Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/40—Colouring or decolouring of foods
  • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
  • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/40—Colouring or decolouring of foods
  • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
  • A23L5/47—Addition of dyes or pigments, e.g. in combination with optical brighteners using synthetic organic dyes or pigments not covered by groups A23L5/43 - A23L5/46
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
  • C09B67/0033—Blends of pigments; Mixtured crystals; Solid solutions
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
  • C09B67/006—Preparation of organic pigments
  • C09B67/0063—Preparation of organic pigments of organic pigments with only macromolecular substances

Definitions

• COLLOIDAL PARTICLES COMPRISING HYDROPHOBIC POLYMER, CURCUMINOID, AND BLUE COLOURANT The present invention relates to colloidal particles comprising hydrophobic polymer, curcuminoid, and blue colourant, and to a method for preparation of these, and to the use of these as a green colourant.
• Green is one of most important secondary colours as far as consumer goods are concerned, because as a colour, it can be associated with being fresh and 'natural'.
• chlorophyll and copper complexes of chlorophylls and chlorophyllins are the only known natural and nature-identical approved green colourants for food applications.
• their use in food products is often limited due to their susceptibility to photodegradation and instability in the acidic pH of most food products like (green) teas and juices.
• Curcumin is a natural dietary ingredient, which is abundantly found in the popular Indian spice turmeric (a spice derived from the perennial herb Curcuma longa L.), which is a member of the ginger family (Zingiberaceae). Curcumin belongs to the curcuminoids; is brightly coloured and may be used as a food colouring; is approved as a food additive, with E-number E100 in Europe. It has been found to have antioxidant, anti-inflammatory, and anti-cancer properties. Curcumin is a hydrophobic compound, and hence is difficult to disperse in aqueous systems, like aqueous food products. As a result the presence of large particle in the product can cause sedimentation, aggregation, sandiness and chalkiness. Curcumin has been incorporated in colloidal particles, in order to be able to disperse it in aqueous compositions. It can be used as a yellow colourant.
• US 5,993,880 discloses a water-soluble, acid-stable green colouring composition comprising copper chlorophyllin.
• WO 2012/000757 A1 disclose nanoparticles comprising a hydrophobic polymer like zein and a hydrophobic phenolic compound, like curcumin.
• WO 2006/120227 discloses an ingestible ink composition comprising a zein binder, a solvent and an ingestible pigment or colourant, e.g. curcumin.
• the zein binder has to be soluble in the solvent and bind the pigment to a substrate upon drying of the ink.
• the dissolved zein may stabilise dispersions of pigment particles smaller than 0.4 micrometer. After drying the ink, particles deposit on a substrate.
• US 2009/0035440 A1 discloses particles comprising a complex of polyphenol and a protein comprising amine groups. This allows incorporation of polyphenols in products at high levels with minimum impact on product sensory properties and/or increased stability of the polyphenol in the product.
• the polyphenol is preferably present as part of a complex with the polymer.
• the complex is present in the composition in the form of particles.
• US 2002/026886 A1 discloses water dispersible compositions containing water-insoluble pigments which are useful for the coating and colouring of edible products and
• Example of such pigments are curcumin, carmine, and a carotenoid.
• the pigments may be present as bodies having a maximum size of
• the pigment can be mixed with a hydrophilic polymer, such as gelatin to coat edible products and pharmaceutical products.
• US 4,368,208 discloses a water-soluble curcumin complex suitable for use as a colouring agent in foods. This is prepared by dissolving and mixing a source of curcumin and gelatin in an aqueous acetic acid solution. The complex comprises up to about 15 percent curcumin by weight.
• WO 91/06286 discloses zein microspheres that can be used as carriers for compounds for subsequent release. These compounds can be biologically active compounds such as pharmaceuticals. Examples show particles having a diameter of 30-35 micrometers.
• WO 2009/1371 12 A1 discloses a method of preparing zein nanoparticles using a pH precipitation method, leading to controlled particle size distribution from about 100 to 400 nanometer. Nanoconjugates can be made for use in a therapeutic method. The nanoparticles may be loaded by compounds, such as 6,7-hydroxy-coumarin.
• the present invention deals with a new approach to prepare green colour by mixing food grade yellow colourant (preferably curcumin) and water-soluble blue colourant (preferably indigocarmine) in colloidal particles generated using hydrophobic polymer comprising prolamin protein (preferably zein) as a carrier.
• a water-soluble blue colourant can be combined with the hydrophobic polymer and the hydrophobic curcuminoid in a particle.
• the particles can be used to provide green colour to aqueous compositions, in particular to aqueous food compositions.
• the coloured particles are stable against acidic pH and against photo-degradation. Moreover they are small such that they disperse in an aqueous composition and provide an even coloured green aqueous composition.
• the present invention provides a composition in the form of a particle comprising a hydrophobic polymer
• the polymer comprises one or more prolamins, selected from the group consisting of zein, gliadin, hordein, secalin, and avenin; and
• the present invention provides a method for preparation of a composition according to the first aspect of the invention, comprising the steps:
• step a) adding the mixture from step a) to water under stirring;
• step b) optionally separating particles precipitated in step b) from the remaining liquid; d) optionally drying the particles to remove water and solvent.
• the present invention provides use of a composition according to the first aspect of the invention or prepared according to the second aspect of the invention as a green colourant for aqueous products, preferably aqueous food products.
• an average particle diameter is generally expressed as the d4,3 value, which is the volume weighted mean diameter.
• the volume based particle size equals the diameter of the sphere that has same the same volume as a given particle.
• the given range includes the mentioned endpoints.
• Hydrophobic in the context of the present invention indicates that the compound is practically not soluble in water (less than 0.1 milligram per milliliter water, at ambient temperature, 20-25 ⁇ ), or slightly soluble in water (0.1 to 1 milligram per milliliter water at ambient temperature, 20-25 °C).
• Curcumin (diferuloylmethane) together with desmethoxycurcumin, and bis- desmethoxycurcumin forms the curcuminoid component obtained from the traditional Indian spice turmeric (Curcuma longa).
• the curcuminoids are phenolic compounds and are responsible for the yellow colour of turmeric.
• Curcumin can exist in at least two tautomeric forms, keto and enol. The enol form is energetically more stable in the solid phase and in solution.
• Molecular formula of curcumin C 2 i H 2 o0 6 ; molar mass 368.38 g/mol; bright yellow-orange powder; melting point 183°C.
• Prolamins are a group of plant storage proteins having a high proline content and are found in the seeds of cereal grains. Examples of these grains are wheat (protein gliadin), barley (protein hordein), rye (protein secalin), corn (protein zein) and as a minor protein, avenin in oats.
• the prolamins are characterised by a high glutamine and proline content and are generally soluble only in strong alcohol solutions. Zein is the alcohol-soluble protein of corn and is classified as a prolamin. Biologically, zein is a mixture of proteins varying in molecular size and solubility.
• alpha ( ⁇ ), beta ( ⁇ ), gamma ( ⁇ ), and delta ( ⁇ ) zein alpha ( ⁇ ), beta ( ⁇ ), gamma ( ⁇ ), and delta ( ⁇ ) zein.
• Alpha-zein is the most abundant, accounting for about 70% of the total. The next most abundant zein is gamma-zein, contributing to about 20% of the total.
• Gluten is a storage protein from wheat and comprises two major protein groups, namely the gliadins (molecular weight 30,000-80,000) and glutenin polymers (molecular weight higher than 100,000). It is classified as prolamin due to the presence of aqueous alcohol soluble gliadin groups.
• Gliadin is a glycoprotein present in wheat and several other cereals within the grass genus Triticum. Gliadins are prolamins and are separated on the basis of electrophoretic mobility and isoelectric focusing. Together with glutenin it forms an important component of wheat gluten. Hordein is a major storage protein from barley. Its a glycoprotein also classified as prolamin based on its solubility characteristics. Secalin, a storage protein found in rye, with high glutamine and proline content and low lysine content is also classified as prolamin. Water-soluble blue colourant
• Indigocarmine is a water-soluble blue colourant that is approved as a food additive in Europe and in the USA, and has E-number E132 in Europe. It is also known as indigotine.
• Indigocarmine is also known as a pH indicator, being blue below pH 1 1 .4, and being yellow above pH 13.0.
• Brilliant blue is another water-soluble blue colourant that is approved as a food additive in
• the present invention provides a composition in the form of a particle comprising a hydrophobic polymer
• the polymer comprises one or more prolamins, selected from the group consisting of zein, gliadin, hordein, secalin, and avenin; and
• the particles have a volume weighted mean diameter of between 10 and 1000 nanometer.
• the hydrophobic polymer comprises zein.
• the curcuminoid comprises curcumin.
• the particles comprise one or more compounds selected from the group of the curcuminoids at a concentration ranging from 0.5 to 25% by weight of the composition, preferably ranging from 1 to 20% by weight. More preferred the particles comprise one or more compounds selected from the group of the curcuminoids at a concentration ranging from 2 to 20% by weight, more preferably from 3 to 18% by weight, more preferred from 4 to 16% by weight. If the ratio of curcuminoid becomes too high, the particles may become too big and be prone to sedimentation.
• the water-soluble colourant comprises indigocarmine.
• the water- soluble blue colourant comprises brilliant blue.
• the particles comprise a water- soluble blue colourant at a concentration ranging from 0.5 to 25% by weight of the composition, preferably ranging from 1 to 20% by weight.
• particles comprise a water-soluble blue colourant at a concentration ranging from 1 to 10% by weight.
• the majority of the weight of the particles consists of the hydrophobic polymer.
• the concentration of curcuminoids and of water-soluble blue colourant By adjusting the concentration of curcuminoids and of water-soluble blue colourant, the shade of green can be varied.
• the particles have a volume weighted mean diameter ranging from 20 to 800 nanometer, more preferably from 50 to 500 nanometer, more preferably from 60 to 300 nanometer. Most preferably the volume weighted mean diameter of the particles ranges from 80 to 300 nanometer. For some purposes a small particle size may be required, for some other application a somewhat larger particle size may be better.
• the morphology of the particles according to the present invention is preferably substantially convex and compact. More preferably their morphology is ellipsoidal, even more preferably it is spheroidal, and still more preferably it is substantially spherical.
• An ellipsoidal particle according to the invention preferably has an aspect ratio of smaller than 10 to 1 .
• a particle is considered substantially spherical if its largest and its smallest cross- sectional diameter differ by less than 25%. Preferably its largest and its smallest cross- sectional diameter differ by less than 20%, preferably less than 10%.
• the particles according to the invention are generally well-dispersible in water: Preferably, at least 75% by weight, more preferably at least 85% by weight and still more preferably at least 95% by weight of the colloidal particles of the invention is dispersed within 5 minutes upon dissolution in a dissolution medium consisting of deionised water with 0.05% of polysorbate 80, using a USP 2 dissolution test.
• the particles according to the invention generally show good pH stability at acidic pH, preferably at a pH ranging from 3 to 5, which is a common pH for many food beverages.
• the particles according to the invention generally show good photostability.
• at least 40% by weight, more preferably at least 50% by weight and even more preferably at least 60% by weight of the curcuminoid as well as indigocarmine comprised in the particles according to the invention is not degraded within 70 minutes, upon irradiation of a colloidal suspension of the particles at a concentration of 0.05%wt curcuminoid and indigocarmine respectively with UV light at a wavelength of 302 nm using a Bio-Rad Gel Doc 1000 mini-transilluminator.
• the morphology of the particles according to the present invention may positively contribute to the enhanced water-dispersibility, pH stability, and photostability.
• morphology is understood as including aspects of the particle shape and size, as well as the internal distribution and mutual interactions of the prolamins and curcuminoid and blue colourant within the particles.
• Such morphological features may for instance be controlled by the method by which the particles are prepared. Therefore, the particles according to the invention are preferably obtainable by the method according to the second aspect of the invention.
• hydrophobic curcuminoids are formulated such that they can be dispersed in aqueous products, with enhanced water dispersibility and stability against aggregation and sedimentation.
• hydrophobic curcuminoid can be combined with a hydrophilic blue colourant, which together can be used to provide green colour to aqueous products.
• compositions comprising the particles
• the particles according to the invention will be dispersed in a supporting medium.
• the supporting medium will make up the bulk of the composition and determine, to a large extent, its sensory and physical characteristics.
• the supporting medium may be any suitable substance and will depend to a large extent on the intended end use of the composition.
• the supporting medium will be a liquid, dispersion (single or duplex emulsion, foam or suspension), gel, solid, or may be a mixture thereof.
• the supporting medium may be aqueous (preferably comprising at least 50% water by weight of the supporting medium) or non-aqueous.
• the properties of the particles, especially their size and surface properties, and those of the supporting medium, especially its viscosity and polarity, are selected such that the particles form a stable colloidal dispersion in the supporting medium.
• the dispersion is preferably stable such that no appreciable sedimentation of the particles occurs over a period of at least 7 days at a storage temperature of 20°C, more preferably over a period of at least 1 month and most preferably at least 6 months.
• the present invention provides a composition comprising a composition in the form of a particle according to the first aspect of the invention, wherein the composition is in liquid, solid, or semi-solid form.
• the composition may be in the form of an emulsion, a foam, and/or gel based.
• the particles of the present invention will generally be incorporated as a dry powder.
• the particles In a liquid composition, the particles will generally be dispersed in a liquid.
• a semi-solid or semi-liquid composition like gels, jellies or paste, particles will preferably be incorporated as aqueous dispersion.
• Non-limiting examples of solid compositions are a nutritional bar and an instant powder for preparing a beverage.
• Non-limiting examples of liquid compositions are beverages.
• Non-limiting examples of semi-solid or semi-liquid compositions are spreads and yoghurt.
• a composition comprising the particles according to the invention is edible.
• Another aspect of the present invention is a food product comprising a composition according to the first aspect of the invention, as this allows for convenient and enjoyable consumption of the compounds.
• Preferred food products are aqueous food products. These food products contain a continuous aqueous phase. It is especially preferred that the food composition is a beverage.
• the food composition is a beverage, it is preferably a coffee-based beverage, a tea-based beverage and/or a cocoa-based beverage.
• the beverage is tea-based.
• the pH of the beverage may be from 2.5 to 8, preferably 3 to 6, more preferably from 3.5 to 5.
• the preferred beverages can be coloured due to the formation of a colloidal dispersion of the composition of the invention in the beverage.
• the concentration of the particles By variation of the concentration of the particles the colour of the beverage can be modified.
• the concentration of the curcuminoid and of the water-soluble blue colourant By variation of the concentration of the curcuminoid and of the water-soluble blue colourant, the shade of green can be controlled.
• the concentration of the composition in the form of particles in a food product preferably varies from to 0.01 to 2% by weight of the food product, preferably from 0.01 to 1 % by weight, preferably from 0.05% to 0.8% by weight, preferably from 0.1 % to 0.6%.
• a hydrophobic colourant curcuminoid
• a hydrophilic blue colourant can be combined to give an even green colour, especially to an aqueous food product.
• the food composition may also be, for example, a margarine, low fat spread,
• confectionery product such as chocolate or cereal bar
• ice cream such as chocolate or cereal bar
• dressing such as chocolate or cereal bar
• mayonnaise sauce
• bakery product such as cereal bar
• shortening or cheese such as chocolate or cereal bar
• the food may be dried and contain less than 40% water by weight of the composition, preferably less than 25%, more preferably from 1 to 15%.
• the food may be substantially aqueous and contain at least 40% water by weight of the composition, preferably at least 50%, more preferably from 65 to 99.9%.
• the food preferably comprises nutrients including carbohydrate (including sugars and/or starches), protein, fat, vitamins, minerals, phytonutrients (including terpenes, organosulfides or a mixture thereof) or mixtures thereof.
• the food may be low calorie (e.g. have an energy content of less than 100 kCal per 100 g of the composition) or may have a high calorie content (e.g.
• the food may also contain salt, flavours, colours, preservatives, antioxidants, non-nutritive sweetener or a mixture thereof.
• the composition according to the invention may be used in pharmaceutical products or in dietary supplements.
• the form of the composition may, among others, be a tablet, pill, lozenge, paste, lotion, gel, cream, liquid (including emulsion and/or suspension), spray (including aerosol spray), foam or powder.
• the pharmaceutical of the present invention may be suitable for any form of administration, preferably oral administration.
• the pharmaceutical composition generally will comprise a
• composition according to the invention may act as a diluent, dispersant or carrier for the particles according to the invention.
• vehicle may be aqueous or anhydrous.
• the present invention provides a method for preparation of a composition according to the first aspect of the invention, comprising the steps:
• step a) adding the mixture from step a) to water under stirring;
• step b) optionally separating particles precipitated in step b) from the remaining liquid; d) optionally drying the particles to remove water and solvent.
• the mixture of water and organic solvent comprises ethanol, preferably at a volumetric ratio of ethanol to water ranging from 60:40 (v/v) to 90:10 (v/v).
• the temperature in step a) ranges from 0 to ⁇ ' ⁇ , preferably 10 to 60 ' ⁇ .
• the hydrophobic polymer comprises zein.
• the curcuminoid comprises curcumin.
• the water-soluble colourant comprises indigocarmine.
• the stirring in step b) can be done with any commonly known stirrer.
• the type of stirrer and stirring speed are not critical, stirring is required to prevent localised precipitation of hydrophobic polymer and/or curcuminoid.
• the particles may be separated from the liquid in step c). This may be done by filtration, decanting, centrifugation, evaporation of the liquid phase, or any other commonly known method.
• the particles may be dried using any commonly known method. This way water and solvent may be removed.
• other stabilisers such as surfactants, emulsifiers, surface active (bio)polymers, and proteins (other than prolamins) can be included in the water phase or if soluble in the organic solvent containing phase.
• An advantage of the method for the preparation of a composition in the form of a particle according to the present invention is that it is especially suitable for controlling the morphology of the particles.
• the density and the shape of the particles may be controlled, leading for example to the formation of particles the morphology of which is substantially convex and compact.
• the method may even yield ellipsoidal, spheroidal, or substantially spherical particles.
• the method is suitable to incorporate a water-soluble colourant into the particle, together with the hydrophobic compounds.
• the dried particles can be used further in formulating compositions in which the particles are incorporated, preferably food products.
• the dried particles do not need to be further formulated into a product, they could also be packed as such before being marketed.
• compositions containing the particles according to the invention may be produced by any normal method for producing the food.
• the particles can be added in liquid and semi-liquid formats in form of powder or (concentrated) dispersion.
• dried particles according to the invention can be mixed in or spray on to powder
• the present invention provides use of a composition according to the first aspect of the invention or prepared according to the second aspect of the invention as a green colourant for aqueous products, preferably aqueous food products.
• a composition according to the first aspect of the invention or prepared according to the second aspect of the invention as a green colourant for aqueous products, preferably aqueous food products.
• One of the advantages of the present invention is that a combination of a hydrophobic colourant (curcuminoid) and a hydrophilic blue colourant can be combined to give an even green colour, especially to an aqueous food product.
• Figure 1 Representative transmission electron microscopy images of A) zein:curcumin
• Figure 2 Particle size distribution curves (in nanometer) for dispersions prepared from zein:curcumin (20:1 wt/wt); zein:curcumin:indigocarmine (20:1 :0.25; 20:1 :0.5; 20:1 :1 ;
• Figure 3 Colloidal dispersions containing (from left to right) zein:curcumin (20:1 wt/wt); zein:curcumin:indigocarmine (20:1 :0.25; 20:1 :0.5; 20:1 :1 ; 20:0.5:1 and 20:0.25:1 wt/wt/wt) and zein:indigocarmine (20:1 wt/wt).
• the total concentration of zein in all cases was 1 % by weight of the dispersion.
• Figure 4 Spectra of composite colloidal dispersion prepared at varying ratios of curcumin to indigocarmine at constant indigocarmine concentration (A) and constant curcumin concentration (B) and plots of absorbance at 510 nm (corresponding to the mid wavelength for spectra of green colour) versus changing proportions of
• indigocarmine against UV irradiation (302 nm) time for molecular solutions of curcumin in ethanol, indigocarmine aqueous solution and colloidal dispersions prepared using zein:curcumin (20:1 wt/wt) and zein:indigocarmine (20:1 wt/wt).
• curve 3 indigocarmine (molecular solution)
• curve 4 zein-indigocarmine particle
• Figure 6 X-ray diffraction graphs (peak intensity as function of the diffraction angle (2 ⁇ (theta)) for curcumin, indigocarmine and colloidal particles formed using
• curve 3 zein-curcumin-indigocarmine (20:1 :1 wt/wt/wt) particle
• Curcumin (95% purity as per supplier's claim), supplier Sanjivani Phytopharma Pvt. Ltd., India;
• Tween 80 Polyethylene glycol sorbitan monooleate (polysorbate 80), emulsifier; supplier Sigma Aldrich Inc.;
• Particle size and surface potential The volume weighted mean particle size and zeta potential of dispersions were determined by dynamic light scattering (DLS) using a Zeta sizer Nano (Malvern Instruments Ltd, UK) after appropriate dilution. All measurements were carried out at 25 ⁇ and the results reported are average of three readings.
• Diameters are expressed as volume weighted mean particle diameter, unless indicated otherwise. Also the zeta-potential (expressed in millivolt) is determined using this apparatus.
• Morphology evaluation The shape of synthesized particles was analyzed by taking TEM photographs using Technai transmission electron microscope (FEI Company, The Netherlands). The dispersion was diluted in demineralised water and one drop of the diluted dispersion was placed on a 200-mesh carbon coated copper grid. The photographs were taken at various magnifications and 100 kV voltage.
• Spectrophotometry analysis Absorbance was measured at wavelength of 426 nm (for curcumin) and 620 nm (for indigocarmine). The value of absorbance was used to calculate the concentration of curcumin and indigocarmine via the standard calibration curve which was plotted beforehand.
• X-ray diffraction Diffraction lines of the samples were obtained with a Bruker AXS (Karlsruhe, Germany) D8 Discover diffractometer. The instrument was equipped with a copper anode that produces Cu Ka X-rays with an accelerating voltage 40kV and a tube current 40mA. The diffractogram was collected with a monocap collimator of 0.3 mm during 300 sec. An angular range of thetal at 4.5° and theta2 at 10° 25° and 40° were use with a theta rocking of 1 ° and XY amplitude of 2 mm resulted in a 2q between 4° and 55° after merging the separate recordings. Rocking and amplitude oscillation were used to obtain an average diffractogram of the sample and minimize a preferred orientation of crystals.
• Stocks of zein:curcumin; zein:indigocarmine and zein:curcumin:indigocarmine mixtures were prepared by dissolving accurately weighed quantities of components in 100 ml_ of ethanohwater (80:20 wt/wt). Colloidal dispersions were prepared by adding these stock solutions to 300 ml_ water with 0.02 wt% Tween 80 under continuous stirring (1000 rpm) using magnetic stirrer (Model EM3300T, Labotech Inc, Germany). To keep the final pH of dispersion around the acidic range (pH 3-4), the pH of water used to prepare stock solutions and the dilution was adjusted to 3 using 0.1 M HCI. Samples for x-ray diffraction were obtained by subjecting the colloidal dispersion to freeze drying at -85 °C using a vacuum of 0.040 mbar (Labconco Freezone 6 plus, Labconco Corporation, USA).
• Colloidal particles were prepared with various ratios of zein to curcumin to indigocarmine, in this way. Particles were prepared with the following weight ratios:
• zein:curcumin:indigocarmine 20:1 :0.25; 20:1 :0.5; 20:1 :1 ; 20:0.5:1 and 20:0.25:1 wt/wt/wt;
• Figure 1 gives representative transmission electron microscopy images of zein:curcumin (20:1 wt/wt) colloidal particles (A); zein:indigocarmine (20:1 wt/wt) colloidal particles (B); and zein:curcumin:indigocarmine (20:1 :1 wt/wt/wt) colloidal particles.
• the dark spheres are the nanoparticles. These images show that the colloidal particles appeared to be almost perfect spheres.
• Table 1 gives some typical parameters of the particles that were obtained.
• Table 1 Average particle size and absolute value of the zeta-potential values of colloidal dispersions of particles.
• the average particle size of these particles was in the range of 75-300 nm (particle size distribution curves are presented as Figure 2) and the absolute value of the zeta potential was between 10 and 30 mV.
• Figure 3 shows the coloured pictures of the dispersions of the particles, showing that the zein:curcumin 20:1 particles were yellow, and the zein:indigocarmine 20:1 particles were blue. In all these cases the concentration of zein is 1 % by weight of the dispersion.
• the zein:curcumin:indigocarmine had various shades of green, with more indigocarmine leading to darker green colour.
• the colour of the dispersions has been determined by colour analysis of the digital images that were made and that are shown in Figure 3. Digital pictures were made using a Fuji FinePix A345 camera with 4.1 megapixels; automatic camera settings were used: The colour analysis of the pictures has been done using the website
• RGB red, green and blue
• Figure 4 shows spectra of the colloidal dispersions prepared at varying ratios of curcumin to indigocarmine at constant indigocarmine concentration (A) and constant curcumin concentration (B). These graphs show the absorbance at a range of wavelengths, in order to show the colour difference between the various colloidal particles. Additionally Figure 4 shows plots of absorbance at 510 nm (corresponding to the mid wavelength for spectra of green colour) versus changing weight proportions of indigocarmine:curcumin (C) and curcumin:indigocarmine (D) (these particles also contain zein). These indicate the change in colour as function of the ratio between indigocarmine and curcumin.
• Particles were prepared of zein-curcumin (20:1 wt/wt) and zein-indigocarmine (20:1 wt/wt), following the procedure in example 1 .
• Curcumin and indigocarmine may both be photolabile and their colour solutions are known to fade under light illumination.
• Individual molecular solutions of curcumin and indigocarmine were irradiated with UV light (302 nm) and the decrease in their concentration (which is an indication of photo-degradation) was noted over time (70 minutes).
• the colloidal dispersion of particles with concentration of individual colourant equivalent to the corresponding molecular solutions was irradiated in similar fashion followed by analyzing the concentration of both curcumin and
• Figure 5 shows a comparison graph of percent of unchanged curcumin and indigocarmine against UV irradiation time for molecular solutions of curcumin, indigocarmine and colloidal dispersions prepared using zein:curcumin and zein:indigocarmine.
• molecular solutions of both curcumin and indigocarmine degraded quite rapidly under UV light with concentration dropping to 35% (curve 1 ) and 55% (curve 3) respectively, of their initial concentration at the end of 70 minutes.
• Crystalline compounds are converted to amorphous forms when encapsulated in colloidal particles.
• the X-ray diffractogram ( Figure 6), showed sharp peaks at diffraction angles (2 theta) of 8.9, 12.1 , 14.4, 17.1 , 18.0, 21 .0, 23.2, 24.3, 25.3, 27.1 and 28.8 for pure curcumin powder (curve 1 ) and at diffraction angles (2 theta) of 15.5, 17.7, 23.2, 24.8, 27.0, 30.8 and 33.6 for pure indigocarmine powder (curve 2), suggesting highly crystalline nature of these pigments.
• the composite particles showed absence of all the peaks (curve 3), suggesting formation of amorphous curcumin and indigocarmine due to the suppression of their crystallisation in the nanoscale confinement and possibly due to the formation of an amorphous complex with protein within the particle matrix.
• nanoparticle with the zein and the curcumin although the indigocarmine is a hydrophilic compound, and the zein and curcumin are hydrophobic compounds.

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