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 (O/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/O) or water-in-oil-in-water (W/O/W) 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. l 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. 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 1 1 . 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. 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 [A], 0.5[D], 2[O] and 4% νν/ν[Δ], respectively. A control experiment was undertaken with 0% polyphenol and 0% WPI[«]. 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 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 3.0:97.0.

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. 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 μηι 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 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 WO200506303. 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. 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 C6-C3-C6 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. 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 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, olactalbumin, β-lactalbumin, bovine serum albumin, acid casein, caseinates, a-casein, β-casein. 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 olactalburmin and β-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/v% of the aqueous phase of the emulsion, preferably between 0.05 and 7.5 w/v% of the aqueous phase of the emulsion, and preferably between 0.05 and 5 w/v% or 0.1 and 4 w/v% 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 4.0wt%. 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 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. 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 50wt% of the total weight of the confectionery product, preferably from about 0.5 to about 30wt% and preferably 1.0 to about 25wt%, e.g. from about 1 to about 10wt%.

In an embodiment of the present invention, the combined amount of emulsion stabilisers present in the emulsion in the confectionery product, is from about 0.00006wt% to about 5.25wt% of the total weight of the confectionery product, preferably from about 0.0001wt% to 3.5wt% and preferably from about 0.015wt% to 1.05wt%.

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. 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 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. 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 limitative 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 (S25N-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 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 μΙ_ 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. 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 I FT) 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 (DataPhysics 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 γ. 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 OCA25 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 μί) 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 (9w) or oil droplet (Θ 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. 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 Δ 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 5 min) only or ultra-Turrax followed by ultrasound bath (2, 5 and 10 min), heat (60-65 °C for 1 h) 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 0.1 1 microns, comparing to the other methods. The particle size distribution plot showed that the curcumin particles were polydispersed under ultra- 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 μηη. 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, Θ 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 Θ 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 Θ 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 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 (γ) 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, was measured between soybean oil and aqueous phase (in the absence of particles) as a control experiment for comparison purposes. The equilibrium γ 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 γ (24.6 and 25.3 mN nr 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 γ (17.1 and 18.0 mN/m for curcumin and quercetin crystals, respectively). Table 1 :

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 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 μηη) than those stabilized by quercetin -1 1 μ 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. 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 Curcumin/ wt% WPI (pH-3)/ wt% Stability/ days

0,08 0 4

0,06 0.01 or 0.05 4

0.06 0.1 - 4 1

0.14 0 4

0.01 or 0,05 or

0,14 2

0.1

0 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 DC, 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 0.1 M 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. Figure 9 displays the images of W/0 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).