FACULTY OF BIOSCIENCE ENGINEERING
INTERUNIVERSITY PROGRAMME (IUPFOOD)
MASTER OF SCIENCE IN FOOD TECHNOLOGY
Major Food Science and Technology
Academic year 2014-2015
Encapsulation efficiency of proteins in water-in-oil-in-water
emulsions
by Zahra Pouri
Promoter: prof. dr. ir. Paul Van der Meeren
Tutor: Lien Vermeir
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 2015
The promotor
the author
Prof. dr. ir. Paul Van der Meeren
Zahra Pouri
Acknowledgements
I would like to express my sincerest gratitude to my promotor, Prof. dr. ir. Paul Van der Meeren,
who has supported me throughout my thesis with his knowledge and useful comments.
Furthermore, I would like to thank my tutor, Lien Vermeir for her patience, valuable guidance and
engagement through the learning process of this master thesis. I am also grateful to all of the
members of the department of Applied Physical Chemistry for their help and support. I take this
opportunity to thank my friends: Azadeh, Darya, Mahtab and all who directly or indirectly
supporting me on the way.
Lastly, I offer my special regards to my lovely parents and my sisters, Sara and Saeedeh, for their
encouragement, support and attention.
Table of Contents
List of bbreviations………………………………………………................................................1
Abstract…………………………………………………………………………………………...2
Chapter 1: Literature review
Classification of emulsions...................................................................................................... 3
1.1.1
Single emulsions ....................................................................................................... 3
1.1.2
Double emulsions...................................................................................................... 3
Preparation of W/O/W emulsions ........................................................................................... 4
Instability and release properties of double emulsions............................................................ 5
1.3.1
Coalescence phenomena ........................................................................................... 6
1.3.2
Diffusion and/or permeation of chemical ingredients .............................................. 7
1.3.3
Gravitational separation ............................................................................................ 8
Baking process......................................................................................................................... 9
Enzyme application in baking ............................................................................................... 10
1.5.1
Enzymes used in baked products ............................................................................ 10
Measurement of the enzyme activity..................................................................................... 12
1.6.1
Continuous assays ................................................................................................... 12
1.6.2
Stopped assays ........................................................................................................ 12
Factors to control enzymatic analysis.................................................................................... 12
1.7.1
Substrate concentration ........................................................................................... 12
1.7.2
pH............................................................................................................................ 13
1.7.3
Temperature ............................................................................................................ 13
1.7.4
Salt content (ionic strength) .................................................................................... 13
Chapter 2: Materials and methods
Materials ................................................................................................................................ 15
Preparation of water-in-oil-in-water (W1/O/W2) double emulsions ...................................... 16
2.2.1
Quasi-isotonic conditions, ∆𝜋 = 𝜋𝑖 − 𝜋𝑒 = 0 ....................................................... 16
2.2.2
Negative osmotic pressure gradient, ∆𝜋 = 𝜋𝑖 − 𝜋𝑒 < 0........................................ 18
2.2.3
Positive osmotic pressure gradient, ∆𝜋 = 𝜋𝑖 − 𝜋𝑒 > 0 ......................................... 19
Protein concentration measurement in W1/O/W2 emulsions ................................................. 19
2.3.1
Schacterle and Pollack method ............................................................................... 20
2.3.2
Nitrogen-determination upon persulfate oxidation ................................................. 21
Water yield determination of W1/O/W2 emulsions ............................................................... 21
2.4.1
Analytical photocentrifugation ............................................................................... 22
2.4.2
Pfg-NMR diffusometry ........................................................................................... 23
WPI encapsulation efficiency determination of W1O/W2 emulsions .................................... 24
Viscosity measurement .......................................................................................................... 24
Enzymatic assay of Alkaline Phosphatase ............................................................................ 25
2.7.1
Standard curve ........................................................................................................ 27
Statistical analysis ................................................................................................................. 28
Chapter 3: Results and discussion
Measurement of whey protein isolate (WPI) in the separated outer aqueous phase using the
Schacterle and Pollack method .............................................................................................. 29
Protein solubility determination using the Schacterle and Pollack method .......................... 31
Measurement of the recovery yield of WPI using the Schacterle and Pollack method ........ 32
3.3.1
Effect of the presence of Tween 80 ........................................................................ 33
3.3.2
Effect of homogenization intensity ......................................................................... 37
Measurement of N-containing compounds using the nitrogen determination method
following persulfate oxidation ............................................................................................... 38
3.4.1
Influence of NaClO3 on the NaNO3 nitrogen content determination ..................... 38
3.4.2
Influence of potassium phosphate buffer on the NaNO3 nitrogen content
determination ......................................................................................................................... 39
3.4.3
Influence of NaN3 on the NaNO3 nitrogen content determination ......................... 40
3.4.4
Influence of NaClO3, potassium phosphate buffer and NaN3 on the WPI nitrogen
content determination ............................................................................................................ 44
3.4.5
Influence of Tween 80 on the WPI nitrogen content determination ....................... 45
3.4.6
Influence of the sodium persulfate concentration on the on the reduction-diazotationabsorption or persulfate digestion reaction ............................................................. 46
3.4.7
Standard curve ........................................................................................................ 47
Effect of storage temperature and concentration of WPI in the W1-phase on its release
kinetics as a function of storage time under quasi-isotonic conditions ................................. 48
3.5.1
WPI release from the W1/O/W2 emulsions prepared by moderate homogenization
intensity ................................................................................................................... 49
3.5.2
Released WPI concentration from the W1/O/W2 emulsions prepared by low-energy
homogenization intensity ........................................................................................ 51
3.5.3
Water yield and WPI yield of W1/O/W2 emulsions prepared by moderate and lowenergy homogenization intensity ............................................................................ 54
Effect of homogenization time duration in the secondary emulsification step on the release
kinetics of WPI from the W1-phase to the W2-phase directly after preparation of the gelled
and non-gelled W1/O/W2 emulsions ...................................................................................... 57
3.6.1
Released WPI concentration from the gelled and non-gelled W1/O/W2 emulsions 58
3.6.2
Water yield and WPI yield of the gelled and non-gelled W1/O/W2 emulsions ...... 59
Effect of the application of mechanical stress (pumping) on the release kinetics of WPI from
the W1-phase into the W2-phase directly after preparation of the W1/O/W2 emulsions ....... 60
Influence of the osmotic pressure gradient (∆𝛑) on the release kinetics of WPI as a function
of storage time ....................................................................................................................... 62
3.8.1
Viscosity measurement of the W1/O/W2 emulsions ............................................... 63
3.8.2
Released WPI concentration from the W1/O/W2 emulsions ................................... 65
3.8.3
Water yield and WPI yield of the W1/O/W2 emulsions .......................................... 67
The effect of emulsification shear intensity and shear temperature on the enzyme activity . 70
General conclusions…………………………………………………………………………….73
References………………...……………………………………………………………………..74
List of abbreviations
Abbreviation
Definition
EV
Enclosed water volume fraction
LHI
Low-energy homogenization intensity
LUM
analytical photocentrifugation
MHI
Moderate homogenization intensity
AP
Centrifuged aqueous phase of an O/W emulsion
OAP
Centrifuged outer aqueous phase of a W/O/W emulsion
pfg-NMR
Pulsed field gradient Nuclear Magnetic Resonance
PGPR
Polyglycerol polyricinoleate
PNP
p-Nitrophenol
PNPP
p-Nitrophenyl Phosphate
SDS
Sodium dodecyl sulfate
T80
Tween 80
W1
Internal water phase
W2
External water phase
W1/O
primary water-in-oil emulsion
W1/O/W2
Water-in-oil-in-water emulsion
WPI
Whey protein isolate
YieldLUM
Water yield measured by analytical photocentrifugation
YieldNMR
Water yield measured by pfg-NMR
1
Abstract
Water-in-oil-in-water (W/O/W) emulsions have promising applications in the food industry.
Due to the presence of two aqueous domains separated by an oil layer, they offer great potential
for encapsulation and controlled release of hydrophilic bioactive ingredients, as well as for the
preparation of a single mix of two water-soluble incompatible compounds. The latter application
might be interesting for liquid bread improvers, e.g. for preparation of a single mix of protease and
lipase. Nowadays, the liquid formulation of bread improvers is preferred for its ease of mixing
with other ingredients and providing accurate dosing, while minimizing potentially allergenic dust
formation. The objective of this research was to determine the encapsulation efficiency of proteins
in W/O/W double emulsions. Whey protein isolate (WPI) was selected as a first model compound.
To that end, two methods for WPI concentration measurement were optimized; the Schacterle and
Pollack method and the nitrogen determination method following persulfate digestion. The protein
solubility and recovery yield were determined using the former method. The water yield of W/O/W
emulsions was measured using pulsed field gradient Nuclear Magnetic Resonance (pfg-NMR)
diffusometry and analytical photocentrifugation. Pfg-NMR is a relatively fast and non-destructive
method which can discriminate between internal and external water based on differences in
molecular diffusion behavior of water. Analytical photocentrifugation is a simpler, straightforward
and less expensive method with simple data-analysis and the ability to process several samples at
the same time. Upon optical detection of the cream volume and subtracting the oil content, the
water yield can be determined.
The release of WPI was compared to the release of water by comparison of their yield values, upon
application of shear, mechanical stress, different temperatures and an osmotic pressure gradient
between the water phases of the W/O/W emulsion. Under certain conditions, it was shown that
WPI protein and water behave differently in W/O/W emulsions. In addition, the alkaline
phosphatase enzyme was chosen as a second protein model compound for investigation of the
influence of emulsification shear intensity and shear temperature on the enzymatic activity.
2
Chapter 1
Literature review
Classification of emulsions
1.1.1 Single emulsions
The basic structure of emulsions consists of two immiscible fluids in which one is dispersed in
another one in the presence of emulsifiers. As emulsions are metastable colloids, energy is required
for preparation (Leal-Calderon et al., 2007), which results in a diameter of the dispersed phase
droplets between 0.1 µm and 0.1 mm depending on the emulsification method (van der Graaf et
al., 2005).
1.1.2 Double emulsions
Double emulsions consist of two main types: water-in-oil-in-water (W/O/W), where a W1/O
emulsion is dispersed as droplets in an aqueous phase and oil-in-water-in-oil (O/W/O), in which
oil droplets are dispersed in water droplets that are located in an oil phase. W/O/W double
emulsions are more frequently studied (van der Graaf et al., 2005). They have promising
applications in food industry including the encapsulation of hydrophilic compounds such as
vitamins or minerals, aroma and flavor release, and the production of low-calories food, e.g. lowfat dressing. In addition, there is a great interest in production of W1/O/W2 double emulsions in
the pharmaceutical industry for controlled release and targeted delivery of drugs (Sapei et al.,
2012). An overview of food applications of W/O/W double emulsions is given in Table 1.1.
3
Table 1.1. Overview of food applications of double emulsions in literature.
Reference
Food application
De Cindio and Cacace, 1995
Low caloric food
Garti, 1997
Sodium chloride encapsulation
Yoshida et al., 1999
Vitamin A encapsulation
Malone et al., 2003
Control sour taste perception in low pH foods
Lobato-Calleros et al., 2008
Reduced-fat cheese-like product
Taki, 2008
Low-fat dressing
Lobato-Calleros et al., 2009
Low-fat stirred yoghurt
Bonnet et al., 2009
Magnesium encapsulation
Carrillo-Navas et al., 2012
Ascorbic acid encapsulation
Preparation of W/O/W emulsions
Double emulsions are generally prepared in a two-stage method (Figure 1.1). First, the inner W1/O
emulsion is produced by conventional high shear emulsification devices such as high pressure
homogenizers and rotor stator systems to gain small droplet sizes and narrow droplet size
distributions. The secondary emulsification step is conducted with less shear intensity to prevent
rupture of the internal droplets (Schuch et al., 2013). For stabilization of the primary emulsion
(W1/O), hydrophobic surfactants with a low hydrophilic–lipophilic (HLB) value (< 10) are used
whereas in the second step, the primary emulsion is stabilized in a second aqueous phase by
surfactants with a high HLB value (typically > 10). Both types of emulsifiers are present in the
interfaces and have an influence on the rate and extent of coalescence (Pays et al., 2002).
The biggest challenge in the production of double emulsions lies in the second emulsification step.
During this step, the droplet size distribution of inner water droplets can be determined, which can
alter the properties and functionality of double emulsions (van der Graaf et al., 2005). Moreover,
applying a higher shear stress migh cause the release of the inner aqueous phase to the outer
aqueous phase (Schuch et al., 2013).
4
Figure 1.1. Two-stage preparation method for W/O/W double emulsions. A high-shear
emulsification step (a) and a low shear emulsification step (b) (van der Graaf et al., 2005).
Different preparation methods for the second emulsification step have been applied. Membrane
emulsification devices directly form droplets of the W1/O emulsions in the continuous water phase.
High encapsulation efficiencies and low release rates of inner water phase can be fulfilled since
this process is very gentle. However, this method is not applicable for large scale production due
to low throughput (in the range of liter per hour) (Schuch et al., 2013).
Instability and release properties of double emulsions
Conventional emulsions are thermodynamically unstable due to instability mechanisms such as
droplet aggregation (coalescence and flocculation), droplet growth (Ostwald ripening),
gravitational separation (creaming and sedimentation) (Piorkowski and McClements, 2014), and
diffusion processes (Figure 1.2) (Mezzenga et al., 2004). As a result, uncontrolled release of active
substances from the inner to the outer aqueous phase might occur, which mainly limits the
commercial application of double emulsions.
However, Pays et al. (2002) showed that there is a possibility to shift from one type of mechanism
to another one by changing the nature and/or proportions of surfactants. In that regard, Sapei et al.
(2012) reported on the use of steric stabilizers, protein-polysaccharide hybrids, gelatin, fat crystals,
5
as well as on the combination of NaCl and polyglycerol polyricinoleate (PGPR) in the primary
emulsion and using sodium caseinate-dextran conjugates as an external emulsifier.
Figure 1.2. Schematic figure of possible instabilities occurring in W/O/W double emulsions
(Mezzenga et al., 2004).
1.3.1 Coalescence phenomena
Coalescence phenomena can occur at several levels in multiple emulsions (Mezzenga et al., 2004;
Leal-Calderon et al., 2007):
1. between small inner water droplets
2. between large oil globules with possibly subsequent creaming
3. between the oil globules and the small dispersed water droplets
In double emulsions with a high initial droplet volume fraction, large nuclei form as a result of
droplet-droplet coalescence. Once they reach the globule surface, coalescence occurs. In the
system with low initial droplet concentration, droplet coalescence is no longer observed. Several
studies confirm that droplet-droplet and droplet-globule coalescence occur in presence of high
amount of hydrophilic surfactants (either ionic or non-ionic) (Leal-Calderon et al., 2007).
Kabalnov and Wennerström (1996) proposed a mechanism that links the hydrophilic surfactant
concentration with the coalescence energy barrier of emulsions.
Pays et al. (2001) investigated the impact of the concentration of hydrophilic surfactant Ch (sodium
dodecyl sulfate) on the release property of sodium chloride from W/O/W emulsion. The results
6
showed slow release at Ch ≤ CMC. The rate decreased when Ch increased and it was minimum
around the CMC.
1.3.2 Diffusion and/or permeation of chemical ingredients
Compositional ripening without film rupturing is a mechanism that can occur by diffusion and/or
permeation of compounds across the oil phase. Migration of water across the oil phase boundary
layer is relatively fast (i.e. minute range) compared to migration of hydrophilic solutes (i.e. days,
weeks, months range) such as proteins and amino acids (Aserin 2008).
Osmotic pressure gradients between internal and external aqueous phases of W/O/W double
emulsions may affect the stability of double emulsions. Depending on the direction of the osmotic
pressure gradient, water may pass from external to internal phase or vice versa (Leal-Calderon et
al., 2007). When the solute concentration of the internal aqueous phase is higher than of the outer
aqueous phase, water molecules migrate to the internal aqueous phase. Hereby, Iqbal et al. (2013)
observed a swelling of the internal water droplets prior to rupture and release of their bioactive
content into the external water phase, whereas gradual emptying of internal droplets occurred in
case of a higher concentration of solute in the external water phase. In both cases, the double
emulsion structure is converted to a simple emulsion (Frasch-Melnik et al., 2010).
The rate of water transport can be influenced by the magnitude of the osmotic pressure gradient
between the aqueous phases, the concentration and nature of surfactants, as well as by the nature
and viscosity of the oil phase.
Kita et al. (1978) established two possible mechanisms for migration of water and soluble materials
(Figure 1.3):
1) Water molecules pass through the thin liquid film formed by internal droplets in contact
with globule surface
2) Water molecules diffuse across the oil phase by being incorporated in reversed micelles
Oil soluble surfactant is recognized as a main factor for water transport by Garti et al. (1997) and
Jager-Lezer et al. (1997) observed that the water migration rate increased with increase in oil
soluble surfactants concentration (Leal-Calderon et al., 2007).
The kinetic stability of W/O/W double emulsions is limited. Different strategies have been
employed to improve the stability of W/O/W double emulsions such as increasing the viscosity of
the double emulsion by different methods. An overview of these methods is given in Table 1.2.
7
Figure 1.3. Schematic representation of (a) micellar transport of water from the internal aqueous
phase to the outer aqueous phase through the oil layer in W/O/W emulsions (b) water transport
through the thin liquid film (Garti, 1997).
Table 1.2. Overview of improving the stability of W/O/W double emulsions by increasing
the viscosity in literature.
Method
Reference
Osmotically driven gelation
Delample et al., 2014
Iqbal et al., 2013
Leal-Calderon et al., 2012
Viscofying agents in external water phase (W2) Özer et al., 2000
O̕ Regan and Mulvihill, 2010
Gelling agents in internal water phase (W1)
Perez-Moral et al., 2014
Surh et al., 2007
1.3.3 Gravitational separation
One of the most common problems reported in emulsions is gravitational separation (i.e. creaming
and sedimentation). The greater density differences between the dispersed phase and the
surrounding phase might accelerate the rate of gravitational separation. According to the Stokes’s
8
law (Equation 1.1), emulsions become more stable to creaming as the particle size decreases and
the difference in density, as well as in viscosity between dispersed phase and continuous phase
decreases (i.e. 𝜂𝐷 ⁄𝜂𝐶 close to unity) (Piorkowski and McClements, 2014).
𝑣=
2𝑔𝑟 2 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 (𝜌𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 − 𝜌0 )
Equation 1.1
9𝜂0
In the Stokes’s equation, 𝑣 is the creaming velocity, 𝑟𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 is the particle radius, 𝜌𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 is the
particle density, 𝜌0 is the continuous phase density, 𝜂0 is the continuous phase viscosity and g is
the gravity acceleration.
Baking process
Man found out the art of breadmaking more than 4000 years ago (Dewettinck et al., 2008). Basic
ingredients are cereal flour, water, yeast or another chemical leavening agent, and salt (Mondal et
al., 2008). The most important crop for breadmaking is wheat (Triticum Aestivum) due to its good
baking properties in comparison with all other cereals.
Different chemical and physical transformations occur during breadmaking which have an
important role in the final bread quality. Some of these reactions can be affected by the presence
of additives such as bread improvers, which can alter the structural and physiochemical
characteristics of the flour constituents and hence, improve their functionality in breadmaking
(Joye et al., 2009). The final bread product is characterized by a solid foam structure containing a
continuous phase made up of an elastic network of cross-linked gluten protein molecules and of
leached starch polymer molecules, mainly amylose with polar lipid molecules and also a
discontinuous phase of entrapped, gelatinized, swollen, deformed starch granules. The
breadmaking process consists of three major stages: mixing, fermentation (resting and proofing)
and baking, all of which affect the properties of final product.
Bread processing begins with the hydration of flour particle during mixing. Depolymerization and
(re)-polymerization processes cause formation of a gluten network during mixing. Further
polymerization of gluten proteins occurs during the resting step. The fermentation is a fundamental
stage in breadmaking in which baker’s yeast belonging to the Sacchromyces cerevisiae species
converts sugar to carbon dioxide. The gluten matrix retains the carbon dioxide produced by the
yeast during the fermentation step and in the beginning of the baking stage. Gas retention of the
9
dough determines the volume of loaf and the crumb structure. The gliadin/glutenin ratio of flour
gluten indicates the bread baking quality. Glutenins have an effect on the cohesiveness and
elasticity of the dough and gliadins contribute to dough viscosity and plasticity (Miguel et al.,
2013).The baking process results in a series of physical, chemical and biochemical changes in the
product including volume expansion, evaporation of water, formation of a porous structure,
denaturation of proteins, gelatinization of starch, crust formation and browning reactions. During
baking, new disulfide cross-links form in the gluten network probably due to the modification in
protein hydrophobicity which results in the formation of a permanent network. The crust browning
is directly related to caramelization and/or Maillard reactions. In addition, different flavor
compounds are produced and give a special smell and taste to bread.
Enzyme application in baking
The restriction on the use of chemical additives has led to an increase in application of enzymes
as one of the important ingredients in fermented products. Enzymes are added to the dough to
control the baking process, allowing the use of different baking processes, reducing process time,
slowing-down staling, compensating for flour variability and substituting chemical additives. As
such, traditional bread improvers typically contain 1000 to 10000 ppm enzyme. However, enzymes
have the potential for causing allergies in bakery workers. Thus, enzyme dust formation must be
limited (Popper et al., 2006).
1.5.1 Enzymes used in baked products
There are three sources for enzymes used in bakery industry:
1. The endogenous enzymes in flour
2. Enzymes associated with the metabolic activity of the dominant microorganisms
3. Exogenous enzymes which are added in the dough
Flour standardization is usually done with the fortification of flour and dough with enzyme
improvers. Enzymes can be added individually or in combination with other enzymes to improve
the effectiveness. The most frequently used enzymes are given in Table 1.3 (Miguel et al., 2013).
10
Table 1.3. Application of enzymes in baking
Enzymes (classification)
Substrate in
Reaction
Application in baked products
bread
Amylolytic enzymes
Starch
Hydrolysis of
linkage
Providing of fermentable
compounds, increase in bread
volume, reduction of
fermentation time, formation of
reducing sugars and subsequent
Maillard reaction products,
anti-staling effect
Cellulases and
hemicellulases
Non-starch
compounds of
cereals
Hydrolysis of
linkage
Providing more soluble dietary
fiber in baked products,
production of prebiotic
oligosaccharides, improvement
of rheological properties of
dough
Protease
Gliadin and
glutenin
Hydrolysis of
peptide bonds
Reduction of mixing time,
control of viscoelastic
properties of gluten, enhance
dough extensibility, formation
of amino acids and flavor,
production of gluten-free
products
Lipases
Lipids
Hydrolysis of
ester bonds
Improvement of bread volume
and dough stability, formation
of emulsifiers, anti-staling
effect, flavor development
Lipoxygenase
Polyunsaturated
fatty acids
Oxidation of
fatty acids
Bleaching of fat-soluble flour
pigments, oxidation of
sulfhydryl group of proteins by
formed hydroperoxides
Glucose oxidase
β-D-glucose
Oxidation of
Control of browning reaction,
β-D-glucose to improvement in crumb
gluconic acid
properties
11
Measurement of the enzyme activity
Enzyme assays can be divided into two groups:
1.6.1 Continuous assays
Continuous assays provide a continuous reading of activity. Therefore, any deviation of initial rate
from linearity can be observed. Changes in absorbance, fluorescence, viscosity, pH or other
possible physical parameters are the simplest continuous assays. In some cases, the enzyme
activity does not make detectable changes in the physical properties. This can be overcome by
using a coupled continuous method. In this method, the products react usually with another enzyme
to cause visible changes. One of the advantages of this process is that the product is removed to
keep a constant rate over a long period by avoiding the reversal reaction (Scopes 2002).
1.6.2 Stopped assays
In stopped assays the reaction is stopped after a fixed time by strong acids, alkali, detergents,
heating or irreversible inhibitors such as heavy metal ions. All these materials result in enzyme
denaturation and consequently the enzyme activity stops. The concentration of substrates/products
can be determined by radiometric assays and chromatographic assays (Scopes, 2002).
Factors to control enzymatic analysis
There are several factors which affect the activity of enzymes including the substrate
concentration, pH, ionic strength, presence of salts and temperature.
1.7.1
Substrate concentration
If the reaction velocity is plotted versus substrate concentration, the curve will have a hyperbolic
response according to the Michaelis-Menten model as shown in Figure 1.4.
As shown in Figure 1.4, the reaction velocity varies linearly in case of small substrate
concentrations (S), while at large concentrations, it reaches a plateau. The maximum theoretical
reaction velocity (Vmax) is reached when all enzyme active sites are saturated with substrate at
infinite substrate concentration (www.Columbia.edu, 2003). Km is the substrate concentration that
yield a half of maximum reaction rate.
12
Figure 1.4. The reaction rate variation with substrate concentration (www.Columbia.edu, 2003).
1.7.2 pH
Within a limited range of optimum pH, the enzymes have their maximum activity (i.e. the highest
value for Vmax). Therefore, an appropriate buffer is used in enzyme assays, characterized by a pH
close to the enzyme’s optimum and meanwhile appropriate for the other components in the
mixture.
1.7.3
Temperature
At a temperature below the denaturation temperature, an increase in temperature generally leads
to an increase in the reaction rate. At higher temperatures, however, the protein denatures and
consequently the enzyme is inactivated.
In general, the loss of enzyme activity is caused by the change in its three-dimensional protein
structure at a too high temperature (Figure 1.5) (Bisswanger 2014).
1.7.4 Salt content (ionic strength)
Most of the enzymes are inhibited at high content of salt (> 0.5 mol/l) due to ion interference with
the weak ionic bonds of proteins (Scopes, 2002).
13
Figure 1.5. Dependence of the enzyme activity on the temperature (Bisswanger, 2014).
14
Chapter 2
Materials and methods
Materials
The materials used in the following experiments are listed in Table 2.1.
Table 2.1. Materials used in experiments.
Material
Company and country
Polyglycerol polyricinoleate (PGPR)
Palsgaard® 4150, Palsgaard A/S, Denmark
Polysorbate 80 (Tween 80)
Sigma-Aldrich, Steinheim, Germany
Sunflower oil
Delhaize group, Brussel, Belgium
NaN3
KH2PO4
Sigma-Aldrich, St-Louis, USA
Merck, KGaA, Darmstadt, Germany
K2HPO4
Alfa Aesar, Karlsruhe, Germany
Whey protein isolate (WPI)
BiPro, Davisco, Le Sueur, USA
(< 0.5 % fat, < 5 % moisture and 1.9 % of ash, >
92.6 % of WPI protein, with 85 % β-lactoglobulin
of the total protein)
CuSO4.5H2O
AnalaR NORMAPUR®, Leuven, Belgium
Na2CO3
AnalaR NORMAPUR®, Leuven, Belgium
NaOH
AnalaR NORMAPUR®, Leuven, Belgium
Folin-Ciocalteous
Merck, KGaA, Darmstadt, Germany
Na2-tartrate
Acros Organics, Geel, Belgium
Sodium dodecyl sulfate (SDS)
Acros Organics, Geel, Belgium
Glycine
Alkaline Phosphatase
Sigma-Aldrich, St-Louis, USA
Sigma-Aldrich, St-Louis, USA
p-Nitrophenyl Phosphate (PNPP)
Sigma-Aldrich, St-Louis, USA
p-Nitrophenol (PNP)
Sigma-Aldrich, St-Louis, USA
MgCl2
ZnCl2
VWR, ProLABO, EC
Sigma-Aldrich, St-Louis, USA
Na2S2O8
Sigma-Aldrich, St-Louis, USA
NaClO3
Sigma-Aldrich, St-Louis, USA
H3BO3
Merck, KGaA, Darmstadt, Germany
15
Preparation of water-in-oil-in-water (W1/O/W2) double emulsions
2.2.1 Quasi-isotonic conditions, ∆𝝅 = 𝝅𝒊 − 𝝅𝒆 = 𝟎
Unless stated differently, the internal aqueous phase (W1-phase) containing WPI (5, 2.5 or 1 wt
%) in potassium phosphate buffer (5 mM, pH 6.6), 0.02 wt % NaN3 (antimicrobial agent) and 0.1
M NaCl was stirred for at least an hour at room temperature using moderate magnetic stirring. The
potassium phosphate buffer solution was prepared based on the Henderson-Hasselbalch equation
to have a solution with pH 7, by mixing KH2PO4 and K2HPO4. The pH of the buffer solution
amounted 6.6 as measured at room temperature by a pH-meter. The difference between the
measured and calculated pH might come from the applied calibration liquids. The oil phase
contained 5 wt % of oil soluble emulsifier polyglycerol polyricinoleate (PGPR) in commercial
sunflower oil and was heated at 60 ℃ for 10 minutes. The external aqueous phase (W2-phase) was
prepared by hydrating Tween 80 (1.67 wt %) in the same buffer used for W1 under moderate
magnetic stirring conditions. The composition of the different species present in the different
phases of the W1/O/W2 emulsions is illustrated in Table 2.2.
Table 2.2. Composition of different phases of W1/O/W2 emulsions.
Materials
Aqueous phases
External water phase
(50 wt %)
Internal water phase
(25 wt %)
PGPR (wt %)
NaCl (M)
0.1
0.1
Sodium azide (wt %)
0.02
0.02
WPI (wt %)
1, 2.5, 5.*
Tween 80 (wt %)
1.67
PO4 buffer (mM)
5
5
* internal aqueous phase were prepared in three concentrations: 5, 2.5 or 1 wt %
Oil phase (25 wt %)
5
The W1/O/W2 double emulsions were prepared using a two-stage emulsification process. The
primary water-in-oil emulsion (W1/O) was homogenized by adding drop-wise the W1-phase (80
g) at 40 C to the oil phase (O) (80 g) at 40-50 C using an Ultra-Turrax S50 N-G45F operating at
5200 rpm for 5 minutes. The secondary emulsion (W1/O/W2) was prepared by mixing the W1/O
emulsion (150 g) with the W2-phase (150 g) using different homogenization intensities:
16
1- Moderate homogenization intensity (MHI): the W1/O emulsion and the W2-phase were
mixed by using an Ultra-Turrax S25-10G at 24000 rpm for 1 minute. The mixture was then
homogenized with a continuous Ultra-Turrax DK25 operating at 24000 rpm for 2 minutes.
2- Low-energy homogenization intensity (LHI): the W1/O emulsion and the W2-phase were
mixed by using an Ultra-Turrax S25-10G at 13500 rpm for 1 minute. The mixture was then
homogenized with a continuous Ultra-Turrax DK25 operating at 13500 rpm for 1 minute.
Unless stated differently, the prepared emulsions were statically stored till end of the storage time.
In quasi-isotonic conditions, the initial osmotic pressure in the internal and the external water
phases was adequately matched to avoid water transfer. To determine the initial osmotic pressure,
the ideal behavior of the solutes was taken into account based on the van’t Hoff approximation:
Equation 2.1
𝜋𝑖,𝑒 = (∑ 𝐶𝑖,𝑒 ) 𝑅𝑇
Where 𝐶𝑖,𝑒 is the osmolar concentration of solutes in the corresponding aqueous phases (“i” and
“e” stand for the internal and external aqueous phases respectively), R is the gas constant (8.314
J/mol/K) and T is the absolute temperature of 278 and 298 K (storage temperature). The summation
in Equation 2.1 mainly encompasses solutes with low molar mass (in this study: NaCl, KH2PO4,
K2HPO4, NaN3). As a result of a high molecular weight and low molar concentration, WPI has a
negligible contribution to the osmotic pressure (Leal-Calderon et al. 2012). From Table 2.3, it is
clear that minor transport of water from the W1-phase to the W2- phase would restore the osmotic
equilibrium.
2.2.1.1 Gelled and non-gelled W1/O/W2 prepared with variable mixing time
Two compositions, a gelled and non-gelled composition, were prepared under quasi-isotonic
conditions using a series of homogenization durations in the second emulsification step of the
W/O/W preparation. Their W1-phase contained 2.5 wt % of WPI and their W1/O emulsion was
made as described in section 2.2.1. In contrast to the non-gelled composition, the W/O emulsion
for the gelled composition was subjected to a heat treatment at 80 ℃ for 1 h in a water bath
(Memmert, Germany), by which gelation of the internal water droplets occurred. The W/O/W
emulsions were prepared by gradual addition of 50 g of W1/O at room temperature to 50 g of W2phase (with same composition as in section 2.1.1.) using an Ultra-Turrax S25-10G at 24000 rpm
for 2 minutes. Directly afterwards, the mixture was homogenized with a continuous Ultra-Turrax
17
DK25 operating at 24000 rpm for different time durations (i.e.1, 2, 4, 6 and 8 minutes). It should
be noted that a cooling device was used during continuous emulsification to prevent temperature
rise.
Table 2.3. Calculation of initial osmotic pressure for the external and internal aqueous
phases under quasi-isotonic conditions at 278 and 298 K (storage temperature).
W1-phase
concentratio
n (g/L)
molar mass
(g/mol)
molarity
(mM)
osmolarity
(osm/m3)
KH2PO4
K2HPO4
NaN3
NaCl
0.4174
0.3366
0.2
5844
136.09
174.14
64.99
58.44
3.07
1.93
3.08
100
6.13
5.80
6.15
200
218.09
W2-phase
concentratio
n (g/L)
molar mass
(g/mol)
molarity
(mM)
osmolarity
(osm/m3)
KH2PO4
K2HPO4
NaN3
NaCl
Tween 80
0.4174
0.3366
0.2
5844
16.7
136.09
174.14
64.99
58.44
1310
3.07
1.93
3.08
100
12.75
6.13
5.80
6.15
200
12.75
230.84
osmotic pressure
(kPa)
at 278 K at 298 K
504.06
540.33
osmotic pressure
(kPa)
at 278 K at 298 K
533.54
571.92
2.2.2 Negative osmotic pressure gradient, ∆𝝅 = 𝝅𝒊 − 𝝅𝒆 < 𝟎
The W1/O/W2 emulsion was prepared using moderate homogenization intensity as described in
part 2.2.1., whereby the W1-phase contained 2.5 wt % of WPI. Solid sodium chloride was added
to the W1/O/W2 emulsion such that the W2-phase had a NaCl concentration of 0.2 M, while the
W1-phase contained 0.1 M NaCl. The prepared emulsion was end-over-end rotated at the minimum
speed of 2 rpm till end of the storage time (Figure 2.1). Due to a viscosity rise after 3 days of
storage, the outer aqueous phase was separated in a one-step centrifugation using ultracentrifuge
operating at 21 °C, at 45000 rpm for 1 h (Beckman Ultracentrifuge L7-55, USA).
18
Figure 2.1. The W1/O/W2 emulsions on the end-over-end rotator at 2 rpm.
2.2.3 Positive osmotic pressure gradient, ∆𝝅 = 𝝅𝒊 − 𝝅𝒆 > 𝟎
The W1/O/W2 emulsion was prepared using moderate homogenization intensity as described in
part 2.2.1., whereby the W1-phase contained 2.5 wt % of WPI and the W2-phase had the same
composition except for the absence of NaCl. The prepared W1/O/W2 emulsion was diluted with
hypotonic solution (5 mM potassium phosphate buffer, pH 6.6) containing 0.02 wt % NaN3 in a
mass ratio of 1:1. The prepared emulsion was placed on the end-over-end rotator operated at the
minimum speed of 2 rpm till end of the storage time.
Protein concentration measurement in W1/O/W2 emulsions
Unless stated differently, the protein measurement required a two-step centrifugation of the
W1/O/W2 emulsions in a benchtop centrifuge (Sigma 3-16P, Germany) at 500 g for 40 minutes at
room temperature to separate the outer aqueous phase. Subsequently, the obtained the outer
aqueous phase was ultracentrifuged at 45,000 rpm for 60 minutes at 21 ℃ to remove small droplets
of W1/O emulsions (Beckman Ultracentrifuge L7-55, USA).
The WPI concentration was measured against a calibration curve. According to the producer, this
BiPro sample contains > 92.6 % of WPI protein, which was taken into account for the protein
determination.
19
2.3.1 Schacterle and Pollack method
The protein concentration was measured by a modified Lowry method as described by Schacterle
and Pollack (1973). The alkaline Cu-reagent and phenol-reagent used in this method were prepared
according to Table 2.4.
Table 2.4. Composition of alkaline Cu-reagent and phenol-reagent in Schacterle and
Pollack method.
Material
CuSO4.5H2O (wt %)
Na2-tartrate (wt %)
Na2CO3 (wt %)
NaOH (N)
Folin-Ciocalteous (mL)
Distilled water (mL)
Alkaline Cu-reagent
Solution A
Solution B
Phenol-reagent
0.05
0.1
10
0.5
ad 250
ad 250
6
96
For preparation of the alkaline Cu-reagent, it should be noted that solution A and B were mixed at
equal proportion, which was stable in a dark place for one month. For measuring the protein
concentration, 1 mL of alkaline Cu-reagent was added to 1 mL of centrifuged outer aqueous phase
in test tubes and mixed with a vortex mixer. After 10 minutes leaving undisturbed, 4 mL of phenolreagent was added and the tubes were turned upside down two times. The tubes were placed in a
hot water bath at 55 ℃ for 5 minutes and then immediately put in an ice water bath until measuring
the absorbance. The absorbance of the samples was measured within at most 30 minutes by a
spectrophotometer (UV 1600 PC, VWR) at 650 nm against the blank solution, which only differed
from samples in its protein content (i.e. 0 mg protein/mL sample).
2.3.1.1 Standard curve in absence of Tween 80
The stock solution was made containing 25 mg WPI protein (27.0 mg BiPro) in 25 mL aqueous
solution of 5 mM potassium phosphate buffer (pH 6.6), 0.1 M NaCl and 0.02 wt % NaN3.
Subsequently, standard series were made containing 0, 40, 80, 120, 160, 200 and 240 mg WPI
protein in 1 L, respectively. The absorbance of known concentrations of WPI was measured with
two methods: the Schacterle and Pollack method (hereafter called the reference method) and the
SDS modified Schacterle and Pollack method. For the modified method with SDS, the mixture of
solution A (10 vol %), solution B (50 vol %) and 1wt % SDS solution (40 vol %) was used instead
of mixture of A and B solutions (50/50 vol %) in the reference protein measurement protocol.
20
2.3.1.2 Standard series in presence of Tween 80
The preparation protocol of the standard series containing Tween 80 was the same as mentioned
for the reference standard series with the difference that the potassium phosphate buffer (5 mM,
pH 6.6) additionally contained 1.67 wt % Tween 80. The absorbance of known concentrations of
WPI was measured with two methods: the Schacterle and Pollack reference method and the
modified method with SDS.
2.3.2 Nitrogen-determination upon persulfate oxidation
The persulfate oxidation process includes several heat induced reactions as shown below. 𝑁𝐻4+ is
a representative of N containing compounds (Zhu, 2011) . The below scheme indicates that other
components, such as chlorides or organic compounds, may interfere as they compete for the
generated oxygen.
S2O82- + 2 OH-  2 SO42- + H2O + 1/2 O2
NH4+ + 2 O2  NO3- + H2O + 2 H+
Cl- + 3/2 O2  ClO3C + O2  CO2
Unless stated differently, 4 mL of N-containing compounds was mixed with 6 mL of reagent
containing 1 M sodium persulfate and 2 M sodium hydroxide. The prepared mixture was digested
in an autoclave at 121 ℃ and 0.5 bar for 1 h, during which N-containing compounds are converted
into nitrate. After cooling, the nitrate determination is based on the Cd-reduction method in a
Skalar automatic continuous flow nitrogen analyzer (the Netherlands). After sample dialysis, the
sample is passed through a column containing granulated Cu-Cd for the reduction of nitrate to
nitrite. The nitrite is diazotized with sulfanilamide and N-(1-naphtyl)-ethylene diamine
dihydrochloride to form a highly colored azo dye which is measured at a wavelength of 540 nm
(Zhu, 2011).
Water yield determination of W1/O/W2 emulsions
The most frequently used method for yield determination of double emulsions is based on
measuring the amount of a water-soluble marker released from the internal water phase into the
external water phase (Surh et al. 2007). Separation methods (i.e. centrifugation and dialysis) and
non-separation methods (i.e. conductometry, flam atomic absorption, and fluorimetry) are used for
21
indirectly determination of the yield. In this study, two direct methods for water yield
determination are used. Pulsed field gradient nuclear magnetic resonance (pfg-NMR) is a relatively
fast and non-destructive method which can discriminate between internal and external water based
on differences in molecular diffusion behavior of water. In the external water phase, water
molecules experience free diffusion, whereas there is restricted diffusion in the internal water
droplets (Vermeir et al., 2014). Analytical photocentrifugation is a simple, straightforward and less
expensive method with simple data-analysis and the ability to process several samples at the same
time. Upon optical detection of the cream volume and subtracting the oil content, the water yield
can be determined (Balcaen et al., in press).
2.4.1 Analytical photocentrifugation
The water yield was evaluated with analytical photocentrifugation (LUMiFuge 116, L.U.M.
GmbH, Berlin, Germany). Rectangular polycarbonate sample cells (type 110-131 xy) were filled
with 0.4 g of the double emulsion. Transmission profiles were measured every 300 s while the
samples were centrifuged for 2 hours at a speed of 3000 rpm (approximately 1200 × g). The
transmission profiles were measured between the top and bottom of the sample cells by radiation
of the NIR (near infrared) light through the cells during centrifugation (Ng et al., 2013). Front
tracking at 30 % transmission was used to calculate the water yield (Equation 2.2 and 2.3). Hereby,
the volume % oil and volume % W1-phase of the prepared W1/O/W2 emulsion amounted to 26.5
% and 24.5 %, respectively. A schematic image of photocentrifugation is shown in Figure 2.2.
𝑉𝑜𝑙. % 𝑐𝑟𝑒𝑎𝑚 =
𝐻𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑟𝑒𝑎𝑚 𝑙𝑎𝑦𝑒𝑟 (𝑚𝑚)
× 100%
𝐻𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑚)
Equation 2.2
𝑉𝑜𝑙. % 𝑐𝑟𝑒𝑎𝑚 − 𝑉𝑜𝑙. % 𝑜𝑖𝑙
× 100%
𝑉𝑜𝑙. % 𝑊1
Equation 2.3
𝑌𝑖𝑒𝑙𝑑 𝐿𝑈𝑀 (%) =
22
Figure 2.2. Schematic figure of the analytical photocentrifugation analyzer (Ng et al. 2013).
2.4.2 Pfg-NMR diffusometry
Pfg-NMR measurements were performed by a Maran Ultra 23 NMR spectrometer (Oxford
Instruments, UK) operating at a frequency of 23.4 MHz. The samples were filled in 18 mm
diameter glass NMR-tubes (Oxford Instruments, UK). Sample tubes were filled for 15 mm height
and measured at +5 ℃ (Set Temperature of the attached water bath was -2.5 ℃). Irrespective of
the storage temperature of the samples, the measurements were performed at +5 °C (equilibration
for 1 h) and at least 24 h after preparation. Upon application of a relaxation filter, the available
spectrometer selectively measures the 1H signal of the water phases in the W1/O/W2 sample. Based
on differences in molecular diffusion behavior between the internal (restricted diffusion of water
molecules) and external water phase (free diffusion of water molecules), pfg-NMR diffusometry
enables to determine the enclosed water volume fraction EV. The EV is the total percentage of
water entrapped as internal water droplets, whereas the water yield is the measured enclosed water
volume comparison to the theoretical maximum value (Equation 2.5).
For calculation of the EV, Equation 2.4 was fitted to the experimentally obtained normalized echo
intensity using Sigma Plot 13 software (SPSS Inc.).
𝐸𝑡𝑜𝑡 = (1 − EV). exp(−a𝐺 2 ) + 𝐸𝑉. exp(−b𝐺 2 )
Equation 2.4
Where 𝐸𝑡𝑜𝑡 is the echo intensity ratio (I/I0) corrected for the oil phase contribution, G in T/m is the
gradient strength and I0 is the echo intensity in the absence of a magnetic field gradient (G = 0
23
T/m). Measurements were performed varying the gradient strength G between 0 and 3.17 T/m
while keeping the gradient duration (δ) and the diffusion delay (∆) fixed constant at 2.5 ms and 60
ms, respectively. Using the same setting, the oil sample containing 5 wt % PGPR, with
proportional mass as present in the double emulsion, was measured for oil phase signal subtraction
from the signal intensity of the double emulsion.
𝑌𝑖𝑒𝑙𝑑 𝑁𝑀𝑅 (%) =
𝐸𝑉𝑁𝑀𝑅
× 100%
𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝐸𝑉
Equation 2.5
For a 25:25:50 W1/O/W2 double emulsion, the theoretical maximum EV amounts to 33.3 %
(i.e.25/(25 + 50) × 100%).
WPI encapsulation efficiency determination of W1/O/W2 emulsions
The WPI encapsulation efficiency (EE) was calculated based on the WPI concentration which was
added to the internal aqueous phase (Equation 2.6).
𝐸𝐸 = 1 −
𝑂𝐴𝑃
𝐶𝑊𝑃𝐼
𝑖𝐴𝑃
𝑚𝑊𝑃𝐼
× [𝑚𝑊2 + 𝑚𝑊1 (1 − 𝑤𝑎𝑡𝑒𝑟 𝑦𝑖𝑒𝑙𝑑)]
Equation 2.6
Where:
𝑂𝐴𝑃
𝐶𝑊𝑃𝐼
Concentration of WPI in centrifuged outer aqueous phase (OAP) (g/g)
𝑖𝐴𝑃
𝑚𝑊𝑃𝐼
Mass of WPI initially added to internal aqueous phase (iAP) of a 100 g
W1/O/W2 emulsion (i.e. 1.25 g, 0.625 g or 0.25 g WPI in 25 g of W1 containing
5, 2.5 or 1 % WPI, respectively)
𝑚𝑊2
Initially applied mass of W2-phase during preparation of 100 g W1/O/W2
emulsion (50 g in a 25:25:50 W/O/W emulsion)
𝑚𝑊1
Initially applied mass of W1-phase during preparation of 100 g W1/O/W2
emulsion (25 g in a 25:25:50 W/O/W emulsion)
Viscosity measurement
A Programmable LV-DV-II+ Viscometer (Brookfield, Stoughton, MA, USA), at speeds ranging
from 30-160 rpm (rotations per minute) was used to measure the viscosity of samples after W/O/W
emulsion preparation as well as after 1 day and 5 days of end-over-end rotation. All measurements
24
were performed at room temperature. Spindle SC4-18 was used in combination with a small
sample adapter (with 6.7 g of sample in the rheometer cell) to measure the viscosity of the samples.
Enzymatic assay of Alkaline Phosphatase
Alkaline Phosphatase is a phospho-mono esterase enzyme yielding phosphate and the
corresponding alcohol by catalyze the hydrolysis of monoesters of phosphoric acid (at alkaline
pH). ALP is found in milk and other body fluids from many organisms at different levels. It shows
the maximum activity in the pH range 9.67 to 10.1 at 37 ℃. Alkaline Phosphatase test is recognized
as a standard assay for rapid evaluation of the pasteurization process of milk to inactivate the target
bacterial pathogens (namely Coxiellaburnetii and Mycobacterium tuberculosis) which are the most
heat-resistant bacteria present in milk (Rankin et al., 2010). ALP hydrolyzes the colorless paranitrophenyl phosphate (pNPP) (substrate) to produce a yellow product of para-nitrophenol (pNP)
(Figure 2.3) (Dean, 2002).
Figure 2.3. Enzymatic reaction of Alkaline Phosphatase in presence of para-nitrophenyl phosphate
(Dean, 2002).
If the reaction velocity is plotted versus the substrate concentration, the curve will have a
hyperbolic response according to the Michaelis-Menten equation 2.7:
𝑣=
𝑉𝑚𝑎𝑥 × 𝑆
𝐾𝑚 + 𝑆
Equation 2.7
For determination of the Km (substrate concentration that yield a half of maximum reaction rate)
and the Vmax (maximum theoretical reaction rate), the linearizing method of Hanes and Woolf was
used. Km is independent of the enzyme concentration. A lower value of Km shows that the enzyme
has got more catalytic efficiency due to a more firm binding of substrate and enzyme. Km values
usually are in the range of 10-2 to 10-5 mol/l (Belitz et al., 2009).
25
However, there are many enzymes which do not obey the simple Michaelis-Menten equation. A
sigmoid shape of response curve is found in enzymes known as allosteric enzymes (Scopes, 2002).
A procedure that linearizes the Michaelis-Menten equation was proposed by Hanes and Woolf
(Figure 2.4 and Equation 2.8) (Belitz et al., 2009):
𝑆
𝑆
𝐾𝑚
=
+
𝑉
𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥
Equation 2.8
Figure 2.4. Hanes-Woolf linearization of the Michaelis-Menten equation (Scopes, 2002).
The enzymatic activity of Alkaline Phosphatase was measured in the presence of different
concentrations of para-nitrophenyl phosphate (pNPP) with reaction volume of 2.5 mL (Table 2.5).
The 0.1 M Glycine-NaOH buffer contained MgCl2 and ZnCl2 salts, both in a concentration of 1
mM. The pH was adjusted to 10.4 with 1 M NaOH. The 6 mM pNPP stock solution was freshly
prepared in 0.1 M salt containing Glycine-NaOH buffer. The enzyme stock solution was prepared
by addition of 1 mg enzyme powder to cold deionized water (corresponding to 0.042 mg enzyme
per 25 mL).
Prepared 2.5 mL reaction mixtures were incubated at 37 C for 10 minutes. Then the enzyme
activity was immediately stopped by adding 7.5 mL of 0.2 M NaOH. The resulting solutions were
incubated at 37 C for 30 minutes. Finally, the absorbance was measured at 420 nm and the
associated para-nitrophenol (pNP) concentration was determined using a standard curve. It should
be noted that the absorbance of the prepared solutions must be in the linear part of the standard
curve. Therefore, enzyme solutions were diluted using an appropriate dilution factor of 1:13.
26
Table 2.5. Composition of solutions for the enzymatic activity measurement of Alkaline
Phosphatase.
Samples
6 mM Substrate pNPP stock solution
(mL)
0.1 M salt containing Glycine-NaOH
buffer (mL)
Enzyme solution (mL)
Deionized water (mL)
Total reaction volume (mL)
pNPP concentration in 2.5 mL
reaction volume (mM)
0.0
0.0
0.1
0.2
0.4
0.6
0.8
1.0
1.5
2.0
2.0
1.9
1.8
1.6
1.4
1.2
1.0
0.5
0.0
0.5
2.5
0.0
0.5
0.0
2.5
0.0
0.5
0.0
2.5
0.24
0.5
0.0
2.5
0.48
0.5
0.0
2.5
0.96
0.5
0.0
2.5
1.44
0.5
0.0
2.5
1.92
0.5
0.0
2.5
2.4
0.5
0.0
2.5
3.6
2.7.1 Standard curve
Standard solutions were prepared as described in Table 2.6. The solutions agreed with product
concentrations of p-nitrophenol (pNP) of 0, 10, 20, 40, 60, 80 and 100 µM in a reaction volume of
2.5 mL. The 1000 µM pNP stock solution in 0.1 M salt containing Glycine-NaOH buffer was
freshly prepared. The 10 mL solutions were incubated at 37 C for 30 minutes and the absorbance
was measured at 420 nm. The standard curve was obtained by plotting the absorbance versus the
concentration of p-nitrophenol (Figure 2.5).
Table 2.6. Composition of standard solutions for enzyme activity measurement of Alkaline
Phosphatase.
Samples
1000 µM pNP stock solution (mL)
0.1 M Glycine-NaOH buffer (mL)
Deionized water (mL)
0.2 M NaOH stock solution (mL)
Total reaction volume (mL)
pNP concentration in 2.5 mL reaction
volume (µM)
0.0
2.0
0.5
7.5
10
0
0.1
1.9
0.5
7.5
10
10
27
0.2
1.8
0.5
7.5
10
20
0.4
1.6
0.5
7.5
10
40
0.6
1.4
0.5
7.5
10
60
0.8
1.2
0.5
7.5
10
80
1.0
1.0
0.5
7.5
10
100
Absorbance at 420 nm
0,8
0,6
0,4
y = 0.0227x - 0.0109
R² = 0.9945
0,2
0
0
10
20
30
40
p-nitrophenol concentration (µM)
Figure 2.5. Standard curve of the product concentration (p-nitrophenol) for determination of the
Alkaline Phosphatase activity.
Statistical analysis
Statistical data were analyzed by a Wilcoxon signed-rank test using S-Plus (Spotfire S+®
8.2,TIBCO Software Inc.) and by linear regression using Excel (Microsoft Office 2013) at a 5 %
significance level. All experiments considering emulsions were done in triplicate.
28
Chapter 3
Results and discussion
Measurement of whey protein isolate (WPI) in the separated outer aqueous phase
using the Schacterle and Pollack method
The WPI concentration was measured by the simplified Lowry method (Schacterle et al., 1973),
which requires a reference WPI-calibration series (section 2.3.1 and Figure 3.2).
Many compounds interfere with the Lowry assay and form a yellow precipitate after adding the
phenol-reagent. In our study, a yellow precipitate was observed during WPI concentration
measurement in samples which were prepared by a ‘low-energy homogenization intensity’ in the
secondary emulsification step. The emulsification procedure was described in section 2.2.1. Figure
3.1 indicates more precipitation with increasing amount of Tween 80 in the associated separated
serum phase. Whereas dilution of samples might reduce the effect of interfering substances
(Johnson, 2012), this is not an option for samples with a low amount of WPI as that might result
in undetectably low protein levels. In order to determine the effect of precipitation on the WPI
concentration determination of samples, an additional standard series containing 1.67 wt % Tween
80 was prepared (Figure 3.2). Figure 3.2 shows that at higher WPI levels, the precipitate formation
caused protein content underestimation.
The addition of sodium dodecyl sulfate (SDS) might prevent precipitate formation caused by
nonionic and cationic detergents without affecting color development (Dulley et al., 1975;Peterson
1977). Therefore, the standard series with Tween 80 was repeated in the presence of SDS, which
was added to the alkaline Cu-reagent (Figure 3.2). However, the addition of SDS could not prevent
the precipitate formation.
29
a
b
c
Figure 3.1. Yellow precipitate formation upon addition of the phenol reagent to the serum phase
of emulsions prepared by low-energy homogenization which contained 1.67 % (a), 1 % (b), and 0.5
% (c) Tween 80 in the external water phase.
Absorbance at 650 nm
0,8
0,7
0,6
0,5
Reference
0,4
with tween 80
0,3
with tween 80 and SDS
0,2
with SDS
0,1
0
0
100
200
300
WPI concentration (mg/L)
Figure 3.2. Polynomial comparison between reference standard series and standard series upon
addition of Tween 80 and/or SDS.
It is worth mentioning that SDS might influence the structure of proteins and possibly change their
solubility. This protein-detergent interaction is used in the analytical measurement of proteins.
SDS is an ionic detergent that binds strongly to proteins often with protein denaturation (Otzen
2002;Fano et al., 2011). This denaturation of globular proteins affects the tertiary structure,
whereas the secondary structure is unaffected (Fano et al., 2011). Globular protein denaturation
typically occurs above the critical micelle concentration (CMC) which is about 7 mM for SDS
(Otez, 2002). In our experiment, the concentration of SDS was 34.68 mM. In order to evaluate the
30
effect of SDS on the protein determination, a standard series was made which only differed from
the reference series in the presence of 1 wt % SDS in the alkaline Cu-reagent (Figure 3.2). Figure
3.2 shows a larger deviation in absorbance at higher WPI concentrations in comparison to the
standard series containing Tween 80, with or without SDS. This indicates that the deviation of the
standard curve in the presence of SDS was strongly affected by the Tween 80 concentration, which
was not measured. For sake of simplicity, the reference standard curve (Figure 3.2) was used for
WPI determination in each following sample after subtracting the absorbance of an identically
made blank solution. Hereby, the absorbance was kept sufficiently low (i.e. lower than the above
mentioned deviating region).
Protein solubility determination using the Schacterle and Pollack method
The WPI concentration was measured in bulk solutions of the internal water phase (W1), the
mixture of internal and external aqueous phases (W1 + W2), and external water phase (W2) with a
composition described in section 2.2.1. The Schacterle and Pollack method (1973) was used (see
section 2.3.1). Hereby, associated blank solutions were made without WPI. WPI measurements in
bulk water phases enable to determine the protein solubility according to:
𝑊𝑃𝐼 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 (%) =
𝑀𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑊𝑃𝐼 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
∙ 100 %
𝐴𝑝𝑝𝑙𝑖𝑒𝑑 𝑊𝑃𝐼 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
Equation 3.1
The protein solubility can give information about the accuracy of the WPI concentration
measurements in the evaluation of WPI encapsulation in W1/O/W2 emulsion using the Schacterle
and Pollack method (Table 3.1).
Table 3.1. The solubility of WPI in the internal water phase (W1), the mixture of internal
and external water phases (W1 + W2), and the external water phase (W2). Each listed values
is the average of triplicate sets of measurements ± standard deviation (n = 3).
Measurable concentration of WPI (%)
Initial concentration of WPI in W1phase (%)
5.0
2.5
1.0
W1
104.9 ± 3.5
102.7 ± 2.6
97.9 ± 2.6
31
W1 + W2
116.8 ± 2.6
114.1 ± 1.3
115.8 ± 1.7
W2
122.7 ± 0.3
120.5 ± 2.4
112.6 ± 1.2
As it can be seen from Table 3.1 the WPI solubility in the different aqueous phases was roughly
around 100 %. A slight overestimation was noticed when Tween 80 (non-ionic surfactant) was
added to the aqueous phases. As described previously, Tween 80 might interfere with the Lowry
assay.
Measurement of the recovery yield of WPI using the Schacterle and Pollack method
The recovery yield of WPI in an emulsion might be defined as the concentration of WPI found in
the water phase recovered from the emulsion which has been separated into a cream phase and an
aqueous phase by centrifugation proportional to the concentration of WPI present in (or added to)
the water phase after emulsion preparation. The recovery yield of a water-soluble marker in an
emulsion is a measure for the partitioning of the marker with the cream phase (O'Regan et al.,
2009). In this section, for determination of the WPI recovery yield, two types of O/W emulsions
(with phase composition as described in section 2.2.1) were prepared:
1. The O/(W1+W2) emulsion: a simple O/(W1+W2) was prepared by simultaneous
homogenization of the same internal aqueous phase (W1), external aqueous phase (W2),
and oil phase (O) as in the W1/O/W2.
2. The (O/W2) + W1 emulsion: a simple O/W2 emulsion was prepared by homogenization of
the oil phase (O) and external water phase (W2). This O/W2 emulsion was diluted with the
same internal aqueous phase (W1) as in the W1/O/W2 emulsion upon gentle mixing directly
after emulsification.
Due to their amphiphilic nature, proteins can adsorb at the fat droplet interface during the
homogenization process. However, protein desorption from the fat surface might occur during
storage time due to a competitive mechanism with low molecular weight emulsifiers (Granger et
al., 2005).
WPI was always present in the W1-phase in a concentration of 5, 2.5, or 1 wt %. For measuring
the WPI concentration, the simple emulsions were separated into a cream phase and aqueous phase
by centrifugation using a benchtop centrifuge at 4000 rpm for 1 h at room temperature. The
concentration of WPI in the recovered subnatant was determined by the Schacterle and Pollack
method (1973). In this experiment, the subnatant obtained from associated simple emulsions
without WPI served as a reference blank.
It should be noted that the recovery yield values are corrected using the protein solubility values
at different WPI concentrations in [W1+W2]-phase (see section 3.2).
32
The recovery yield of WPI in the O/W emulsion is calculated as follows (Equation 3.2) (O'Regan
and Mulvihill 2009):
𝑅𝑦(%) =
𝐴𝑃
𝐶𝑊𝑃𝐼(𝑡)
𝐶𝑊𝑃𝐼(𝑡=0)
∙
1
∙ 100 %
𝑊𝑃𝐼 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 (−)
Equation 3.2
𝐴𝑃
Where 𝐶𝑊𝑃𝐼(𝑡)
is the measured concentration of the WPI in the aqueous phase (AP) recovered on
centrifugation of the emulsion at time t and 𝐶𝑊𝑃𝐼(𝑡=0) is the WPI concentration initially added to
𝐴𝑃
the water phase of the O/W emulsion. 𝐶𝑊𝑃𝐼(𝑡)
and the WPI protein solubility fraction are measured
using the Schacterle and Pollack method (section 2.3.1 and 3.2, respectively).
3.3.1 Effect of the presence of Tween 80
This experiment was performed in the presence and absence of Tween 80 as a hydrophilic
surfactant in the W2-phase. In the latter case, a smaller recovery yield is expected as more WPI
might adsorb at the oil-water interface. In the O/(W1+W2) emulsion, WPI is present before
emulsion preparation, whereas it is added afterwards for the (O/W2) + W1 emulsion. We postulated
that the difference in WPI results between O/(W1+W2) and (O/W2)+W1 might give information
about the difference in migration of WPI during preparation and with storage time. Therefore, the
recovery yield was measured as a function of storage time at room temperature (i.e. 0, 1, 2, 3, 5,
7, 14 and 12 days).
The recovery yield for WPI in the O/(W1+W2) and (O/W2)+W1 is shown in Figure 3.3. Figure 3.3
shows that in most cases the recovery yield did not change as a function of time. This indicates
that WPI did not migrate from to the cream phase in the post-preparation storage time.
In the presence of Tween 80 and when using 5 % and 1 % WPI, the recovery yield was smaller for
the O/(W1+W2) emulsion than for the O/(W1+W2) emulsion, albeit this was not the case for 2.5 %
WPI. In the absence of Tween, an even lower recovery yield of the O/(W 1+W2) emulsion was
observed for all applied WPI concentration in comparison to the O/(W1+W2) emulsion. This shows
that partitioning of WPI with the cream phase occurred during emulsification, especially in the
absence of other hydrophilic surfactants.
33
5 % WPI, without T80
100
Recovery yield (%)
Recovery yield (%)
5 % WPI, with T80
80
60
40
O/(W1+W2)
20
(O/W2)+W1
100
80
60
40
O/(W1+W2)
(O/W2)+W1
20
0
0
0
5
10
15
20
Storage time (days)
25
0
2.5 % WPI, with T80
Recovery yield (%)
Recovery yield (%)
25
80
60
40
O/(W1+W2)
(O/W2)+W1
100
80
60
40
O/(W1+W2)
20
0
(O/W2)+W1
0
0
5
10
15
20
Storage time (days)
25
0
1.0 % WPI, with T80
Recovery yield (%)
80
60
40
O/(W1+W2)
(O/W2)+W1
20
5
10
15
20
Storage time (days)
25
1.0 % WPI, without T80
100
Recovery yield (%)
10
15
20
Storage time (days)
2.5 % WPI, without T80
100
20
5
0
100
80
60
40
O/(W1+W2)
(O/W2)+W1
20
0
0
5
10
15
20
Storage time (days)
25
0
5
10
15
20
Storage Time (days)
25
Figure 3.3. The recovery yield of the WPI in the recovered aqueous phase of the centrifuged
O/(W1+W2) and (O/W2)+W1 emulsions containing 5%, 2.5 % and 1% WPI in W1-phase. Moderate
homogenization intensity was applied with or without Tween 80 (T80). The error bars represent
the standard deviation of triplicate sets of measurements.
34
The protein adsorption at the fat globules might be influenced by the nature of the low molecular
weight emulsifiers, the protein type, and/or the fat characteristics (Granger et al., 2005).
In our study, for stabilization of the W1/O/W2 emulsion, the non-ionic hydrophobic surfactants
PGPR and the non-ionic hydrophilic surfactant Tween 80 were used.
Gülseren and Corredig (2012) reported that the addition of PGPR in the presence of 0.1 % βlactoglobulin (β-lg) lowered the interface elasticity, suggesting that PGPR and β-lg interacted
possibly through hydrophobic interactions, resulting in the interference of the protein-protein
interactions at the surface or displacement of β-lg. In addition, they presented that even at low
concentration of PGPR (0.008 %), the interfacial elasticity was mostly affected by PGPR rather
than proteins. Whey proteins such as β-lg have globular structures, which need a longer time to
adsorb at the interface due to a less flexible structure. In general, proteins mainly adsorb in a
diffusion controlled manner at the water-oil interface in the absence of surfactants (Miller et al.,
2000). The adsorption process of proteins at water-oil interface may be classified in three main
steps:
1. Diffusion of protein molecules from bulk solution to the near surface region
2. Adsorption of the proteins to the interface
3. Rearrangement of adsorbed protein molecules
However, conformational stability and surface hydrophobicity have an influence on the rate and
extent of interfacial protein adsorption (Tripp et al., 1995).
Miller et al. (2000) reported on the hydrophobic interaction between proteins and non-ionic
surfactants. At low concentration of non-ionic surfactants the adsorption layer was mainly formed
by competitive adsorption, whereas with increasing surfactant concentration, the interface was
almost exclusively covered by non-ionic surfactants. Figure 3.4 schematically shows the effect of
an elevated amount of non-ionic surfactants in a protein solution. Therefore, a high concentration
of non-ionic surfactants might avoid protein adsorption at the interface due to almost entire
coverage of the interface by non-ionic surfactants. In our experiment, the prepared emulsion
contained a relatively high concentration of PGPR (5 wt % in oil phase) and Tween 80 (1.67 wt %
in external aqueous phase). Gülseren and Corredig (2012) revealed that the presence of PGPR
inhibited the formation of a β-lg film at the interface.
35
Figure 3.4. Schematic image of interaction between protein and non-ionic surfactant: free
protein molecules (a), free surfactant molecules (b), first interaction via hydrophobic
interaction (c), increased hydrophobic interaction by increasing surfactant concentration (d)
(e) (Miller et al., 2000).
O/W emulsions are rapidly destroyed in the presence of oil-soluble surfactants such as PGPR since
the edge of the possible formed holes in the surfactant film is curved against the direction favored
by the spontaneous curvature resulting in phase separation (Leal-Calderon et al., 2007). A
schematic image of spontaneous curvature in W/O emulsion is shown in Figure 3.5.
Figure 3.5. Schematic figure of spontaneous curvature in W/O emulsions (Leal-Calderon et al.,
2007).
36
3.3.2 Effect of homogenization intensity
Based on the fact that the homogenization intensity influences the oil droplet size, higher recovery
yield values are expected when less homogenization intensity is applied due to less surface area
created for protein adsorption. Therefore, the simple emulsions were also prepared with a lowenergy homogenization intensity (see section 2.2.1) and the recovery yield of WPI in the
O/(W1+W2) and (O/W2)+W1 emulsions is shown in Figure 3.6. In contrast to the phases mixed
with moderate homogenization intensity in section 3.3.1, the recovery yield values of WPI for
most emulsions depicted in Figure 3.6 are similar for the O/(W1+W2) and (O/W2)+W1 emulsions.
A plausible explanation could be inhibition of protein adsorption due to the high concentration of
both hydrophilic (1.67 wt % in external water phase) and hydrophobic (5 wt % in oil phase)
surfactants at the oil-water interface.
5 % WPI, with T80
5 % WPI, without T80
100
Recovery yield (%)
Recovery yield (%)
100
80
60
40
O/(W1+W2)
20
(O/W2)+W1
0
80
60
40
O/(W1+W2)
20
0
0
5
10
15
20
Storage time (days)
25
0
2.5 % WPI, with T80
5
10
15
20
Storage time (days)
25
2.5 % WPI, without T80
100
100
Recovery yield (%)
Recovery yield (%)
(O/W2)+W1
80
60
40
O/(W1+W2)
(O/W2)+W1
20
0
80
60
40
O/(W1+W2)
(O/W2)+W1
20
0
0
5
10
15
20
Storage time (days)
25
0
37
5
10
15
20
Storage time (days)
25
1.0 % WPI, without T80
100
Recovery yield (%)
Recovery yield (%)
1.0 % WPI, with T80
80
60
40
O/(W1+W2)
20
(O/W2)+W1
0
100
80
60
40
O/(W1+W2)
20
(O/W2)+W1
0
0
5
10
15
20
Storage time (days)
25
0
5
10
15
20
Storage time (days)
25
Figure 3.6. The recovery yield of the WPI in the recovered aqueous phase of the centrifuged
O/(W1+W2) and (O/W2)+W1 emulsions containing 5%, 2.5 % and 1% WPI in W1-phase. Lowenergy homogenization intensity was applied with or without Tween 80 (T80). The error bars
represent the standard deviation of triplicate sets of measurements.
Measurement of N-containing compounds using the nitrogen determination method
following persulfate oxidation
Since the W1/O/W2 emulsions contained different chemical compounds and they might interfere
with this method, the effect of the double emulsion composition on the determination of the
nitrogen content of NaNO3 solutions were evaluated. Hereby, NaNO3 was selected as a simple Ncontaining compound.
3.4.1 Influence of NaClO3 on the NaNO3 nitrogen content determination
In section 2.2, sodium chloride was used in the double emulsion preparation for adjusting the
osmotic pressure. Due to O2 consumption by NaCl, the osmotic agent was replaced by NaClO3 and
its influence on the nitrogen content determination was investigated. To that end, two series of
NaNO3 concentrations ranging from 0 to 3.575 mM were measured in deionized water and in 0.1
M NaClO3 solution (Table 3.2). The slope with 95 % confidence interval of 1.00  0.06 in Figure
3.7 indicates that there was no interference of the presence of NaClO3 on the response for the
nitrogen determination.
38
Table 3.2. NaNO3 standard series in deionized water or in 0.1 M NaClO3 solution.
Samples
5 mM NaNO3 in either deionized water or in
0.1M NaClO3 solution (mL)
Deionized water or 0.1M NaClO3 solution (mL)
NaNO3 concentration (mM)
Reagent (mL)
Total volume (mL)
Calculated N (ppm)
0.0
0.71
1.43
2.14
2.86
4.0
0.0
6
10
0.0
3.29
0.89
6
10
4.97
2.57
1.79
6
10
10.01
1.86
2.68
6
10
14.98
1.14
3.58
6
10
20.02
Response value NaNO3 in NaClO3
(a.u.)
70
60
50
40
y = 1,0048x
R² = 0,9962
30
20
10
0
0
10
20
30
40
50
60
70
Response value NaNO3 in deionized water (a.u.)
Figure 3.7. Correlation between NaNO3 as measured in deionized water and in an aqueous 0.1 M
NaClO3 solution.
3.4.2 Influence of potassium phosphate buffer on the NaNO3 nitrogen content
determination
For assessment of the effect of potassium phosphate buffer on the nitrogen content determination,
two standard series of NaNO3 were made with NaNO3 concentrations ranging from 0 to 0.715
mM; one series in 0.1 M NaClO3 solution and another in additionally 5 mM potassium phosphate
buffer (pH 6.6) (Table 3.3). The slope with 95 % confidence interval of 0.97  0.03 in Figure 3.8
indicates that there was no interference of the presence of 5 mM potassium phosphate buffer on
the response for the nitrogen determination.
39
Table 3.3. NaNO3 standard series in 0.1 M NaClO3 solution with or without 5 mM
potassium phosphate buffer solution.
Samples
Response value for NaNO3 in NaClO3
and potassium PO4 buffer (a.u.)
1 mM NaNO3 in 0.1 M NaClO3 in with or without
5 mM potassium PO4 buffer (mL)
0.1 M NaClO3 in with or without 5 mM potassium
PO4 buffer (mL)
NaNO3 concentration (mM)
Reagent (mL)
Total volume (mL)
Calculated N (ppm)
0.0
0.71
1.43
2.14
2.86
4.0
3.29
2.57
1.86
1.14
0.0
0.18
0.36
0.54
0.72
6
10
0.0
6
10
0.99
6
10
2.00
6
10
3.00
6
10
4.00
33
y = 0,9717x
R² = 0,9956
31
29
27
25
23
21
19
17
15
15
20
25
30
35
Response value for NaNO3 in NaClO3 (a.u.)
Figure 3.8. Correlation between NaNO3 as measured in aqueous 0.1 M NaClO3 and in an aqueous
solution of 0.1 M NaClO3 and 1 mM potassium phosphate buffer.
3.4.3 Influence of NaN3 on the NaNO3 nitrogen content determination
The NaNO3 solutions with concentrations ranging from 0 to 0.715 mM were measured in the
presence and absence of NaN3 in an aqueous NaClO3 solution (Table 3.4), whereby for both
deionized water was used. An overestimation of about 24 a.u. was observed in the presence of
NaN3 (Figure 3.9), indicating that NaN3 affected the persulfate digestion and/or reductiondiazotation-absorption step. From the slope of the calibration curve in the absence of NaN3, it
40
follows that the overestimation due to the presence of NaN3 was about 14 ppm. Hence, NaN3 is
only partly digested.
Table 3.4. NaNO3 standard series are prepared in 0.02 wt % NaN3 and 0.1 M NaClO3 in
deionized water.
Samples
1 mM NaNO3 in 0.1 M NaClO3 and 0.02 wt %
NaN3 in deionized water (mL)
0.0
0.71
1.43
2.14
2.86
0.1 M NaClO3 and 0.02 wt % NaN3 in deionized
water (mL)
4.0
3.29
2.57
1.86
1.14
NaNO3 concentration (mM)
0.0
0.18
0.36
0.54
0.72
Reagent (mL)
6
6
6
6
6
Total volume (mL)
10
10
10
10
10
Calculated N from NaNO3 (ppm)
0.0
0.99
2.00
3.00
4.00
NaN3 concentration (wt %)
0.008
0.008
0.008
0.008
0.008
Calculated N from NaN3* (ppm)
0 - 52
0 -52
0 -52
0 -52
0 -52
* depends on the persulfate digestion of NaN3
50
Response value (a.u.)
45
40
NaNO3 in deionized water
35
30
y = 1,4387x + 13,578
R² = 0,9935
25
20
NaNO3 in solution of NaClO3
and NaN3
15
10
y = 1,6742x + 37,607
R² = 0,9289
5
0
0
1
2
3
4
5
Calculated N (ppm)
Figure 3.9. Comparison of nitrogen content determination of NaNO3 standard series with or
without 0.02 % NaN3 in the 0.1 M NaClO3 solution.
41
3.4.3.1 Influence of NaN3 on the reduction-diazotation-absorption or persulfate digestion
reaction
Constant NaNO3 concentration (0.143 mM) in 0.1 M NaClO3 solution were made. Different final
NaN3 concentrations ranging from 0 to 0.8 wt % were added to the first series after digestion (Table
3.5). To the second series, these NaN3 concentrations were added before the digestion step (Table
3.6). A change in response with increasing post-digestion NaN3 concentration is expected to
specifically indicate an effect of NaN3 on the absorption step, whereas a response change with predigestion NaN3 concentration indicates an effect on the absorption and/or digestion step. Figure
3.10 clearly indicates an effect of NaN3 on the absorption step. Since the response change differs
for Figure 3.10a and 3.10b, there is also an effect of NaN3 on the digestion reaction. Figure 3.10a
further indicates that a dilution solution without NaN3 should be used for samples that require a
post-digestion dilution (i.e. because of a too high absorbance). Figure 3.10b shows that the
comparison of differently pre-digested diluted samples containing NaN3 is not possibly and hence,
the same dilution factor should be applied for the NaN3-containing samples, assuming that the
inner and outer aqueous NaN3 concentration of the W/O/W emulsion remains constant. Based on
the complexity of the NaN3 effect, the water phases of the double emulsions, as analyzed with the
nitrogen determination method, were made without NaN3.
Table 3.5. Series with constant NaNO3 concentration in 0.1 M NaClO3 and Milli-Q water
to which different NaN3 concentration are added after digestion.
Samples
1 mM NaNO3 in 0.1 M NaClO3 in Milli-Q water
(mL)
0.1 M NaClO3 in Milli-Q water (mL)
Reagent (mL)
Total volume (mL)
Digestion
Post-digestion dilution
10 wt % NaN3 in 0.1 M NaClO3 in Milli-Q water
(mL)
0.1 M NaClO3 in Milli-Q water (mL)
Final volume (mL)
NaN3 concentration (wt %)
NaNO3 concentration (mM)
Calculated N from NaNO3 (ppm)
42
0.0
1.57
1.57
1.57
1.57
1.57
4.0
2.43
2.43
2.43
2.43
2.43
6
10
6
10
6
10
6
10
6
10
6
10
0.0
0.0
0.11
0.33
0.55
0.88
1.0
1.0
0.89
0.67
0.45
0.12
11
0.0
0.0
0.0
11
0.0
0.14
2.00
11
0.1
0.14
2.00
11
0.3
0.14
2.00
11
0.5
0.14
2.00
11
0.8
0.14
2.00
Table 3.6. Series with constant NaNO3 concentration in 0.1 M NaClO3 and Milli-Q water
to which different NaN3 concentration are added before digestion.
Samples
1 mM NaNO3 in 0.1 M NaClO3 in Milli-Q water
(mL)
10 wt % NaN3 in 0.1 M NaClO3 in Milli-Q water
(mL)
0.1 M NaClO3 in Milli-Q water (mL)
Reagent (mL)
0.0
1.43
1.43
1.43
1.43
1.43
0.0
0.0
0.10
0.30
0.50
0.80
4.0
6
2.57
6
2.47
6
2.27
6
2.07
6
1.77
6
Total volume (mL)
10
10
10
10
10
10
NaN3 concentration (wt %)
NaNO3 concentration (wt %)
Calculated N from NaNO3 (ppm)
0.0
0.0
0.0
0.0
0.14
2.00
0.1
0.14
2.00
0.3
0.14
2.00
0.5
0.14
2.00
0.8
0.14
2.00
a
b
400
y = 401,54x + 26,023
R² = 0,9978
350
25
Respose value (a.u.)
Response value (a.u.)
30
20
15
10
5
300
250
200
150
100
50
0
0
0
0,2
0,4
0,6
0,8
0
0,5
1
Pre-digestion addition of NaN3 (final
conc. g/100 mL)
Post-digestion addition of NaN3 (final
conc. g/100 mL)
Figure 3.10. N content determination of NaNO3 standard series with NaN3 addition after (a) and
before (b) the digestion step.
43
3.4.4 Influence of NaClO3, potassium phosphate buffer and NaN3 on the WPI nitrogen
content determination
Analogously to the nitrogen determination of NaNO3 solutions, the effect of different aqueous
compounds (NaClO3, potassium phosphate buffer pH 6.6 and NaN3) on the nitrogen determination
of a WPI series was evaluated (Table 3.7). In order to calculate the nitrogen content of WPI in
ppm, a molecular mass of 18363 g/mol was assumed (Farrell et al., 2004) and a nitrogen content
in WPI of 15.77 % (Mariotti et al., 2008). As such, the calculated number of moles of N-atoms in
one mol of WPI amounts to 207. Figure 3.11 shows that the presence of NaN3 resulted in an
overestimation of the nitrogen content as described earlier for NaNO3 (see section 3.4.3).
Table 3.7. WPI standard series in different dilution liquids (either Milli-Q water, 0.1 M
NaClO3 in Milli-Q water, 5 mM potassium phosphate buffer in Milli-Q water, or 0.02 wt
% NaN3 in Milli-Q water).
Samples
0.02 wt % WPI in dilution liquid (mL)
Dilution liquid (mL)
WPI concentration (mM)
Reagent
Total volume (mL)
Calculated N (ppm)
0.0
4.0
0.0
6
10
0.0
0.4
3.6
0.001
6
10
1.26
0.8
3.2
0.002
6
10
2.52
1.2
2.8
0.003
6
10
3.79
1.6
2.4
0.004
6
10
5.05
80
Response value (a.u.)
70
WPI in MilliQ
y = 1,3273x + 26,125
R² = 0,8542
60
50
30
WPI in NaClO3, MilliQ
y = 2,1392x + 22,434
R² = 0,9992
20
WPI in PO4 buffer, MilliQ
40
y = 2,1974x + 21,643
R² = 0,9996
10
0
0
1
2
3
4
Calculated N (ppm)
5
6
WPI in NaN3, MilliQ
y = 2,13x + 64,898
R² = 0,9984
Figure 3.11. Comparison of nitrogen content determination of WPI standard series in Milli-Q
water as well as in NaN3, NaClO3 or potassium phosphate buffer containing aqueous solutions.
44
As it can be observed in Figure 3.11, there was an overlap between the curves obtained from the
WPI standard series in Milli-Q water and the ones containing NaClO3 or potassium phosphate
buffer. In fact, the slopes of all curves were similar, which indicates that WPI provoked the same
response, irrespective of the dilution liquid. The slope of the calibration curve obtained in distilled
water was smaller; figure 3.11 shows that this underestimated slope was mainly due to an
overestimation of the blank reading.
3.4.5 Influence of Tween 80 on the WPI nitrogen content determination
Since Tween 80 might consume O2, the effect of its presence on the nitrogen content determination
of WPI was evaluated. Samples with constant concentration of WPI were made, to which
increasing concentrations of Tween 80 in Milli-Q water were added. A second series additionally
contained a constant amount of NaClO3, NaN3 and potassium phosphate buffer (pH 6.6) in
deionized water (Table 3.8 and 3.9). It should be noted that dissolved Tween 80 was heated for 10
minutes at 50-55 ℃ (used after cooling). The linear regression analysis of Figure 3.12a and 3.12b
resulted in a slope (with 95 % confidence interval) of -0.08  1.98 and -4.37  5.50, which means
that the slopes are not significantly different from zero. Hence, Tween 80 in the applied
concentration range did not interfere with the nitrogen determination method. It should be noted
that the larger response value in Figure 3.12b is due to the effect of NaN3 on the nitrogen
determination method.
Table 3.8. WPI series in the presence of Tween 80 (T80) in Milli-Q water.
Samples
0.02 wt % WPI in Milli-Q water (mL)
0.25 wt % T80 in Milli-Q water (mL)
Milli-Q water (mL)
WPI concentration (mM)
Reagent (mL)
Total volume (mL)
Tween 80 concentration (mM)
Calculated N from WPI (ppm)
0.0
0.0
4.0
0.0
6
10
0.0
0.0
0.44
0.0
3.56
0.001
6
10
0.0
1.39
45
0.44
0.5
3.06
0.001
6
10
0.10
1.39
0.44
1.5
2.06
0.001
6
10
0.29
1.39
0.44
2.5
1.06
0.001
6
10
0.48
1.39
0.44
3.5
0.06
0.001
6
10
0.67
1.39
Table 3.9. WPI series in the presence of a mixture of Tween 80, NaClO3, NaN3 and
phosphate buffer.
Samples
0.02 wt % WPI in 0.1 M NaClO3, 0.02 wt %
NaN3 and 5 mM potassium PO4 buffer (mL)
0.25 wt % Tween 80 in 0.1 M NaClO3, 0.02
wt % NaN3 and 5 mM potassium PO4 buffer
(mL)
0.1 M NaClO3, 0.02 wt% NaN3 and 5 mM
potassium PO4 buffer (mL)
WPI concentration (mM)
Reagent
Total volume (mL)
Tween 80 concentration (mM)
Calculated N from WPI (ppm)
0.0
0.44
0.44
0.44
0.44
0.44
0.0
0.0
0.30
0.60
1.72
2.80
4
3.56
3.26
2.96
1.84
0.76
0.001
6
10
0.0
1.39
0.001
6
10
0.0
1.39
0.001
6
10
0.06
1.39
0.001
6
10
0.11
1.39
0.001
6
10
0.33
1.39
0.001
6
10
0.53
1.39
a
b
45
Response value (a.u.)
Response value (a.u.)
30
25
y = -0,0795x + 25,2
R² = 0,0054
20
15
10
5
40
35
30
y = -4,3692x + 39,882
R² = 0,6808
25
20
15
10
5
0
0
0
0,2
0,4
0,6
0
0,8
0,2
0,4
0,6
Tween 80 concentration (mM) in 10
mL
Tween 80 concentration (mM) in 10
mL
Figure 3.12. Comparison of nitrogen content of WPI in the presence of Tween 80 (a) or a mixture
of Tween 80, NaClO3, NaN3, and potassium phosphate buffer (b).
3.4.6 Influence of the sodium persulfate concentration on the reduction-diazotationabsorption or persulfate digestion reaction
Based on the study of Zhu (2011), an increase in the Na-persulfate concentration resulted in an
overestimation of the N content. In order to specify the action of sodium persulfate on the digestion
and/or absorption step of the nitrogen determination, two series were made. In the first series,
different sodium persulfate reagent concentrations ranging from 0.1 to 0.6 M as dissolved in 0.1
46
M NaClO3 were digested (Table 3.10). A change in response is expected to indicate an effect of
sodium persulfate on the absorption and/or digestion. A second series of a constant sodium
persulfate concentration of 0.6 M in 0.1 M NaClO3 was digested and afterwards with 0.1 M
NaClO3 in order to obtain different concentrations of sodium persulfate prior to the absorption step
(Table 3.11). A change in response is expected to indicate a selective effect of sodium persulfate
on the digestion.
The response obtained for pre-digestion and post-digestion difference in sodium persulfate
concentration was linearly proportional with the sodium persulfate concentration in the final
solution with slopes of 36.13 and 36.74, respectively.
Table 3.10. Series containing different concentrations of reagent prior to digestion.
0.1 M NaClO3 in Milli-Q water (mL)
Reagent (mL)
Total volume (mL)
Calculated N (ppm)
sodium persulfate concentration (M)
9
1
10
0
0.1
8
2
10
0
0.2
Samples
7
6
3
4
10
10
0
0
0.3
0.4
5
5
10
0
0.5
4
6
10
0
0.6
4
6
10
0.6
4
6
10
0.6
2
12
1.2
0.5
0
10
1
0.6
Table 3.11. Series with fixed reagent concentration diluted after digestion.
0.1 M NaClO3 in Milli-Q water (mL)
Reagent (mL)
Total volume (mL)
sodium persulfate concentration (M)
Digestion
Post-digestion dilution
0.1 M NaClO3 in Milli-Q water (mL)
Final volume (mL)
Dilution factor
sodium persulfate concentration (M) in 10 mL
4
6
10
0.6
4
6
10
0.6
Samples
4
4
6
6
10
10
0.6
0.6
50
60
6
0.1
20
30
3
0.2
10
20
2
0.3
5
15
1.5
0.4
3.4.7 Standard curve
The standard WPI series for nitrogen content determination was constructed using WPI in NaClO3
and potassium phosphate buffer (pH 6.6) (Table 3.12 and Figure 3.13). Based on the complexity
of the NaN3 effect as described in section 3.4.3, the water phases of the standard curve and of the
double emulsions, as analyzed with the nitrogen determination method, were made without NaN3.
47
Table 3.12. WPI standard series in 5 mM potassium phosphate buffer and 0.1 M NaClO3
solution.
Samples
0.02 wt% WPI in 0,1 M NaClO3 and 5 mm PO4
buffer (mL)
0.1 M NaClO3 and 5 mm PO4 buffer (mL)
WPI concentration (mM)
Reagent (mL)
Total volume (mL)
Calculated N (ppm)
0.0
1.0
1.6
2.5
3.2
4.0
3.0
2.4
1.5
0.8
0.0
6
10
0.003
6
10
0.004
6
10
0.007
6
10
0.009
6
10
0.0
3.15
5.05
7.89
10.1
70
Response value (a.u.)
60
50
y = 3,1932x + 31,299
R² = 0,9992
40
30
20
10
0
0
2
4
6
8
10
12
Calculated N (ppm)
Figure 3.13. WPI standard curve in the presence of NaClO3 and potassium phosphate buffer (pH
6.6). The error bars represent the standard deviation of triplicate sets of measurements.
Effect of storage temperature and concentration of WPI in the W1-phase on its release
kinetics as a function of storage time under quasi-isotonic conditions
In quasi-isotonic conditions, the concentration of sodium chloride was chosen so that the two
aqueous phases of the W/O/W emulsion had approximately the same initial osmotic pressure (with
composition as described in section 2.2.1). Two types of W/O/W emulsions were made, which
differed in the applied homogenization intensity in the secondary emulsification step, i.e. the
moderate and low homogenization intensity procedure (see section 2.2.1). The double emulsions
were stored at 5 ℃ or at room temperature. The WPI release into the external water phase was
48
measured directly after preparation and during 21 days of storage. In addition, the effect of
different WPI concentrations (5 %, 2.5 % or 1 %) in the inner water phase on the exchange kinetics
between the internal and external aqueous phase was measured as a function of storage time. It is
worth mentioning that the thermal denaturation temperature of whey protein is about 74 ℃ (Surh
et al., 2007). In our experiments, the temperature of the W1/O raised to about 60 ℃ during the first
emulsification step. Hence, gelation of the internal aqueous phase was not expected to occur.
3.5.1 WPI release from the W1/O/W2 emulsions prepared by moderate homogenization
intensity
In this experiment, creaming of the multiple droplets was slowed down by decreasing their size.
To that end, a moderate homogenization intensity was applied in the secondary emulsification step
(see section 2.2.1). Figure 3.14 shows the concentration of WPI in the external water phase
separated from the emulsions with different concentrations of WPI (5, 2.5 and 1 % in the internal
water phase).
As shown in Figure 3.14, the absolute WPI release increased with increasing WPI concentration.
The concentration of WPI in the outer aqueous phase did not significantly change as a function of
time (Table 3.13).
Regarding the storage temperature, there was no clear trend of the effect on the release with time;
for 5 % WPI, there was a significantly larger release at room temperature (p = 0.027), whereas for
2.5 % and 1 % WPI, there was a significantly larger release at 5 °C (both p = 0.004). Coalescence
and diffusion phenomena might be involved in the destabilization process of double emulsions.
Encapsulated hydrophilic compounds can migrate between two aqueous phases with and without
film rupturing. The concentration gradient of the species dissolved in the two aqueous phases is
generally responsible for the transport leading to equilibration of the concentrations (Delample et
al. 2014). As there is no osmotic pressure driving force between the water phases under quasiisotonic conditions and the WPI concentration was almost constant during storage time, it can be
concluded that some WPI release from the internal water phase into the external water phase is
mainly due to the turbulent mixing produced by the second emulsification step. More intensive
homogenization produces smaller oil droplets and increases the possibility to rupture the internal
water droplets.
49
WPI concentration in OAP (mg/L)
4000
3500
3000
5 % WPI, 25°C
2500
5 % WPI, 5°C
2000
2.5 % WPI, 25°C
1500
2.5 % WPI, 5°C
1 % WPI, 25°C
1000
1 % WPI, 5°C
500
0
0
5
10
15
20
25
Storage time (days)
Figure 3.14. WPI concentration in the separated outer aqueous phase (OAP) obtained from the
W1/O/W2 emulsions containing 5%, 2.5 % or 1% WPI in W1-phase. The intact emulsions were
(statically) stored at 5 °C or at room temperature (25 °C) and were prepared using moderate
homogenization intensity in the secondary emulsification step. The error bars represent the
standard deviation of triplicate sets of measurements.
Bonnet et al. (2009) reported that the magnesium leakage from double emulsions increased over 1
month, irrespective of the oil nature. Moreover, the rate of magnesium release increased at 25 ℃
in comparison to 4 ℃ due to the thermally activated nature of the diffusion or permeation
phenomena. They proposed that magnesium release was not due to droplet-globule coalescence
but rather to diffusion and/or permeation. The molecular weight of magnesium is 24.30 g/mol
while WPI has got a molecular weight of 18363 g/mol. Therefore, the rate of WPI diffusion and/or
permeation might be lower and less influenced by the storage temperature as a result of the bigger
molecular size (Figure 3.14).
Table 3.13. Change in WPI concentration as a function of time at room temperature and
5 ℃ for emulsions prepared with moderate homogenization intensity. Each listed value is
the slope ± 95 % confidence interval.
5%
Storage T
25 °C
5 °C
Change in WPI
with time (mg/L
WPI/day)
5.56 ± 5.87
- 4.52 ± 7.43
WPI concentration
2.5 %
25 °C
5 °C
0.12 ± 2.16
50
1.76 ± 2.06
1%
25 °C
5 °C
- 0.25 ± 0.41
- 0.86 ± 1.13
Figure 3.15 shows the percentage of disrupted water droplets during the second emulsification as
a main mechanism for WPI release into external water phase, assuming that all WPI in the external
phase is due to broken internal water droplets. Overall, the fraction of disrupted internal water
droplets is about 15% in all cases. The W1/O/W2 emulsions containing 1 % WPI had higher
percentage of disrupted water droplets (especially the sample stored at 5 °C) in comparison to the
W1/O/W2 emulsions containing a higher amount of WPI, suggesting that it might be harder to
break up the W1/O emulsion when more protein is present in the internal water phase. Surh et al.,
(2007) reported that emulsions containing WPI (gelled or non-gelled) had larger mean droplet
Percentage of disrupted water droplets
diameters.
25
20
5 % WPI, 25°C
15
5 % WPI, 5°C
2.5 % WPI, 25°C
10
2.5 % WPI, 5°C
1 % WPI, 25°C
5
1 % WPI, 5°C
0
0
5
10
15
20
25
Storage time (days)
Figure 3.15. Percentage of disturbed water droplets from the W1/O/W2 emulsions containing 5%,
2.5 % or 1% WPI in the W1-phase. The intact emulsions were (statically) stored at 5 °C or at room
temperature (25 °C) and were prepared using moderate homogenization intensity in the secondary
emulsification step. The error bars represent the standard deviation of triplicate sets of
measurements.
3.5.2 Released WPI concentration from the W1/O/W2 emulsions prepared by low-energy
homogenization intensity
Using high-shear stresses in the production of double emulsions might cause disruption of the
W1/O emulsion and release of inner water into the external aqueous phase (van der Graaf et al.
2005). On the other hand, large multiple globules in the double emulsion might result in
unsatisfactory properties such as a higher susceptibility towards creaming, coalescence, and
51
flocculation (Surh et al., 2007). Thus, a balance between both effects should be favored. Figure
3.16 shows two emulsions prepared with moderate and low-energy homogenization intensity. It
can be seen from Figure 3.16 that after 24 h settling, both emulsions separated into a cream layer
at the top and aqueous phase at the bottom. However, the subnatant aqueous phase in the emulsion
prepared with low-energy homogenization intensity was almost clear while it was more turbid in
the emulsion prepared by the moderate homogenization intensity procedure. The latter might
indicate the presence of smaller oil droplets in the serum.
bb
aa
Figure 3.16. Double emulsion prepared with moderate homogenization intensity (a) and double
emulsion prepared with low-energy homogenization intensity (b) after 24 h settling.
Figure 3.17 represents the concentration of WPI in the outer water phase separated from emulsions
with different concentrations of WPI (5, 2.5 and 1 % in internal water phase) as prepared with lowenergy homogenization intensity. The WPI concentrations in the outer water phases were lower
than 25 mg/L in all cases, indicating that less than 0.25 % of the internal water droplets underwent
rupture during preparation, irrespective of the applied WPI concentration. The concentration of
WPI in the outer aqueous phase did not show a clear trend as a function of time (Table 3.14),
possibly mediated by a low sensitivity of the modified Lowry method for measuring low
concentrations of WPI.
Regarding the storage temperature, there was a significantly larger WPI concentration with time
at room temperature than at 5 °C using 5 % and 2.5 % WPI (p = 0.027 and p = 0.004, respectively),
whereas the opposite was observed for 1 % WPI (p = 0.004).
52
Table 3.14. Change in WPI concentration as a function of time at room temperature and
5 ℃ for emulsions prepared with low-energy homogenization intensity. Each listed value
is the slope ± confidence interval
WPI concentration
5%
2.5 %
1%
Storage T
25 °C
5 °C
25 °C
5 °C
25 °C
5 °C
Change in WPI
with time (mg/L
WPI/day)
0.43 ± 0.25
0.17 ± 0.09
0.06 ± 0.07
0.26 ± 0.08
0.03 ± 0.08
0.11 ± 0.09
WPI concentration in OAP
(mg/L)
a
40
30
20
5°C
Room temperature
10
0
0
5
10
15
20
25
Storage time (days)
WPI concentration in OAP
(mg/L)
b
40
30
20
5°C
Room temperature
10
0
0
5
10
15
Storage time (days)
53
20
25
WPI concentration in OAP
(mg/L)
c
40
30
20
5°C
Room temperature
10
0
0
5
10
15
20
25
Storage time (days)
Figure 3.17. WPI concentration in the separated outer aqueous phase (OAP) obtained from
W1/O/W2 emulsions containing 5% (a), 2.5 % (b) and 1% (c) WPI in W1-phase. The intact
emulsions were (statically) stored at room temperature and 5 ℃ and were prepared using lowenergy homogenization intensity in the secondary emulsification step. The error bars represent the
standard deviation of triplicate sets of measurements.
3.5.3 Water yield and WPI yield of W1O/W2 emulsions prepared by moderate and lowenergy homogenization intensity
In this experiment, pfg-NMR diffusometry and analytical photocentrifugation were applied to
determine the water yield of the W/O/W emulsions after 14 days of storage at room temperature
and 5 ℃, as shown in Table 3.15-3.16 and Table 3.17, respectively.
The emulsions prepared by moderate homogenization intensity MHI (Table 3.15 and Table 3.17)
were characterized by a smaller water yield compared to the emulsions produced by low-energy
homogenization intensity (Table 3.16). Probably, during the second emulsification step, more
water was expelled from the multiple globules upon increase of the shear intensity. The EV in
emulsions prepared by low-energy homogenization intensity LHI was close to the maximum
theoretical EVmax of 33.3 %.
Table 3.15, Table 3.17 and Figure 3.17 indicate that the double emulsions prepared with moderate
homogenization intensity resulted in a lower water yield as determined by the pfg-NMR than by
the analytical photocentrifugation method. Under certain conditions, the pfg-NMR might
underestimate the yield due to exchange between the water phases during the course of the analysis
(Vermeir et al. 2014). In this work, water exchange effects were minimized by measuring at a low
temperature (5 ℃) and by using a small diffusion delay time (∆=60 ms). Recent investigation of
our research group showed that the analytical photocentrifugation method might result in an
overestimation of the yield due to the contribution of interstitial water. Interstitial water can be
54
accounted for by measuring an O/W emulsion with the analytical photocentrifugation method, but
this is not a straightforward approach on account of possible differences in compressibility of the
multiple droplet in the W/O/W emulsion and the oil droplet in the O/W emulsion (Balcaen et al.,
in press). In this work, the resulting overestimation was limited by performing intensive
centrifugation (2h at 3000 rpm) and by prohibiting rebound. Nevertheless, Table 3.15, Table 3.17
and Figure 3.18 show a similar trend in the water yield for both methods as a function of applied
WPI concentration, whereby the NMR-result of the 5 % WPI sample as stored at 25 °C (in Fig.
3.18) might be an outlier. The water yield of the MHI-emulsions, when combining the water
yieldNMR and water yieldLUM for all WPI concentrations, was significantly larger at 5 °C than at 25
°C (p = 0.0024). In contrast, an effect of the WPI concentration or storage temperature on the water
yield of LHI-emulsions was difficult to observe, as their average water yield (with 95 % confidence
and when combining the water yieldNMR and water yieldLUM for all WPI concentrations) was about
100 %, i.e. 100.0  1.8 % at 5 °C and 102.0  2.1 % at 25 °C.
Table 3.15. Enclosed water volume fraction (EV) and water yield for emulsions
prepared by moderate homogenization intensity in the secondary emulsification step
(Pfg-NMR method) after 14 days of storage at room temperature and 5 ℃. Each listed
value is the average of triplicate sets of measurements ± standard deviation (n = 3).
WPI
concentration (%)
5.0
2.5
1.0
EVNMR(%)
25 °C
24.6 ± 0.3
27.8 ± 0.4
27.2 ± 0.2
5 °C
29.5 ± 0.7
28.6 ± 1.2
28.2 ± 1.0
Water yieldNMR (%)
25 °C
74.0 ± 0.8
83.5 ± 1.3
81.8 ± 0.5
5°C
88.7 ± 2.1
85.8 ± 3.7
84.6 ± 3.0
Table 3.16. Enclosed water volume fraction (EV) and water yield for emulsions
prepared by low-energy homogenization intensity in the secondary emulsification step
(Pfg-NMR method) after 14 days of storage at room temperature and 5 ℃. Each listed
value is the average of triplicate sets of measurements ± standard deviation (n = 3).
WPI
concentration (%)
5.0
2.5
1.0
EVNMR(%)
25 °C
5 °C
31.6 ± 1.3
31.3 ± 0.6
34.5 ± 0.6
33.1 ± 0.5
35.5 ± 0.6
33.5 ± 1.2
55
Water yieldNMR (%)
25 °C
5 °C
94.8 ± 3.8
94.0 ± 1.7
103.5 ± 1.7
99.4 ± 1.4
106.6 ± 1.8
100.6 ± 3.6
Table 3.17. Water yield determination for emulsions prepared by moderate and lowenergy homogenization intensity in the secondary emulsification step (analytical
photocentrifugation method) after 14 days of storage at room temperature and 5 ℃.
Each listed value is the average of triplicate sets of measurements ± standard deviation
(n = 3).
WPI
concentration (%)
5.0
2.5
1.0
Water yieldLUM (%)
Moderate homogenization
Low-energy homogenization
intensity
intensity
25 °C
5 °C
25 °C
5 °C
98.0 ± 0.9
102.7 ± 0.8
103.9 ± 0.5
101.7 ± 1.7
97.3 ± 1.7
97.5 ± 0.7
102.1 ± 0.8
101.5 ± 0.8
93.1 ± 1.2
93.3 ± 2.2
101.0 ± 1.5
102.7 ± 1.3
100
Yield (%)
80
NMR-1% WPI
60
NMR-2.5% WPI
NMR-5%WPI
40
LUM-1% WPI
20
LUM-2.5% WPI
LUM-5% WPI
0
5 °C
25 °C
storage temperature
Figure 3.18. Water yield of W1/O/W2 emulsions prepared by moderate homogenization intensity
in the secondary emulsification step at storage temperature of 5 ℃ and 25 ℃. NMR = Pfg-NMR
diffusometry, LUM = analytical photocentrifugation. The error bars represent the standard
deviation of triplicate sets of measurements.
The WPI yield was determined after 14 days of storage using the water yieldNMR and Equation 2.6
(Figure 3.19). Analogously to the water yield of the LHI-emulsions, the WPI yield was close to
100 % for both storage temperatures. In contrast to the water yield of the MHI emulsions, a similar
WPI yield was obtained for different applied WPI concentrations.
56
WPI yield (%) after 14 days of
stoarge
100
80
1 % WPI, LHI
60
2.5 % WPI, LHI
5 % WPI, LHI
40
1 % WPI, MHI
20
2.5 % WPI, MHI
5 % WPI, MHI
0
5 °C
25 °C
Storage temperature
Figure 3.19. WPI yield of W1/O/W2 emulsions prepared by moderate and low-energy
homogenization intensity in the secondary emulsification step after 14 days of storage at 5 ℃ and
25 ℃. The error bars represent the standard deviation of triplicate sets of measurements.
Effect of homogenization time duration in the secondary emulsification step on the
release kinetics of WPI from the W1-phase to the W2-phase directly after preparation
of the gelled and non-gelled W1/O/W2 emulsions
As described by Vermeir et al. (2014) the encapsulation efficiency of water in the W/O/W
emulsion showed a decrease upon increasing homogenization duration. In this section, the W/O/W
emulsions (∆π = 0) were prepared using different homogenization time durations in the second
emulsification step (1, 2 or 4 min using a continuous Ultra-Turrax, see section 2.2.1.1) in an effort
to evaluate the release of the marker compound (WPI) upon application of shear. Hereby, the effect
of thermal gelation of WPI, as contained within the inner aqueous phase of the W/O emulsion, on
the WPI release kinetics into the external water of the double emulsions was evaluated. Since
gelation of the inner water phase in the W/O emulsion results from WPI denaturation, the modified
Lowry method might be hampered. Therefore, measurement of WPI concentration in the separated
outer aqueous phase was performed by means of nitrogen content determination following
persulfate oxidation (see section 2.3.2) and the standard curve in section 3.4.7.
Also the water yield was measured, which allows the comparison of the behavior of water and a
marker compound under shear conditions. The water yield was evaluated by analytical
photocentrifugation directly after double emulsion preparation, as well as by pfg-NMR
diffusometry for which the samples were stored for at least 24 h at 5 °C.
57
3.6.1 Released WPI concentration from the gelled and non-gelled W1/O/W2 emulsions
The encapsulation efficiency is dependent on the homogenization duration and intensity applied
during production. We postulated that the gelation of the internal water droplets within the W/O
emulsions used to prepare a W/O/W emulsion might reduce the tendency for water loss during the
second emulsification step. Thus, it might be possible to apply longer emulsification duration to
prepare a W/O/W emulsion with smaller droplet size in order to have higher creaming stability.
The concentration of WPI in the outer aqueous phase separated from the emulsions contained nongelled and gelled WPI is shown in Figure 3.20.
Concentration WPI in OAP (mg/L)
6000
5000
4000
3000
2000
gelled W/O/W
non-gelled W/O/W
1000
0
1
2
4
Emulsification duration (min)
Figure 3.20. WPI concentration in the separated outer aqueous phase (OAP) obtained from the
W1/O/W2 emulsions containing non-gelled WPI (shaded bars) or gelled WPI (solid bar) for
different emulsification durations. The WPI concentration was very low (≅ 𝟎) for the gelled double
emulsions prepared at 1 and 2 min homogenization time. The error bars represent the standard
deviation of triplicate sets of measurements.
From Figure 3.20 it is clear that the WPI concentration in the separated outer aqueous phase of the
gelled double emulsions was significantly lower than the non-gelled double emulsions. Heating of
W/O emulsions above the critical temperature leads to thermal denaturation of WPI which is
known to occur around 80 to 90 °C. Unfolded proteins form a gel-like network through
hydrophobic and disulfide bond formation which can lead to an increase in apparent shear viscosity
of the emulsions (Iqbal et al. 2013). Surh et al. (2007) reported that it may be harder to break up
the W/O phase into droplets when a gelled internal aqueous phase is present. Therefore the
disruption of the W/O phase in the second emulsification step might be harder when WPI gelation
was applied.
58
3.6.2 Water yield and WPI yield of the gelled and non-gelled W1/O/W2 emulsions
Regarding analytical photocentrifugation (Figure 3.21) and pfg-NMR diffusometry, the non-gelled
double emulsions were characterized by a smaller water yield compared to the gelled double
emulsions. The normalized NMR echo decays are shown in Figure 3.22. In contrast to section
3.5.3, the water yield values from NMR were higher than from the centrifugation method. For the
non-gelled emulsions, the average yieldNMR (and standard deviation of a triplicate set) amounted
to 102  3 %, 92  4 % and 52  1 % after 1, 2 and 4 minutes of mixing. The yieldNMR of the gelled
emulsions amounted to 115  2 %, 115  1 % and 77  2 %, respectively. The effect of gelation
on the relaxation time was not investigated and hence, a possible correction factor of the yieldNMR
was unknown. Therefore, the WPI yield was determined using the water yieldLUM according to
Equation 2.6 (Figure 3.21). Figure 3.21 shows a difference in behavior of WPI in the gelled and
non-gelled W/O/W emulsion upon application of shear. The yield of WPI in the gelled emulsion
remains unchanged, whereas the WPI yield in the non-gelled emulsion decreases with increasing
100
80
80
60
60
40
20
20
(%)
40
water, LUM
100
Yield
Yield WPI (%)
shear application.
0
WPI yield gelled W/O/W
WPI yield non-gelled W/O/W
water yield gelled W/O/W
water yield non-gelled W/O/W
0
0
1
2
3
4
Emulsification duration (min)
5
Figure 3.21. Comparison of the yield of WPI (black markers) as measured by the nitrogen content
determination method following persulfate digestion and the yield of water (empty markers) as
measured by pfg-NMR diffusometry at different emulsification durations for gelled (squares) and
non-gelled (circles) double emulsions. The error bars represent the standard deviation of triplicate
sets of measurements.
59
1,0
0,9
0,8
0,7
I/I0
0,6
0,5
non-gelled 1 min
0,4
non-gelled 2 min
0,3
non-gelled 4 min
0,2
0,1
0,0
0
2
4
6
8
10
12
G² (T²/m²)
1,0
0,9
0,8
0,7
I/I0
0,6
0,5
gelled 1 min
0,4
gelled 2 min
0,3
gelled 4 min
0,2
0,1
0,0
0
2
4
6
8
10
12
G² (T²/m²)
Figure 3.22. Normalized NMR echo decay of the non-gelled and gelled W1/O/W2 emulsions as
prepared using different mixing time durations in the secondary emulsification step.
Effect of the application of mechanical stress (pumping) on the release kinetics of WPI
from the W1-phase into the W2-phase directly after preparation of the W1/O/W2
emulsions
Bursting of encapsulated droplets can occur by osmotic swelling after dilution of double emulsions
or by application of mechanical stress (Muguet et al., 2001). Hence, in order to mimic the
mechanical stress applied on double emulsions when pumped through pipelines of a production
plant, the W/O/W emulsion was pumped in recycle mode using a peristaltic pump (Sartorius) with
60
a flow rate for water in the applied set-up of 7.4 mL/s. The pump was set at a fixed pumping speed
value (“high”) for different time durations (i.e. 5, 10 and 15 minutes). It should be noted that
displacement pumps (e.g. peristaltic pump) work more gentle in comparison to centrifugal pumps.
The W1/O/W2 emulsion under quasi-isotonic conditions was prepared using moderate
homogenization intensity as described in section 2.2.1, whereby the W1-phase contained 2.5 wt %
of WPI. The concentration of WPI in the centrifuged outer aqueous phase was measured directly
after W1/O/W2 emulsion preparation by the Schacterle and Pollack method (section 2.3.1). The
water yield was evaluated by analytical photocentrifugation directly after double emulsion
preparation, as well as by pfg-NMR diffusometry after storage for at least 24 h keeping at 5 ℃ (see
section 2.4).
Table 3.18 shows the concentration of WPI in the outer aqueous phase (OAP), the EVNMR, the
water yieldNMR and the water yieldLUM.
Table 3.18. WPI concentration in OAP, EVNMR, water yieldNMR and water yieldLUM values
measured for W/O/W emulsions pumped for different time durations.
WPI concentration in OAP (mg/L)
EVNMR(%)
Water yieldNMR (%)
Water yieldLUM (%)
Time duration of pumping (min)
5
10
1094 ± 37
1089 ± 25
31.5 ± 1.4
30.0 ± 0.6
94.6 ± 4.0
90.0 ± 1.7
104.1 ± 5.1
97.7 ± 0.8
15
1094 ± 40
29.9 ± 0.5
89.6 ± 1.4
98.8 ± 1.1
Table 3.21 shows that pumping under the applied conditions did not have a significant influence
on the WPI and water release from the inner to the outer aqueous phase. Muguet et al. (2001)
reported that the electrolyte release (MgSO4.7H2O) from multiple emulsion was low and remained
constant under shear rates lower than 1600 s-1. The critical shear rate responsible for the first
bursting was 2200 s-1 in their experiments.
The WPI yield was calculated using Equation 2.6 and the water yield as obtained from the NMR
method (Figure 3.22). Figure 3.23 shows a similar behavior of water and WPI in the W/O/W
emulsion that was pumped in recycle mode for up to 15 minutes.
61
100
95
Yield (%)
90
85
80
WPI yield
Water yield NMR
75
70
0
5
10
15
Pumping time duration (min)
20
Figure 3.23. Comparison of the yield of WPI (black markers) as measured by the Schacterle and
Pollack method and the yield of water (empty markers) as measured by pfg-NMR diffusometry
for different pumping time durations. The error bars represent the standard deviation of triplicate
sets of measurements.
Influence of the osmotic pressure gradient (∆𝝅) on the release kinetics of WPI as a
function of storage time
In order to elucidate the role of the osmotic pressure gradient on the kinetic release of WPI from
the double emulsion, different osmotic pressure gradients (∆𝜋) between the encapsulated (W1) and
the continuous aqueous phase (W2) were created (Table 3.19). This osmotic pressure was
calculated according to Equation 2.1, using 2.5 wt % WPI in the W1-phase. In absence of an
osmotic pressure gradient (i.e ∆𝜋 = 𝜋𝑖 − 𝜋𝑒 = 0), no water is expected to be transported between
the internal and external aqueous phase. When ∆𝜋 is positive, the concentration of NaCl is greater
in the W1-phase than in the W2-phase. In this case, the water might be transported into the W1
droplets. On the other hand, a negative ∆𝜋 might result in water transport from the W1 to the W2
aqueous phase (when the concentration of NaCl is greater in W2).
Leal-Calderon et al. (2012) postulated that 24 hours was long enough for osmotic equilibration
through water transport between the two aqueous phases but it was not sufficient for co-solutes
transportation. Therefore, the characteristics of the W/O/W emulsions in our study were monitored
for a longer period of time (i.e. 0, 1, 2, 3, 5, 7, 14, 21 days at room temperature). The W/O/W
emulsions were prepared as described in section 2.2.2 and 2.2.3 and the concentration of WPI in
the centrifuged outer aqueous phase was measured as a function of storage time by the Schacterle
62
and Pollack method (section 2.3.1). The water yield was evaluated by analytical
photocentrifugation and pfg-NMR diffusometry (see section 2.4).
Table 3.19: The osmotic pressure gradients between encapsulated (W1) and continuous
aqueous phase (W2) at a storage temperature of 298 K
Osmotic pressure
gradient, ∆𝜋 (kPa)
Molar concentration
of solute
Concentration of
NaCl in W2-phase
(M)
0.0
Preferential transport
of water
+464
𝑊1 > 𝑊2
0
𝑊1 ≅ 𝑊2
0.1
None
− 527
𝑊1 < 𝑊2
0.2
Toward the W2-phase
Toward the W1-phase
3.8.1 Viscosity measurement of the W1/O/W2 emulsions
The W/O/W emulsions did not show a good gravitational stability under static conditions as
creaming was observed in a few hours after preparation. Therefore, the W/O/W emulsions
prepared under positive and negative osmotic pressure gradient were submitted to end-over-end
rotation over storage time to avoid phase separation. The latter might lead to a decrease in
interfacial area for water transport.
Although W1/O/W2 emulsions were fluid-like mixture after the preparation process, the emulsions
under positive and negative osmotic pressure gradient conditions started to show a gel-like
structure after 1 day of end-over-end rotation at room temperature. At first the produced gel-like
structure was not very firm, but over storage time, it turned into a relatively viscous fluid. In order
to exclude compositional effects, also the double emulsion under quasi-isotonic conditions was
subjected to end-over-end rotation and the viscosity was measured immediately after preparation,
as well as after 1 day and 5 days of end-over-end rotation (Figure 3.24). One problem with the
shear viscosity measurement was that it was difficult to introduce the semi-solid samples formed
during end-over-end rotation into the rheometer measurement cell. Thus, we were not able to track
the increase in viscosity of the samples for more than 5 days of storage. Figure 3.23 shows that the
double emulsions exhibited shear thinning behavior and the viscosity increased significantly after
5 days of end-over-end rotation.
63
a
Viscosity (Pa.s)
0,02
0,015
0,01
after preparation process
0,005
after 1 day rotation
0
0
50
100
150
200
Speed (rpm)
b
10
Viscosity (Pa.s)
8
6
4
after 5 day rotation
2
0
0
50
100
150
200
Speed (rpm)
Figure 3.24. Viscosity of the W/O/W emulsion prepared under quasi-isotonic condition
Immediately after preparation and after 1 day (a) and 5 days of end-over-end rotation (b). The
error bars represent the standard deviation of triplicate sets of measurements.
Leal-Calderon et al. (2012) and Delample et al. (2014) observed a gelation phenomenon in W/O/W
emulsions within a few minutes. This was based on an osmotically driven water transfer process
from the external water to the internal water due to high solute concentration in the internal water
phase. However, in our experiment gelation was observed in all emulsions even for emulsions
prepared in the absence of an osmotic pressure gradient. A possible explanation could be the coldset gelation of heat-denaturated WPI in the presence of sodium chloride. Cold-set gelation consists
of a two-step process. The first step includes the heating of a protein solution at a pH sufficiently
far from the isoelectric point of the proteins resulting in partial unfolding of the proteins. The
64
second step consists of the addition of monovalent or divalent salts (i.e. CaCl2 and NaCl) after
cooling. This reduces the electrostatic repulsion between charged protein molecules and finally
results in the formation of a gel. Low ionic strength and low concentration of proteins are required
for cold-set gelation of proteins (Bryant et al., 2000;Kuhn et al., 2010). On the other hand, Iqbal
et al. (2013) reported that if the globular proteins remained entirely in the internal droplets, their
gelation (by heating at 90 ℃ for 20 minutes after W/O/W emulsion preparation) should not cause
a major influence on the overall rheological properties of the system as the texture characteristics
of a colloidal suspension containing the same concentration of liquid or solid particles is fairly
similar. In our experiment, some of the released WPI into the external aqueous phase might have
become partially unfolded due to heat produced during the first and second emulsification step and
might interact with NaCl to form subsequent aggregates over storage time. It might be possible
that the chance for interaction between charged proteins and NaCl is greater due to mixing under
end-over-end rotation. In fact, this experiment shows that it might be possible to produce a
gravitationally stable W1/O/W2 emulsion with a gel-like structure in the presence of a small
amount of WPI and NaCl in the external water phase under the application of end-over-end
rotation.
3.8.2 Released WPI concentration from the W1/O/W2 emulsions
The concentration of WPI in the outer aqueous phase recovered from the W1/O/W2 emulsions with
different osmotic pressure gradients is shown in Figure 3.25. The values for ∆π > 0 were corrected
for the applied hypotonic dilution. For separation of the outer aqueous phase of fluid-like W1/O/W2
emulsions, low-speed centrifugation (500 g for 40 minutes) followed by ultracentrifugation was
applied on the resulting subnatant to remove small droplets of W1/O emulsion. However, with
increasing viscosity over storage time, collecting the continuous phase from the gel-like W1/O/W2
emulsions required direct ultracentrifugation (45000 rpm for 60 minutes) (Figure 3.26). This might
lead to globule compression and possibly partial destruction of the double emulsion structure.
65
WPI concentration in OAP (mg/L)
1600
1200
negative osmotic pressure gradient
800
positive osmotic pressure gradient
no osmotic pressure gradient
400
0
0
5
10
15
20
25
Storage time (days) at room temperature
Figure 3.25. WPI concentration in the recovered outer aqueous phase (OAP) of end-over-end
rotated W1/O/W2 emulsions under negative and positive osmotic pressure gradients (𝝅𝒊 − 𝝅𝒆 <
𝟎, 𝝅𝒊 − 𝝅𝒆 > 𝟎) as well as non-end-over-end rotated quasi-isotonic conditions (𝛑𝐢 − 𝛑𝐞 = 𝟎) as a
function of storage time at room temperature. The error bars represent the standard deviation of
triplicate sets of measurements.
Figure 3.26. Test tube of direct centrifugation of viscous W1/O/W2 by ultracentrifuge.
As it can be seen in Figure 3.25, the concentration of WPI in the outer aqueous phase was not
influenced by the various osmotic pressure gradients during the first week of storage time at room
temperature. In case of the negative osmotic pressure gradient, the concentration of WPI in outer
aqueous phase increased after the first week of storage.
66
3.8.3 Water yield and WPI yield of the W1/O/W2 emulsions
After one day of storage, it was not possible to measure the water yield analytical
photocentrifugation since some emulsion particles stuck to the measurement cell due to a gel-like
structure of the emulsion (Figure 3.27). Therefore, the water yield was just measured after the
preparation process when the W1/O/W2 emulsion still had a fluid-like structure. All mentioned
values for ∆π > 0 were corrected for the applied hypotonic dilution. The water yield was 113.5 ±
4.3 % and 48.6 ± 1.9 % for emulsions prepared under the positive and negative osmotic pressure
gradient, respectively. These values are lower than their counterpart by pfg-NMR (179.2 ±
5.8 % and 61.8 ± 0.8 %, respectively) for reasons stated in section 3.5.3. The very high values
obtained under the positive osmotic pressure gradient clearly show the transport of water from the
external to the inner water phase.
Figure 3.27. Double emulsion particles were stuck to the LUMiFuge sample cell after storage of
one day as prepared under negative and positive osmotic pressure gradient.
Table 3.20 shows the enclosed water volume of the end-over-end rotated W1/O/W2 emulsions
prepared under a positive and negative osmotic pressure gradient as measured by pfg-NMR. The
entrapped water of the former double emulsion was gradually released over storage time. In case
of the negative osmotic pressure gradient (i.e. 𝜋𝑖 − 𝜋𝑒 < 0), the enclosed water volume directly
after preparation was lower in comparison to the (non end-over-end rotated) quasi-isotonic
condition. Hence, a fraction of inner water might have been released directly during emulsion
preparation. Its enclosed water volume fraction hardly changed with storage time.
67
After 14 days of storage, the EVNMR and yieldNMR for the W1/O/W2 emulsion prepared under (non
end-over-end rotated) quasi-isotonic conditions, amounted to 27.8 ± 0.4 % and 83.5 ± 1.3 %,
respectively.
Table 3.20. Enclosed water volume fraction (EV) of the W1/O/W2 emulsions prepared under
positive and negative osmotic pressure gradient as a function of storage time at room temperature
and as measured by pfg-NMR at 5 ℃. Each listed value is the average of triplicate sets of
measurements ± standard deviation (n = 3). On the day of preparation, the EV was measured at
least 2 hours after emulsification.
Storage time (days)
2 days
5 days
Preparation
day
1 day
EVNMR (%)
𝜋𝑖 − 𝜋𝑒 > 0
59.7 ± 1.9
62.3 ± 1.5
50.7 ± 1.4
𝜋𝑖 − 𝜋𝑒 < 0
20.8 ± 0.3
21.1 ± 0.7
20.7 ± 1.3
7 days
14 days
48.6 ± 1.3
44.3 ± 0.7
36.3 ± 2.8
22.6 ± 0.5
20.5 ± 0.6
19.6 ± 0.4
Also Leal-Calderon et al. (2012) observed a release of the inner droplets through coalescence in a
W1/O/W2 emulsion with a positive osmotic pressure gradient between the water phases. LealCalderon et al. (2012) and Delample et al. (2014) proposed that droplet-droplet and droplet-globule
coalescence may occur due to tightly packed inner droplets upon swelling and large compressive
forces which leads to the full release of the internal droplet content into the external water phase.
Droplet-globule coalescence is more likely to occur for large internal droplets due to a higher
surface area of contact with the globule surface.
Some Tween 80 in the external water phase might also be transported to the internal water phase.
Wen and Papadopoulos (2000) expressed that water transport rates increased in the presence of
Tween 80 in the internal water phase. They suggested that water-soluble surfactants can solubilize
the oil-soluble surfactants from the oil phase thus could enhance the rupture of the oil film between
the W1-phase and W2-phase. In addition, they reported that Tween 80 may promote water transport
by facilitating the hydration of surfactants, formation of reverse micelles and spontaneous
emulsified droplets.
Figure 3.28 shows the water yield and WPI yield of the W1/O/W2 emulsion with a positive and
negative osmotic pressure gradient. Hereby, the WPI yield was determined using the water
yieldNMR and Equation 2.6. From Figure 3.28 it is clear that the WPI yield of both types of
W1/O/W2 emulsion was not affected by the storage time at room temperature. In the W1/O/W2
68
emulsion as prepared with a higher salt concentration in the external water phase, water molecules
and WPI behaved similarly as a function of time. In the W1/O/W2 emulsion as prepared with a
higher salt concentration in the internal water phase, water was released faster than WPI from the
inner to the outer water phase.
a
200
180
160
Yield (%)
140
120
100
80
60
40
WPI yield, ∆π > 0
20
water yield, ∆π > 0
0
0
Yield (%)
b
5
10
Storage time (days) at room temperature
100
90
80
70
60
50
40
30
20
10
0
15
WPI yield, ∆π < 0
water yield, ∆π < 0
0
5
10
Storage time (days) at room temperature
15
Figure 3.28. Water yield and WPI yield of end-over-end rotated W1/O/W2 emulsions. Emulsions
were prepared (with 2.5 % WPI in W1) under positive (a) and negative (b) osmotic pressure
gradient as measured by pfg-NMR. The error bars represent the standard deviation of triplicate
sets of measurements.
69
The effect of emulsification shear intensity and shear temperature on the enzyme
activity
Aiming at encapsulation of proteins with enzyme activity in double emulsions, the effect of
emulsification shear intensity and shear temperature should be evaluated. In this study, the enzyme
solutions were sheared at different intensities (0, 6000, 13500, 24000 rpm for 3 minutes) using an
Ultra-Turrax S25-10G at room temperature or at 50 C. Hereby, alkaline phosphatase (ALP) was
used as a simple test enzyme as it only requires one substrate to measure its activity. In this singlesubstrate reaction the enzyme catalyses the conversion of the substrate to a measurable product,
whereas for many enzymes (e.g. oxidoreductase and ligase) two or more substrates are involved.
In order to measure the enzyme activity, the substrate concentration (colorless para-nitrophenyl
phosphate or pNPP) was varied. The composition of solutions with different concentration of
pNPP is shown in section 2.7. The reaction rate was measured according to the Michaelis-Menten
equation and the Hanes-Woolf linearization method (Equation 2.7 and 2.8). A stop assay was used,
i.e. the reaction was stopped following a given period of time, after which the product
concentration was measured (yellow para-nitrophenol or pNP).
Figure 3.29 shows the hyperbolic relationship between the reaction rate and substrate
concentration for various applied shear intensities at room temperature and at 50 °C. Figure 3.30
shows the calculated Km and Vmax at room temperature and 50 ℃. The associated reaction rate
constant Vmax/Km is shown in Figure 3.30. In comparison to all other applied shear intensities at
room temperature and at 50 °C, Figure 3.31 indicates a much larger reaction rate constant for the
protein solution sheared at 24000 rpm at room temperature. However, that might be an outlier as
a result of an error in enzyme concentration. Data about the denaturation temperature of alkaline
phosphatase (ALP) indicate that this enzyme could be inactivated upon increasing the holding time
or increasing the temperature, i.e. 56 °C for 30 min or 72.5 °C for 15 s (Rankin et al. 2010;Pinho
et al., 2011). Rankin (2010) reported on the inactivation of milk APL by high-pressure processes
due to mechanical forces such as shear, cavitation and impact. Conflicting results have been
reported about the effect of high shear intensity on protein stability and its function (Di Stasio et
al., 2010). Jaspe and Hagen (2006) observed no evidence of denaturation of small proteins
(cytochrome c with 104 amino acids) by strong shear stress (𝛾 . > 105 𝑠 −1 ) under well-defined flow
conditions. In addition, they reported that both folding and unfolding of cytochrome c is rapid
70
(time scale of approximately microseconds to milliseconds) and fully reversible under applying
high shear stress.
4
Reaction velocity (µM
[P]/min)
a
3
0 rpm
2
6000 rpm
13500 rpm
1
24000 rpm
0
0
b
1000
2000
3000
4000
Substrate concentration (µM)
Reaction velocity (µM
[P]/min)
4
3
0 rpm
2
6000 rpm
13500 rpm
1
24000 rpm
0
0
1000
2000
3000
4000
Substrate concentration (µM)
Figure 3.29. Hyperbolic relationsh between the reaction rate of APL and the substrate
concentration by applying different shear intensities for 3 min at room temperature (a) and 50 ℃
(b).
However, Figure 3.31 indicates that heating of the enzyme solution to 50 °C prior to shear
application at 24000 rpm for 3 minutes minimally affected the enzyme activity of ALP.
71
a
8
Vmax (µM/min)
Km (µM)
3000
2000
1000
0
6
4
2
0
0 rpm
6000
13500 24000
rpm
rpm
rpm
Shear intensity for 3 min
0 rpm
6000 rpm 13500
24000
rpm
rpm
Shear intensity for 3 min
b
8
Vmax (µM/min)
Km (µM)
3000
2000
1000
0
0 rpm
6000
13500 24000
rpm
rpm
rpm
Shear intensity for 3 min
6
4
2
0
0 rpm
6000 rpm 13500
24000
rpm
rpm
Shear intensity for 3 min
Figure 3.30. Calculated Km and Vmax at room temperature (a) and 50 ℃ (b) for different applied
shear intensities for 3 minutes.
Vmax/Km (min-1)
0,008
0,006
0,004
room temperature
50 °C
0,002
0
0 rpm
6000 rpm 13500 rpm 24000 rpm
Shear intensity for 3min
Figure 3.31. Calculated reaction rate constant Vmax/Km at room temperature and 50 ℃ for different
applied shear intensities for 3 minutes. Error bars denote the 95 % confidence intervals. The
enzyme concentration was 1mg/50 mL.
72
General conclusions
The encapsulation efficiency of proteins in W/O/W double emulsions was investigated. Hereby,
whey protein isolate (WPI) was used as a model compound. To that end, two methods for WPI
concentration measurement were optimized; the Schacterle and Pollack method and the nitrogen
determination method following persulfate digestion. The hydrophilic surfactant Tween 80
interfered at high WPI concentrations with the Schacterle and Pollack method, whereas the
antimicrobial agent NaN3 strongly influenced the nitrogen determination method. The Schacterle
and Pollack method was also applied to determine the partitioning of WPI in the cream phase,
expressed as the recovery yield. Only in the absence of Tween 80 and upon application of moderate
homogenization intensity, some WPI partitioned with the cream phase of an emulsion.
The release of WPI was compared to the release of water by comparison of their yield values, upon
application of shear, mechanical stress, different temperatures and an osmotic pressure gradient
between the water phases of the W/O/W emulsion.
When the osmotic pressure gradient between the two water phases of the W/O/W emulsions was
approximately nihil, a difference in behavior of WPI and water was observed as a function of
applied WPI concentration after 14 days of storage at room temperature. When more salt was
present in the inner water phase of the W/O/W emulsion, the release kinetics of water were higher
in comparison to WPI, of which the encapsulation was unaffected upon storage at room
temperature up to 14 days after preparation. When more salt was present in the external water
phase, probably a drop in water and WPI yield occurred directly after preparation, after which it
remained constant with time.
Gelation of the inner water phase of the W/O/W emulsion also showed a different behavior of WPI
and water release. The application of shear clearly expelled water in gelled and non-gelled
emulsions, whereas the release of WPI was strongly reduced upon gelation.
Finally, aiming at entrapping an enzyme in the W/O/W emulsion, the effect of shear intensity and
shear temperature on the enzyme activity of alkaline phosphatase was evaluated. Hereby, heating
of the enzyme solution to 50 °C prior to shear application at 24000 rpm for 3 minutes minimally
affected the enzyme activity of ALP.
73
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