katholieke universiteit leuven...sodium caseinate (1.25%,w/v) 3.4.6 selection of the filter block 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

Chapter 1: Literature review

1.1 Water-in-oil-in-water emulsions

1.1.1 Water-in-oil-in-water technology

1.1.2 Preparation of a w/o/w-emulsion

1.1.3 Encapsulation efficiency

1.1.4 Instability of w/o/w-emulsions

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

1.1.4.2 Water transport between the w1 and w2-phase

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

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

1.2 Traditional dairy whipping cream

1.3 Changes during whipping of cream

1.3.1 Three stages during whipping of cream

1.3.2 Foam stabilization by partial coalescence

1.3.2.1 Determination of partial coalescence

1.3.2.2 Distinction between partial coalescence and complete coalescence

1.3.3 Alternatives to partial coalescence

1.4 Mimicing whipping cream

1.5 Quality characteristics of whipped cream

1.5.1 Overrun

1.5.2 Whipping time

1.5.3 Textural analysis

1.5.4 Physical stability

1.5.4.1 Coarsening of foam

1.5.4.2 Drainage

1.6 Factors determining functional properties of whipping cream

1.6.1 Temperature

1.6.1.1 Tempering

1.6.1.2 Heat treatment

1.6.2 Fat content

1.6.3 Homogenization

1.6.4 Miscellaneous factors

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Table of contents

Chapter 2: Materials and methods

2.1 Commercial butters

2.1.1 Preparation of butter samples

2.1.2 Analysis of butter samples

2.2 Water-in-oil emulsions

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

2.2.3 Composition of the w/o-emulsions

2.2.4 Preparation of the w/o-emulsions

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

2.3.1 Pulsed field gradient-Nuclear Magnetic Resonance

2.3.2 The pfg-NMR experiment

2.3.2.1 Calibration procedure

2.3.3 Restricted diffusion in w/o emulsions

2.3.3.1 Data procesing by the Droplet Size application

2.3.3.2 Data processing by Excel

2.3.3.3 Data processing by Matlab

2.3.4 Statistical methods

2.3.5 Important issues during pfg-NMR analysis

2.3.5.1 Temperature of the emulsion during pgf-NMR analysis

2.3.5.2 Assumption of a lognormal size distribution

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

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

2.4.1 Fluorescence microscopy

2.4.2 Fluorescence

2.4.3 EosinY

2.4.4 Fluorimetric analysis of w/o emulsions

2.4.4.1 Fluorimeter

2.4.4.2 Determination of a suitable concentration of eosinY

2.4.4.3 Determination of maximum excitation and emission wavelength

2.4.5 Confocal laser scanning microscopy


2.5.1 Composition of the w/o/w-emulsions

2.5.2 Preparation of the w/o/w-emulsions

2.5.2.1 Method A

2.5.2.2 Method B

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2.5.2.3 Method C

2.5.2.4 Method D

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

emulsions

2.6.1 The CPMG-experiment

2.6.2 Determination of the enclosed water volume

2.6.3 Determination of the yield of a double emulsion

2.6.4 Statistical methods

2.7 Fat globule analysis by laser diffraction

2.8 Profilometric analysis of double emulsions

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

2.9.1 Commercial dairy cream

2.9.2 W/o/w emulsions

2.9.2.1 First attempt to prepare whippable double emulsions

2.9.2.2 Second attempt to prepare whippable double emulsions

2.9.2.3 Third attempt to prepare whippable double emulsions

2.9.2.4 Fourth attempt to prepare whippable double emulsions

2.9.3 Determination of the whipping time

2.9.4 Determination of the overrun of a whipped emulsion

2.9.5 Physical destabilization (drainage) of whipped emulsions

Chapter 3: Results and discussion

3.1 Optimization of wayer droplet size analysis

3.1.1 Temperature experiment by using a thermocouple

3.1.2 Repeatability experiment

3.2 Quantitative particle size analysis of water droplets in commercial butters

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

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

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

P0.5/2

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

P0.5/2

3.3.2.2 Difference between different data processing methods

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

3.3.2.4 Influence of the emulsifier on the mean water droplet size

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Table of contents

3.3.3 Elevation of the water fraction of emulsions based on Hozol

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

3.3.5 Analysis of emulsions with the hydrophilic surfactant whey protein isolate

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

microscopy

3.4.1 Preliminary investigation of eosinY-solutions by fluorimetry

3.4.2 Determination of the maximum excitation and emission wavelength of eosinY

(5µg/mL) in water


(5µg/mL) in an aqueous phosphate buffer (pH6.7)


(5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v)


(5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v) and

sodium caseinate (1.25%,w/v)

3.4.6 Selection of the filter block in the fluorescence microscope

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

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

microscopy

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

3.5.1 Method optimization

3.5.1.1 Optimization of the preparation method

3.5.1.2 Finding an appropriate concentration of MnCl2

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

experiment

3.5.1.3.1 Analysis of the area under the curve at different temperatures

3.5.1.3.2 Analysis of the signal amplitude at different temperatures

3.5.1.3.3 Analysis of the relaxation time at different temperatures

3.5.1.4 Variation of the duration of mixing of the double emulsions

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

10G

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

3.5.2 Method application

3.5.2.1 Variation of the composition of the fat phase

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

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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 T2-distribution of

double emulsion made by Method B


yield of double emulsions made by Method C

3.5.2.1.4 Effect of the variation of the fat phase on the T2-distribution of

double emulsion made by Method C


yield of double emulsions made by Method D

3.5.2.2 Effect of the concentration of the hydrophilic emulsifier in the external

water phase on the enclosed water volume

3.6 Fat globule analysis by a Malvern Mastersizer S

3.7 Visualization of double emulsions by light microscopy

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

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

3.8.2 Profilometric analysis of double emulsions

3.9 Whipping of a commercial dairy cream

3.9.1 Whipping time of a whipped commercial dairy cream

3.9.2 Overrun of a whipped commercial dairy cream

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

3.10 Whipping of w/o/w-emulsions

3.10.1 First attempt

3.10.2 Second attempt

3.10.3 Third attempt

3.10.4 Fourth attempt

General conclusions

List of references

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Abstract

1

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 T2-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 T2-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

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Chapter 1

Literature review


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).

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


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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 w1/o-emulsion

and w1/o/w2-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 (w1) and coalescence of the w1-droplets

with the outer water phase (w2) (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).

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

Mostly, the mixing of the w1/o-emulsion and the w2-phase is done at a lower temperature than

the preparation of the w1/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.


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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 w1-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 w1-phase and its detection in the w2-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).


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

Reference

Oil phase

Lipophilic surfactant in the oil-phase

Concentration of lipophilic surfactant in the oil phase

Compounds in the w2-phase

Concentration of the hydrophilic surfactant in w2-phase

Compounds in the w1-phase

Concentration of compounds in the w1-phase

Ratio of the w1/o/w2- emulsion

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, 2010

Median chain triglyceride (MCT) PGPR 2wt%

Na caseinate/ Na caseinate-maltodextrin 1wt% protein gelatin and NaCl 5wt% and 0.6wt% 8/32/60

Lutz et al., 2009 Median chain triglyceride (MCT) PGPR 10wt% WPI and pectin

4% and 0.5wt% - - 12/18/70

Frasch-Melnik et al., 2010 Saturated MG, tripalmitate and 0.44wt%+0.88wt% Na caseinate 1wt% - - 3/17/80

Sunflower oil PGPR 0.35wt%

GMO and PGPR 2.2wt% and 8.9wt% WPI 6.7wt% Glycerol 3wt% 4/16/80 Benichou et al., 2007

Median chain triglyceride (MCT)

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% CaCl2 0.38-1.5wt% 8/32/60

CaCO3 1.5wt%

Ca lactate 1.5wt%


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Table 1.2: Overview of different markers and techniques of detection

Reference Marker Measurement

Sela et al., 1995 2wt% NaCl/NaF/NaBr/NaI in w1-phase

1wt% ephedrine HCl in w1-phase

2wt% KNO3 in w1-phase

Conductometry of serum phase

Lutz et al., 2009 4.4wt% NaCl in w1-phase

15wt% Na ascorbate in w1-phase

Conductometry of the serum phase

Lindenstruth and Müller, 2004 0.5%(w/v) Na diclofenac in w1-phase HPLC after ultrafiltration

Frasch-Melnik et al., 2010b 1.6wt% KCl in w1-phase Conductometry of the serum phase

Benichou et al., 2007 2wt% glucose in w1-phase Glucometry

0.33-3g/100g emulsion vitamine B1 Differential pulse polarography

Tirnaksiz and Kalsin, 2005 1.5wt% caffeine + 0.03wt% NaCl Spectrophotometry (271nm) after dialysis

0.3wt%NaCl in w1-phase Conductometry of the serum phase

Wolf et al., 2009 0.02wt% vitamin B12 in w1-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 w1-phase, no phase alteration and no phase

separation during 8 month at +7°C are the characteristics of a high storage stability

emulsion.


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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 w2-phase phase, whereas the kinetics of the coalescence

between inner water droplets is related to the type of hydrophilic emulsifier in the w1-

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).


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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 w2-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 w2-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 w1-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 w1/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).


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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 w1 and w2-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).


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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 w2-phase decreases the release rate (Muschiolik, 2007).

Addition of gelled gelatin to the w1-phase slowed down the movement of the w1-

phase to the w2-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 w1-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 w1-phase (Muschiolik, 2007).


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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 w2-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 w1/o-interface and not at the o/w2-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 w2-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 Co. After a while, the emulsion

separates into three distinguishable layers: a serum layer with a lower droplet

concentration than Co, an emulsion layer with a multiple droplet concentration equal

to Co and a cream layer with a larger multiple droplet concentration than Co. 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


12

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).

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


13

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 23rd, 1934.

The Netherlands Min 30% Commodities Act Decree of dairy products, Article 16, 1994

United Kingdom Min 35% Food Labeling Regulations, Cheese and Cream Regulations, No. 52, 1996

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-


14

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).

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).


15

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).

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).


16

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).

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).


17

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


18

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% CaCl2 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

w1-phase and the increase of consistency due to the osmotic gradient and by the

interaction of released calcium with soybean proteins at the o/w2-interface, which

results in the flocculation of w/o droplets.


19

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.


20

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.,


21

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.


22

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).


23

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).


24

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


25

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).

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


26

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, 1932

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

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).


27

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).

Figure 1.13: Effect of whipping temperature (in °C) on the firmness (yield stress, g/cm2) of whipped cream of different fat contents (%) (Mulder and Walstra, 1974).

1.6.1.1 Tempering

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


28

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.


29

The newly formed membrane gradually disintegrates and the liquid fat is released and

cements the remaining fat globules.

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).


30

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

Chapter 2 Materials and methods

31

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.

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.


32

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 KH2PO4 (Merck KGaA, Darmstadt, Germany) and

K2HPO4 (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.


33

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.

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.

Palm Oil

Stearin fraction

Olein fraction

Hard stearin

Soft PMF

Super Olein

Hard PMF

Mid olein


34

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 (%) 0.02 0.02 0.02 0.02 0.02 0.02

Sodium caseinate (%) 0.00 0,50 0.00 0,50 0,50 0.00

Buffer solution (pH 6.7) (%) ad 100 ad 100 ad 100 ad 100 ad 100 ad 100

Oil phase

PGPR (%) 1 1 1 1 2 1

Hozol (%) 99 99 0 0 0 49.5

Soft PMF (%) 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.


35

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.

Piston


36

Flow chart 2.1: A procedure for handling the Microfluidizer M110S

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 syringethen execute STEP 2, 3 and 4*

To collect next samples with the same composition, execute STEP 2, 3 and 4*:

To collect the first sample: execute STEP 3then 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)

Repeat twice STEP 3 and 4

STEP 4Fill 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 andsuck the remaining liquid out of the reservoir with a syringe

STEP 3Fill the reservoir with 10mL premixed emulsion -> empty without circulation, by opening the valve until the liquid level in the reservoir is ~1 cm,


STEP 2Fill 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,


STEP 1Fill the reservoir with 20mL oil phase -> empty without circulation, empty by opening the valve until the liquid level in the reservoir is ~1 cm,


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

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


37

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.


38

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-1s-1, and the bulk diffusion coefficient of water

in the dispersed fluid (D) at 5°C, which differs among different composed water


39

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

katholieke universiteit leuven...sodium caseinate (1.25%,w/v) 3.4.6 selection of the filter block in...

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