapment or yield percentage can be determined by conductometry and is determined by the formula:

Yield percentage= (Ci-Ct)100% / Ci where Ci and Ct are the conductivity of the internal aqueous phase and of the multiple emulsion at a given time t (Tirnaksiz and Kalsin, 2005). The release of marker compounds from the inner to the outer water phase depends on its diffusion coefficient, its initial concentration, the sphere radii (surface area of oil globule), the viscosity of the oil phase and the solubility of the marker in the oil phase (Sela et al., 1995).

Chapter 1 Literature review

Table 1.1: Overview of compositions of double emulsions in literature.

   

Lipophilic surfactant in the oil-phase

Concentration of lipophilic surfactant in the oil phase

 

Concentration

Compounds in the w 1 -phase

Concentration of compounds in the

Ratio of the

Reference

Oil phase

Compounds in the w 2 -phase

of the

 

hydrophilic

 

w

1 -phase

1 /o/w 2 - emulsion

w

surfactant in

 
 

w

2 -phase

 

Muschiolik et al., 2006

Sunflower oil

PGPR

4%(w/v)

WPI

1%(w/v)

NaCl or gelatin

0.6 or 3%(w/v)

3/12/85

Su et al., 2006

Soybean oil

PGPR

2%(w/v)

Na caseinate

0.5%(w/v)

Na caseinate

0.5%(w/v)

4/16/80

O'Regan and Mulvihill,

Median chain triglyceride

   

Na caseinate/ Na caseinate-maltodextrin

       

2010

(MCT)

PGPR

2wt%

1wt% protein

gelatin and NaCl

5wt% and 0.6wt%

8/32/60

 

Median chain triglyceride (MCT)

     

4% and

     

Lutz et al., 2009

PGPR

10wt%

WPI and pectin

0.5wt%

-

-

12/18/70

Frasch-Melnik et al., 2010

Sunflower oil

Saturated MG, tripalmitate and

0.44wt%+0.88wt%

Na caseinate

1wt%

-

-

3/17/80

PGPR

0.35wt%

Benichou et al., 2007

Median chain triglyceride (MCT)

GMO and PGPR

2.2wt% and 8.9wt%

WPI

6.7wt%

Glycerol

3wt%

4/16/80

WPI and xanthan (4/0.5)

6.7wt%

Glycerol

3wt%

Mun et al., 2010

Soybean oil

PGPR

4; 6; 8wt%

WPI

2/4/6wt%

-

-

8/32/60

Márquez and Wagner, 2010

Sunflower oil

PGPR

0.5 to 2wt%

xanthan gum in soy milk

0.2wt%

CaCl 2

0.38-1.5wt%

8/32/60

CaCO 3

1.5wt%

Ca lactate

1.5wt%

5

Chapter 1 Literature review

Table 1.2: Overview of different markers and techniques of detection

Reference

Marker

Measurement

Sela et al., 1995

2wt% NaCl/NaF/NaBr/NaI in w 1 -phase 1wt% ephedrine HCl in w 1 -phase

Conductometry of serum phase

2wt% KNO 3 in w 1 -phase

Lutz et al., 2009

4.4wt% NaCl in w 1 -phase 15wt% Na ascorbate in w 1 -phase

Conductometry of the serum phase

Lindenstruth and Müller, 2004

0.5%(w/v) Na diclofenac in w 1 -phase

HPLC after ultrafiltration

Frasch-Melnik et al., 2010b

1.6wt% KCl in w 1 -phase

Conductometry of the serum phase

Benichou et al., 2007

2wt% glucose in w 1 -phase 0.33-3g/100g emulsion vitamine B1

Glucometry Differential pulse polarography

Tirnaksiz and Kalsin, 2005

1.5wt% caffeine + 0.03wt% NaCl 0.3wt%NaCl in w 1 -phase

Spectrophotometry (271nm) after dialysis Conductometry of the serum phase

Wolf et al., 2009

0.02wt% vitamin B12 in w 1 -phase

Spectrophotometry (361nm) after centrifugation and filtration of the serum phase

1.1.4 Instability of w/o/w-emulsions

1.1.4.1 Thermodynamic instability of a w/o/w-emulsion

Applicability of multiple emulsions is limited by their thermodynamic instability (Ursica et al., 2005), which is caused by the large surface free energy between fat and water. Hence, emulsions will strive to lower the interfacial free energy by minimization of the contact area between the phases until water is completely separated from the fat (Jiao and Burgess, 2008; Goff, 1997). Destabilization of a double emulsion can occur by coalescence of internal water droplets or multiple droplets and water diffusion between the two water phases, as represented in Figure

1.3.

Muschiolik et al. (2006) defined long–term stability of double emulsions as a period of at least 12 months. A high yield of the w 1 -phase, no phase alteration and no phase separation during 8 month at +7°C are the characteristics of a high storage stability emulsion.

Chapter 1 Literature review

Chapter 1 Literature review Figure 1.3: Overview of the possible destabilization mechanisms in w/o/w emulsions (Mezzenga

Figure 1.3: Overview of the possible destabilization mechanisms in w/o/w emulsions (Mezzenga et al., 2004)

Coalescence involves rupture of the film between two adjacent water or oil droplets: a hole is formed that grows under the action of surface tension and results in the fusion of two adjacent droplets. Rising the temperature might activate coalescence and decrease the entrapment of water in the multiple droplets. Larger inner water droplets promote coalescence between the interfaces of the inner water droplets and the multiple droplets (Bibette et al., 1999). The kinetics of coalescence of inner water droplets with the multiple droplet interface is related to the concentration of the hydrophilic surfactant in the w 2 -phase phase, whereas the kinetics of the coalescence between inner water droplets is related to the type of hydrophilic emulsifier in the w 1 - phase (Garti and Bisperink, 1998).

Coalescence can be measured by measuring the turbidity. Since the larger droplets scatter light less effectively than smaller ones, the emulsion may appear less turbid. It can also be measured by particle size analysis. A more time consuming method is the following method: an oil droplet can be released from a capillary tube, placed on the bottom of an aqueous phase with at the top a planar oil-water interface. The droplet moves upwards, reaches the oil-water planar interface and the time to merge with the planar oil-water interface is determined with an optical microscope (McClements,

2007).

Water diffusion or Ostwald ripening from the outer to the inner water phase can result in a bigger average inner water droplet diameter (coarsening) and a reduction in number and is due to diffusion of water molecules across the oil layer in both directions, which doesn’t disrupt the interfacial film (Bibette et al., 1999).

Chapter 1 Literature review

The composition of a double emulsion can affect its stability. Garti and Aserin (1996) reported that stable double emulsions are obtained by the use of an inner hydrophobic emulsifier in great excess (10-30wt% of the w/o-emulsion) and an external hydrophilic emulsifier in lower concentrations (0.5-5wt%). Increasing the latter concentration above a certain threshold concentration resulted in a lower encapsulation efficiency, due to an excess of osmotic pressure in the outer water phase and the formation of reversed micelles that pumps the hydrophobic surfactant outside into the w 2 -phase (Shima et al., 2004; Bibette et al., 1999). Consequently, the w/o/w-emulsion turns into a o/w-emulsion. However, absence of hydrophilic surfactants in the w 2 -phase, might not be able to prevent flocculation and coalescence (Shima et al., 2004).

Other approaches to influence the stability of a double emulsion are the increase of the viscosity of the inner water or oil phase, strengthening and rigidifying the interfaces with polymeric emulsifiers and reduction of the inner water droplet size (Garti and Aserin, 1996; Leal-Calderon et al., 2007). The viscoelasticity of the film can be the result of the interaction between surface active lipids and folded and unfolded proteins at the (w/o)/w-interface (Rousseau, 2000). Addition of sodium caseinate to the w 1 -phase improves the stability of PGPR-based double emulsions, which might be due to its effect on the water-oil interface (Hindmarsch et al., 2005). The combination of a polymeric surfactant (BSA) and monomeric lipophilic surfactant (Span 80) formed a thick interfacial layer w 1 /o, which improves its elasticity and resistance to rupture (Garti, 1997).

Stability testing of a multiple emulsion can be performed by video microscopy, whereby multiple emulsions are covered on micro slides, which makes the inner water droplets to coalesce inside the oil droplet. This results in a dimpled structure (Figure 1.4), because inner water droplets are pushed to the edge of the multiple droplets, whereby the inner water droplets are still separated from the oil phase by a thin film as long as the interfacial film is strong enough. Hence, a double emulsion is more stable if there is a larger resistance to coverslip pressure (Jiao et al., 2002).

Chapter 1 Literature review

Chapter 1 Literature review Figure 1.4: Schematic representation of typical changes occurring to a multiple droplet

Figure 1.4: Schematic representation of typical changes occurring to a multiple droplet on application of a cover slip pressure (Jiao et al., 2002).

1.1.4.2 Water transport between the w 1 and w 2 -phase

Benichou et al. (2004) reported three mechanisms of water transport in double emulsions: transport through thin lamellae of surfactants, transport in reverse micelles and transport via hydrated surfactants (Figure 1.5). Reverse micelles are formed in the oil phase of a w/o/w-emulsion in the presence of monomeric hydrophobic and hydrophilic surfactants, such as Span and Tween, respectively, whereas for polymeric surfactants (e.g. BSA) this is unknown in literature as they serve as a mechanical barrier for the transport of solutes (Sela et al., 1995; Benichou et al., 2007). Hence, the release can be slowed down by the complex formation of biopolymers and hydrophobic surfactants at the inner w/o-interface, which reduces the rate of solubilization by reverse micelles (Bibette et al., 1999). Reverse micelle formation can occur without the presence of an osmotic gradient and doesn’t disrupt the emulsion, whereas transport through thin layers of oil destroys the interfacial films and releases entire inner water droplets (Garti, 1997; Lutz et al., 2009). Especially when there is an osmotic pressure difference between the two aqueous phases, thin layer transport of water happens (Florence and Whitehill, 1982). The stability of the oil film can be improved by increasing the concentration of the hydrophobic surfactant (Bibette et al., 1999). Transport via hydrated surfactants occurs by slow emulsification of water droplets in the oil phase (Wen and Papadopoulos, 2000).

Chapter 1 Literature review

Chapter 1 Literature review Figure 1.5: Schematic representation of three transport mechanisms of water in double

Figure 1.5: Schematic representation of three transport mechanisms of water in double emulsions. (a) transport via reverse micelles. (b) transport through thin lamellae. (c) water transport via hydrated surfactants (Benichou et al., 2004).

Transport rates can be reduced by increasing the viscosity of the oil phase, by polymerization of the interfacial adsorbed surfactant molecules, by gelation of the oil or water phases and by the presence of the right concentration of surfactants (Schmidts et al., 2009). For example, the water permeation through the oil layer has been reported to decrease if the oil-soluble surfactant Span 80 was present in high concentrations (10 to 50wt% of the oil phase) in virtue of the high viscosity in the oil phase (Wen and Papadopoulos, 2000). Complex formation between proteins and polysaccharides in the w 2 -phase decreases the release rate (Muschiolik, 2007). Addition of gelled gelatin to the w 1 -phase slowed down the movement of the w 1 - phase to the w 2 -phase (Muschiolik et al., 2006). In the study of Muschiolik et al. (2006) about multiple emulsions, an increase in PGPR concentration had a reducing effect on the release of components from the w 1 -phase. Limited release was obtained with 4%(w/v) PGPR. This concentration could be reduced to 2%(w/v) by addition of 0.5%(w/v) sodium caseinate in the w 1 -phase (Muschiolik, 2007).

Chapter 1 Literature review

1.1.4.3 Gravitational instability of a w/o/w-emulsion

Regarding gravitational instability, according to the law of Stokes, creaming depends on the density difference between the multiple droplets and the external water, the multiple droplet size and the viscosity of the w 2 -phase. The density of the oil phase can be changed by altering the type of oil or by adding fat crystals (McClements, 2007). However, sub-micron fat crystals can stabilize a w/o-emulsion, but regarding an o/w or a w/o/w-emulsion, the control of the concentration of these fat crystals is crucial. A scraped surface heat exchanger can be applied to selectively bring the crystals at the w 1 /o-interface and not at the o/w 2 -interface. If they would be located at the latter position, aggregation and coalescence might occur. In order to render fat crystals amphiphilic or adsorbable at the interface, surfactants (monoglycerides, lecithin) are needed. On addition of PGPR, the crystals are displaced and the interface consists of mainly PGPR and some crystals, which increases the permeability of the interface and allows rapid swelling when an osmotic gradient is applied. Hence, the presence of crystals become superfluous (Frasch-Melnik et al., 2010b). Monoglycerides and diglycerides will form the membrane around the fat globule and can increase the density depending on the adsorbed amount, which can decrease the rate of creaming (Goff, 1997). The viscosity of the w 2 -phase can be increased by the use of polymeric compounds (Lutz and Aserin; 2008). Creaming can be monitored by measurement of the separation of the serum and cream layer (Figure 1.6). At time zero, the multiple droplets are homogeneously distributed in the sample and characterized by a concentration C o . After a while, the emulsion separates into three distinguishable layers: a serum layer with a lower droplet concentration than C o , an emulsion layer with a multiple droplet concentration equal to C o and a cream layer with a larger multiple droplet concentration than C o . Besides visual observation of the boundary of the layers, also light scattering measurement can be applied, although in a concentrated emulsion (>10% droplets) this does not change greatly with increasing droplet concentration. Alternatively, creaming can be studied by the physical sectioning method, which is a destructive method that requires freezing of the emulsions. As such the droplet concentration in each frozen section is measured. The sample can also be divided into pieces by collecting successive aliquots of the emulsion stored in a burette or separation funnel. When the creaming is

Chapter 1 Literature review

monitored as a function of time, e.g. by video imaging or by making photographs over time, the creaming rate can be determined (McClements, 2007).

the creaming rate can be determined (McClements, 2007). Figure 1.6: Schematic representation of creaming as a

Figure 1.6: Schematic representation of creaming as a function of time in a (w/o)/w emulsion (McClements, 2007).

1.1.5 Desired characteristics of a whippable w/o/w-cream

A whipping cream should be quiescently or perikinetically stable prior to whipping

and unstable when sheared during whipping. The resistance against shear induced destabilization is defined as orthokinetic stability (Goff, 1997; Davies et al., 2001). In view of sensory resemblance to whippable dairy cream, the size of multiple droplets of a w/o/w-emulsion should be similar to the oil globules of the dairy cream (Frash-Melnik et al., 2010).

1.2 Traditional dairy whipping cream

Dairy cream, an oil-in-water emulsion can be defined as the part of milk that is rich in fat, separated from raw milk by centrifugation at speeds of 4700-6500 rpm, resulting

in cream and skimmed milk (Hoffmann, 2002). Besides a heat treatment, the cream

can be homogenized at a low pressure, which prevents it from creaming and thus

Chapter 1 Literature review

destabilization. Non-homogenized fat globules are surrounded by a milk fat globule membrane (MFGM), which prevents them from coalescing (Mulder and Walstra, 1974). The MFGM consists of approximately 25 to 60% of proteins (e.g. butyrophillin and xanthin oxidase), lipids (e.g. phospholipids) and other compounds, which results in a thickness between 10 and 50nm (Hui et al., 2007). A homogenization process disintegrates the MFGM and the fat globules will be surrounded by mainly caseins. In the absence of stabilizing additives, the process of whipping of cream is usually performed in situ by the consumer with a domestic electric mixer. After whipping, the cream already collapses after 24 to 48h (Allen et al., 2008; Leal-Calderon et al.,

2007).

With regard to the legal aspect of defining cream as a whipping cream, the minimum fat content matters and differs across countries (Table 1.3).

Table 1.3: Different national agreements of the definition of whipping cream.

Country

Fat %

Law

Belgium

Min 40%

Regulation trade of cream, Royal Decree May 23 rd , 1934. Commodities Act Decree of dairy products, Article 16, 1994 Food Labeling Regulations, Cheese and Cream Regulations, No. 52, 1996

The Netherlands

Min 30%

United Kingdom

Min 35%

1.3 Changes during whipping of cream

Whipping and the introduction of air destabilize the oil-in-water emulsion, because the coalescence of fat globules is favored by agitation (Leal-Calderon et al., 2007). The resulting foam is an emulsion in which the dispersed phase is a gas, from which the air-water interface is stabilized by fat globules (Schmitt et al., 2005). However, also in absence of air, cream can be whipped.

1.3.1 Three stages during whipping of cream

Three stages can be distinguished during whipping of cream. In the first stage, most of the air is incorporated and the foam is protein-stabilized. In the second stage, the bubbles are coated by a layer of fat globules. The high packing density of fat globule-

Chapter 1 Literature review

coated gas bubbles inhibits further incorporation of gas. A simultaneous disruption and coalescence of air bubbles take place. However, the result is a reduction of bubble size (mostly 10-100 µm in diameter) and distribution (van Aken, 2001; Mulder and Walstra, 1974) until maximum foam strength is achieved (Stanley et al., 1996). During air bubble coalescence, the adsorbed fat globules are pushed together at the bubble surface, flocculate and partially coalesce in clumps at the air-water interface (Allen et al., 2008b) (Figure 1.7 and Figure 1.8).

interface (Allen et al., 2008b) (Figure 1.7 and Figure 1.8). Figure 1.7: A floccule and a

Figure 1.7: A floccule and a clump of fat globules. Note that the identity of the original globule in the clump is still retained (Mulder and Walstra, 1974).

In the third stage, on prolonged whipping, the formed aggregates of fat globules become so large that they are expelled into the continuous phase and hence the gas bubbles are quickly destabilized, which results in a bimodal bubble size distribution (Jakubczyk and Niranjan, 2006). The consequence is a rapid loss of air and a phenomenon called churning, which is the formation of butter grains present in a phased inversed emulsion (van Aken, 2001). The rate and efficiency of beating determine the balance between whipping and churning. If beaten too slowly, the cream may churn before a satisfactory foam has been formed. Vigorous beating leads to high overrun and a foam with small air bubbles (Mulder and Walstra, 1974).

Chapter 1 Literature review

1.3.2 Foam stabilization by partial coalescence

As discussed in section 1.2.1, partial coalescence of fat takes place in the second stage

of whipping. It results in a rigid network in which bubbles are linked to one another

and liquid is entrapped (Mulder and Walstra, 1974). It prevents the full coalescence into bigger fat globules that are not capable of structure-building (Mulder and Walstra, 1974). The fat crystals break and penetrate the interfacial layer around the fat globules in the emulsion, allowing globules to clump irreversibly together into a network, whilst the identity of the original globule is retained (Allen et al., 2008a) (Figure 1.8).

globule is retained (Allen et al., 2008a) (Figure 1.8). Figure 1.8: (Left) Rigid network due to
globule is retained (Allen et al., 2008a) (Figure 1.8). Figure 1.8: (Left) Rigid network due to

Figure 1.8: (Left) Rigid network due to partial coalescence of fat globules (Goff, 2011). (Right) Fat droplets flocculate and partially coalesce into clumps (A) at the protein-covered air bubble. If the air bubble bursts, a partially coalesced fat clump remains (C). If the air bubble remains stable, fat clumps from the bulk may partially coalesce with the adsorbed fat clump (B) (Hotrum et al., 2005a).

Hereby, the presence of partial crystalline fat, which is of a needle or platelet shape in milk fat, is a crucial factor. The shape of the fat crystals is related to the cooling rate.

A slow cooling rate, e.g. 0.1°C/min, creates a small number of irregularly shaped

crystals, which are characterized by poor coverage of droplet interfaces and are beneficial for partial coalescence. Rapid cooling, e.g. 10°C/min, promotes the formation of a lot of fine crystals which are able to form a rigid dense coverage of the

interface and to stabilize the emulsion against coalescence (Frasch-Melnik et al.,

2010a).

Chapter 1 Literature review

However, almost completely solid fats, e.g. 80%, are not able to practice a spreading function at the air-water interface, nor can form clumps and hence are not efficient at stabilizing foam (Dalgleish, 2006; Mulder and Walstra, 1974). Davies et al. (2000) concluded that partial coalescence of fat droplets is maximized when the solid fat content is approximately 10-50% (Figure 1.9). Besides the amount of crystalline fat, also the fat volume fraction is of importance. The rate of partial coalescence is proportional to the squared fat volume fraction (Fredrick et al., 2010).

to the squared fat volume fraction (Fredrick et al., 2010). Figure 1.9 (Left) rate of partial
to the squared fat volume fraction (Fredrick et al., 2010). Figure 1.9 (Left) rate of partial

Figure 1.9 (Left) rate of partial coalescence as a function of solid fat content (McClements, 2007). (Right) Correlation between whipping time (emulsion stability) and solid fat content of a 30% fat emulsion as a function of storage time (ageing time) at 10°C. Black dots refer to the liquid fat content and white dots refer to whipping time (Darling, 1982).

The fat globule-stabilized air bubble layer (Figure 1.10) will remain intact as long as the crystals don’t melt. A partial liquid part of fat globules is needed to make adhesion and partial wetting of the air bubble surface possible. A too high liquid fraction of fat is characterized by too rapid churning during whipping. Moreover, the excessive spreading of the liquid fat over the air bubbles prevents the formation of sufficiently small air bubbles (Mulder and Walstra, 1974).

Chapter 1 Literature review

Chapter 1 Literature review Figure 1.10: Scanning electron micrograph of an air bubble which is stabilized

Figure 1.10: Scanning electron micrograph of an air bubble which is stabilized by fat droplets in whipped cream. The scale bar represents 20 µm (Dalgleish, 2006).

1.3.2.1 Determination of partial coalescence

There are different methods to determine partial coalescence. Firstly, partial coalescence can be measured by a dye solution absorbance method. It comes down to the determination of the change in absorbance of a lipophilic dye solution due to the dilution by free fat. During partial coalescence aggregates of fat globules, which are considered as free fat, are formed. The Palanuwech method uses the dye Oil Red O (0.0015wt% in oil), which absorbs maximally at 520nm. Pure oil phase is taken as blank. The dye solution is poured onto the surface of the emulsion under investigation, gently mixing and allowing the colored oil to float at the surface under gentle centrifugation. The free fat in the emulsion will be dissolved in the colored oil, but the emulsified fat remains in its droplets. After transfer of the diluted-dye solution fraction from the surface, its absorbance is measured. Out of the change in absorbance, the free fat fraction in the emulsion can be calculated (Palanuwech et al.,

2003).

Secondly, turbidity can be measured to investigate the aggregates of fat globules (Kiokas et al., 2004). Thirdly, partial coalescence can be investigated by measuring the size of clumped fat droplets. In a study of Kiokas et al. (2004) this was done with pfg-NMR and supplemented with scanning electron microscopy imaging.

1.3.2.2 Distinction between partial coalescence and complete coalescence

Regarding completely coalesced fat globules, the identity of original fat globules is no longer retained. Partial coalescing results in an increase of volume fraction and hence

Chapter 1 Literature review

viscosity, whereas the viscosity doesn’t increase when two droplets coalesce completely. Another difference lies in the fact that an applied velocity can increase the rate of partial coalescence without affecting the rate of complete coalescence (Fredrick et al., 2010).

1.3.3 Alternatives to partial coalescence

Like stated above, stabilization of the foam structure can be done by partial coalescence of semi-crystalline fat globules, though this is not the only mechanism (Allen et al., 2008a). An alternative foam-stabilizing method concerns fat globule aggregation induced by acidification in the presence of a small molecule weight lipophilic surfactant LACTEM (lactic acid esters of monoglycerides)(0.25wt%). In protein-stabilized emulsions, a reduction of pH towards the protein’s IEP, diminishes

the electrostatic stabilization and enhances protein-protein interactions. This results in

a protein-stabilized fat globule network, in which the fat droplets are not partially

coalesced but occur as distinct entities, which is similar to the production of yoghurt. The addition of an emulsifier partially displaces the adsorbed casein at the oil-water interface, which induces the formation of strong interdroplet crystal–crystal interactions and fat droplet aggregates upon whipping. The advantage of this method

is that the amount of solid fat enabling whipping can be reduced. Still, its presence is

essential in terms of achieving a similar rigidity as traditional whipped cream (Allen

et al., 2008a).

Márquez and Wagner (2010) concluded that an unwhipped w/o/w emulsion based on liquid oil, PGPR and 0.12wt% CaCl 2 or calcium lactate in the primary emulsion and 0.2wt% xanthan gum in soybean milk as an external water phase, offers an alternative

for whipped dairy cream because of its creamy texture obtained directly after preparation, which is similar to whipped dairy cream. This is explained by the

swelling of the internal water droplets in the presence of soluble calcium salts in the

w 1 -phase and the increase of consistency due to the osmotic gradient and by the

interaction of released calcium with soybean proteins at the o/w 2 -interface, which results in the flocculation of w/o droplets.

Chapter 1 Literature review

1.4 Mimicing whipping cream

Recombined cream is an emulsion of butterfat or a higher melting fat stabilized by skimmed milk products (Hotrum et al., 2005a). If a reconstituted cream contains other non-dairy sources of fat, it is denoted as a filled cream (Hui et al., 2007). In order to mimic a whipping cream, partial coalescence can be influenced by the type and amount of emulsifier in the formulation. For example, proteins in a certain concentration can reduce the susceptibility of the emulsion to partial coalescence by formation of a thick layer around the fat globules, which increases the repulsive forces and the resistance to penetration of fat globules by fat crystals (McClements, 2007). Segall and Goff (1999) reported a too high stability against whipping for WPH or SMP-stabilized emulsions. Hydrolysis of whey proteins (WPH) exposes previously buried hydrophobic sites, which increases its surface activity in comparison to WPI. SMP consists of large casein micelles which result in a thick layer of surface coverage, which makes it more resistant to shear. A similar change in surface activity can be noticed by denaturation of whey proteins. Consequently, an increased fat membrane integrity and hence, increased stability against partial coalescence can be observed (Goff, 1997). WPI and sodium caseinate-stabilized emulsions resulted in a larger susceptibility to partial coalescence than SMP-stabilized emulsions (Goff, 1997; Leser and Michel, 1999). However if more than 0.5% WPI was present, a too strong film around the fat globules was formed and partial coalescence was impaired (Leser and Michel, 1999). Van Lent et al. (2008) investigated the differences between recombined creams made of different protein sources: skimmed milk powder (SMP) and cream residue powder (CRP) (Table 1.4). In SMP-creams casein micelles act as surface active material, whereas in CRP-creams small molecular weight surfactants and whey proteins stabilize the interface of the o/w emulsion. Moreover, more stabilization by CRP- surface material per unit weight emulsifier is achieved than casein micelles do in SMP-cream. With regard to fresh cream, smaller fat droplets and a larger whipping time were observed than for SMP-creams and the freeze thaw serum leakage was smaller than for SMP- and CRP-creams. It was concluded that CRP-creams mimicked fresh cream better than SMP-creams.

Chapter 1 Literature review

Table 1.4: Differences in characteristics between recombined creams made of skimmed milk powder or cream residue powder (Van Lent et al., 2008)

Characteristics

SMP-cream

CRP-cream

Fat droplet diameter Free protein content Whipping time Freeze-thaw serum leakage Air phase fraction Apparent viscosity Creaming rate

 

>

 

<

<

>

<

>

>

Scott et al. (2003) proved that the separation temperature in obtaining the emulsifying components, skim milk or sweet buttermilk and butter derived aqueous phase, which are generated after churning cream that is obtained by separation at 49 or 55°C, mattered. Mimicked creams manufactured from components at a 55°C separation temperature were more stable than those coming from a 49°C separation temperature, perhaps because of the more efficient separation and inactivation of agglutinins that might cause cold agglutination. Regarding the sensory analysis, the flavor of creams formulated with sweet butter milk or butter derived aqueous phase were said to be rich and creamy (Scott et al., 2003).

Besides the type and concentration of protein in the emulsion, the presence of small molecular weight surfactants influences the susceptibility to partial coalescence. Examples of small molecule surfactants are monoglycerides, diglycerides, and polysorbates. Also phospholipids such as soy lecithin, exceeding a minimum concentration, can render an emulsion more susceptible to partial coalescence upon shearing action. Small molecule surfactants act as protein displacers, due to their better surface active properties than proteins, which results in preferential migration to the fat-water interface (Dalgleish, 2006). Consequently, the fat globules are no longer covered by a thick stabilizing layer, break easier during whipping and more extensive partial coalescence takes place (Segall and Goff, 2002). In conclusion, the fat globule membrane should not be too strong because the aim of whipping is to destabilize the emulsion (Van Lent et al., 2008; Mulder and Walstra, 1974), which can be obtained by addition of small molecule surfactants, which are potential destabilizers of the emulsion.

Small molecule surfactants might also act on the morphology of the fat crystals, e.g. mono-olein (unsaturated MAG), which doesn’t show much protein displacement, but it results in a cream that is more susceptible to partial coalescence (Fredrick et al.,

Chapter 1 Literature review

2010). Hotrum et al. (2005b) noticed shorter whipping times upon addition of small molecule surfactants. In comparison to non-isolated whey products, caseins are more readily displaced by surfactants, whereby water soluble surfactants are more effective in doing this than oil-soluble surfactants (Cornec et al., 1998).

Instead of the application of small molecule surfactants, Goff (1997) promoted partial coalescence by preparing an emulsion stabilized by milk proteins in such a concentration that a weak yet sufficient interfacial layer stabilized the emulsion during storage, which could be destabilized during a whipping process (Goff, 1997). Additionally, additives that act on the properties of the continuous phase can be added. Carrageenan in a concentration of 0.02% can prevent creaming by increasing the viscosity. It reduces the drip volume in whipped creams without affecting the whipping properties (Kováčová et al., 2010). Xanthan gum, an anionic polysaccharide, increases the viscosity of the emulsion. In a concentration of 0.1% it doesn’t affect the overrun (Zhao et al., 2009). Concerning the stability of mimicked creams, Parkinson and Dickinson (2007) suggested that the change of a 3wt% beta-lactoglobulin stabilized emulsion (45vol% n-tetredecane, o/w-emulsion) into a 2.97wt% beta-lactoglobulin stabilized emulsion to which 0.03wt% sodium caseinate was added, can improve the shelf-life of imitation cream. The researchers observed an enhancement of long-term (up to 1.5 years) stability, which was measured by looking at the phase separation.

1.5 Quality characteristics of whipped cream

Quality parameters of whipped cream are the increase in volume or overrun, the whipping time or whipping rate, the texture of the whipped cream, its physical stability (i.e. the extent of drainage and coarsening), and the chemical and microbiological stability (De Meulenaer et al., 2009; Templeton and Sommer, 1932; Lampert, 1975). An overview of the changes of some quality parameters during whipping of cream is given in Figure 1.11.

Chapter 1 Literature review

Chapter 1 Literature review Figure 1.11: Changes of some quality parameters during whipping of cream. The

Figure 1.11: Changes of some quality parameters during whipping of cream. The parameter of firmness is related to the time needed to lower a weight into the product. Leakage is the amount of liquid drained from a certain volume in a certain time. Between the broken lines the product is acceptable. Curves are approximate (Mulder and Walstra, 1974).

1.5.1

Overrun

Overrun depends on the effectiveness of the introduction of gas during the first stage of whipping. According to Graf and Müller (1965) a well whipped cream should have an overrun of approximately 50-60%. Overrun can be determined after a fixed time period or the cream can be whipped until maximum overrun. Maximum overrun corresponds with maximum stability and stiffness of the foam, and all air bubbles at this point are encapsulated by coalesced fat droplets which adsorbed at the air-serum interface (Jakubczyk and Niranjan, 2006). The amount of emulsifier can affect the overrun, e.g. in a study of Zhao et al. (2008) the highest overrun was obtained with 0.7% sodium caseinate in a recombined cream that consisted of hydrogenated palm kernel oil, stabilizers, proteins, sucrose esters and sugar slurry. As soon as the concentration exceeded 0.9% sodium caseinate, the foam of the cream collapsed and hence the overrun decreased. Adding stabilizers, e.g. 0.05wt% λ-carrageenan or 0.1wt% locust bean gum (LBG) to a casein based dairy cream decreases air incorporation capacity of the cream in comparison to creams without these stabilizers. Despite this disadvantage, they also increase the serum phase viscosity and interact with proteins in the cream, leading to a decrease of the serum drainage and whipping time (Camacho et al., 1998).

Chapter 1 Literature review

1.5.2 Whipping time

The whipping time depends on the rapidity with which the partially coalesced network of fat globules can be built up. Shorter whipping times are achieved when the cream is whipped at higher rate (rotational speed of the whisks) (Hotrum et al., 2005b). Templeton and Sommer (1932) defined the whipping time as the time needed until desired stiffness, which was determined by observing the torque (load) on the drive shaft of the whipping apparatus. In van Aken’s (2001) experiment, the endpoint of whipping was set at the maximum of a peak in the electrical current, related to a maximum of stiffness. Whipping time in the study of Van Lent et al. (2008) was based on visual inspection, until a maximum overrun was reached. Ihara et al. (2010) defined the endpoint of whipping as when a certain cone penetration depth was reached. A clear correlation exits between the whipping time (emulsion stability) and the solid fat content of the emulsion (Darling, 1982) as shown in Figure 1.9. Just like for overrun, the whipping time can be affected by the type and concentration of emulsifier and stabilizer in the cream. In a study of van Hotrum et al. (2005b), shorter whipping times were obtained with 1wt% whey protein isolate stabilized emulsions than with 1wt% sodium caseinate. The mimicked cream consisted of 40wt% fat. Whey protein isolate (WPI) consists of mainly beta-lactoglobulin, which forms brittle adsorbed layers at the air-water interface, whereas beta-casein forms more fluid like layers. Addition of 2.7mM and 5.5mM Tween 20 to the mimicked cream reduced the whipping time to such an extent that it was similar to the whipping time of natural cream at the same rotational speed. The lower concentration of Tween 20 resulted in a higher overrun than the higher concentration (Hotrum et al., 2005b). The lowest whipping time was obtained without locust bean gum or λ-carrageenan in dairy cream, suggesting that these stabilizing additives cause kinetic hindrance to the cream foaming, which could be due to not only the increase in the viscosity of the liquid phase, but also to stabilizer-protein interactions that could partially inhibit the foaming properties of milk proteins (Camacho et al., 1998).

Chapter 1 Literature review

1.5.3 Textural analysis

Whipping converts the viscous liquid emulsion into a stiff aerated viscoelastic solid (Allen et al., 2008b). The viscoelastic behavior can be analyzed by measuring rheological properties, the storage (G’) and loss modulus (G”), which both increase when air is introduced (Jakubczyk and Niranjan, 2006). Also the viscosity, cohesiveness and consistency are texture parameters. A back extrusion test and a flat base plate rheometer can be used to study the structure of semisolid food and to quantify the shelf life of whipped creams (Piazza et al., 2009; Allen et al., 2008b). Differences in firmness and viscosity can exist between creams made of different proteins. Zhao et al. (2008) found that creams with whey proteins were characterized by a smaller increase in viscosity during whipping and a lower firmness after whipping than sodium caseinate-whipped creams. The foam structure of dairy cream can be better preserved during chilling when higher concentrations (>0.05%) of additives such as λ-carrageenan or locust bean gum are applied. Higher cream viscosities can be obtained with λ-carrageenan in comparison to locust bean gum, due to protein-polysaccharide interactions (Camacho et al., 1998).

1.5.4 Physical stability

Just like emulsions, foams are never thermodynamic stable, the destabilization can only be kinetically slowed down. Two (physical) destabilization processes can take place: coarsening and drainage (Indrawati et al., 2008; Butt et al., 2006).

1.5.4.1 Coarsening of foam

During Ostwald ripening, which is a thermodynamically favourable process, diffusion of gas through liquid films from small to large compartments is taking place, which is driven by the pressure difference between them (Butt et al., 2006; Dalgleish, 2006). The resulting phenomenon of coarsening or disproportionation can be slowed down by having equally sized air bubbles, but this is not feasible with normal kitchen equipment. Other options are increasing the viscosity of the liquid phase with

Chapter 1 Literature review

hydrocolloids or strengthening the interfacial layer around the gas bubbles with surface-active proteins. Although globular proteins favor foam formation and stability due to interfacial unfolding, the most successful way is to coat air bubbles with semi- crystalline fat globules, undergoing partial coalescence at the air-water interface (Dalgleish, 2006; Schmitt et al., 2005; Indrawati et al., 2008). Coarsening can be studied by measuring the increase in transmitted intensity in a laser light scattering-CCD camera experiment (Saint-Jalmes et al., 2005). Coarsening can also be evaluated by microscopical image analysis, facilitated by low T (for preservation of the original foam structure), scanning electron microscopy (SEM) and computer assisted quantitative stereology (Smith et al., 1999).

1.5.4.2 Drainage

According to Belitz (2009), no serum separation should occur at 18°C after 1h in order to have qualitative whipped cream. Due to the pressure difference between the inside of a bubble and the liquid film, serum drainage or syneresis occurs. The pressure inside the liquid film is significantly smaller than in the air compartments, resulting in liquid being sucked into the ‘Plateau border’, which is the channel at the contact line between liquid films (Figure 1.12).

channel at the contact line between liquid films (Figure 1.12). Figure 1.12: Plateau border in foam

Figure 1.12: Plateau border in foam (Butt et al., 2006)

Chapter 1 Literature review

Once the liquid reaches a Plateau border, the downward flow, driven by gravity, becomes substantial (Butt et al., 2006). Liquid flow can be slowed down by increasing the viscosity of the liquid, e.g. by adding xanthan gum, or by keeping the temperature low (Mulder and Walstra, 1974; Indrawati et al., 2008). In Table 1.5 a summary of different drainage analysis methods mentioned in literature is given. The serum leakage can also be determined after a freeze-thaw experiment, which is an example of an accelerated stability test. In Van Lent et al. (2008) cups with whipped cream were frozen at -18°C for 24h and placed upside-down on an aluminum dish at 16-18°C. After 15 min, the cup was removed and the cream weight was determined. The serum was poured out and weighted after 150 minutes and the serum leakage (ftSL) was determined as the mass of serum over the mass of whipped cream, times 100% (Van Lent et al., 2008).

Table 1.5: Overwiew of drainage analysis methods in literature

Reference

Drainage analysis method

Allen et al., 2008b

 

Serum drainage stability is the amount liquid drained under gravity from 10 g

 

sample over 5h period.

Templeton

and

Sommer,

The drainage time is the time between placing the cream in a funnel and the first

1932

drop of serum falling from the end of the glass stem of the glass funnel. The drain

amount is gravimetrically determined.

Van Aken, 2001

 

Serum loss was measured by placing 30g whipped cream on a sieve and measuring

 

the volume of serum leaking that passes through the sieve during 2h at 20°C.

Shamsi et al., 2002

 

40g whipped cream is immersed into a warm water bath at different temperatures

 

for 6h. Measurement of separated serum in mm.

Van Lent et al., 2008

 

The drainage stability was determined by placing 50g whipped cream on a sieve

 

with openings of 1mm. After 1h, at 16-18°C, serum leakage (SL) was determined

as the mass serum over the mass whipped cream, times 100%.

1.6

Factors determining functional properties of whipping cream

1.6.1

Temperature

During whipping of cream, the presence of a certain proportion of fat solidification is crucial for partial coalescence of fat globules and firmness of the whipped cream, hence, temperature is important (Figure 1.13).

Chapter 1 Literature review

Milk fat is a mixture of many different triacylglycerols (about 98%) with individual melting points and thus it has an extensive melting range of -40°C to 40°C (Smet, 2010). Generally the fat should be partially solid at 5°C, solid enough at ambient temperature and melt at temperatures below 37°C (Shamsi et al., 2002). Prior to whipping, it is advised to keep cream, metal bowl and beaters below 7.2 °C (Lampert,

1975).

cream, metal bowl and beaters below 7.2 °C (Lampert, 1975). 1.6.1.1 Tempering Figure 1.13: Effect of

1.6.1.1 Tempering

Figure 1.13: Effect of whipping temperature (in °C) on the firmness (yield stress, g/cm 2 ) of whipped cream of different fat contents (%) (Mulder and Walstra, 1974).

The stability of whipped cream, based on crystallizable oils with a large melting range, can be enhanced by a process called cycling or tempering. This treatment implies a temperature increase of the whipped cream to 25-30°C immediately after whipping and subsequently the cream is cooled down. A clearly stiffened foam, storable at 4°C for several weeks without any visible structural change (no fat or gas segregation) can be observed, especially for native dairy creams with 20-40wt% fat (Leal-Calderon et al., 2007). By contrast, non-tempered whipped cream collapses already after 48h (Gravier et al., 2006). In detail, the increase in temperature and a holding time of 5 minutes doesn’t melt all of the crystals, hence the foam will not be collapsed. Upon cooling, the remaining crystals serve as catalytic impurities, which results in a controllable nucleation and increase in crystal size, which can be achieved without deep supercooling (Fredrick et al., 2010). Drelon et al. (2006) could not relate tempering with polymorphism of fat nor with an increase of the solid fat content. Though they could report a higher increase of partial coalescence of whipped tempered creams upon whipping in comparison to non-tempered whipped creams, which was measured by laser

Chapter 1 Literature review

diffraction after application of a heating step to transform the partially coalesced fat globules into fully coalesced spherical fat globules (Drelon et al., 2006).

1.6.1.2 Heat treatment

The thermal treatment might have an effect on the whipping properties of cream. Higher temperature pasteurization denatures whey proteins, which become unfolded and interact with kappa-casein on the fat globules in order to form a complex that makes the fat globules stable against partial coalescence (Bruhn and Bruhn, 1988; Smith et al., 1999). Regarding gravitational stability, the complex formation is related to a decreased creaming rate due to an increase of the density of the fat globules (Parkinson & Dickinson, 2007). UHT-treated cream exhibited larger fat globules than HTST-treated creams. The former was associated with lower overrun and lower foam stability (Smith et al.,

2000).

1.6.2

Fat content

Whipped dairy cream should not contain less than 30% fat in order to form a stable network capable of enclosing air bubbles. As illustrated in Figure 1.14, an increase in fat content results in a shorter whipping time, a higher firmness and less serum leakage (Mulder and Walstra, 1974), but a fat content of more than 38% decreases the overrun and doesn’t improve the foam firmness any longer (Lampert, 1975). Whipped creams with less 30% fat are characterized by a long whipping time and an increase of the rate and extent of serum drainage (Templeton and Sommer, 1932).

1.6.3 Homogenization

During whipping of homogenized dairy cream, the fat globule membrane is disintegrated twice. The first time happens when the cream is homogenized, resulting in a newly formed membrane, the second time occurs when the cream is whipped.

Chapter 1 Literature review

The newly formed membrane gradually disintegrates and the liquid fat is released and cements the remaining fat globules.

fat is released and cements the remaining fat globules. Figure 1.14: Properties of whipped cream. From

Figure 1.14: Properties of whipped cream. From left to right: whipping time (minutes), overrun

(%), firmness, leakage of liquid (ml) as a function of fat content for conventional whipping cream

(

) and for a cream with surfactants ( ----). Data are approximate (Mulder and Walstra,

1974).

Apart from the beneficial effect on the color (more white) (Hui et al., 2007) and the creaming rate of cream by reduction of the fat globule size, homogenization impairs whipping properties, particularly for a low fat cream: whipping time is much longer and the foam is less firm. The clumping tendency of the homogenized fat globules is probably too low, so more fat globules are needed to form a clump (Mulder and Walstra, 1974). Two-stage homogenization is used for the manufacture of recombined creams. The first stage at higher pressure creates smaller fat globules, increases the rate of fat globules collision and promotes the formation of heat-stable clusters (Figure 1.15), which are more pronounced with increasing fat content and will separate much quicker than non-aggregated fat globules (Mulder and Walstra, 1974). In the second stage at lower pressure fat globules clusters are broken up, while avoiding the destruction of the single fat globules.

1.6.4 Miscellaneous factors

Aging affects the rearrangement of the composition of the protein membrane in homogenized cream (Darling, 1982) and it allows time for fat crystallization (Segall and Goff, 2002).

Chapter 1 Literature review

Chapter 1 Literature review Figure 1.15: Homogenization clusters in homogenized cream (Mulder and Walstra, 1974) 30

Figure 1.15: Homogenization clusters in homogenized cream (Mulder and Walstra, 1974)

Chapter 2 Materials and methods

Chapter 2

Materials and methods

2.1 Commercial butters

Two types of commercial butters with a fat content of 82% were analyzed with the Maran Ultra 23 spectrometer: an organic churned butter, Bio Karneboter (Delhaize) and a cream butter, (Zachte) Ardense Roomboter (Carlsbourg), as illustrated in Figure 2.1. Bio Karneboter contains less than 0.1% salt and on the package of Ardense Roomboter nothing is mentioned about the salt content. The salt content of salted butters ranges from 0.5 to more than 3%, so the commercial butters used in this experiment can be assumed to be non-salted.

used in this experiment can be assumed to be non-salted. Figure 2.1: Analyzed commercial butters: Ardense
used in this experiment can be assumed to be non-salted. Figure 2.1: Analyzed commercial butters: Ardense

Figure 2.1: Analyzed commercial butters: Ardense Roomboter and Bio Karneboter

2.1.1 Preparation of butter samples

Calibration of pfg-NMR requires a pure fat sample and a dispersed phase sample. The separation of the phases was obtained by melting 125g of butter in an oven at 45-50°C and by centrifugation at 2800g for 20 minutes, resulting in clarified butter oil at the top and serum at the bottom of the recipient. Two glass NMR-tubes were filled to the marked line (8mL) with fat and dispersed phase respectively, by using a syringe with long needle to reach the separated phases.

Chapter 2 Materials and methods

Three glass NMR-tubes of each commercial butter were filled to the marked line (8mL) with butter samples by using a cheese trier.

2.1.2 Analysis of butter samples

The samples at 5°C were analyzed in a Maran Ultra 23 spectrometer, which was set at -7°C in order to get +5°C in the probe of the spectrometer. Water droplet size was obtained with the software Droplet Size application (Resonance Instruments Ltd), Excel and Matlab (see 2.3.3).

2.2

Water-in-oil emulsions

2.2.1

Materials needed for the preparation of w/o-emulsions

The materials used are a lipophilic emulsifier (polyglycerol polyricinoleate, Palsgaard® 4150, Palsgaard A/S, Denmark), a hydrophilic emulsifier (sodium caseinate, kindly provided by Armor Protéines, Saint Brice en Cogles, France), high oleic sunflower oil (Hozol, Contined B.V., Bennekom, The Netherlands), soft- palmitine mid fraction (Figure 2.2) (soft PMF, Unigra Sp., Conselice, Italy), a buffer solution and an anti-microbial agent (sodium azide, Acros Organics, Geel, Belgium). The buffer solution was made, according to the Henderson-Hasselbalch equation and an aimed pH of 7, by mixing KH 2 PO 4 (Merck KGaA, Darmstadt, Germany) and K 2 HPO 4 (Alfa Aesar, Karslruhe, Gemany) solutions of equal molarity (0.1M) in a volume ratio 1:0.63. This pH avoids flocculation of sodium caseinate in the water phase (I.E.P. is 4.6). By means of a pH-meter, the pH of the buffer solution at room temperature was measured and amounted to 6.7. The difference in measured and intended pH might be explained by the applied calibration liquids. Some emulsions were made with whey protein isolate (WPI, BiPro, Davisco, Le Sueur, USA) instead of sodium caseinate.

Chapter 2 Materials and methods

2.2.5 Composition of the w/o-emulsions

Different emulsions were obtained by varying the concentration of hydrophilic emulsifier, lipophilic emulsifier, the composition of the oil phase and the water-to-oil ratio.

the composition of the oil phase and the water-to-oil ratio. Palm Oil Stearin Olein fraction fraction
Palm Oil Stearin Olein fraction fraction Hard Soft Super stearin PMF Olein Hard Mid PMF
Palm Oil
Stearin
Olein
fraction
fraction
Hard
Soft
Super
stearin
PMF
Olein
Hard
Mid
PMF
olein
Figure 2.2: Fractionation scheme of palm oil

The composition of different emulsions with 20% water (w/w) is represented in Table 2.1. In Table 2.2 shows the composition of emulsions with water to oil-ratios (w/w) varying from 20% to 60%. The codes of the emulsions start with a letter, corresponding to the first letter of the composition of the oil phase, followed by two numbers, separated by a slash. The first number refers to the percentage (w/v) of sodium caseinate in the water phase and the second refers to the percentage (w/v) of PGPR in the oil phase. The water phase was made by weighing sodium azide (all emulsions) and sodium caseinate (some emulsions) in a volumetric flask and was filled with buffer solution to the marked line. The oil phase was made by weighing PGPR in a volumetric flask and filling with Hozol (at room temperature) or soft PMF (heated to 60°C) or both (at 60°C) to the marked line.

Chapter 2 Materials and methods

Table 2.1: Composition of the 20% (w/w) W/o-emulsions (components in %, w/v)

W/o-emulsion

H0/1

H0,5/1

P0/1

P0,5/1

P0,5/2

M0/1

Water phase Sodium azide (%) Sodium caseinate (%) Buffer solution (pH 6.7) (%)

0.02

0.02

0.02

0.02

0.02

0.02

0.00

0,50

0.00

0,50

0,50

0.00

ad 100

ad 100

ad 100

ad 100

ad 100

ad 100

Oil phase PGPR (%) Hozol (%) Soft PMF (%)

1

1

1

1

2

1

99

99

0

0

0

49.5

0

0

99

99

98

49.5

Table 2.2: Composition of w/o-emulsions with different water to oil ratios. The oil phase consists

 

of Hozol. The water phase contains sodium azide (0.02%, w/v) and a phosphate buffer (pH 6.7). Percentage water (%, w/w) in the w/o-emulsion

 

20

30

40

50

60

Water phase Sodium caseinate (%,w/v)

0.50

0.75

1.00

1.25

1.50

Oil phase PGPR (%, w/v)

1.00

1.50

2.00

2.50

3.00

2.2.6 Preparation of the w/o-emulsions

An Ultraturrax (type TV45, IKA) and a Microfluidizer (type M110S, Cobra Engineering NL) (Figure 2.3) were used to premix and homogenize the emulsion at 60°C, respectively. To comply with the demands from good practicing when using the Ultraturrax, about 75mL of emulsion was prepared, which comes down to weighing 52g oil phase and 13g water phase in a beaker. The Ultraturrax was set at position 1 to 2 and the emulsion was premixed for 1 minute. The emulsion was homogenized in the Microfluidizer at an air pressure of 6bar for 1.5minutes. All emulsions were analyzed in triplicate with a fresh emulsion prepared for each replicate. When preparing an emulsion containing soft PMF or a mix, special care should be given to preheat the Microfluidizer with boiling water, in order to prevent obstruction, as presented in Flow chart 2.1.

Chapter 2 Materials and methods

Piston
Piston

Figure 2.3: Schematic representation of the Microfluidizer M110S: premixed emulsions (A) were filled into the reservoir (B) and cycled through the dissipation zone (C), cooled by passing the heat exchange coil (D) and then collected at the outlet by opening the valve (E) .

To assure that the ratio of water to oil in the very first collected emulsion samples (after preheating), was 1:4, an oven test was conducted. The first six weighted collected samples in weighted metal recipients with coverage were kept overnight at 105°C in an oven, by this removing the water phase. The difference in weight before and after placing the samples in the oven, made it possible to calculate the w/o-ratio. The outcome of the oven test suggested to start collecting the sample after preheating and executing the first four steps of the Flow chart 2.1, followed by repeating twice the third and fourth step. In other words, just after preheating, the first three collected samples should be rejected. The next emulsions of the same and different composition were collected by operating as illustrated in the Flow chart 2.1. The collected samples in glass NMR-tubes (18mm outer diameter) were kept in the refrigerator for 24h and covered with parafilm. After 24h all transport processes occurring are under equilibrium conditions (Hindmarsch et al., 2005). Some hours before analysis, the samples were kept at 5°C in a water bath (Julabo F12) filled with a mixture of glycol and water.

Chapter 2 Materials and methods

Flow chart 2.1: A procedure for handling the Microfluidizer M110S

Preheat with boiling deionised water -> circulate -> empty by opening the valve until the
Preheat with boiling deionised water -> circulate -> empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe
STEP 1
Fill the reservoir with 20mL oil phase -> empty without circulation, empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe
STEP 2
Fill the reservoir with 20mL premixed emulsion -> circulate for 1.5 min -> empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe
STEP 3
Fill the reservoir with 10mL premixed emulsion -> empty without circulation, by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe
STEP 4
Fill the reservoir with 20mL premixed emulsion -> circulation for 1.5 min -> empty, by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe
Repeat twice STEP 3 and 4
To collect the first sample: execute STEP 3
then STEP 4* :
Fill the reservoir with 20mL premixed emulsion -> circulate for 1.5 min -> collect the sample from the second stroke of the piston onwards
(reject the liquid from the first stroke of the piston)
To collect next samples with the same composition, execute STEP 2, 3 and 4*:
To collect samples with a different composition, execute STEP 1*:
Fill the reservoir with 10mL premixed emulsion-> empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and suck the remaining liquid out of the reservoir with a syringe
then execute STEP 2, 3 and 4*

2.4 Quantitative particle size analysis of water in w/o-emulsions

2.4.1 Pulsed field gradient-Nuclear Magnetic Resonance

In order to characterize a w/o-emulsion, its particle size distribution was determined by pulsed field gradient-Nuclear Magnetic Resonance (pfg-NMR). Pfg-NMR measurements were conducted with a Maran Ultra 23 spectrometer. In the next paragraphs, essential highlights of this method are given, clarifying its application to determine the water droplet size distribution through its ability to measure restricted molecular self-diffusion, as described by Packer and Rees (1972), Lönnqvist et al. (1991), Lönnqvist et al. (1997), Johns (2009), Voda and van Duynhoven (2009), van

36

Chapter 2 Materials and methods

Duynhoven et al. (2002), Balinov et al. (1994) and Calliauw (2009). Reference is made to the latter study for more detailed information about pfg-NMR. Besides information with regard to characterization of the emulsion, one should notice the relationship between water droplet size and sensorial properties and microbial activity. The microbial activity is bigger in larger droplets, because more nutrients are present. Further elaboration of this aspect is beyond the scope of this thesis.

2.3.2 The pfg-NMR experiment

NMR-spectrometry detects the emission of electromagnetic radiation by nuclei. The nucleus of an atom absorbs energy of an applied magnetic field. Here, the nucleus of the proton is studied for determination of the water droplet size distribution. Pfg-NMR consists of a 90° and 180° radio frequency pulse and two pulsed field gradients, as illustrated in Figure 2.4. Applying a 90° radio frequency pulse, which is a rotating magnetic field, supplies energy to be absorbed, with a continuous spectrum of frequencies. The pulse results in the fact that protons resonate at their Larmor frequency, which isn’t the same for each nucleus, due to the difference in chemical environment. The obtained NMR-signal will decrease as signals of protons dephase. The 180° radio frequency pulse reverses the direction of the rotation of the protons, which is called rephasing, which will be followed by dephasing. Together, the rephasing and dephasing signal are called the spin echo signal. If protons resonate at the same frequency before and after the 180° radio frequency pulse, the NMR-signal will be maximum. Diffusion of protons attenuates the echo signal, because their frequency changes due to the different perceived magnetic field. The application of pulsed field gradients is needed to influence the spin echo signal, due to the diffusion of protons, more pronounced. The gradients enlarge the differences of perception of magnetic field and fasten up the dephasing. The stronger the gradients, the greater the reduction in echo intensity. If there wouldn’t be any diffusion, after the second pulsed field gradient, protons rephase at time 2τ, resulting in a maximum echo signal, identical to the signal without gradient pulses. Hereby, τ represents the time between the 90° and the 180° radio frequency pulse. Pulsed field gradients (Figure 2.4) are characterized by a duration δ (s) and a strength g, which was fixed at 1.73953 Tesla/meter.

Chapter 2 Materials and methods

Chapter 2 Materials and methods Figure 2.4: Schematic representation of the pfg-NMR diffusion experiment (Hindmarsh et

Figure 2.4: Schematic representation of the pfg-NMR diffusion experiment (Hindmarsh et al.,

2005).

In the RINMR-software the δ-value was varied in 17 or 18 steps, which was done by using the DSD.RIS.tif script and DSD_Lien.RIS.tif script. The latter script is an adapted version of the DSD.RIS.tif script, from which the Droplist is altered. In the Droplist of DSD_Lien.RIS.tif originally 19 δ-values (µs) were contained, but only for 18 values an output was given, because at δ=10000 µs, the duration of the gradient pulse was too long with regard to the time interval of the two radio frequency pulses. Hence, the δ-value of 10000 µs was dropped out of this Droplist. Running the script with 18 δ-values takes about 10 minutes.

Droplist of DSD.RIS.tif script with 17 δ-values in µs: 400; 600; 800; 1000; 1250; 1500; 1750; 2000; 2250; 2500; 2750; 3000; 3250; 3500; 3750; 4000; 4500

Droplist of DSD_Lien.RIS.tif script with 18 δ-values in µs: 500; 750; 1000; 1250; 1500; 1750; 2000; 2250; 2500; 2750; 3000; 3250; 3500; 4000; 4500; 5000; 6000;

8000

Samples in glass NMR-tubes with outer diameter 18mm were filled until the marked line (8mL) and analyzed at an aimed temperature of 5°C in the Maran Ultra 23 spectrometer. In the performed experiments the time between two gradient pulses () (Figure 2.4) amounted of 0.2s. As discussed in 2.3.3, two other parameters are needed to calculate the water droplet size: the magnetogyric or gyromagnetic ratio (γ), which for H-atoms is equal to 267518000 T -1 s -1 , and the bulk diffusion coefficient of water in the dispersed fluid (D) at 5°C, which differs among different composed water

Chapter 2 Materials and methods

phases of the w/o-emulsion and was experimentally determined in the calibration procedure.

2.3.2.1 Calibration procedure

Before analysis, a calibration procedure is needed, which is included in the mentioned scripts in the RINMR software. First, a pure fat sample (without emulsifier) is used in order to suppress NMR-signals coming from the fat phase. Secondly, pure deionized water is used to be able to calculate the magnetic field gradient strength (g) from the known bulk diffusion coefficient of deionized water (D w =1.3253E-09 m 2 /s at 5°C) and different values of δ. This is based on a pfg-NMR diffusion experiment that acquires a set of data for each δ-value. Thirdly, a dispersed phase sample is used to determine the bulk diffusion coefficient of water in this environment at 5°C, which may be smaller than the diffusivity of pure water due to the presence of dissolved molecules, such as emulsifiers and salts. Hereby D is calculated from the obtained gradient strength (g) and a pfg-NMR diffusion experiment that results in a set of data for each δ-value.

2.4.3 Restricted diffusion in w/o-emulsions

With regard to w/o-emulsions, the water molecules inside the dispersed phase are characterized by restricted diffusion. This means that the mean displacement of the liquid molecules inside the emulsion droplets is of the same order of magnitude or larger than the diameter of the droplet. There is a limit to the amount of diffusion and hence to the echo intensity reduction. If the movement of a molecule over relatively short times is observed, the diffusion will be unrestricted. Observations over a longer time, will result in a restricted diffusion because the molecule cannot move further than the droplet diameter. By measuring the attenuation ratio of the NMR-signal at different times, it is possible to identify when the diffusion becomes restricted and estimate the droplet size distribution.

A

function of the ratio (E) of the intensity of the echo signal I (with gradient pulses)

to

I 0 (without gradient pulses) has been described by Murday and Cotts (1968). The

Chapter 2 Materials and methods

function contains the following parameters: δ (duration of the gradient pulse), γ (the magnetogyric or gyromagnetic ratio), (time between two gradient pulses), g (gradient strength), D (bulk diffusion coefficient of water in the dispersed fluid) and additional parameters λ m (the m th -square root of a Bessel function) and R (the radius of the water droplet):

E(R,

)

=

exp

(

2

2

g

2

2

m

D

2

1

)

m

2

+

2

m

R

2

m

2

D (

2)

2

m

))

= 1 (

exp(

*

2 exp(

2

m

D

)

2 exp(

2

m

D

)

+

exp(

2

m

D (

+

))

(

2

m

D

)

2

Equation 2.1

The echo attenuation is smaller as the droplet size decreases (Figure 2.5). The use of the bulk diffusion coefficient D of the dispersed phase is justified by the fact that only two or three layers adjacent to the emulsion droplet interface have altered properties as compared to the bulk.

1,0 0.25µm 0,9 0.75µm 1.25µm 0,8 1.75µm 0,7 2.25µm 0,6 2.75µm 0,5 3.25µm 0,4 3.75µm
1,0
0.25µm
0,9
0.75µm
1.25µm
0,8
1.75µm
0,7
2.25µm
0,6
2.75µm
0,5
3.25µm
0,4
3.75µm
4.25µm
0,3
4.75µm
0,2
sample1
H0.75/1.50
0,1
free H2O
diffusion
0,0
0
0,001
0,002
0,003
0,004
0,005
0,006
0,007
0,008
0,009
(s)
Echo attenuation ratio Ep (-)

Figure 2.5: Calculated decay of the echo intensity of w/o-emulsions characterized by an R-value, δ (0s to 8ms) and D=1.30E-09 m 2 /s, and obtained from Equation 2.1 assuming γ=267518000T -1 s -1 , g=1.73953T/m and =0.2s. Experimental decay of the echo intensity versus magnetic gradient pulse width of pure deionized water (D w =1.3253E-09 m 2 /s) and of a w/o-emulsion (H0.75/1.5, sample 1) (D=1.289E-09 m 2 /s), both measured at 5°C (γ, g and are identical as for pure deionized water).

Depending on the applied method of data processing, different equations are used in order to get the water droplet size distribution, based on the measured echo intensities.

Chapter 2 Materials and methods

The three methods that are used, are the data processing method by the Droplet Size application (Resonance Instruments Ltd), by Excel or by Matlab.

2.3.3.1 Data processing by the Droplet Size application

This method gives directly the volume-weighted arithmetic mean radius R 43 (denoted in the software as R 33 ), the number-weighted arithmetic mean radius R 10 (denoted in the software as R 00 ), the median radius R med of the number-weighted distribution (denoted in the software as R 0 ) and the distribution width σ. Assuming a lognormal number-weighted distribution, P n (R) is introduced, which is the probability distribution of each droplet radius:

P n (R) = (Rσ√(2π)) -1 exp[-(lnR-lnR med ) 2 /(2σ 2 )]

Equation 2.2

where R is the radius (µm), σ is the distribution width and R med is the median radius considering a lognormal number-weighted distribution, which equals the number- weighted geometric mean radius. In addition, the distribution width σ corresponds to the natural logarithm of the geometric standard deviation σ g of the particle radius distribution:

σ = lnσ g

Equation 2.3

In order to determine the droplet size distribution, the measured echo attenuation ratios (I/I 0 ), which are denoted as Ep(δ), are compared to the calculated echo intensity ratios (Equation 2.4). This is done for each magnetic gradient pulse width δ, and by means of a least-squares analysis the best estimates for R med and σ are obtained.

Ep(δ) = ( (0-) R 3 P n (R)E(R,δ) dR) /( (0-) R 3 P n (R) dR )

Equation

2.4

where Ep is the total attenuation of the NMR-signal of the population of possible spherical droplet radii and E(R,δ) can be calculated by using Equation 2.5.

Chapter 2 Materials and methods

E(R,

)

=

exp

(

2

2

2

g

2

D

)

m = 1

2

D

R

(

2

m

)

2

R

4

*

4

m

(

2

m

2 exp(

2)

2

m

D

R

2

)

+

2 exp(

2

m

D (

)

R

2

)

2.3.3.2 Data processing by Excel

2 exp(

2

m

D

R

2

)

+

2 exp(

2

m

D (

+

)

R

2

)

Equation 2.5

This method gives the volume-weighted geometric mean diameter D 33 and uses the exported data from the Droplet Size Application and consists of 6 steps. Firstly, echo attenuation ratios are calculated by implementing a certain droplet radius, a δ-value, a set of λ m values as well as values for D, γ, and g in Equation 2.1. This is repeated for all δ-values that were used in the analysis, while keeping the droplet radius constant. Secondly, the echo attenuation ratios are calculated for another droplet radius for the different δ-values, obtaining attenuation ratios as function of the droplet radius and δ. This yields curves as shown in Figure 2.5, in which the relative positioning of the measured and the calculated data can be observed. In the Excel file, the set of radii is 0.25; 0.75; 1.25; 1.75; 2.25; 2.75; 3.25; 3.75; 4.25 and 4.75 µm. Choosing a different set of radii is possible, but time consuming. In Appendix A, a part from a data sheet in Excel that represents the calculated echo attenuation ratios as a function of the radius and δ of the emulsion H0.75/1.50 (sample 1) is represented. Thirdly, in prospect of expressing the water droplet size in diameter and since the lognormal volume-weighted probability distribution as a function of droplet radius is equal to the lognormal volume-weighted probability distribution as a function of droplet diameter, the latter will be used in the next equations. The relative frequencies (P(ɸ)) for each droplet diameter ɸ are calculated and normalized (P norm (ɸ)=P norm (Rx2)) by dividing it by the sum of the relative frequencies by using Equation 2.6.

Chapter 2 Materials and methods

P

v,norm

(R)

=

P

v,norm

(

)

=

P (R)

v

(

2 )
2
)

-1

exp[-(ln( )- ln(R

33

2

x2) /(2

2

)]

=

(P (R))

v

(P (

v

))

Equation 2.6

Hereby, P v represents the lognormal volume-weighted distribution and the following parameters are brought in: the distribution width (σ), whose starting value is directly obtained from the Droplet Size application, and the volume-weighted geometric mean diameter D 33 (or R 33 x2), whose initial guess is calculated by putting the number- weighted geometric mean radius (R med ) in Equation 2.7 and converting it to diameter. The radius R med is directly obtained from the output of the Droplet Size application. Equation 2.6 also needs an arbitrary diameter (ɸ).

R 33 = R med exp(3σ 2 )

Equation 2.7

Fourthly, the echo attenuation ratios (Ep(δ)), that will be compared to the ones measured, are obtained by multiplying the matrix consisting of rows with E values corresponding to different δ-values and columns with E values corresponding to different droplet radii (Appendix A) by the vector consisting of normalized relative frequencies for the different droplet diameters (or radii). This operation boils down to executing Equation 2.8, which is a modified version of Equation 2.4. The main modification is the replacement of a number-weighted distribution in Equation 2.4 by a volume-weighted distribution in Equation 2.8.

Ep(δ) = Σ [P v, norm (ɸ) E(R,δ)]

Equation 2.8

Fifthly, the sum of the squared differences between the calculated and the measured echo attenuation ratios is calculated. Sixthly, through ‘solver’ of Excel, the best fitting volume-weighted geometric mean diameter D 33 (or R 33 x2) and the distribution width (σ) are determined, so that the sum of squared differences between the calculated and experimental echo attenuation ratio is the smallest. The volume-weighted arithmetic mean diameter D 43 is obtained by using Equation 2.9. Also here, the distribution width (σ) is converted to the geometric standard deviation (σ g ) by using Equation 2.3.

Chapter 2 Materials and methods

R 43 = R 33 exp(σ 2 /2)

Equation 2.9

Figure 2.6 illustrates relative normalized frequencies of a lognormal volume-weighted distribution of the emulsion H0.75/1.50 (sample 1) as a function of particle diameter

class, which are obtained by calculation in Excel and by usage of R 43 from the output

in the Droplet Size application, which is first converted to R 33 by Equation 2.9 and

then included into Equation 2.6. A discrepancy between the lognormal volume- weighted distribution by Excel and the Droplet Size application can be observed, which might be due to the difference in Equation 2.1 and Equation 2.5.

Figure 2.7 illustrates the experimental and the best fitting calculated echo attenuation ratios versus magnetic gradient pulse width (δ). To get a better fit, the product of calculated normalized relative frequencies and calculated echo attenuation ratios is multiplied by a factor (the multiplication factor MF (Equation 2.10)) which is also included as a freely adjustable parameter in the solver.

Ep(δ) = Σ (MF)[P v, norm (ɸ) E(R,δ)]

Equation 2.10

The highest measured echo intensity at the smallest value for δ is considered as I o (the echo intensity without gradient). This results in the fact that the ratio (E) of the echo intensity I relative to I o , associated with the smallest δ-value, is allocated a value of unity. As it is not possible to have an experimental point corresponding to a δ-value of 0µs, the measured relative echo decay data (E i ) are slightly overestimated, which can be compensated by multiplication of the calculated data by a multiplication factor, which is generally slightly larger than unity.

A major drawback of the Excel approach is that it includes only a limited number of

particle size classes, which are quite broad (especially in the low size region). This will surely be problematic for samples with a rather narrow particle size distribution.

Chapter 2 Materials and methods

0.8 0.7 0.6 Excel 0.5 DSA Relative 0.4 normalized frequency 0.3 0.2 0.1 0 Particle
0.8
0.7
0.6
Excel
0.5
DSA
Relative
0.4
normalized
frequency
0.3
0.2
0.1
0
Particle diameter class
0 - 1 1 µm
- 2 2 µm
- 3 3 µm
- 4 4 µm
- 5 5 µm
- 6 6 µm
- 7 7 µm
- 8 8 µm
- 9 9 - µm
10 µm

Figure 2.6: Relative normalized frequencies of the lognormal volume-weighted distribution of the emulsion H0.75/1.50 (sample 1) measured at 5°C as a function of particle diameter class, obtained by calculation in Excel according to section 2.3.3.2 and by inclusion of the output (σ and the R 43 ) of the Droplet Size application (DSA) in Equation 2.6 after conversion of R 43 to R 33 via Equation 2.9 (γ=267518T -1 s -1 , g=1.73953T/m, =0.2s, D=1.289E-09 m 2 /s).

1 0.9 Ep calculated Ep measured 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
1
0.9
Ep calculated
Ep measured
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
Ep (-)

δ (s)

Figure 2.7: Measured and calculated (using R 33 =3.0277µm, σ=0.0782 and MF=1.0448) echo attenuation ratio versus magnetic gradient pulse width (δ) of the emulsion H0.75/1.50 (sample 1) at 5°C (γ=267518T -1 s -1 , g=1.73953T/m, =0.2s, D=1.289E-09 m 2 /s).

Chapter 2 Materials and methods

2.3.3.3 Data processing by Matlab

Also this method uses the exported data from the Droplet Size Application. The difference between applying the Droplet Size application or Excel, lies in the fact that this method is less cumbersome in terms of time consumption to result in an outcome. Data processing by Matlab is based on the ‘inverse’ approach. Instead of calculating the (lognormal) probability for a certain droplet size, which is done in Excel and the Droplet Size application, a set of radii is calculated from the cumulative probability (Figure 2.8), which ranges from 0.01 up to 0.99 with a fixed interval width of 0.01 units so that each radius has the same probability. In contrast, as stated in 2.3.3.2, Excel uses only ten droplet diameters to calculate the lognormal probabilities (starting from 0.5 µm in steps of 1 µm up to 9.5 µm diameter). This limitation results in the fact that in Figure 2.8 only a few diameters out of ten are represented. For the sake of clarity of Figure 2.8, only interval widths of 0.1 units are represented and to allow comparison with Excel, the probability as a function of diameter instead of radius is given. In order to calculate the water droplet size, three files are used: Simulation_data.m, Fitlien.m and Minim_Lien.m (Appendix B), where the latter actually refers to Fitlien.m. The values for the free diffusion coefficient of water in the dispersed phase and the gyromagnetic constant (γ), the reference to the Excel file, which contains the values of the magnetic gradient pulse width (δ), the magnetic field gradient (g), the measured intensities I and I 0 , are used in Simulation_data.m. Firstly, the echo intensities I(δ,R) (where I refers to the echo intensity and R refers to the radius that corresponds to the cumulative probability of a lognormal distribution), are calculated by multiplying Equation 2.1 by I 0 . As initial guess for the parameter I 0 , the experimental echo intensity obtained at the smallest δ is used. The radius is calculated (and not arbitrarily chosen as in Excel or the Droplet size application) by means of the inverse approach (Figure 2.8), based on the natural logarithm of the geometric mean radius R 33 of the lognormal volume-weighted particle radius distribution (µ) and the natural logarithm of the geometric standard deviation σ g of the particle radius distribution (σ), according to Equations 2.11 and 2.12.

µ

= ln(R 33 ) = ln[R 43 2 /(stdev 2 + R 43 2 )]

Equation 2.11

σ = ln(σ g ) = [ln(stdev 2 / R 43 2 +1)]

Equation 2.12

Chapter 2 Materials and methods

Thereby, stdev and R 43 represent the arithmetic standard deviation and arithmetic mean radius of the volume-weighted particle radius distribution.

radius of the volume-weighted particle radius distribution. Figure 2.8: Cumulative distribution of the diameter in

Figure 2.8: Cumulative distribution of the diameter in micrometer (assuming a median diameter of 1.62µm and stdev of 0.2µm) with graphical representation of the two approaches: the horizontal arrows illustrate the inverse approach (used in Matlab) and the vertical arrows illustrate the direct approach (used in Excel).

Secondly, the echo intensities (Raccum(δ)) are obtained by the transformation of the I(δ,R). Thirdly, the file Fitlien.m calculates the best fitting volume-weighted arithmetic mean radius (R 43 ) (which is denoted as ‘mean’ in the Matlab file) and the arithmetic standard deviation of the particle radius distribution (which is denoted as ‘stdev’ in the Matlab file), so that the squared difference between the experimental echo intensities I(δ) and calculated echo intensities (Raccum(δ)) is the smallest. Figure 2.9 is a graphical representation of the experimental and best fitting calculated echo intensities versus magnetic gradient pulse width (δ). A logarithmic transformation of the least squared difference results in the so-called maximum likelihood (MLH). Matlab examines the possible combinations of means and standard deviation and ceases its action when the lowest MLH is found. The best fitting arithmetic standard deviation of the particle radius distribution is converted to σ g by using Equation 2.12.

Chapter 2 Materials and methods

8000 I(small delta) Raccum(small delta) 7000 6000 5000 4000 3000 2000 1000 0 1 2
8000
I(small delta)
Raccum(small delta)
7000
6000
5000
4000
3000
2000
1000
0
1
2
3
4
5
6
7
8
9
Small delta (s)
x 10 -3
Echo intensity

Figure 2.9: Experimental (I) and best fitting calculated (Raccum) echo intensity (assuming

= 1.0860 and I 0 = 7,72E+03) versus magnetic gradient pulse width (δ) of the

emulsion H0.75/1.50 (sample 1), measured at 5°C. (γ=267518T -1 s -1 , g=1.73953T/m, =0.2s, D=1.289E-09 m 2 /s).

R 33 =1.5596µm, σ

g

2.4.4 Statistical methods

It is assumed that water droplet sizes in w/o-emulsions are characterized by a lognormal distribution. Whether a confidence interval of the mean of three volume- weighted geometric mean diameters of three samples can be constructed, depends on the distribution of the volume-weighted mean diameters. A small scale experiment (Kolmogorov Smirnov-test, S-Plus) of replica’s with the same composition revealed no rejection of the hypothesis of a normal distribution of the volume-weighted geometric mean diameters on the 5% significance level. Hence, the construction of a 95% confidence interval of the mean of three volume-weighted mean diameters of three samples is possible. The average of three samples for an emulsion, the 95% confidence interval of the mean and the standard deviation of three samples are calculated with Excel (Microsoft Office 2003).

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Furthermore, parametric tests, which rely on the normal distribution, can be applied. In order to compare the values of D 33 between the three data processing methods or compositions of emulsions, two-sample paired and two-sample independent sample t- tests (Excel) are used, respectively. A significance level of 5% is applied. If more than two groups need to be compared simultaneously, a Bonferroni correction (S-Plus) was applied. R-squared values are calculated in Excel and indicate the variability in a measured data set that is accounted for by the calculated data set. For the Droplet Size application and Excel, the calculated and measured echo attenuation ratios are compared, whereas for Matlab, the echo intensities are compared. Whenever multiple outcomes for the same sample were obtained, the outcome characterized by the highest R-squared value was used in the statistical analysis.

2.4.5 Important issues during pfg-NMR analysis

2.3.5.1 Temperature of the emulsion during pgf-NMR analysis

Measurements should be done at a stabilized temperature, because diffusion data are highly temperature sensitive. The Stokes-Einstein equation shows the connection between temperature and diffusion coefficient:

D=kT/6πηR

where k is the Boltzmann constant, T is temperature, η is the viscosity and R is the radius of a spherical particle. The diffusion coefficient increases at higher temperatures because of the temperature effect on the viscosity. Samples are analyzed at 5°C for having a maximum microstructural stability of the sample. At this temperature, interdroplet water diffusion is minimized and only intradroplet diffusion results in the attenuation of the NMR-signal (Van Lent et al., 2008a; Price, 1997; Voda & van Duynhoven, 2009). Fourel et al. (1994) noted that as temperature decreases, the standard deviation is approximately constant, but there is a large decrease in mean diameter of the distribution, because then the condition of restricted

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diffusion is better obtained. At higher temperatures, the continuous phase becomes more permeable for the water and interdroplet diffusion becomes more interfering. In conclusion, as long as the temperature is around 5°C and the calibration step is performed at the same temperature, then the exact temperature is of less importance than the stability of the temperature due to the adaption of the output to the calibration.

2.3.5.2 Assumption of a lognormal size distribution

Systematic deviations between results of the fit and the experimental data can be due to random errors or to the not completely appropriate assumed size distribution, namely the lognormal size distribution. A way to cope with the fact that the assumption of log normality cannot hold, is by modification of the equations. In a study of Fourel et al. (1994) an unrestricted diffusion term was added to the equation of Ep (Equation 2.4) to get better results in the case of no perfect lognormal distribution of droplet size or in the case of existence of possible unrestricted diffusion due to poor emulsification.

2.3.5.3 Advantages of pfg-NMR analysis for determination of water droplet size

In comparison of other methods, pfg-NMR has the advantage that the same samples can be tested repeatedly, and dilution and centrifugation are not necessary. This non- invasive technique is fast (about 10 min) and able to test opaque emulsions in which the continuous phase is not conducting. However, there is a lack of a reference for validation (Peña and Hirasaki, 2006).

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2.5 Qualitative particle size analysis of water in w/o-emulsions

2.4.1 Fluorescence microscopy

Water droplets of w/o-emulsions are visualized with the water-soluble fluorescent agent eosinY (MP Biomedicals Inc., Illkirch, France) and a fluorescence microscope (Leitz Diaplan Incident light fluorescence 3-λ Ploemabak illuminator, Wild Leitz, Germany) (Figure 2.10) equipped with Cell-software (Olympus Soft Imaging Solutions GmbH, Germany).

(Olympus Soft Imaging Solutions GmbH, Germany). Figure 2.10: Incident light fluorescence 3- λ Ploemabak

Figure 2.10: Incident light fluorescence 3-λ Ploemabak illuminator. 1: lamp housing; 2:

lamp housing mount; 3: BG38 red-absorption filter; 4:

excitation light blocking slide; 5:

field diaphragm; 6: filter block interchange control; 7: stop for twin wavelength study; 8: 3- lambda Ploemabak; 9:

localization of the camera.

In this fluorescence microscope, light from a mercury lamp is filtered by an excitation filter in the filter block, which reflects the light under an angle of 90° through the objective lens on the sample. The excitation filter selects the incoming light or excitation wavelength range. EosinY absorbs the light and emits fluorescent light. The latter passes through the same objective and straight through an emission filter of the same filter block towards the camera or binocular tubes. The emission filter selects the emission wavelength range. The choice of filter block depends on the maximum excitation and emission wavelength of the applied fluorescent agent. The applied filter block (I2/3) passes excitation wavelengths between 450 to 490nm (BP450-490 or band pass filter) and emission wavelengths above 520nm (LP520 or long-pass filter). Depending on the settings, the microscope can be used as a fluorescence or polarized

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light microscope. The fluorescence mode requires opening of filters nr.4 in Figure 2.10 and centration of the mercury or metal halide light by adjustment of the lamp housing. Filter nr.3 (BG38) is a short-pass filter that passes excitation wavelengths around 500nm, hence this resembles the excitation filtration of filter block I2/3.

2.4.2 Fluorescence

The fluorescence process comprises three steps (Figure 2.11). Firstly, the fluorescent molecule is excited by absorption of a photon, which comes from the light source in the fluorescence microscope or fluorimeter, by which the molecule moves from the ground state to one of the vibrational levels of the exited state in 10 -15 to 10 -16 s. Secondly, vibrational relaxation (or internal conversion) occurs towards a lower energy vibrational excited state without emission of light. This step takes approximately 10 -12 s. Thirdly, by emission of a photon, the fluorescent molecule returns to one of the levels of the ground state, which results in different emitted photon energies. As a consequence, light is emitted over a range of wavelengths in a time span of 10 -9 s.

over a range of wavelengths in a time span of 10 - 9 s. Figure 2.11:

Figure 2.11: The fluorescence process. S 0 , S 1 and S 2 stand for ground singlet state, first excited singlet state and second excited singlet state, respectively. Wavy arrows denote that the timescale is larger than for straight arrows.

The emitted light is characterized by a smaller energy and hence in accordance to the law of Planck (E = hʋ = hc/λ), a longer wavelength than the light that leads to

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excitation of the molecule. The difference in energy or wavelength between the absorbed and emitted photon is called the Stokes shift. A large Stokes shift is beneficial with regard to the sensitivity of the detector, because in this way by the usage of filters, the excitation light that reaches the detector can be limited.

2.4.3 EosinY

EosinY or tetrabromo fluorescein is a red dye. Besides the usage as a coloring agent in histological research and as a pharmaceutical product that dehydrates varicella- blisters, eosinY can be used as a fluorescent agent (Figure 2.12). The letter Y is an abbreviation of ‘yellowish’ due to the slightly yellowish shade. In contrast, eosin B has a blue shade and is a dibromodinitro fluorescein derivate.

a blue shade and is a dibromodinitro fluorescein derivate. Figure 2.12: Chemical structure of eosinY

Figure 2.12: Chemical structure of eosinY (tetrabromofluorescein)

Although the aqueous solubility is 400g/L, the dissolution rate is quite low. Therefore, a magnetic stirrer was used. Repeating the analysis of the same sample in the fluorimeter or illumination by UV and visible light results in repetitions of the excitation and emission cycle until the fluorescent agent is destructed or photo bleached. Illumination decreases both absorption and fluorescence. In a study of Kola (2010), within one day, fluorescence of eosinY-solutions, which were stored in colorless glass in a light room, was found to be destroyed for 98% and completely after 2 days. Even when the eosinY-solutions were stored in brown colored flasks in a light room, 92% of the fluorescence intensity is gone after 3 days. When the aqueous solutions were kept in brown flasks in the shade, only 15% reduction of the fluorescence of eosinY was observed after 3 days

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(Kola, 2010). Hence, the investigated eosinY-solutions in this study are covered with aluminum foil. Above pH 5.58, the fluorescence intensity is constant and higher than below this pH. Regarding temperature, Kola (2010) demonstrated that the fluorescence intensity is stable within the studied temperature range of 10°C to 41°C. Stockert et al. (1994) found that the addition of sodium azide to eosinY-solutions might create new absorption (520nm) and emission (478nm) peaks. Seema and Babulal (2009) reported on the interaction between eosinY and some surfactants other than sodium caseinate. Therefore, the spectrofluorimetric behavior of eosinY in the presence of sodium caseinate was investigated.

2.4.4 Fluorimetric analysis of w/o-emulsions

2.4.4.1 Fluorimeter

A fluorimeter (Varian fluorescence spectrophotometer, Cary Eclipse) was used to perform preliminary investigation of aqueous eosinY-solutions in the presence of different agents. As such, the influence of addition of a phosphate buffer, sodium azide and sodium caseinate to the aqueous solution on the maximum excitation and emission wavelength of eosinY was examined.

2.4.4.2 Determination of a suitable concentration of eosinY

A suitable concentration of eosinY in distilled water that is associated with high fluorescence intensity was looked for. This concentration is used in the water phase of the w/o-emulsion (50:50, w/w) for fluorescence microscopic investigation. The software tool ‘Concentration’ of Cary Eclipse was applied. The limitation that the fluorimeter becomes saturated at very high fluorescence intensities must be taken into account and forces the quest for a suitable concentration of eosinY to be done at sub maximum excitation and emission wavelengths. On the basis of Figure 2.13, an eosinY concentration of 10µg/mL was chosen for the water phase in emulsions. Linear regression on the first nine data points results in y=537277x-11 and R 2 =0.93.

Chapter 2 Materials and methods

1000 100 10 1 1.0E-08 1.0E-07 1.0E-06 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01 1.0E+00 0.1 Fluorescence
1000
100
10
1
1.0E-08
1.0E-07
1.0E-06
1.0E-05
1.0E-04
1.0E-03
1.0E-02
1.0E-01
1.0E+00
0.1
Fluorescence intensity (a.u.)

Eosin Y concentration (g/100mL)

Figure 2.13: Fluorescence intensity as a function of the logarithm of eosinY concentration in water. Excitation wavelength=514nm. Emission wavelength= 564nm.

2.4.4.3 Determination of maximum excitation and emission wavelength

By application of the 3D Mode and selection of the Excitation or Emission-button in the software tool ‘Scan’ of Cary Eclipse, the maximum excitation or emission wavelength can be determined, respectively. During the scan, the fluorescence intensity, which is related to a predetermined range of excitation wavelengths for a predetermined range of emission wavelengths, is recorded. At an emission wavelength of 480nm the intensity is measured for excitation wavelengths from 480 to 600nm. The next scan measures the intensity at an emission wavelength of 482nm. Scanning is proceeded until the emission wavelength of 600nm. The highest recorded intensity corresponds to the scan at the maximum emission wavelength. An example of an excitation spectrum scan is represented in Figure 2.14, in which excitation and emission range from 480 to 600nm in steps of 2nm. Fluorescence intensity is indicated by different colored regions, which is defined as the y-axis of the spectrum. High fluorescence intensity (or red colored regions) gives an idea about the range of the maximum excitation and emission wavelength and can be read from the x-axis and z-axis, respectively. The z-axis represents the number of steps between the lowest and highest analyzed emission wavelength. In this example, the maximum unit on the z-axis is 60, since the difference between 600 and 480nm amounts of 120nm and the size of the steps is 2nm. Conversion of units on the z-axis to the emission wavelength in nanometer is done by Equation 2.13. Analogously, in an emission spectrum, the

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excitation wavelength can be calculated from the units on the z-axis. Hereby, the x- axis and y-axis represent the emission wavelength and fluorescence intensity, respectively.

Emission wavelength (nm) = [(Z-axis units)(size of the steps to go from the first to the last selected emission wavelength) + lowest analyzed emission wavelength (nm)]

Equation 2.13

1000 60.00 55.00 962.92 888.75 50.00 800 814.58 45.00 740.41 40.00 600 666.24 35.00 592.07
1000
60.00
55.00
962.92
888.75
50.00
800
814.58
45.00
740.41
40.00
600
666.24
35.00
592.07
30.00
517.91
400
25.00
443.74
369.57
20.00
200
295.40
15.00
221.23
10.00
147.07
5.00
0
72.90
480
500
520
540
560
580
600
-1.27
480.00
490.00
500.00
510.00
520.00
530.00
W avelength (nm)
540.00
550.00
560.00
570.00
580.00
590.00
600.00
Wavelength (nm)
Z Axis
Intensity (a.u.)
518.05 , 132.512

Figure 2.14: Contour plot of the scan for excitation and emission wavelength ranging from 480 to 600nm in 60 steps of 2nm. X-axis: excitation wavelength (nm). Z-axis: number of steps that are used to go from 480 to 600nm emission wavelength. Red color denotes high fluorescence intensity, green color denotes low fluorescence intensity. Eosin concentration in distilled water is

5µg/mL.

Concerning the determination of the maximum excitation and emission wavelength, a concentration of eosinY in water of 5µg/mL was used and can be performed in two ways. In the case that the maximum excitation wavelength must be determined, the first method consists of narrowing down the emission wavelength range which lies symmetrically around the maximum emission wavelength. However for determination of the maximum excitation and emission wavelength, Sometimes a lower concentration of eosinY must be analyzed to avoid approaching the maximum recordable fluorescence intensity. The second method comprises the measurement of the fluorescence intensity by shifting the narrowed range of emission wavelengths to sub maximum regions. Hence, the maximum recordable fluorescence intensity is not reached. The eosinY-concentration is left unchanged. In the case that the maximum

Chapter 2 Materials and methods

emission wavelength must be determined, the same reasoning applies, but now the excitation wavelength range must be narrowed down.

An example of such an excitation spectrum is given in Figure 2.15, for which the maximum excitation wavelength matches 517nm. As the emission wavelength was located at sub maximum regions, this is an example of the second method. Eleven different cultured lines can be observed because the emission wavelength is elevated from 554nm to 564nm in steps of 1nm.

wavelength is elevated from 554nm to 564nm in steps of 1nm. Figure 2.15: Graphical representation of

Figure 2.15: Graphical representation of the determination of the maximum excitation wavelength of eosinY at a concentration of 5µg/mL. Excitation wavelength range: 490-540nm. Emission wavelength range: 554-564nm.

In order to demonstrate that the maximum excitation or emission peak at different suboptimal emission or excitation wavelengths, respectively, is located at the same wavelength, the maximum emission was recorded at different ranges of sub maximum excitation wavelength as depicted in Table 2.3. No deviating trends could be observed. The average and standard deviation of the nine maximum emission wavelengths is 547nm and 0.7nm, respectively. Hence, identical outcomes can be achieved at excitation wavelengths that are 67nm (527nm minus 460nm) lower than the maximum excitation wavelength. In order to test the repeatability of the fluorimetric analysis, the same sample was repeatedly analyzed at the same excitation wavelength range of 480 to 490nm. The average maximum emission wavelength and standard deviation of ten repetitions was 547.2nm and 0.7nm, respectively.

Chapter 2 Materials and methods

Table 2.3: Recorded maximum emission wavelengths at different excitation wavelength ranges of eosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%,w/v) and sodium caseinate (1.25%,w/v).

Excitation range (nm)

Max. emission wavelength (nm)

500-510

547

495-505

548

490-500

548

485-495

547

480-490

547

475-485

546

470-480

546

465-475

547

460-470

547

2.4.5 Confocal laser scanning microscopy

Two major differences between fluorescence microscopy and confocal laser scanning microscopy (CLSM) are the depth selectivity and the applied light source. Whereas fluorescence microscopy detects fluorescence of all layers in the specimen, images by CLSM can be taken of a selected layer or depth of the specimen. This is achieved by focusing the laser beam, which is the light source, by an objective lens into a certain volume of a certain layer of the specimen whereby the emitted light from other layers does not reach the detector due to the presence of a detector-aperture (Figure 2.16).

due to the presence of a detector-aperture (Figure 2.16). Figure 2.16: Schematic representation of the principle

Figure 2.16: Schematic representation of the principle of confocal laser scanning microscopy

Chapter 2 Materials and methods

2.6 Water-in-oil-in-water emulsions

2.9.3 Composition of the w/o/w-emulsions

Depending on the preparation method, 70g or 100g of w/o-emulsion (50:50, w/w) was prepared. Addition of the external water phase to a w/o-emulsion in a ratio 40:60 (w/w) gave rise to a w/o/w emulsion. The water phase of the w/o-emulsion contained 1.25% (w/v) sodium caseinate, 0.02% (w/v) sodium azide, 0.001% (w/v) eosinY and a phosphate buffer (pH6.7). The fat phase consisted of 2.5% (w/v) PGPR in soft PMF or a mix of soft PMF and Hozol. Here, eosinY is added to ameliorate the visualization of the internal and external water phase. Unless stated differently, the external water phase only differs from the internal water phase in the concentration of sodium caseinate (1%, w/v) and the absence of eosinY.

2.9.4 Preparation of the w/o/w-emulsions

Four manufacturing methods were attempted, which differed in the utilized apparatus and the modus of sampling.

2.9.4.1 Method A

This method entails subsequent application of an Ultraturrax TV45 (until visual satisfaction) and a Microfluidizer M110S (at 6bar for 1.5 minutes) in order to fabricate 100g of w/o-emulsion (50:50, w/w) with soft PMF as the fat phase. W/o- emulsions were kept in a water bath of 50°C before mixing of the external water phase with an Ultraturrax (for 0.5 minutes) and Microfluidizer (at 1bar for 1.5 minutes) in prospect of making a double emulsion. The samples were collected in glass NMR-tubes with an outer diameter of 18mm, filled for 40mm height (or 8mL), covered with parafilm and cooled in an ice bath. Prior to NMR-analysis, samples were vortexed and meanwhile maximum 100µL 10mM MnCl 2 (Normapur, VWRinternational, Fontenay sous Bois, France) was added to 8mL emulsion.

Chapter 2 Materials and methods

2.9.4.2 Method B

This method comprises the generation of 70g w/o-emulsion by an Ultraturrax TV45. Fourty grams of w/o-emulsion were blended with 60g of external water phase with an Ultraturrax S25-10G (Figure 2.17) at 24000rpm for 2 minutes, unless stated differently. In a few samples, the concentration of sodium caseinate was 1.25% (w/v) instead of 1% (w/v) (2.5.2) in the external water. The type of fat phase is mentioned for each experiment. Afterwards, double emulsions were further mixed with a continuous Ultraturrax DK25 (Figure 2.17) at 24000rpm for a specified time. The advantage of the latter type of Ultraturrax is the reduction of foam formation and hence higher availability of surfactant to stabilize the emulsion instead of air. Unless stated differently, samples were cooled in an ice bath. For NMR experiments, the samples were filled in NMR-tubes up to 40mm with an outer diameter of 18mm and received MnCl 2 directly after production or just before NMR-analysis. The latter required vortexing.

or just before NMR-analysis. The latter required vortexing. Figure 2.17: Construction of a continuous Ultraturrax DK25

Figure 2.17: Construction of a continuous Ultraturrax DK25 flow chamber (bottom) and an Ultraturrax S25- 10G (top)

Chapter 2 Materials and methods

2.9.4.3 Method C

The third method differs from the previous methods only in the filling of the NMR- tubes. NMR-tubes with an outer diameter of 18mm, were filled in such a way that the detection zone of the spectrometer with a length of 25mm (Zhu, 2011) (Figure 2.18) comprises the complete sample. It is chosen to fill the tube for 15mm, which means that it needs to be elevated for 27mm in the spectrometer by means of wrapping a rubber band around the tube. Right after production, 28µL 10mM MnCl 2 is added to 3mL of emulsion and vortexed. In this manner, errors owing to improper homogenization of the creamy layer by vortexing are avoided.

homogenization of the creamy layer by vortexing are avoided. Figure 2.18: The detection zone (red) of

Figure 2.18: The detection zone (red) of a Maran Ultra 23 spectrometer lies between 22mm and 47mm height (Zhu, 2011). Hence, incompletely filled tubes (e.g. considering 15mm of sample) must be elevated (e.g. over 27mm) to enable the detection of their whole contents.

Regarding the effect of the elevation of a tube in the Maran Ultra 23 spectrometer on the T 2 -distribution, Figure 2.19, 2.20 and 2.21 illustrate the location of the detection window. Elevation for 0cm, 0.75cm, 1.5cm, 3cm, 4cm, 5cm and 6cm of an NMR-tube in the Maran Ultra 23 spectrometer was realized by means of a rubber band. To a double emulsion with P1.25/2.5/1, 75µL of 10mM MnCl 2 was added to a 4cm height filled NMR tube (about 8mL) right after production. Twenty four hours later, analysis was performed on the phase separated sample, that was not vortexed.

Chapter 2 Materials and methods

According to Zhu (2011), the detection window of the Maran spectrometer is situated between 2.2 and 4.7cm above the bottom. As long as the sample is located in the detection zone of the spectrometer and the signal is maximized by optimization of the receiver gain, the total area under the curve stays constant. Upon elevation, the area under the curve that is related to the fat phase and internal water decreases, whereas the area under the curve of external water increases. The fat globules with internal water are enriched at the top of a phase separated sample, whereas the external water content is higher at the bottom of a tube.

external water content is higher at the bottom of a tube. Figure 2.19: Schematic representation of

Figure 2.19: Schematic representation of the experiment concerning the elevation of the NMR- tube in the Maran Ultra 23 spectrometer. P1.25/2.5/1 with 75µL of 10mMMnCl 2 in a 4cm filled (about 8mL) NMR-tube of 1.8cm diameter, added directly after production. (P/A 1-3/03/11)

The fact that at an elevation of 5cm a signal could still be received, might be due to the inaccurate elevation by means of the rubber band or a broader detection window than 2.5cm. In order to assess the detection zone a small experiment was performed. An NMR-tube of 18mm outer diameter was gradually filled with water from 0 to 12mL with a pipet. For each water volume the area under the curve in the T 2 - distribution was plotted versus the water volume in the tube (Figure 2.22). By linear

Chapter 2 Materials and methods

regression it was found that the area under the curve linearly increases with a water volume of 3.7 to 9.2mL, which can be converted to a detection window of 20.5 to 50.5mm (5.51mm/mL). Based on this experiment, the width of the detection window is 3.0cm, which can explain the recorded signal at 5cm elevation in Figure 2.21. However, close to the detection window margins, more inaccuracy can be noticed (Zhu, 2011), hence the detection zone of 2.2cm to 4.7cm can still be applied.

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Figure 2.20: T 2 -distribution of samples at different heights in the Maran Ultra 23 spectrometer. Black arrows denote the direction of change upon elevation. P1.25/2.5/1, 75µL of 10mM MnCl 2 after production. (P/A 1-3/03/11).

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Figure 2.21: Area under the curve for different relaxation modes and elevation of the NMR-tube. P1.25/2.5/1 with 75µL of 10mM MnCl 2 directly added after production. (P/A 1-4/03/11)

Chapter 2 Materials and methods

18000 16000 14000 12000 10000 8000 6000 4000 2000 0 0 2 4 6 8
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Figure 2.22: Area under the curve in the T 2 -distribution versus water volume in an NMR-tube of 18mm outer diameter. Receiver gain of the spectrometer was kept constant (RG=1.64).

2.9.4.4 Method D

Method D is a combination of Method B and C. This method is similar as Method B

regarding the preparation of a w/o-emulsion, but besides an Ultraturrax TV45 (until

visual satisfaction), also a Microfluidizer (at 6bar for 1.5 minutes) is utilized in order

to make a w/o-emulsion. The w/o/w emulsion is made exactly like in Method B. The

filling of the NMR-tube is done as in Method C.

2.10 Quantitative analysis of the enclosed water volume and yield of water-in-oil-in-

water emulsions

2.10.1 The CPMG-experiment

The enclosed water volume was estimated by transverse relaxation time constant (T 2 )

distribution measurements via CPMG-experiments (Carr Purcell Meiboom Gill) in the

presence of MnCl 2 with a Maran Ultra 23 spectrometer. The temperature of the

samples was +5°C and the Set Temperature of the spectrometer was -7°C. One radio

Chapter 2 Materials and methods

frequency pulse of 90° is applied and N radio frequency pulses of 180°, which results in N spin echoes or CPMG spin-echo trains (Figure 2.23).

in N spin echoes or CPMG spin-echo trains (Figure 2.23). Figure 2.23: The CPMG-experiment The basis

Figure 2.23: The CPMG-experiment

The basis of an NMR experiment is the change of the total magnetic moment or magnetization of a sample, which can be represented in a xyz-space. Without pulses, the magnetization vector is aligned with and precesses about the static field B 0 or z- axis. Application of a 90° pulse (radio frequency pulse or B 1 ) puts the magnetization vector (M 0 ) into the xy-plane (Figure 2.24). All spins experience the same applied pulse and are in phase along the y-axis.

the same applied pulse and are in phase along the y-axis. Figure 2.24: Change of the

Figure 2.24: Change of the magnization by a 90°pulse, without 180°pulses. The magnetization vector aligns back with the static field B 0 over time scales of T 2 .

Without subsequent 180° pulse, the magnetization vector is completely restored along the z-axis. More into detail, over time scales of T 2 , the spins lose phase coherence in the xy-plane due to differences in magnetic field in the sample (spin spin interactions) or inhomogeneity in the magnetic field. Because the dephasing is measured in the xy- plane, which is perpendicular to the z-axis, this is called a transverse magnetization experiment. Alternatively, the loss of coherence can also be observed along the z-axis,

Chapter 2 Materials and methods

which happens over time scales of T 1 and is denoted as the recovery of the longitudinal magnetization. Application of a 180° pulse flips the magnetization vector around the y-axis (Figure 2.25). The so-called spin isochromats are bundles of spins that experience the same magnetic field. The sum of the spin isochromats is equal to M 0 or magnetization vector at equilibrium, which is equal to the magnetization vector M when aligned with the static field B 0 . As each spin isochromat continues to precess with its frequency, all isochromats will rephase. This rephasing removes the influence of inhomogeneity of the magnetic field on the spins, hence the change of magnitude of the echo will be due to the spin spin interactions only. Application of multiple 180° pulses, results in multiple echos, from which the magnitude further decreases as more pulses are applied (Figure 2.23). The amplitudes of the decaying spin echoes as a function of time yield an exponentially decaying curve, which is characterized by a time constant T 2 .

curve, which is characterized by a time constant T 2 . Figure 2.25: A 90°x pulse

Figure 2.25: A 90°x pulse puts M 0 into the Y-direction (a). Spin isochromats fan out with time (b). A 180°y pulse rotates the spins around the Y-axis (c). Rephasing of the spins results in an echo (d). The difference of magnitude of M in (d) and (a) is a direct measure of T 2 .

After running a script (Appendix C), this (experimental) relaxation time curve (Figure 2.23) is recorded by the software RINMR. Unless stated differently, the receiver gain (RG), which is a parameter that maximizes the signal, is optimized automatically prior to analysis. The calculated NMR is obtained after uploading the experimental data in the software WinDXP and exporting them to Excel (Appendix D). The x-axis or time- axis of the relaxation time curve (Figure 2.26) increases from 403.95µs in steps of 400µs to 3276804µs. The y-axis, which represents the relaxation magnitude, differs

Chapter 2 Materials and methods

among samples. The decay can be mono or bi-exponential, depending on the different water phases present and their differentiation with the paramagnetic agent MnCl 2 . A stock solution of 10mM MnCl 2 in water was prepared. This compound reduces the T 2 - relaxation time of water in contact with it and therefore decays faster. In a double emulsion, MnCl 2 will only end up in the external water phase, on the condition that there is no exchange of water between both water phases through the fat phase. If no distinction between the internal and external water phase of a double emulsion is made, e.g. in the absence of MnCl 2 (Figure 2.26, red curve), or if only an internal water phase is present, e.g. in w/o-emulsions, then a mono exponential decay is acquired. A bi-exponential decay is achieved for w/o/w emulsions after addition of MnCl 2 . The fast and slow decaying part of the bi-exponential curve (Figure 2.26, black curve) are associated with the external and internal water of the double emulsion, respectively.

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Figure 2.26: CPMG T 2 -relaxation decay profile of a w/o/w emulsion P1.25/2.5/1. Before (-MnCl 2 ) and after (+MnCl 2 ) addition of 100µL 10mM MnCl 2. to 8mL sample, P/A: 25-26/02).

By means of illustration, the CPMG T 2 -decay for various emulsions is represented in Figure 2.27. Fast decays are associated with T 2 -analysis of the fat phase. Irrespective of the fat phase, a bi-exponential decay is obtained for double emulsions upon addition of 75µL 10mM MnCl 2 to 8mL sample. A mono-exponential decay can be observed for w/o/w emulsions with an insufficient amount of MnCl 2 , which comes down to an addition of less than 75µL 10mM MnCl 2 to 8mL sample. Contin analysis of the relaxation time curve creates a T 2 -distribution (Figure 2.28) time scale goes up from 100µs to100s, where the increase occurs in steps of (t 3 -

Chapter 2 Materials and methods

t 2 )=(t 2 -t 1 )*1.056. A T 2 -distribution can be constructed directly with the raw data from the relaxation time curve in the software WinDXP or can be rebuilt by exporting the data from WinDXP to Excel (Appendix D). Irrespective of the volume of the sample in the NMR-tube, the total area under the curve is approximately constant. Figure 2.28 (red curve) reveals that without MnCl 2 , only one peak associated with water is observed at a T 2 of about 1s. This peak comprises both the external and the internal water. By addition of MnCl 2 the water peak is split into two peaks corresponding to the external and internal water (Figure 2.28, black curve): the transverse (T 2 ) relaxation time will be longer for water molecules within the fat droplets of the double emulsions than when present in the external water phase to which MnCl 2 is added.

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Figure 2.27: CPMG T 2 -decay profiles for various emulsions on a linear scale (Top) and a logarithmic scale (Bottom). A w/o/w emulsion with insufficient MnCl 2 signifies an addition of less than 75µL 10mM MnCl 2 to 8mL sample. In w/o/w emulsions P1.25/2.5/1 and M1.25/2.5/1 (ratio (soft PMF/Hozol) =3/1), 100µL and 28µL 10mM MnCl 2 was added to (vortexed) 8mL and (non- vortexed) 3mL, respectively.

Chapter 2 Materials and methods

Typical T 2 -relaxation times for water in the absence of MnCl 2 amount to 1s, whereas the relaxation time of the water in contact with MnCl 2 can be found at about 100ms. An additional peak at about 10ms embodies the hydrogen atoms in the liquid part of the fat phase. Hence, a T 2 -distribution of a double emulsion is defined as a trimodal distribution, which is characterized by three modal relaxation times (small, medium and large) and a signal amplitude of the modes.

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