Note: Descriptions are shown in the official language in which they were submitted.
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Emulsion in Foods
Field of the invention
The present invention relates to the field of emulsions, more particularly to
the stabilisation
of emulsions by food ingredient particles.
Background of the invention
In general emulsions are widely used in food technology, for instance as a
means to improve
the nutritional profile of food products by enabling fat content reduction,
and/or the
incorporation of water soluble nutrients and flavourings.
An emulsion is conventionally a dispersion of one immiscible liquid in another
the most
common example being water and oil. The first liquid which is distributed as
droplets in the
second liquid is known as the dispersed, discontinuous or internal phase. The
second liquid
into which the first is dispersed is known as the continuous or external
phase.
The main types of emulsions that are known in the art are oil-in-water (0/W)
emulsions
whereby the oil droplets are dispersed in water and examples include salad
dressings,
mayonnaise, soups. The other type are water-in-oil (W/O) emulsions whereby
water
droplets are dispersed in oil and examples include butter, margarine. Multiple
emulsions
also exist and these include for example oil-in-water-in-oil (0/W/0) or water-
in-oil-in-water
(W/O/VV) emulsions.
The lack of stability of the emulsion systems presents one of the common
challenges in this
field this is because emulsions are thermodynamically unstable systems and are
prone to
phase separation over time. Coalescence, sedimentation, flocculation, Ostward
ripening
are all physical indications of emulsions destabilisation. Emulsion stability
is usually
indicated by the ability of an emulsion to resist changes in its properties
over time.
Thus, it is usually necessary to use emulsifying agents or emulsifiers, which
are surface
active agents to produce stable emulsions. Typically, emulsions are normally
obtained using
different molecular emulsifying agents like emulsifiers, proteins or
amphiphilic polymers
(also called stabilizers). These ingredients are essential to the manufacture
of stable
commercially acceptable emulsion based products.
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Efficient stabiliser and emulsifier systems already exist, but these are often
based on
chemically modified ingredients. Emulsifiers and stabilizers are generally
considered as
additives which under many countries' health regulations must be declared in
the product
label by their respective E-numbers and some are considered "synthetic"
ingredients, i.e.
obtained by chemical processing. There is a growing demand from consumers for
products,
which are free from undesirable artificial additives or so-called "E numbers".
Thus, there is a continued need for replacing synthetic or artificial
emulsifiers with natural
emulsifier systems that can provide the necessary tensioactive properties
whilst not
compromising on the product quality.
Natural ingredients with emulsifying properties are known, but they are
usually not as
efficient as synthetic emulsifiers and/or present other drawbacks.
In general it is known that emulsions may be stabilised by particles, and
particle-stabilised
emulsions are known as Pickering emulsions [S.U. Pickering, J. Chem. Soc.
Trans., 91,
2001 (1907)]. Pickering Stabilization is commonly known to arise once the
dispersed
particles accumulate at the water-oil interface forming a mechanical (steric)
barrier that
protects the emulsion droplets against coalescence.
Thus, it is well established in the scientific literature that solid particles
may also be
employed to stabilize emulsions (see for instance Bernard P. Binks, Current
Opinion in
Colloid & Interface Science, 7 (2002), 21-41). By using solid particles, the
concentration of
conventional emulsifying agents can be reduced and in some cases, emulsifying
agents
can even be completely replaced. Until now, most of the particles selected to
produce
particle-stabilized emulsions have been synthetic (polymer lattices, silica,
metal oxides,
polymeric microgel particles, etc.). The use of naturally occurring
stabilizers represents an
interesting extension. However, only very few naturally occurring stabilizers
have been
described in the literature. F.Leal-Calderon et al., Current Opinion in
Colloid & Interface
Science 13 (2008) 217-227 mentions the use of bacteria and cowpea mosaic
virus. More
recently naturally occurring spore particles of Lycopodium clavatum have also
been shown
to act as efficient stabilizers for emulsions (Bernard P. Binks et al.,
"Naturally occurring
spore particles behaviour at fluid interfaces and in emulsions", Langmuir
2005; 21:8161-7).
Accordingly, there is a need to provide natural, clean label emulsifier
systems, which can
replace synthetic emulsifiers in food applications.
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Furthermore, it would be advantageous to provide an emulsifier system, which
can replace
synthetic emulsifiers in particular in the manufacture of foodstuffs,
preferably confectionery
products, while not compromising on the product quality.
Any discussion of the prior art throughout the specification should in no way
be considered
as an admission that such prior art is widely known or forms part of common
general
knowledge in the field.
The object of the present invention is achieved by the subject matter of the
independent
claims. The dependent claims further develop the idea of the present
invention.
Summary of the invention
It was surprisingly found by the inventors that water-in-oil emulsions were
stabilised using
a combination of oil in water emulsion stabilisers, preferably flavonoid
particles and whey
protein particles.
The emulsion for use in the present invention is described in claims 1 to 11.
Applications,
uses and methods for the production of the stabilising system of the present
invention and
the emulsion of the present invention are described in claims 12 to 15.
Accordingly, the present invention utilises in an aspect an emulsion
composition comprising
flavonoid or polyphenol particles, whey protein particles, a continuous oil
phase and
dispersed water droplets, wherein the emulsion is preferably stabilised by a
complex
comprising flavonoid or polyphenol and whey protein.
The present invention also provides the use of a combination of flavonoid or
polyphenol
particles and whey protein, preferably particles, as the emulsifier system for
the stabilisation
of a water-in-oil or oil-in-water emulsion. In a preferred embodiment,
provided is the use
according to the present invention wherein the emulsion is for a confectionery
product.
A confectionery product, comprising, preferably consisting of, an emulsion
according to
present invention, comprising flavonoid and whey protein particles or a
flavonoid and whey
protein complex as the emulsifying agent, in the absence of any synthetic or
artificial
emulsifiers or structuring agents.
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A process for preparing a food product, preferably a confectionery product,
comprising an
emulsion according to present invention comprising the steps of:
(i) mixing ingredients of the fat phase, preferably oil phase,
(ii) mixing ingredients of the aqueous phase,
(iii) dispersing the at least two emulsion stabilisers, preferably food
ingredient, particles, in
one or both of the aqueous phase, or the fat phase, preferably oil phase,
(iv) homogenizing the two phases to form an emulsion.
Without wishing to be bound by theory, in a preferred embodiment, it was found
according
to an aspect of the present invention that there was a formation of a complex
of whey protein
and flavonoid at the interface. Thus, the inventors advantageously found a
novel way to
stabilize water droplets inside an oil phase via a complex formation between
flavonoids and
biopolymers, for example whey protein at the interface.
Additional features are described herein and will be apparent from the
following detailed
description and the figures, which are not intended to be limiting the scope
of the invention.
Brief description of the Figures
Additional features and advantages of the present invention are described in,
and will be
apparent from, the description of the presently preferred embodiments, which
are set out
below with reference to the drawings in which:
Figure 1 shows a water in oil stabilised emulsion stabilised by flavonoid
particles
Figure 2 shows a water in oil emulsion stabilised by flavonoid and whey
protein particles
Figure 3 illustrates the size of the emulsions stabilised by flavonoid
particles over time
Figure 4 shows the size of the emulsions stabilised by flavonoid and whey
protein particles
(biopolymer) over time
Figure 5 shows particles-stabilized (Mechanism 1) and particles/biopolymer-
stabilized
(Mechanism 2) emulsions. On Mechanism 1, no any WPI is presented in the
aqueous phase
(pH 3 or 7). On Mechanism 2, water droplets are stabilized by both particles
in the oil phase
and different WPI concentrations in the aqueous phase (pH 3 or 7).
Figure 6 shows interfacial shear viscosity at W-0 interface of 0.14% w/w
curcumin (a) and
quercetin (b) particles dispersed in purified oil and different WPI
concentrations; 0 [=], 0.05
[=], 0.5[o], 2[0] and 4% w/v[A], respectively. A control experiment was
undertaken with 0%
polyphenol and 0% WPI[=].
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Figure 7 shows a water in oil stabilised emulsion stabilised by polyphenol and
whey protein
particles
Figure 8 shows a water in oil emulsion stabilised by polyphenol and whey
protein particles
Figure 9 shows a water in oil stabilised emulsion stabilised by polyphenol and
whey protein
particles
Detailed description of the invention and the preferred embodiments
Emulsion System
Without wishing to be bound by any theory it is believed that the emulsifying
capacity of the
combination of emulsion stabilisers has been found to exhibit the observed
sufficient
emulsion stabilisation effects without requiring the addition of any other
conventional
emulsifier, stabilising agent, or structuring agent, and without requiring any
activation of the
particles.
Conventional emulsifiers include for instance sugar esters, polyglycerol fatty
acid esters,
polyglycerol polyricinoleate (PGPR), polysorbates (polyoxyethylene sorbitan
esters),
monoglycerides/diglycerides and their derivatives, sodium stearoyl lactylate
(SSL),
phospholipids, glycerol monooeleate, amongst others. Advantageously, the
present
invention uses the claimed components to stabilize emulsions without the need
of addition
of such emulsifiers or stabilizing agents.
Advantageously, an embodiment of the present invention enables the preparation
of food
products, in particular confectionery products, based on emulsions that are
free of artificial
or synthetic emulsifiers. Advantageously, the present invention enables the
preparation of
food products that are free of monoglycerides, diglycerides and their
derivatives.
Advantageously, the present invention enables the preparation of food
products, in
particular confectionery products, based on emulsions that are free of
glycerol monooleate,
polyglycerol esters and polyglycerol esters of polyrincinoleic acid.
In a preferred embodiment of the present invention, the emulsion is a water in
oil emulsion.
In an embodiment, the pH of the aqueous phase of the emulsion is below 7.0,
preferably
the pH is between 1.5 and 5.0, preferably between 2.0 and 4.0, for example
2.5, 2.75, 3,
3.25, 3.50 or 3.75. In an embodiment, the pH is measured at 20.0+ 2 C. In an
embodiment,
the pH of the emulsions of the present invention may be controlled by addition
of the
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appropriate amount of acidic or alkaline component, preferably food grade
acids or alkaline
compounds.
An emulsion according to any one of the proceeding claims wherein the aqueous
phase-in-
oil, preferably water-in-oil, ratio (i.e. the weight ratio between the aqueous
phase and the
oil phase) is between 0.5:99.5 and 20:80, preferably between 1.0:99.0 and
15.0:85.0,
preferably between 1.0:99.0 and 10.0:90, preferably between 2.0:99.0 and
7.0:93.0,
preferably 5:95 or 0.75:99.25 to 3.0:97Ø
In a preferred embodiment, the oil phase of the present invention comprises a
liquid oil
(preferably an oil that is liquid at 20 C + 2 C).
In a preferred embodiment, the oil comprises an edible oil, preferably an
edible liquid oil.
In an embodiment, the oil is selected from the group consisting of sunflower
oil, rapeseed
oil, olive oil, soybean oil, fish oil, linseed oil, safflower oil, corn oil,
algae oil, cottonseed oil,
grape seed oil, nut oils such as hazelnut oil, walnut oil, rice bran oil,
sesame oil, peanut oil,
palm oil, palm kernel oil, coconut oil, and emerging seed oil crops such as
high oleic
sunflower oil, high oleic rapeseed, high oleic palm, high oleic soybean oils &
high stearin
sunflower or combinations thereof. In a preferred embodiment, the oil is
selected from the
group consisting of palm oil, coconut oil, soybean oil, sunflower oil and
mixtures thereof.
In any embodiment, a composition comprising the emulsion and/or the emulsion
of the
present invention comprises cocoa butter. In an embodiment, the oil phase
comprises
cocoa butter. In an embodiment, the cocoa butter is present in combination
with an edible
and/or liquid oil, as mentioned above.
In an embodiment, emulsion droplet size distributions were measured using
static light
scattering (SLS) via a Mastersizer Hydro SM small volume wet sample dispersion
unit
(Malvern Instruments, UK). In an embodiment, average droplet size was measured
in terms
of Sauter mean diameter, d3;2, or volume mean diameter, d4;3, preferably the
volume
mean diameter, d4;3. The refractive indices of water and soybean oil were
taken as 1.330
and 1.474, respectively. The emulsion droplet size was monitored over a period
of storage,
and change in droplet diameter has been used as a measure of stability. No
change or a
small increase in droplet size shows a stable emulsion whereas as a
significant increase in
droplet size is evidence of droplet coalescence and therefore an unstable
emulsion.
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In an embodiment, the aqueous phase contains particles, preferably water
droplets, that
have an average diameter of between 5 and 250 microns, preferably 10 and 200
microns,
preferably 10 and 100 microns, and preferably between 20 and 60 pm or between
10 and
50 microns. In an embodiment, the sizes relate to the d3,2 value.
In an embodiment, the aqueous phase contains particles, preferably water
droplets, that
have an average diameter of between 5 and 250 microns, preferably 10 and 200
microns
and preferably between 10 and 150 microns, 15 and 100 microns or 20 and 60
microns. In
an embodiment, the sizes relate to the d4,3 value.
A process for preparing an emulsion for use in the present invention
comprising the steps
of:
(i) mixing ingredients of the oil phase,
(ii) mixing ingredients of the aqueous phase,
(iii) dispersing the at least two emulsion stabilisers in one or both of the
aqueous phase or
the oil phase, and
(iv) homogenizing the two phases to form an emulsion.
In a preferred embodiment, the first emulsion stabiliser is dispersed in the
oil phase. In a
preferred embodiment, the second emulsion stabiliser is dispersed in the
aqueous phase.
In an embodiment, the second emulsion stabiliser is dissolved in the aqueous
phase to
ensure complete hydration, for example for at least one hour or at least two
hours and
optionally less than four hours.
In an embodiment, the aqueous phase may comprise a sugar or sugar alcohol or
any
mixture of two or more thereof. It should be understood that it would be
possible to have
some or all the sugars or sugar alcohols as crystalline material in the fat
phase whereupon,
on mixing the fat phase with the water phase, the sugar or sugar alcohol in
the fat phase
would dissolve into the water phase.
The mixture of sugars and/or sugar alcohols may be chosen to provide bulk, a
reduction in
water activity and an appropriate viscosity as well as serving as sweeteners.
There is a
spectrum of materials that can be used for this purpose, but broadly speaking
smaller
molecules such as monosaccharides and small sugar alcohols are more effective
at
reducing the water activity and make a lower contribution to viscosity than
the larger
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molecular weight materials such as higher polymers of glucose found in low
dextrose
equivalent (DE) corn syrups. Suitable mixtures of sugars and sugar alcohols
can comprise
corn syrup, sucrose, maltitol syrup, polydextrose, dextrins, inulin, sorbitol,
glycerol, fructose
and dextrose.
The amounts of the components of the water phase (by weight based on the
weight of the
water phase) may be, for example,
Sugar alcohol 0-40%, preferably 10 - 30%; and/or
Sugar 0-70%, preferably 15- 60%; and/or
added water 1- 30%, preferably 5-17%.
Optionally, flavourings or salt can be added to the water phase. The
flavouring may be, for
example, strawberry, raspberry, orange, lemon, mint, coffee, etc. but is
preferably
chocolate.
In an embodiment of the present invention, after the emulsion has formed, it
is held in a
vessel with stirring, advantageously using a gate-arm mixer and then fed to an
aeration
system to form the mousse. Aeration is carried out by injecting a gas, which
does not react
with the ingredients of the emulsion as it flows through the emulsion. The gas
flow is
increased or decreased relative to the material flow rate to achieve the
desired density. The
aeration may be carried out by using any of several known continuous aeration
equipments,
for example, a Mondomix machine or the aeration and depositing system
described in
W0200506303. In a batch process, whipping could be used, possibly under
pressure as in
a Morton pressure whisk. Any gas commonly used for aerating foodstuffs,
preferably
confectionery could be used, for example, air, nitrogen, carbon dioxide or
nitrous oxide.
In an embodiment, the density of the aerated emulsion is from 0.4 to 1.2
g/cm3, preferably
0.6 to 1.0 g/cm3, more preferably 0.8 to 0.9g/cm3.
In an embodiment, the emulsion of the present invention preferably has a water
activity (Aw)
of less than 0.70, preferably less than 0.60 and optionally greater than 0.10,
greater than
0.20.
In an embodiment, a composition comprising the emulsion of the present
invention may
have any desirable flavour, e.g. fruit, mint, caramel, hazelnut, coffee, etc.
but preferably
chocolate.
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Polyphenols and Flavonoids (First Emulsion Stabiliser)
In an embodiment, the present invention includes at least one emulsion
stabiliser that
comprises a polyphenol, optionally a flavonoid. In an embodiment, the emulsion
stabiliser
may be a source of a flavonoid or a polyphenol or alternatively may consist
essentially of a
flavonoid or a polyphenol.
Phenolic or polyphenol molecule is often characteristic of a plant species or
even of a
particular organ or tissue of that plant and have received significant
attention in recent years
due to their reported biological activities and general abundance in the diet.
More than 8000
phenolic structures are currently known, which 4000 of them are flavonoids.
Fruits,
vegetables, leaves, seeds and other types of foods and beverages such as tea,
chocolate
and wine are rich sources of polyphenols. These compounds are classified into
different
groups depending on the number of phenol rings that they contain and the
structural
elements involved for the binding of phenol rings to one another. Examples of
polyphenols
include curcumin. Any polyphenol known in the art may be used in the present
invention.
Flavonoids are polyphenols secondary metabolites derived from plants but they
can be
characterised by their 06-03-06 basic backbone. They can be sub-divided into
two main
groups; anthocyanins (glycosylated derivative of anthocyanidin) and
anthoxanthins.
Anthoxanthins are composed of several categories, such as flavones, flavonols,
isoflavones, flavanols, flavanones, and their glycosides. These flavonoids are
sub-classified
according to their substitution patterns, conformations and oxidation states.
Examples of
flavonoids include quercetin. Any type of flavonoid known in the art may be
used in the
present invention.
In an embodiment, the polyphenol comprises a compound selected from the group
consisting of flavonoids (for example, flavones, flavonols, flavanones,
isoflavones,
anthocyanidins, chalcones, catechins and mixtures thereof), stilbenes, lignans
and phenolic
acids (hydroxybenzoic acids, hydroxycinnamic acids and mixtures thereof) and
mixtures
thereof.
It is appreciated that the term "polyphenol" is broader than the term
"flavonoid". Accordingly,
in the present invention at least one of the emulsion stabilisers may be a
flavonoid or may
be a non-flavonoid polyphenol or a mixture thereof.
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In an embodiment, the polyphenol is selected from the group consisting of
tannic acid,
ellagtanin, (epi)catechin, (pro)anthocyanin, tiliroside, resveratrol,
quercetin, curcumin and
mixtures thereof. In a preferred embodiment, the emulsion stabiliser comprises
curcumin,
quercetin or mixtures thereof.
In an embodiment, at least one of the emulsion stabilisers, preferably the
flavonoid or
polyphenol particles, is present at a level between 0.01 and 0.50 wt% of the
oil phase of the
emulsion, preferably between 0.02 and 0.20 wt% of the oil phase of the
emulsion, and
preferably between 0.06 and 0.14 wt% of the oil phase of the emulsion. This
relates to the
total amount of the emulsion stabiliser in the oil phase, for example, when
there are multiple
stabilisers present.
In an embodiment, at least one of the emulsion stabilisers, preferably the
flavonoid or
polyphenol particles, is present at a level between 0.01 and 0.475 wt% of the
emulsion,
preferably between 0.02 and 0.20 wt% of the emulsion, and preferably between
0.06 and
0.14 wt% of the emulsion. This relates to the total amount of the emulsion
stabiliser, for
example, when there are multiple stabilisers present. The person skilled in
the art will
realise that although the above ranges overlap, the percentage present in the
emulsion
cannot be higher than the percentage present in the individual phase.
In an embodiment, the first emulsion stabiliser has a preferred particle size
of 0.05 microns
to 10.0 microns, preferably from 0.075 microns to 7.5 microns, preferably from
0.10 microns
to 7.0 microns.
In an embodiment, when the polyphenol is a flavonoid, preferably quercetin,
the preferred
particle size is in the range of 3.5 microns to 7.0 microns, for example
between 3.75 microns
and 6.75 microns.
In an embodiment, when the polyphenol is a non-flavonoid, preferably curcumin,
the
preferred particle size is in the range of 0.05 microns to 0.25 microns, for
example between
0.90 microns and 0.25 microns.
In an embodiment, particle size distributions were measured at a low angle
laser diffraction
particle size analyser (LS 13 320 series Beckman Coulter, Inc, UK) utilising
the Fraunhofer
optical model. Average sizes were assessed using d4,3, the volume mean or d3,2
the
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surface area mean (Sauter mean diameter). In an embodiment, these sizes were
measured
with soy bean oil as a dispersant.
In an embodiment, the emulsion stabilisers may be treated by known methods,
e.g. jet
homogenisation, in order to arrive at the above particle sizes.
In an embodiment, the polyphenol may be provided as a component of a
composition. In a
preferred embodiment, the composition is an edible composition comprising a
polyphenol
as defined above. For example, the emulsion stabiliser of the present
invention may
comprise cocao, peppermint, cloves, spearmint, blueberry, blackcurrant, hazel
nuts, pecan
and mixtures thereof. In a preferred embodiment, the emulsion stabiliser is a
powder form
of the above.
In an embodiment, suitably the particles of the composition comprising the
polyphenol can
have a particle size (otherwise referred to as a mean particle diameter) with
an average
particle size of from about 1 to about 200 microns, preferably of from about 1
to about 100
microns. In some embodiments, the particles have an average particles size of
from about
1 to about 50 microns, such as of from about 5 to about 40 microns. In certain
embodiments,
the particles have an average particles size of from about 10 to about 20
microns. In other
embodiments, the particles have an average particles size of less than 10
microns, even
less than 5 microns, such as from about 0.1 to about 5 microns.
Biopolymer (Second Emulsion Stabiliser)
In an embodiment, the present invention includes at least one emulsion
stabiliser that
comprises a biopolymer, preferably a protein, preferably a food protein.
In an embodiment, preferably the biopolymer is any food-grade protein such as
milk and/or
whey proteins, soy proteins, pea proteins, caseinate, egg albumen, lyzozyme,
gluten, rice
protein, corn protein, potato protein, pea protein, skimmed milk proteins or
any kind of
globular and random coil proteins as well as combinations thereof. In one
preferred
embodiment the protein is one or more milk and/or whey derived protein.
Preferred milk proteins or milk protein fractions in accordance with the
present invention
comprise, for example, whey proteins, a-lactalbumin, 13-lactalbumin, bovine
serum albumin,
acid casein, caseinates, a-casein, 13-casein.
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As far as whey proteins are concerned, the protein source may be based on acid
whey or
sweet whey or mixtures thereof and may include a-lactalbumin and 13-
lactalbumin in any
proportions. The proteins may be intact or at least partially hydrolysed.
In an embodiment of the invention, the second emulsion stabiliser may comprise
a protein
or protein derived material such as whey protein, egg white, casein
hydrolysate or mixtures
of these.
In a preferred embodiment, the food protein is isolated from a dairy source,
preferably from
milk. In a preferred embodiment, the protein is selected from the group
consisting of whey
isolate, whey concentrate, or whey hydrolysate. In a preferred embodiment, the
protein is a
whey protein isolate. In a preferred embodiment, the protein consists
essentially of whey
protein isolate, preferably is substantially free from lactose, carbohydrate,
fat, and
cholesterol.
In an alternative embodiment, the protein may be provided as a component of a
composition. In a preferred embodiment, the composition is an edible
composition
comprising said protein, such as skimmed milk powder.
In a preferred embodiment, the at least one of the emulsion stabilisers,
preferably the whey
protein particles, is present at a level between 0.01 and 10.0 w/e0 of the
aqueous phase
of the emulsion, preferably between 0.05 and 7.5 w/e0 of the aqueous phase of
the
emulsion, and preferably between 0.05 and 5 w/e0 or 0.1 and 4 w/e0 of the
aqueous phase
of the emulsion. This relates to the total amount of the emulsion stabiliser,
for example,
when there are multiple stabilisers present.
In an embodiment, the at least one of the emulsion stabilisers, preferably the
whey protein
particles, is present at a level between 0.01 and 10.0 wt% of the emulsion,
preferably
between 0.7 and 7.5 wt% of the emulsion, and preferably between 0.1 and 4 wt%
of the
emulsion. For example, between 0.5 and 4wt% or 4.0wt%, between 0.1 and 4wt% or
4.0wr/o. This relates to the total amount of the emulsion stabiliser, for
example, when there
are multiple stabilisers present. The person skilled in the art will realise
that although the
above ranges overlap, the percentage present in the emulsion cannot be higher
than the
percentage present in the individual phase.
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In an embodiment, the second emulsion stabiliser is incorporated into the
aqueous phase
of the emulsion during the preparation of the emulsion.
Food Products
The present invention provides a foodstuff that comprises the emulsion of the
present
invention.
The term "foodstuff" encompasses food, beverage and nutritional products for
humans and
animals, including but not limited to baby and infant nutrition products,
water, water-based
beverages, juices and other beverages, cereals, chocolate and confectionery,
coffee, tea,
chocolate or milk based beverages, culinary, chilled and frozen food, dairy,
drinks, food
service, healthcare nutrition, ice cream, sports nutrition, weight management,
pet health &
nutrition, liquid food and beverages for human (including infant) or animal
consumption,
foods for special medical purposes, medical food, foods for special dietary
use, dietary
supplements, medical nutrition, clinical food and functional food.
For example, the present invention provides a food product selected from the
group
consisting of confectionery products, ice cream, sauces (e.g. hollandaise
sauce), salad
dressings (e.g. vinaigrette or salad cream), mayonnaise, soups, processed meat
(e.g.
sausages), butter, and margarine that comprises the emulsion of the present
invention.
Confectionery Products
Surprisingly, the inventors of the present invention have found that the
emulsion systems
of the invention are able to remarkably stabilise water-in-oil emulsions. This
is particularly
advantageous for applications in confectionery products. Accordingly, in one
preferred
aspect the invention provides the use of the combination of emulsion
stabilisers as the
emulsifier system for the stabilization of a water-in-oil emulsion.
According to one aspect of the invention there is provided a confectionery
product
comprising an emulsion comprising the first and second emulsion stabilisers as
the
emulsifying agent, preferably in the absence of any synthetic or artificial
emulsifiers or
structuring agents.
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The confectionery product comprising an emulsion may be a chocolate, a
chocolate-like
(e.g. comprising cocoa butter replacers, or cocoa-butter equivalents), a
chocolate spread,
a chocolate sauce, a coating chocolate, a coating chocolate for ice-creams, a
praline, a
chocolate filling, a fudge, a chocolate cream, a refrigerated chocolate cream,
an extruded
chocolate product, or the like. The confectionery product may be in any
conventional form,
such as in the form of an aerated product, a bar, a spread, a sauce or a
filling, among others.
It may also be in the form of inclusions, chocolate layers, chocolate nuggets,
chocolate
pieces, chocolate drops, or shaped chocolates and the like. The confectionery
product may
further contain inclusions e.g. cereals, like expanded or toasted rice or
dried fruit pieces and
the like.
The amount of emulsion stabilisers included as the emulsifier will depend on
the desired
properties of the emulsion product and the amount of emulsion present in the
final product
will depend on the final product.
In an embodiment of the present invention, the emulsion is present in amount
of from about
0.1 to about 50wr/0 of the total weight of the confectionery product,
preferably from about
0.5 to about 30wr/0 and preferably 1.0 to about 25wr/o, e.g. from about 1 to
about 10wr/o.
In an embodiment of the present invention, the combined amount of emulsion
stabilisers
present in the emulsion in the confectionery product, is from about 0.00006wW0
to about
5.25wt% of the total weight of the confectionery product, preferably from
about 0.0001wr/0
to 3.5wW0 and preferably from about 0.015wr/0 to 1.05wr/o.
The confectionery product may comprise sugars. These sugars include sucrose,
fructose,
sugar replacers such as polyols (e.g., maltitol, lactitol, isomalt,
erythritol, sorbitol, mannitol,
xylitol) or bulking agents like polydextrose or other sweeteners like tagatose
or high intensity
sweeteners like saccharin, aspartame, acesulfame-K, cyclamate, neohesperidin,
thaumathin, sucralose, alitame, neotame or any combination thereof.
The confectionery product may comprise ingredients such as flavouring agents,
colorants,
or milk ingredients. Typically flavouring agents are used to add flavours such
as vanilla,
raspberry, orange, mint, citrus, strawberry, apricot, lavender flavours, etc,
and any other
fruit, nutty or flower flavouring agent, among others. Milk ingredients can be
liquid milk or
milk powder, either full fat, partially defatted or defatted, and
delactosylated or not.
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In the confectionery product the fat phase is typically cocoa butter, a cocoa
butter substitute,
cocoa butter replacer, cocoa butter improver and/or cocoa butter equivalent,
among others.
Cocoa butter substitute is a lauric fat obtained from the kernel of the fruit
of palm trees
obtained by fractionation and/or hydrogenation of palm kernel oil. It
comprises about 55%
lauric acid, 20% myristic acid and 7% oleic acid, cocoa butter substitutes
cannot be mixed
with cocoa butter. Cocoa butter equivalents are vegetable fats with similar
chemical and
physical characteristics to cocoa butter, which are obtained by blending
different fractions
of other fats or by intersterification, and can be used interchangeably with
cocoa butter in
any recipe. Cocoa butter replacers are formed by non lauric vegetable fats
which may be
mixed with cocoa butter but only in limited proportions: they have similar
physical, but not
chemical characteristics to cocoa butter. Cocoa butter replacers can be used
in recipes
partially based on cocoa mass or cocoa butter. Cocoa butter improvers are
harder cocoa
butter equivalents which are not only equivalent in their compatibility but
also improve the
hardness of some of the softer qualities of cocoa butter.
Advantageously the present invention allows the preparation of confectionery
products
based on emulsions having very good stability properties, in the absence of
any added
emulsifiers, structuring agents or other stabilizing agents. Advantageously
the present
invention allows the preparation of emulsion-based confectionery products
having very
good emulsion stability properties, which stabilised by the emulsifying agents
of this
invention, without the addition of any other emulsifier and without the need
for carrying out
any activation step/treatment on the emulsifying agents.
General Definitions
Unless otherwise specified % in the present description correspond to wt%.
The terms "substantially", "consists of" and "consist essentially" as used
herein may refer
to a quantity or entity to imply a large amount or proportion thereof. Where
it is relevant in
the context in which it is used these terms can be understood to mean
quantitatively (in
relation to whatever quantity or entity to which it refers in the context of
the description)
there comprises an proportion of at least 80%, preferably at least 85%, more
preferably at
least 90%, most preferably at least 95%, especially at least 98%, for example
about 100%
of the relevant whole. By analogy the term "substantially-free" or alike may
similarly denote
that quantity or entity to which it refers comprises no more than 20%,
preferably no more
than 15%, more preferably no more than 10%, most preferably no more than 5%,
especially
is
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no more than 2%, for example about 0% of the relevant whole. Preferably, where
appropriate (for example in amounts of ingredient) such percentages are by
weight.
In the present specification, the term "fat phase" is understood as including
any solid and/or
liquid ingredient miscible with oil or fat or that has the capacity to
dissolve in oil or fat, and
"aqueous phase" as including any solid and/or liquid ingredient miscible with
water or that
has the capacity to dissolve in water.
In the present description, what is meant by "natural ingredients" is
ingredients of natural
origin. These include ingredients which come directly from the field etc. They
may also
include ingredients which are the result of a physical or
microbiological/enzymatic process
(e.g. extraction, fermentation etc.). However, they do not include ingredients
which are the
result of a chemical modification process.
In the present description, "food-ingredients" refers to ingredients of
natural origin
containing nutrients that are consumed to provide nutritional support for the
body.
Unless defined otherwise, all technical and scientific terms used herein have
and should be
given the same meaning as commonly understood by one of ordinary skill in the
art to which
this invention belongs.
Unless the context clearly indicates otherwise, as used herein plural forms of
the terms
herein are to be construed as including the singular form and vice versa.
In all ranges defined above, the end points are included within the scope of
the range as
written. Additionally, the end points of the broadest ranges in an embodiment
and the end
points of the narrower ranges may be combined.
It will be understood that the total sum of any quantities expressed herein as
percentages
cannot (allowing for rounding errors) exceed 100%. For example the sum of all
components
of which the composition of the invention (or part(s) thereof) comprises may,
when
expressed as a weight (or other) percentage of the composition (or the same
part(s)
thereof), total 100% allowing for rounding errors. However where a list of
components is
non exhaustive the sum of the percentage for each of such components may be
less than
100% to allow a certain percentage for additional amount(s) of any additional
component(s)
that may not be explicitly described herein.
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It should be understood that various changes and modifications to the
presently preferred
embodiments described herein will be apparent to those skilled in the art.
Such changes
and modifications can be made without departing from the spirit and scope of
the present
invention and without diminishing its attendant advantages. It is therefore
intended that such
changes and modifications be covered by the appended claims.
Examples
The following examples are illustrative of the products and methods of making
the same
falling within the scope of the present invention. They are not to be
considered in any way
!imitative of the invention. Changes and modifications can be made with
respect to the
invention. That is the skilled person will recognise many possible variations
in these
examples covering a wide range of compositions, ingredients, processing
methods and
mixtures and can adjust the naturally occurring levels of the compounds of the
invention for
a variety of applications.
Materials
Curcumin (orange-yellow powder) from turmeric rhizome (95% total curcuminoid
content)
was obtained from Alfa Aesar (UK). Quercetin (95%) in the form of yellow
crystalline solid
was purchased from Cayman Chemicals (USA). Both polyphenols were used without
further
purification. Whey protein Isolate (WPI) containing 96.5% protein was obtained
from
Fonterra (New Zealand). Soybean oil (KTC, UK) was purchased from local store.
Water
purified by treatment with Milli-Q apparatus (Millipore, Bedford, UK) with a
resistivity not
less than 18 M cm was used for the preparation of the emulsions. Few drops of
hydrochloric
acid (0.1 M HCI) or sodium hydroxide (0.1 M NaOH) were used to adjust the pH
of the
emulsions.
Methods
Preparation of Pickering Particle Dispersion
The curcumin or quercetin particles were firstly dispersed in the continuous
phase (soybean
oil) using an Ultra-Turrax T25 mixer (Janke & Kunkel, IKA-Labortechnik) with a
13 mm mixer
head (525N-10 G) operating at 9,500 rpm for 5 minutes.
For the assessment of particles, the particle dispersion was sonicated in an
ultrasonic bath
(KERRY, Guyson International LtD, UK) at different times (2, 5 or 10 minutes),
heated at
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60-65 C for 1 hour whilst being agitated with a magnetic stirrer and also
homogenized
using a high pressure jet homogenization twice, operating at 300 bar.
Preparation of Aqueous Phase
The aqueous phase was prepared without (0% w/v) or with WPI (0.05, 0.5, 2 and
4% w/v).
WPI (4% w/v) was dissolved in aqueous phase for at least 120 min at room
temperature to
ensure complete hydration. Then, a number of dilutions were performed in order
to reach
the desired WPI concentration (0.05, 0.5 and 2% w/v) and 0.02 g sodium azide
was added
as a preservative. The pH of the aqueous phase was maintained at 3 or 7,
depending on
each experiment, by adding few drops of 0.1 M HCI or 0.1 M NaOH.
Preparation of Emulsions
Coarse emulsions were prepared by homogenising 5% w/w of the aqueous phase
with 95%
w/w oil phase using an Ultra-Turrax mixer for 2 minutes at 13,500 rpm. Fine
emulsions were
prepared by passing the coarse emulsions through a high pressure jet
homogenizer, twice,
operating at 300 bar. Immediately after preparation, emulsions were sealed in
a 25 ml
cylindrical tube (internal diameter = 17 mm) and stored at room temperature in
a dark place.
Particle and Emulsion Droplet Measurements
Emulsion droplet size distributions were measured using static light
scattering (SLS) via a
Mastersizer Hydro SM small volume wet sample dispersion unit (Malvern
Instruments, UK).
Average droplet size was measured in terms of Sauter mean diameter, d3;2, or
volume
mean diameter, d4;3. The refractive indices of water and soybean oil were
taken as 1.330
and 1.474, respectively. All measurements were made at room temperature on at
least
three different samples.
Confocal Microscopy
The emulsion micro-structure was observed using confocal microscope (Zeiss
LSM880
inverted with Airyscan, Germany). Rhodamine B (excitation/emission maxima _
568/600-
700 nm) was used in aqueous phase. Approximately 80 pL of sample was placed
into a
laboratory-made welled slide and a coverslip (0.17 mm thickness) was placed on
top,
ensuring that there was no air gap (or bubbles) trapped between the sample and
coverslip.
The samples were scanned at room temperature (25 + 1 C) using 20x/0.8
objective lenses.
Fluorescence from the sample was excited with the 488 nm Ar and 633 nm He¨Ne
laser
lines. Images were processed using the image analysis software Image J.
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Potential Measurements
The potential measurements of WPI solution (0.5% w/v) over different pH values
were
performed using a Nanoseries ZS instrument (Zetasizer Nano-ZS, Malvern
Instruments,
Worcestershire, UK). The instrument software was used to convert the
electrophoretic
mobility into potential values using the Smoluchowski model. The pH of freshly
prepared
WPI solutions was adjusted from pH 2 to pH 8 using various concentrations of
HCI and
NaOH. Two readings of zeta potential were made per sample.
Interfacial Tension Measurements
Interfacial tension (y or IFT) measurements were performed between the soybean
oil with
or without the presence of polyphenol crystals and Milli-Q water (pH 3) using
the pendant
drop method in a Dataphysics OCA tensiometer (Data Physics Instruments,
Germany). The
apparatus includes an experimental cell, an optical system for the
illumination and the
visualization of the drop shape and a data acquisition system. An upward
bended needle
was used to immerse a drop of a lower density liquid into a higher density
one. Thus, a drop
of soybean oil or oil suspension (0.14% w/w curcumin or quercetin dispersed in
soybean
oil) was formed at the tip of the needle and suspended in the cuvette
containing Milli-Q
water, at pH 3. The contour of the drop extracted by the SCA 20 software was
fitted to
Young-Laplace equation to obtain y. All measurements were carried out in
triplicate and
error bars represent the standard deviations.
Wettability Measurements
The hydrophilic/hydrophobic character of the particles was evaluated in terms
of their
wettability. The wettability measurements were carried out at room temperature
using
00A25 drop-shape tensiometer (DataPhysics Instruments, Germany) fitted with a
micro-
syringe and high-speed camera. Static contact angles were measured using the
sessile
drop method. Water or oil droplets (3 pL) were spotted onto compressed
particle disc
surfaces via the micro-syringe. The video camera was used to video-record
droplet
formation. The initial droplet contour was mathematically described by the
Young-Laplace
equation using the SCA software and the contact angles between the particle
substrate and
water droplet (Ow) or oil droplet (A o) were measured. The compressed particle
discs were
prepared by placing 0.3 g of the pure powdered particles between the plates of
a hydraulic
bench press (Clarke, UK) using a 1.54 cm diameter die under a weight of 3
tonnes for 30 s.
All measurements were carried out in triplicate and error bars represent + 1
standard
deviation.
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The results and discussion section will be separated in two main parts. The
first part involves
the assessment of particles as Pickering stabilizers according to their size,
contact angle
and interfacial tension measurements. The second part involves the preparation
of W/O
emulsions, which is further divided into 2 main subsections; the results from
particle-
stabilized emulsions and those from particle/biopolymer stabilized emulsions
on both
curcumin and quercetin particles at pH 3 and 7 in the aqueous phase. Two
mechanisms
are taking place as shown in Figure 5. The Mechanism 1 includes the particle-
stabilized
emulsions where the water droplets are stabilized only with
polyphenol/flavonoid particles
in the absence of any WPI in the aqueous phase (pH 3 or 7). The Mechanism 2
involves
the particle/biopolymer stabilized emulsions where the water droplets are
stabilized by
particles in the oil phase and different WPI concentrations in the aqueous
phase (pH 3 or
7). Five different WPI concentrations were used; low (0.05 % w/v), medium (0.5
% w/v) and
two high concentration (2 and 4 % w/v).
Curcumin and Quercetin particles were characterised in terms of their size,
wettability and
interfacial behaviour, in an attempt to assess their potential as Pickering
stabilisers.
Curcumin and Quercetin were selected not only due to their high logP values,
4.31 and 2.16
respectively, but also due to their availability and potential health
benefits.
Effect of Particle size
The size of particles dispersed in the continuous phase is an important
parameter on the
Pickering functionality. It is used for the estimation of the amount of
surface active particles
require for surface coverage in order to form stable emulsions. Additionally,
the overall
stability of an emulsion is inversely proportional to particle size, with
smaller particles giving
a higher packing efficiency and therefore providing a more homogeneous layer
at the
interface preventing coalescence. On the other hand, particle size has a
direct effect on the
energy of desorption (AGd), and if adsorption occurs, smaller particles
provide lower A Gd.
This cause the detachment of smaller particles from the oil-water interface
more easily than
larger ones. On this experiment, the size of curcumin and quercetin particles
dispersed in
an oil medium (soybean oil) was measured after treatment with ultra-Turrax
(9,500 rpm for
min) only or ultra-Turrax followed by ultrasound bath (2, 5 and 10 min), heat
(60-65 C for
1h) or jet homogenizer (twice, operating at 300 bar).
According to the results of the present invention only heat treatment reduced
the size of
curcumin particles, from 0.16 to 0.11 microns, comparing to the other methods.
The particle
size distribution plot showed that the curcumin particles were polydispersed
under ultra-
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Turrax, ultrasound bath and jet homogenizer treatment whilst under heat it
became bi-
dispersed. These results indicate that the powder curcumin was not fully
dispersed in oil
phase under the different treatments used but curcumin dispersed very well
when it was
heated at 60 deg C for 1 h. Moreover, the colour of the sample with the heated
dispersion
was more transparent than the other ones. On the other hand for quercetin
particles neither
ultrasound bath or heat change the size of particles significantly. Only
treatment with jet
homogenizer significantly reduced the size of quercetin particles from 6.43 to
4.15 pm. This
suggests that the jet homogenizer may help to break up the flavonoid crystals
or aggregates
into smaller entities. Consequently, not a huge difference was observed on the
size of both
particles under different treatments. Quercetin particles were much bigger in
size (6.43
microns) and monodispersed than curcumin particles (0.16 microns).
Additionally, larger
particles (queretin) have higher delta Gd values and therefore expected to
detach from the
oil-water interface more difficult than smaller particles (curcumin).
Contact Angle and Particle Wettability
The hydrophilic/hydrophobic character of particles can be identified through
particles
wettability (the tendency of one liquid to spread on a solid surface) in
aqueous and oil
phases. This can be determined by measuring the contact angle formed between
particles
and water (w) or oil (o) phase. It can be used as an indicator of the type of
emulsion that
these particles would favour to stabilize. Therefore, when the w significantly
exceeds o for
particles, they can be categorised as hydrophobic, with the reverse being true
for hydrophilic
particles.
On this experiment, both curcumin and quercetin particles had w value that
exceeded their
o value and indicating that both possess a hydrophobic character. It was
observed that
different pH values in the aqueous phase did not significantly affect the
contact angle of
curucmin particles. On the other hand, for quercetin the w at pH 3 was much
smaller than
that at pH 7. The contact angle, 0 is directly related to the relative
strength of the cohesive
(such as hydrogen bonding and Van derWaals forces) and adhesive forces (such
as
mechanical and electrostatic forces). At pH 7 the 0 is larger than that at pH
3, thus there is
a larger strength of the cohesive force relative to the adhesive one and the
liquid tends to
resist separation. Besides, at pH 3 the 0 is smaller so the relative strength
is smaller and
the adhesive force causes the liquid to cling to the surface on which it
rests.
Interfacial Tension
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It was determined that both polyphenol crystals are hydrophobic and can
stabilize W/O
emulsions. However, to fully understand if the stabilization was arising from
particles, the
interfacial tension was measured. Interfacial tension (y) decreases
dramatically on
surfactant or biopolymer adsorption but in the case of Pickering
stabilization, it does not
change significantly. The effects of the presence of curcumin or quercetin
particles
dispersed in oil on the interfacial tension are shown in the Table 1. Firstly,
y was measured
between soybean oil and aqueous phase (in the absence of particles) as a
control
experiment for comparison purposes. The equilibrium y for such a system was
25.8 mN. As
expected, addition of low concentration (0.14% w/w) of curcumin or quercetin
particles in
the oil phase did not alter the y (24.6 and 25.3 mN m-1, respectively)
significantly. However,
addition of 0.5% w/v concentration of WPI in the aqueous phase in the presence
of curcumin
and quercetin crystals showed a significant decrease in the y (17.1 and 18.0
mN/m for
curcumin and quercetin crystals, respectively).Table 1:
I I II
.
1.
õ
Moreover, interfacial shear viscosities at W-0 interface of 0.14% w/w curcumin
(a) and
quercetin (b) particles dispersed in purified oil and different WPI
concentrations; 0, 0.05, 0.5
2.0 and 4.0 w/v, respectively, were tested. A control experiment was
undertaken with 0%
w/w polyphenol and 0 w/v% WPI. The pH of the aqueous phase was adjusted to pH
3. Error
bars represent standard deviation of at least two independent experiments.
Results are
shown in Figure 6.
W/O Emulsions
Particle-Stabilized Emulsions
For particles-stabilized emulsions two particle concentrations, 0.06 and 0.14%
w/w, were
tested. It was observed that the emulsions prepared by 0.06% w/w of both
curcumin and
quercetin particles were not as stable as those with 0.14% w/w. The emulsions
prepared by
0.06% w/w curcumin were phase separated within 1 day, in contrast to those
stabilized by
quercetin where their size increased dramatically over time but they phase
separated within
2 days. At concentration of 0.14% w/w for both particles, the size of water
droplets did not
change significantly over time, but they phase separated within 2 days due to
sedimentation
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effect caused by gravity. The lack of stability of emulsions stabilized by
0.06% w/w in
comparison to those stabilized by 0.14% w/w of particles indicates a rapid
droplet
sedimentation and coalescence. This is potentially due to incomplete coverage
of droplets
surface by particles leading to droplet-droplet coalescence. On the other
hand, curcumin
stabilized emulsions (0.14% w/w) were much smaller in size (3 pm) than those
stabilized
by quercetin (11 p m) possibly due to the smaller size of curcumin dispersion
in the
continuous phase, promoting smaller droplet formation during processing.
Particle-stabilised emulsions according to the present invention
To improve further upon the above-mentioned particle-stabilised emulsions whey
protein
was added in the aqueous phase in order to improve the stability due to the
significant
reduction of the interfacial tension. WPI acts as an emulsifying agent due to
the formation
of viscoelastic adsorbed layer at the interface of the emulsions. Once
adsorbed at the
interface, it unfolds and rearrange its secondary and tertiary structure to
exposed
hydrophobic residues to the hydrophobic phase. The high concentration of
protein at the
interface leads to aggregation and formation of interactions. The mechanical
properties of
the adsorbed layer influence the stability of emulsions, which it depends on
the structure of
the adsorbed protein and the strength of the interactions between them.
According to the invention, the WPI was used at pH 3 because at this pH the
protein is
unfolded and acquired a positive charge. It was observed that addition of
small amount of
WPI in the aqueous phase (0.05% w/v), the stability of water droplets did not
significantly
improve over time showing a very similar effect with the particle-stabilized
system (without
WPI). Thus, it was phase separated within 1-2 days. On the other hand,
addition of at least
0.5% w/v WPI, the stability was improved significantly and the emulsions were
stable for
more than 3 weeks. The particle-size distribution plots of particle/biopolymer-
stabilized
emulsions for both curcumin and quercetin on the first day of the preparation
(0 day). In
both cases, the size of water droplets without (0% w/v) and very small
concentration of WPI
(0.05% w/v), was smaller than those with at least 0.5% w/v WPI. Over time, the
size of
emulsions without and low concentration of WPI was increased dramatically
within 24 h and
phase separated. On the other hand, the size of emulsions with medium (0.5%
w/v) and
high (2 and 4% w/v) concentration of WPI, was stable over time (more than 3
weeks) without
a significant change on size. It was observed a sedimentation from particles
and, possibly,
water droplets but no coalescence was observed as a single layer of water on
the bottom.
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Confocal microscope images from particle/biopolymer- stabilized emulsions for
both
curcumin and quercetin particles. On curcumin/WPI-stabilized emulsions, the
ring around
the water droplets was not visible as it was discussed before, in contrast to
the
quercetin/WPI emulsions where there is an obvious layer of particles at the
interface.
Unfortunately, the position of WPI within the water droplets was not possible
to be detected
using dyes. These results give an indication of complex formation between
particles and
WPI at the interface. WPI at pH 3 is unfolded and exposed its hydrophobic and
positively
charged groups at the interface. On the same time, polyphenol/flavonoid
particles possess
a weak charge in the oil phase, as the soybean oil is relatively polar and
particles acquires
many hydroxyl groups that easily ionised. Thus, it is proposed that particles
with the weak
negative charge interact with the positively charge groups of WPI through
hydrogen bonding
and possibly electrostatic interactions, improving the stability of the
emulsions over time.
Effect of the pH in the Aqueous Phase
The influence of the pH of the aqueous phase on the stability of the emulsions
was tested.
Quercetin/WPI stabilized emulsions were prepared with a pH 7 in the aqueous
phase. The
results showed that the protein at this pH acquires a negative charge.
As before, the emulsions without and small concentration of WPI had smaller
water droplets
on the first day of the preparation but they phase separated within 3 days.
The emulsions
with medium and high WPI concentration were similar in size as those at pH 3
but they
phase separated within 7 days. According to the confocal microscope images
(the
emulsions were partially coalescence some days after the preparation
indicating an
unstable system. Moreover, the particles were aggregated and did not form a
uniform layer
at the interface.
To conclude, it was identified that at pH 7 the emulsions were very unstable
and
coalescence over time. At this pH, WPI possess a negative charge same with the
particles
in the oil (due to the ionised hydroxyl groups in a polar oil). In that case,
particles cannot
interact with WPI at the interface, since both are negatively charge and
probably some
repulsive interactions are taking place. Therefore, these results agree with
the initial
hypothesis that the emulsions will be more stable at pH 3 instead of 7 where a
complex is
formed at the interface between WPI and particles and the main driving force
is arising from
the WPI charge resulting on the formation of electrostatic interactions.
Table 2 stabilised emulsion compositions according to the present invention
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Curcumn. WPI H -3), Stabillty' days
0.06 0 4
0.06 0.01 or 0.05 4
0.06 0.1 - 4 14
0.14 0 4
0.01 0_05 or
0.14 or 2
0.1
0.14 0.5-4 14
Preparation of Oil Dispersions and W/O Emulsions
Polyphenol dispersions were prepared by dispersing 0.14 % w/w of quercetin
crystals in the
continuous phase (soybean oil) using an Ultra-Turrax T25 mixer (Janke &
Kunkel, IKA-
Labortechnik) with a 13 mm mixer head (S25N-10 G) operating at 9,400 rpm for 5
min. The
aqueous phase was made with whey protein particles (0.5 and 1 % w/v). Whey
protein
particles were prepared by dissolving whey protein isolate (10 % w/v) in
aqueous phase for
at least 120 min at room temperature. The protein was heated at 90 E C,
followed by Jet
homogenization, twice, operated at 300 bar. Then, dilution was performed in
order to reach
the desired whey protein particle concentrations and 0.02 g sodium azide was
added as a
preservative. The pH of the aqueous phase was adjusted to 3 or 7, depending on
each
experiment, by adding few drops of 0.1 M HCI or 0.1M NaOH. Coarse emulsions
were
prepared by homogenising 10 % w/w of the aqueous phase with 90 % w/w oil phase
using
an Ultra-Turrax mixer for 2 min at 13,400 rpm. Fine emulsions were prepared by
passing
the coarse emulsions through a high pressure Leeds jet homogenizer, twice,
operated at
300 bar.
Figure 7 displays the mean droplet size distributions of the W/O emulsions
(10:90 % w/w
w:o ratio) stabilized by quercetin crystals (0.14 % w/w) dispersed in the oil
phase at different
whey protein particle concentrations (0.5 and 1 % w/v). The pH was adjusted to
pH 3.
Figure 8 displays the mean droplet size of water droplets (d3,2) stabilized by
quercetin
crystals (0.14 % w/w) at different concentrations of whey protein particles
(0.5 and 1 % w/v),
over time.
CA 03068897 2019-12-31
WO 2019/008059 PCT/EP2018/068131
Figure 9 displays the images of W/O Pickering emulsions (10:90 % w/w w:o
ratio) stabilized
by quercetin crystals (0.14 % w/w) and whey protein particles (0.5% w/v). The
pH of the
aqueous phase was adjusted to pH 3. The brightness in the images is caused by
auto-
fluorescence of quercetin particles (405 nm excitation).
26
