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    • 专利摘要:
    The present invention relates to a particle stabilized emulsion or foam comprising at least two phases and solid particles, said solid particles being starch granules and said starch granules, or a portion thereof, being situated in the present invention. interface between the two phases providing the particle stabilized emulsion or foam. The invention further relates to the use of said particle stabilized emulsion or foam for encapsulating substances chosen from biopharmaceuticals, proteins, probiotics, living cells, enzymes and antibodies, sensitive food ingredients, vitamins, and lipids in food products. , cosmetic products, skin creams, and pharmaceutical formulations.
    公开号:BR112013014574B1
    申请号:R112013014574-9
    申请日:2011-12-15
    公开日:2019-10-15
    发明作者:Petr Dejmek；Anna Timgren；Malin Sjöö；Marilyn Rayner
    申请人:Speximo Ab；
    IPC主号:
    专利说明:

    EMULSION OR FOAM STABILIZED BY PARTICLE, USE
    OF A EMULSION STABILIZED BY PARTICLE AND FORMULATION
    Technical Field of the Invention
    The present invention relates to a particle-stabilized emulsion or foam which comprises at least two phases and solid particles, a dry particle-stabilized emulsion or foam which comprises at least two solid phases and particles and the use of said emulsion or foam stabilized by particle in different applications.
    Background Technique
    Emulsions are a mixture of two or more immiscible phases where one is dispersed in the other as small droplets. Emulsions can be drops of oil in a continuous phase of water or drops of water in a continuous phase of oil, in the case of foams, one of the phases consists of a gaseous phase like air, but in both cases, the droplets or bubbles need to be stabilized to prevent them from coalescing again. The surfactants adsorbed by the interface of the two phases decrease the interfacial tension and can increase steric impedance or electrostatic repulsion, which increases the stability of the emulsion. Proteins and surfactants are usually used as emulsifiers in food emulsions. However, polysaccharides are also used to stabilize emulsions, especially gum arabic and modified celluloses and starches. When used as an emulsion stabilizer, starch is usually gelatinized and / or dissolved. Food emulsions are, in general, stabilized by surfactants, proteins and hydrocolloids; however, recently, the use of particles to stabilize • emulsions has attracted substantial research interest due to their distinct characteristics and potential technological applications.
    Particulate-stabilized oil droplets
    2/79 dispersed, known as Pickering emulsions, were originally observed independently by Ramsden (1903) and Pickering (1907). Emulsions stabilized by solid particles are usually more stable in relation to Ostwald coalescence and aging (Ostwald ripening) compared to systems stabilized by surfactants. They exhibit extreme long-term stability, even with large droplet sizes, and without the addition of surfactants. The particles are often inorganic particles such as silica, titanium oxide or clays, latex, fat crystals, proteins and aggregated hydrocolloids. The particle size used for Pickering emulsions varies from nano to micron and the droplet size decreases with decreased particle size, but only as long as other properties, such as wettability, shape, surface, etc., are the same.
    There is a recognized technological need for edible delivery systems that encapsulate, protect and release bioactive ingredients in, for example, food and pharmaceutical products and other applications. It is desirable to avoid the use of surfactants in emulsions due to effects such as air trapping, foaming, irritation and biological interactions. There is also a need for topical systems as well as other technical products, where improved stabilized foams or emulsions are advantageous.
    starch is abundant, relatively economical, and is obtained from botanical sources. There is a great deal of natural variation related to size, shape and composition. Starch has an intrinsic nutritional value and is a non-allergenic source in contrast to other common food emulsifiers that are derived from egg or soy.
    Depending on the botanical origin, the size and shape distribution of starch granules can differ substantially, as well as the ratio between the two starch polymers,
    3/79 amylopectin and amylose. Starch granules can exist in a variety of forms: smooth, rough or sharp surface and the shape can be spherical, ellipsoidal, flat as discs, polygonal or as rods.
    Document No. W02010 / 01 12216 discloses flour made from amaranth or quinoa and its use in food products. Said patent specification refers to a flour.
    WO96 / 04316 discloses thermally inhibited starch and pre-gelatinized granular flour. Said patent specification refers to a flour.
    The document No 2 WO96 / 22073 pretreatments thermal starch discloses and defines this pre-heat treatment such as thermal inhibition, which is characterized mainly by its effect on the starch viscosity behavior when starch is subjected to a heating pattern above sequence gelatinization and cooling temperature, the Brabender test. While it reveals the use of inhibited starch, and even hydrophobically modified inhibited starch in emulsions, the examples in the description describe that the emulsion must be produced at 80 ° C. The heat pretreatment can damage the starch so that it does not gelatinize. The use of gelatinized starch is the generally recognized manual for the use of hydrophobized starch for emulsification. Heat treatments of starch granules such as those described in WO96 / 22073, and the hydrophobization of starch granules described in the prior art do not form part of the present invention, as will become apparent below. US Patent 4,587,131 discloses the use of granules of native starch, which are not used in accordance with the present invention in view of the fact that native starch does not provide the necessary desired effects.
    There is still a need for information systems
    4/79 delivers edible foods that encapsulate, protect and release bioactive ingredients in, for example, food and pharmaceutical products and other applications. There is also a need for topical formulations with high stability without the use of surfactants that use particles that have a low allergy content and are biodegradable, for example, in cosmetic products, pharmaceuticals for topical delivery and other such applications. The present invention aims to meet the needs mentioned above.
    Summary of the Invention
    The present invention relates, in one aspect, to a particle-stabilized emulsion or foam comprising at least two phases and solid particles, said solid particles being starch granules and said starch granules or a portion thereof they are located at the interface between the two phases that generate the emulsion or foam stabilized by particle. In figure 0-1, it is shown that an oil starch (red in color) and an aqueous phase can form an emulsion after a high speed shear. It is the starch granules at the interface of the two phases that cause the stabilizing effect and not the starch molecules or a primary roughage effect of starch granules in the continuous phase as was the case in the prior art techniques. A schematic illustration explaining the difference between a particle-stabilized emulsion, a starch molecule-stabilized emulsion and a surfactant-stabilized emulsion is provided in figure 0-2. An advantage of the present invention is the flexibility of the system. The added starch granules could be present in the interface at a small or large concentration as long as there is a stabilizing effect. In this way, the interface is stabilized by the added starch granules and not by any other component that may be present in the emulsion.
    5/79 or foam. Figure 0-3 is a micrograph showing how intact starch granules effectively stabilize oil droplets that create Pickering-type emulsions by covering the surface of emulsion droplets. Their hydrophobic capacity allows them to be adsorbed on the oil and water interface, which prevents recoalescence and, therefore, droplet stability. Starch is one of the most prevalent food ingredients, having shown innovative and useful emulsifying properties.
    The present invention relates, in another aspect, to an emulsion or foam stabilized by dry particle, an emulsion or foam stabilized by particle according to the present invention was subjected to the removal of water as through drying, for example, freeze drying, spray drying and / or vacuum drying.
    The present invention relates, in another aspect, to a particle-stabilized emulsion or foam, said particle-stabilized emulsion having undergone a heat treatment in order to intensify or adjust barrier properties and / or rheological properties of the emulsion. stabilized by particle. By carrying out this heat treatment, the shelf life can be extended or adjusted and, in some applications, controlled release or targeted delivery is permitted.
    The present invention relates, in yet another aspect, to the use of a particle-stabilized emulsion to replace fat in food products.
    The present invention relates, in yet another aspect, to the use of a particle-stabilized emulsion for encapsulation of substances chosen from biopharmaceuticals, proteins, probiotics, living cells, enzymes, antibodies, sensitive food ingredients, vitamins and lipids.
    6/79
    The present invention relates, in yet another aspect, to the use of a particle-stabilized emulsion in food products, cosmetic products, skin creams, lotions, and pharmaceutical formulations such as topical formulations, capsules, suppositories, inhalation formulation, suspensions oral, oral solutions, products for intramuscular and subcutaneous injection, and consumer products such as ink.
    The present invention relates, in another aspect, to a formulation comprising a dry particle stabilized emulsion according to the present invention and a substance chosen from biopharmaceuticals, proteins, probiotics, living cells, enzymes, antibodies, food ingredients sensitive, vitamins, and lipids. The dry particle stabilized emulsion is also suitable for food products, cosmetics, skin creams, lotions, and consumer products. The formulation can be a pharmaceutical formulation. Thus, surprising results have been achieved in accordance with the present invention, that is, that hydrophobized, non-gelatinized starch granules are suitable for emulsification at temperatures below the gelatinization temperature. What has been described above is not known from the prior art.
    Description of the Figures
    Figure 0-1: are photographs of samples with 33.3% (v / v) of oil in buffer and 100 mg of starch / ml of oil, emulsification at 11,000 rpm. Left: a non-emulsified sample that includes (from top to bottom) oily phase, aqueous phase, starch; Right: emulsion with OSA modified quinoa starch produced by high shear homogenization. 1 mg of oil-soluble dye (Solvent Red 26) was added to the samples.
    Figure 0-2: in starch Pickering emulsions, they are
    7/79 starch granules are found at the oil / water interface that stabilizes the emulsion. There may be cases where the starch granules coexist with other emulsifiers or surfactants in other emulsion-based products, however, they are not responsible for droplet stabilization. For example, in a starch molecule or emulsions stabilized by surfactant, the granules could be added in the bulky continuous (aqueous) phase, but are not attached to the oil and water interface or act as stabilizing particles in Pickering emulsions. In that case, the starch granules may provide other properties to the product, but are outside the scope of that invention.
    Figure 0-3: intact starch granules effectively stabilize oil droplets that create Pickering-type emulsions covering the surface of emulsion droplets.
    Figure 0-4A: conventional surfactant stabilized emulsion (left); here, small surfactants stabilize the oil and water interface. To increase the thickness of the emulsion, viscosity modifiers are added. The particle-stabilized emulsion (right); here, starch granules stabilize the oil and water interface and are in a weak state of aggregation. This builds the microstructure providing viscoelastic behavior even at low levels of oily phase.
    Figure 0-4B: microscopic image of emulsion stabilized by granule of quinoa starch, 286 mg of starch / ml of oil (scale bar = 100 microns). The rheological and microstructure measurement indicates aggregation between droplets that form a gel-like network. Figure 0-5A shows an important physicochemical property of starch, namely, its ability to gelatinize in the presence of water and heat. First, an emulsion consisting of oil drops covered with starch is then formed through the
    8/79 careful addition of heat, partial gelatinization of the granules is induced to form a cohesive starch layer anchored at the oil and water interface. This intensified barrier can be useful in several ways. This technique was also applied to allow oil droplets to be retained during drying, thus producing powder from oil-laden starch capsules.
    Figure 0-5B: principle of encapsulation of water-soluble substances by double emulsions (A) and oil encapsulation with other substances dispersed in (B). Heat treatment can also be applied to increase the barrier properties of the starch layer and further improve the encapsulation capacity (C and D). Using Starch Pickering emulsions, the droplets are large enough to contain the internal droplets or crystals and the starch layer is cohesive enough to maintain drop stability.
    Figure 0-6 Left: ordinary emulsion, Right: double emulsion. Dual emulsions with high stability can be prepared to protect sensitive water-soluble ingredients. Dual emulsions are attractive for protecting sensitive water-soluble ingredients from an oily phase.
    Figure 1-1: particle size distributions of quinoa starch granules (D 43 3, 45 pm) after high shear mixing in a Ystral D-79282 at 22,000 rpm for 30 seconds (solid line). The resulting quinoa-stabilized emulsion droplets (D 43 50, 6 pm), 6.65 ml continuous phase, 0.35 ml dispersed and 100 mg of 2.9% OSA starch / ml of oil after mixing high shear under the same conditions (dashed line). Microscopic image of an emulsion stabilized by starch (insert).
    Figure 1 -2: droplet size (D 43 ) and volume of
    9/79 relative occlusion as a function of the amount of starch added per ml of oil measured after 1 and 7 days. The concentrations marked up to j correspond to emulsion images in figure 1 -3. The vertical dashed line indicates the theoretical droplet size cutout for neutral buoyancy droplets.
    Figure 1—3: images of cream / emulsion after 1 day (top) and after 7 days (bottom), starch to the left of zero and 5% oil, oil to the right of zero and 1,250 mg of starch. The letters correspond to marked concentrations shown in the plot in figure 1-2.
    Figure 2-1: drop size as a function of the amount of starch added for 4 varieties of starch: quinoa, rice, more, and waxy barley, all of which have been modified by OSA and in a NaCl phosphate buffer at 0, 2 Μ. The amount of added starch corresponds to 1.1, 2.2, and 3.9% by volume of the total system.
    Figure 2-2. specific surface area measured from starch-stabilized emulsions versus estimated surface area that could be stabilized by a given starch granule size and concentration. Solid represents the case in which the measurement is the same as expected.
    Figure 3-1: emulsions were produced using different processing techniques, the purpose being to demonstrate that starch granule stabilized emulsions can be produced using a variety of methods. Images (from top to bottom) of emulsions produced by: level 2 sorvall 300 s, level 8 sorvall 300 s, laboratory scale high pressure homogenizer, circulating using a peristaltic pump. The images on the left are micrographs of emulsions (100 x magnification). The images on the right are characteristic of general emulsion.
    Figure 4-1: emulsions produced with 214 mg of
    10/79 starch / ml of oil with varying amounts of oil volume fraction. Effect of storage time, and oil concentration in visual appearance and (left) and emulsion index (right).
    Figure 4-2: elastic modulus as a function of complex shear stress in four oil concentrations.
    Figure 5-1: in vitro skin penetration of methyl salicylate through pig skin at 32 ° C, from 55% oily starch pickering emulsions; paraffin oil (circles), Miglyol (squares) and chestnut oil (triangles).
    Figure 6-1: elastic modulus (G ', Pa) as a complex force function for starch-stabilized emulsions in various proportions between starch and oil that have 40% of total dispersed phases (oil and starch).
    Figure 7-1: micrographs of an emulsion not treated with 7% Mygliol oil stabilized with 214 mg of starch per ml of oil (top left), corresponding emulsion frozen with a jet freezer and thawed (top right), corresponding emulsion frozen with thawed liquid nitrogen (bottom left), and corresponding heat-treated emulsion 1 minute at 70 ° C (bottom right).
    Figure 7-2: micrographs of a double emulsion before (left) and after (right) freezing and thawing. Liquid nitrogen was used for freezing.
    Figure 7-3: particle size distribution of thermo-treated and untreated double emulsions before and after thawing freeze.
    Figure 8-1: SEM micrograph of freeze-dried emulsion drops containing gelatinized starch layer and
    11/79 Miglyol oil. The emulsions were thermo-treated before freeze drying. The intact drops and partially collapsed drops that left empty starch pockets were obtained.
    Figure 8-2: SEM micrograph of freeze-dried emulsion drops containing chestnut butter. The emulsions were not thermo-treated before freeze drying. The intact aggregated and non-aggregated drops were obtained and the images show the presence of free oil.
    Figure 8-3: SEM micrograph of freeze-dried emulsion drops containing chestnut butter and gelatinized starch layer. The emulsions were thermo-treated before freeze drying. The aggregated and non-aggregated drops were obtained.
    Figure 8-4: SEM micrograph of spray-dried emulsion drops containing chestnut butter and starch granules. The oil-laden starch-covered spheres remain intact after spray drying.
    Figure 8-5: particle size distribution (D43) of emulsions before (left) and after (center) freeze drying, and of a double freeze-dried emulsion (right). The dried emulsions were rehydrated before measurement. The larger particle size of heated emulsions after drying was caused by aggregation.
    Figure 9-1: micrographs with polarized light from unheated emulsion drops (upper figure) and heated (lower figure). The crystalline parts of starch granules are birefringent as seen by the bright color on the entire surface at (upper figure) and close to the oil surface (lower figure). The diffuse area outside the drops (bottom figure) shows partial gelatinized starch.
    Figure 9-2: lipase activity as a function of heat treatment temperature after emulsification.
    12/79
    Figure 10-1: micrographs of an emulsion recently prepared with 10% fish oil stabilized with 500 mg of starch per ml of oil (left), which corresponds to after 1 week of storage (center), or thermo-treated and stored for 1 week (right).
    Figure 11-1: foam stabilized by a starch granule with a rigid structure.
    DETAILED DESCRIPTION OF PREFERENTIAL MODALITIES
    In an embodiment of the invention, the starch granules used in the particle-stabilized emulsion or foam are native or have undergone physical modification and / or chemical modification to increase the hydrophobic capacity of the starch granules. Starch can be chemically modified by treatment with different alkenyl succinyl anhydrides, for example, octenyl succinyl anhydride (OSA), which is approved for food applications in an added amount of up to 3% based on the dry weight of starch. Propenyl succinyl anhydride can also be used. The hydrophobic octenyl group and the carboxyl or sodium carboxylate group increased the starch's ability to stabilize emulsions. It is also possible to make the starch granules more hydrophobic by grafting with other chemicals with a hydrophobic side chain, for example, by esterification with dicarboxylic acids. The modified starch particles have a fairly uniform surface, at least in relation to the hydrophobic capacity, so the droplets covered with starch granules have surface properties similar to those of the individual starch granules. When the granule surface properties allow strong adsorption at the oil and water interface (a contact angle not so far from 90 °), the particles, when dispersed in the aqueous phase, are also in a state of poor aggregation. In this case, a barrier to
    13/79 steric particle base consists of more than a single, densely packed layer of starch granules on the droplet surface, but also extends as a disorderly network / layer of granules between droplets (some of which can be seen in figure 0 -4B), which have the entire aggregate structure maintained by attractive forces between particles, creating asso, the weak gel-like structure observed.
    In the present context, a particle-stabilized emulsion means an emulsion that has at least two phases, with the starch granules or a portion thereof being arranged at the interface between the at least two phases, for example, at the interface between an oil phase and a water-based phase, and thereby stabilizing the emulsion.
    In an embodiment of the invention, the emulsion starch granules or particle-stabilized foam are more hydrophobic by physical modification, for example, by dry heating or by other means, such as a change in pH, high pressure treatment, irradiation, or enzymes . Dry heating causes the surface proteins of the starch granule to change in nature from hydrophilic to hydrophobic. An advantage of thermal modification is that no specific markings are required when used in food applications. Furthermore, the hydrophobic change occurs explicitly on the granule surface.
    In an embodiment of the invention, the emulsion starch granules or particulate stabilized foam preferably have a small granular size in the range of approximately 0.2 to 20 microns, preferably 0.2 to 8 microns, more preferably , 0.2 to 4 microns, more preferably 0.2 to 1 micron.
    In one embodiment of the invention, the emulsion starch granules or particulate stabilized foam are obtained at
    14/79 from any botanical source. Starch granules have been shown to stabilize oil-in-water emulsions. In contrast to the particles commonly used for Pickering emulsions, starch (including hydrophobically modified starch) is an accepted food ingredient. Starch granules are abundant, relatively inexpensive and are obtained from many botanical sources. There is a great deal of natural variation related to size, shape and composition. Starch has intrinsic nutritional value and is a non-allergenic source in contrast to other common food emulsifiers that are derived from egg or soy. The emulsion starch granules or particulate stabilized foam are, for example, obtained from quinoa, rice, plus, amaranth, barley, unripened sweet corn, rye, triticale, wheat, buckwheat, tifa, filipendula, durian, teff , oats, parsnips, small millet, wild rice, birdseed, cow's leg, yam, and taro including high and waxy varieties of the above.
    At least two phases of the particle-stabilized emulsion or foam are chosen from the oil-based phase / water-based phase, and the gas phase / water-based phase. In an embodiment of the invention the emulsion is an oil-in-water emulsion or a water-in-oil emulsion, or a foam.
    In one embodiment of the invention, the amount of starch granules added to the particle-stabilized emulsion or foam corresponds to approximately 0.005 to 70% by volume of the total emulsion. The amount of added starch granules is preferably determined by the droplet coverage and the coverage should be greater than 10%.
    According to the present invention, the possibility of preparing emulsions of a given droplet size depends on
    15/79 critically of the availability of a sufficient quantity of starch granules to stabilize the resulting surface. The sufficient amount can be described in terms of the area that the starch granules can cover when spread in a single layer in relation to the surface area of the emulsion at a given packing density. Specifically, if the emulsion contains a volume of oil (VO) and contains droplets of cover diameter (D 32 ) of D o , then the total interfacial surface of the oil droplets (So) is given by;
    To stabilize this S o area interface, a S s starch layer that occupies the same area is required.
    The area occupied by a starch granule is assumed to be spherical in diameter D s , and attached to the oil and water interface at a 90 ° contact angle with a fraction of interfacial packaging φ.
    number of starch granules (assuming they are D s in diameter) for a given starch weight, W s , and starch density, p 5 .
    The total area they occupy S s = n s a s , or equal to:
    61V S = --— * AÍ-4D,
    The fraction of interfacial packaging φ is the inverse of the amount of space between the particles, and reaches a theoretical limit of φ - 0.907, that is, hexagonal packaging. However, there are many cases where it is slightly higher (1,2) or even
    16/79 significantly lower (0.10) and for extremely pure systems as low as (0.002) and still depend on the system (Gautier et al. 2007, Tcholakova et al. 2008). For practice, it determined that the range would be between 0.10 and 1.2.
    Thus, to cover an area of oil Sq, an area of starch S s is required. By defining S 0 = S s and redistributing, the following is achieved:
    s
    This has units of mg / ml (or kg / m 3 ).
    Example: Topical Cream
    An emulsion with an average drop size (D32) D o 4 9 pm must be produced and granules of quinoa starch are used to stabilize this, which has an average diameter D s = 2.27 m and a solid density p s = 1.550 kg / m 3 with an interfacial packing density φ = 0.73, The amount of starch required per volume of oil is:
    «4 _ _ 4 - 0.72 1550 · 2.27F - 6
    D g ~ 49E - 6
    214 mg / ml
    In one embodiment of the invention, the particle-stabilized emulsion or foam was subjected to heat treatment in order to alter the barrier properties of the particle-stabilized emulsion. There is a need for delivery systems to encapsulate, protect and release bioactive ingredients in food and pharmaceutical products. Many of the ingredients or compounds used in such applications are lipophilic or are intended to be contained in or dispersed in the lipid phase. The starch granules that have been used in the emulsion of the invention have been shown to stabilize the interface against coalescence. However, in some situations, there is a need to further improve
    17/79 the barrier properties. This was also accomplished and improved barrier properties of particle stabilized emulsions or foams were provided with the application of heat, leading to an emulsion with partially gelatinized starch layers. A schematic figure of this concept is shown in figure 0-5A. In general, delivery systems could achieve a number of different functions, for example, an emulsion-based food that delays lipid digestion and induces satiety or, perhaps, targeted and controlled release of bioactive components into the gastrointestinal tract. To quantify the impenetrability of the partially gelatinized starch layer, the decrease in the rate of lipolysis was measured, under the premise that surfaces rigidly covered with starch granules that are difficult to displace from the interface, will reduce the lipase's ability to digest the present lipids emulsified oil.
    In one embodiment of the invention, the particle stabilized emulsion or foam has been subjected to drying, freeze drying, spray drying and / or vacuum drying, whereby an emulsion or dry particle stabilized foam is obtained. Dry emulsions can be added to food, creams and pharmaceuticals as an ingredient and can be used for powdered spray formulations such as inhalers. The emulsion system can be diluted without loss and displacement of the starch. This means that the dry particle stabilized emulsion or foam can be added to other processes in small quantities, at the desired point in the process. This enhances the functionality of sensitive ingredients.
    In one embodiment of the invention, the particle-stabilized emulsion is used to control the density of emulsion droplets. The parameters that influence what was mentioned above are the density of the oil, the density of the
    18/79 liquid, the concentration of the starch as well as the size of the starch granules. The rheological properties of the emulsion can be varied by varying the starch-to-oil ratio. The resulting emulsion will change the flow properties of a low viscosity cream to a particle gel loaded with easily dispersed and fractured droplet that exhibits yield stress at low concentrations. It is possible to form an oil gel / space loading particle at a low volume concentration of 0.5% starch and 5% oil. In highly dispersed phase volumes (more oil and starch granules), the emulsion becomes more rigid and more solid. This is a useful property considering that products with a range of textures can be produced without the use of additional viscosity modifiers (such as polymers) since the particles act as emulsifiers and a thickener (illustrated in figure 0-4A).
    In one embodiment of the invention, particle stabilized emulsions are used to replace fat in food products. Due to the high caloric content of fat, it has been found that replacing fat with the emulsions of the invention is beneficial for the food industry. In one embodiment of the invention, a particle-stabilized foam can replace fat crystals in whipped cream.
    In one embodiment of the invention, particle-stabilized emulsions are used to encapsulate substances chosen from probiotics, living cells, biopharmaceuticals, proteins, enzymes, antibodies, sensitive food ingredients, vitamins, and lipids. Particle stabilized emulsions are also beneficial for concealing the taste of flavor or aroma substances in an unacceptable way such as fish oil and antibiotics. In one embodiment, the particle-stabilized emulsion is used as a double emulsion. Dual emulsions are characterized
    19/7 9 for having a primary emulsion dispersed as droplets of a secondary emulsion. For example, water droplets within oil droplets dispersed in a second aqueous phase (see figure 0-6). A double emulsion of satisfactory stability has an initial encapsulation efficiency of 95% and, after 4 weeks of storage, it still has 70 to 80%. When using Starch Pickering Emulsions, the droplets are large enough to contain the drops and the starch layer is cohesive enough to maintain drop stability. The test showed an initial encapsulation effectiveness> 98.5% and, after 4 weeks of storage, it still has> 90%. Even after a freeze-thaw cycle, only <1% of the internal phase was lost.
    In one embodiment, the particle-stabilized emulsion is used to encapsulate substances poorly soluble in the oil phase. In some medical applications using conventional emulsions, an active substance poorly soluble in oil, the substance crystallizes. These crystals are too big for the small drops causing instability. When using Starch Pickering Emulsions, the droplets are large enough to contain the crystals and the starch layer is cohesive enough to maintain drop stability (see figure 5B-right).
    In one embodiment of the invention, particle-stabilized emulsions are used in food products, cosmetics, skin creams, lotions and pharmaceutical formulations. The particle-stabilized emulsion according to the present invention is a non-allergenic emulsifier that can be used in cosmetics and skin creams as moisturizers and sunscreens.
    In an embodiment of the invention, it is desired to increase the barrier properties to release, in a more satisfactory way, profiles for the skin or to prevent destabilization
    20/79 of the active ingredient / emulsions. The heating step is used in order to partially gelatinize the starch and thereby create a fairer film. For certain applications, the heating step mentioned above is performed.
    The present invention will be exemplified by several non-limiting experiments which are presented below.
    Experimental description
    Experiment 1
    In experiment 1, the ability of starch granules to stabilize oil-in-water emulsions was studied.
    starch was isolated from Quinoa (Biofood, Sweden) by a wet grinding process and modified by OSA to 2.9%. Quinoa was chosen due to its small unimodal and slightly small granule size distribution. The continuous phase of the emulsions was a phosphate buffer with pH 7 with 0.2 M NaCI, density 1009.6 kg / m 3 , at 20 ° C, the dispersed phase was the medium chain triglyceride oil Miglyol 812, density 945 kg / m 3 at 20 ° C (Sasol, Germany).
    Methods
    Quinoa starch isolation
    The Quinoa seeds were ground with water in a mixer (Philips HR7625, Netherlands) and filtered through a sieve cloth. The starch was allowed to settle and the supernatant was removed. Fresh water was added to the starch, which, after laying and removing water, was dried in a vacuum dryer at 20 ° C for 4 days. The proteins in the dried starch were removed by washing the starch twice with 3% NaOH solution, once with water and once with citric acid (pH 4.5) before the starch was dried with air at room temperature and flushed with mortar and pestle.
    Modification by ΟΞΑ starch was perfectly suspended in the double part by weight of water with the use of stainless steel helix and pH
    21/79 was adjusted to 7.8. Four equal amounts of OSA (totally based on 4% by weight of starch) were added with an interval of 15 minutes and the pH was maintained at 7.4 to 7.9 by adding 1M NaOH solution dropwise. When the pH was stable for at least 15 minutes, the starch solution was centrifuged at 3,000 * g for 10 minutes, washed twice with water and once with citric acid (pH 4.5) before the starch was air dried. at room temperature for at least 48 hours.
    The replacement of OSA was determined by a titration method. Briefly, 5 g (dry weight) of starch were dispersed in 50 ml of 0.1 M HCl and stirred for 30 minutes. The aqueous paste was centrifuged at 3,000 * g for 10 minutes, washed once with 50 ml of ethanol (90%) and twice with water before the starch was suspended in 300 ml of water, boiled in a boiling water bath for 10 minutes and cooled to 25 'C. The starch solution was titrated with 0.1 M NaOH at pH 8.3. A blank material was simultaneously titrated with native starch from the same source as OSA starch as a sample. The percentage of OSA carboxyl groups in the starch granules was calculated by:
    X 130 where V is the volume (ml) of NaOH required for the sample and a blank titration, M is the molarity of NaOH (0.1 M), W is the dry weight (mg) of the starch and 210 is the weight molecular structure of the octenyl succinyl group.
    Emulsification
    The emulsions were prepared in glass test tubes, combining 6.65 ml of continuous phase, 0.35 ml of dispersed phase and starch in varying amounts (12.5 mg to 1,250 mg) and emulsified by a high shear mixture. in
    22/79 a Ystrol (D- 79282, Ballrechten-Dottingen, Germany) a
    22,000 rpm for 30 seconds. The emulsified samples subjected to vortex treatment were then photographed 1 day and 1 week after emulsification and the images of the samples were analyzed in ImageJ to determine the volume of the cream / layered layer. The emulsification capacity of the starch and the stability of the emulsions were expressed as the relative occlusion volume, ROV.
    = , ^ emuls --- oil : starch where V erauis is the volume of the emulsion observed (ie, the unclear fraction), U oil is the volume of the oil phase and V amid0 is the volume occupied by the added starch. In a completely separate phase system, ROV = 1, that is, there is no increase in the emulsion layer beyond its constituent phases.
    Particle size measurements of granules and starch emulsions
    Particle size distributions were measured one day and one week after emulsification using laser diffraction with Mie optical mode (Coulter LS 130, Coulter Electronics Ltd, Luton Beds, England) for starch and starch-covered emulsions. the refractive index and 1.54. A small volume of sample was added to the flow system and pumped through the optical chamber for measurements.
    Microscopy
    The emulsions were diluted 5 times with the continuous phase and then the samples were placed in a 100 micron VitroCom square channel (CMS Ltd., Ilkley, UK). Microscope images of the emulsions were obtained using an Olympus BX50 camera (Tokyo, Japan) and a digital camera (DFK 41AF02, Imaging source, Germany).
    Results
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    The starch granules absorb and stabilize the oil and water interface.
    Quinoa starch granules (average diameter 1, .34 pm) were observed to stabilize the oil and water interface in a tightly packed layer (see insert in Figure 1-1) in what appears to be Pickering emulsions. The size distribution of (mean volume diameter D 43 ) is plotted in Figure 1-1 for starch granules (solid line) and starch stabilized emulsions (dashed lines). The measured particle size distribution of the starch granules indicated some aggregation that has sizes in the range of 4 to 10 pm. It is inferred that they are aggregated, since SEM images do not show such a wide range of individual granule sizes. In the resulting emulsion, some aggregates of starch were observed under the microscope and they were also perceived in the particle size distribution of the emulsion (dashed line in figure 1-1) as a minus shoulder at the main peak.
    The droplet size can be controlled by the amount of added starch
    The final emulsion droplet size was decreased as the amount of starch per ml of oil increased. Droplet-sized emulsions ranging from 64 pm (with 36 mg of added starch / ml of oil) to 9.9 pm (3,600 mg of added starch / ml of oil) were observed. The concentration effect on size has a decreasing effect on higher concentrations (see figure 1-2, note logarithmic scale).
    To estimate the degree of possibility of a new execution, two emulsification conditions were performed three times and once in duplicate. The conditions with 71 mg of starch per ml of oil showed an average volume diameter D43 ± standard error of the mean equal to 58.4 ± 1.13, n = 3, conditions
    24/79 with 571 mg of starch per ml of oil showed D 43 ± standard error of the mean equal to 26.9 ± 3.26, n = 3, and conditions with 1,714 mg of starch per ml of oil showed D 43 ± error mean standard equal to 12.3 ± 0.014, n = 2.
    The droplet size was measured after 1 day and after 7 days and it was found to show little change (in some cases, the droplet size still decreased, but at a level within the variability between replicates), with the exception of a tendency for slightly larger droplet sizes after 7 days at the two lower starch concentrations. (See figure 1-2). This could be expected as there may not be enough starch to stabilize the interface at the lower concentration allowing for easier coalescence. Subsequently, it was observed that they remained
    unchanged even after several months of storage The room temperature. There was no significant change at the size in droplet measured since the oil fraction was increased (The
    a constant ratio between starch and oil). In 12.5% oil, D 43 was 36.6 ± 1.98 pm, 16.6% oil D 43 was 36.9 ± 0.240 pm, 25.0% oil D 43 was 35, 9 ± 0 , 156 pm, and 33.3% D 43 oil was 36.4 ± 2.16 pm. This is in line with the above observations that the droplet size is determined by the amount of starch added.
    Droplet density can be controlled by the amount of added starch
    Due to differences in density between starch oil and water, the emulsion covered with a starch particle will not form cream at such a high rate as the buoyancy effects are significantly reduced. From the geometric analysis, and known phase densities (pamido 1.550 kg / m 3 , oil 945 kg / m 3 ) and volumes (Vamido, Vóleo, Vgoticulas) assume fair packaging of starch at the interface of
    25/79 oil and water and that the starch is small compared to the droplet diameter, it is possible to calculate in which droplet sizes the emulsions covered with starch granules should float or sink.
    q _ P amido P amido ^ ó / eo Poleo drip τλ r drip
    As the starch concentration increases, the droplet size decreases and the effective density of starch-covered droplets increases until they eventually become denser than the continuous phase and begin to sink. This level is shown as the vertical line in figure 1-2 and corresponds to the observations of the present invention and photographs figure 1-3 where the emulsion droplets are, in the majority, sinking to concentrations over 200 mg / ml of oil. Since the amount of added starch was increased (expressed as mg of starch per ml of oil), the droplet size decreased, the density increased due to the fact that there is a smaller relative volume of oil in relation to the covering starch layer that. Neutral buoyancy emulsions are not subjected to the formation of cream or settlement and therefore have greater stability.
    Emulsion phase properties
    The properties of the emulsion vary with the starch-to-oil ratio, from a low-viscosity cream to a charged particle gel (possibly bridged with oil) with weak fractured and easily dispersed droplet that exhibits yield stress. The relative occlusion volume of the emulsion phase crosses a maximum of almost 9 to intermediate starch-oil ratios, that is, it is possible to form an oil gel / space loading particle at a volume concentration of 1.7% starch and 5.5% oil.
    Storage properties
    26/79
    No changes were observed during cold storage of emulsions for 1 year.
    Conclusions of Experiment 1
    Experiment 1 showed that intact starch granules effectively stabilize oil droplets that create Pickering-type emulsions. The droplet size was found to be dependent on the concentration of added starch with lower marginal changes at higher starch concentrations. At this point, other factors, such as the level of mechanical treatment, could be decisive. Although many of the emulsions can be subjected to the formation of cream or settlement, they are stable against coalescence which shows little change in the appearance and height of the emulsion layer after the formation of cream or initial settlement. It was observed that they remain unchanged even after several months of storage at room temperature. This type of starch granule Pickering emulsion system can have applications in addition to food, for example, in cosmetics, and for pharmaceutical drug formulations where starch is an approved excipient.
    Experiment 2
    In experiment 2, the effect of the type of hydrophobic treatment and the degree of hydrophobic capacity on the resulting emulsion properties is illustrated.
    Materials
    In this experiment, grains of starch isolated from quinoa (Biofood AB, Sweden, density 1,500 kg / m 3 ) were used. The isolated starch granules were thermo-treated or modified by OSA with n-octenyl succinyl anhydride (CAS: 26680-54-6 Ziyun Chemicals Co., Ltd, China). In the emulsion studies, the dispersed phase was the medium chain triglyceride oil Miglyol 812 (Sasol, Germany, density 945 kg / m 3 ) and the continuous phase was a 5 mM phosphate buffer with pH 7 NaCl a
    27/79
    0.2M (density 1,009.6 kg / m 3 ). The other chemicals used in the study were analytical grade. Small granular starch was isolated from quinoa grains as described in experiment 1. Before use, the starch granules were ground into a fine powder by grinding them with mortar and pestle.
    Osa-modification of starch
    The water content of the starch powders was determined using an IR-balance at 135 ° C, from that mass of starch powders, the equivalent to 50 g of dry weight was measured. The starch was completely suspended in the double part by weight of water using a stainless steel helix and the pH was adjusted to 7.6. OSA was added at 3% (or 6%, 10%) based on the dry weight of the starch, and added in four portions with a 15 minute delay between additions. The pH was adjusted with 25% HCI and / or 1 M NaOH. Then, an automatic titration equipment with a pH meter and 1 M NaOH was used to maintain the pH at 7.6. The process was interrupted when the pH remained stable for at least 15 minutes, that is, no further pH adjustments were needed to keep it at 7.6.
    The starch-water-OSA solution was centrifuged at 3,000 g for 10 minutes and the water was poured. The starch was mixed with distilled water and was centrifuged twice. The starch was mixed with citric acid, pH 4.5 to 5, before being centrifuged and rinsed. The starch was dispersed in stainless steel trays and dried at room temperature for at least 48 hours.
    The determination of the degree of substitution of modified starch by OSA was carried out by a titration method as described in experiment 1. The determination was carried out in duplicate for the starch modified by OSA and the control starch, which has the same batch of origin as OSA modified starch. The dry weight of the starch was determined by an IR-equilibrium at 135 ° C. To this end, an amount of
    28/79 sample of approximately 1 g was used in duplicate. Then, 2.5 g of starch based on a dry substance were weighed and added to a 50 ml beaker. The starch was wetted with a few drops of ethanol before 25 ml of 0.1 M HCl was added and then stirred with a magnetic stirrer for 30 minutes. The aqueous slurry was centrifuged at 3,000 g for 10 minutes and the supernatant was discarded. The starch was mixed with 25 ml of ethanol before centrifugation in order to wash the starch. Then, the supernatant was discarded. The starch was washed as before, but twice with distilled water. The starch was added to a 500 ml beaker and mixed with 150 ml of distilled water. The mixture was heated in a boiling water bath at 95 ° C for 10 minutes before being cooled to 25 ° C. The mixture was titrated with 0.1 M NaOH until the pH was 8.3. The volume of NaOH used was observed. The percentage of OSA carboxyl groups (see table 1-1) in the granules was calculated by:
    % OSA —- ir ' 3 ^ ), A / ' 210 . j Οσ , / ο
    W
    Where V is the volume (ml) of NaOH required for the sample and the control titration, M is the molarity of NaOH (0.1 M), W is the dry weight (mg) of the starch and 210 is the molecular weight of the octenyl succinyl group.
    TABLE 1-1: Verification of the degree of modification by
    OSA expressed in%
    % OSA added V (ml) % of carbonyl groups of OSA in the granules, that is, the degree of modification expressed in% 0 0.325 0 3 2.64 1.95 6 4.15 3.21 10 5.87 4.66
    Thermal modification of starch
    29/79
    The dry starch (10 g) was placed in an open petri dish in a layer 1 to 2 mm thick. The samples were heated to 120 ° C for different durations in a furnace (30, 60, 90, 120 and 150 minutes). The heated samples were left at room temperature for several hours before using them. This treatment was carried out in order to hydrophobically modify the surface of the starch granules and, thus, achieve a greater affinity in relation to the oil and water interface.
    Emulsification
    The emulsions were prepared with a total volume of 6 ml in glass test tubes. All emulsions were produced in triplicate. The emulsions contained 7% Miglyol (ie 0.4 g) as a dispersed phase, a starch quantity of 214 mg / ml of oil (ie 0.089 g) and, as a continuous phase, a 5 mM phosphate buffer solution , pH7, with 0.2 M NaCI (i.e., 5.63 g). All experiments were conducted at room temperature without any temperature control. Starch, oil and buffer were weighed into test tubes, and vortexed (VM20, Chiltern Scientific Instrumentation Ltd, UK) for 5 seconds before mixing at 22,000 rpm for 30 seconds with a Ystral (D -79282, Ballrechten-Dottingen, Germany).
    Characterization of light scattering emulsions
    A laser diffraction particle size analyzer (Mastersizer 2000 Version 5.60, Malvern, United Kingdom) was used to determine the particle size distribution of the oil droplets. The emulsion was added to the flow system containing milliQ-water and was pumped through the optical chamber. In order to reduce the amount of aggregate drops, the pump speed was 2,000 rpm. The refractive index (RI) of the particle was set at 1.54, which corresponds to the starch that covers the droplets. The index of
    30/79 continuous phase refraction was set to 1.33 which is the RI of the water. The sample was added until the obscuration was between 10 and 20%. The average droplet sizes D43 and D32 as well as the mode of the emulsion droplet size distributions were determined.
    Conclusions as a result of Experiment 2
    All treatments allowed the production of emulsions stabilized by starch granules and, although the drops are of varying size and there are some starch-free granules; once formed, visual observations indicated that they remained as drops. However, the untreated granules showed significantly more unsatisfactory emulsification capacity and had the greatest dispersion in the droplet size distribution with a peak (mode) at 127 pm. Table 1-2 lists the measured droplet sizes. It appears to be an ideal level of modification by OSA of around 3% or a heat treatment of 30 to 90 minutes at 120 ° C. A very low level of modification may not provide the granules with sufficient affinity to adsorb at the oil and water interface - where a very high level of hydrophobic capacity can result in aggregated droplets. The hydrophobic modification of intact starch granules causes them to function satisfactorily as particles to stabilize Pickering-type emulsions with many useful properties is further illustrated in the following examples.
    TABLE 1-2: Emulsion particle size measurements produced with starch granules with different hydrophobic modifications using 214 mg starch granules / ml of oil.
    D3.2 weighted average diameter of D4.3 weighted average diameter of Mode (peak) (pm)
    31/79
    area (pm) istdev area (pm) istdevNative Starch 3.71 + 0.486 59.6 + 9.50 127 1.95% OSA 9.96 + 0.335 43.3 ± 1.79 50.9 3.21% OSA 13.5 + 0.991 42.0 + 3.92 42.7 4.66% OSA 19.4 + 1.97 54.6 ± 1.79 54, 9 30 minutes of heat (120° C) 2.95 ± 0.560 28.3 + 22.7 43.4 60 minutes of heat (120 ° C) 3.58 + 1.08 46, 1 + 26, 7 43, 4 90 minutes of heat (120 ° C) 3.41 ± 0.425 41.5 + 9.45 40, 3 120 minutes of heat (120 ° C) 5.11 + 3.01 65.8 + 35.7 88.4 150 minutes of heat (120 ° C) 4.42 + 1.24 62.4 + 31.1 91.8
    Experiment 3
    In experiment 3, the stabilization capacity of 7 different intact starch granules to generate oil-in-water emulsions was studied.
    The following commercial starches were investigated in this screening study: rice, waxy rice, more, more waxy, more high in amylose (HylonVII) and waxy barley (all from Lyckeby-Culinar AB, Sweden). Starch isolated from quinoa grains (Biofood, Sweden) by wet grinding as 10 in experiment 1 was also included in the study. Starches were studied in their native form, thermo-treated and modified by OSA. The modification by OSA was performed as in experiment 1. The continuous phase was a phosphate buffer at 5mM with pH 7 with and without 0.2M NaCl, the dispersed phase was the medium chain triglyceride oil Miglyol 812 (Sasol, Germany ).
    Heat treatment of starch dried starch was placed on glass plates and
    32/7 9 thermo-treated in a furnace at 120 ° C for 150 minutes in order to hydrophobically modify the surface proteins of the starch granules and thus achieve a superior oil-binding capacity.
    Particle size measurements of starch granules
    Starch particle size distributions were measured using laser diffraction (Coulter LS130, Beckman Coulter, UK) in a continuous flow cell (as described in experiment 1).
    Emulsification
    The emulsions were prepared in glass test tubes with 4 ml of the continuous phase, 2 ml of the oil phase and 100 to 400 mg of starch by mixing with a Ystrol (D-79282, Ballrechten-Dottingen, Germany) at 11,000 rpm 30 seconds.
    Dye Stability Test
    Approximately 1 mg of the oil-soluble dye Solvent Red 26 was added to the top of the emulsions after 24 hours and the test tubes were gently rotated 3 times. After another 2 hours, the emulsions were stirred with a vortex mixer for 5 seconds and stored at room temperature for 6 days. The color change of the emulsion was observed. The color after vortex is a measure of the stability of the drops formed. The stable drops do not exchange with the lipophilic dye; therefore, the emulsion phase will remain white. An increased red emulsion phase indicates that the drops have been less stabilized by the absorption starch granules or there is a free oily phase in the system. See table 2-1.
    Microscopy
    For microscopy of Olympus BX50 microscope emulsions (Japan) and digital camera was used. The images
    33/79 were processed in ImageJ (version 1.42m).
    Analyze
    The phase separation of the continuous layer and the emulsion was monitored as follows: the emulsions were stored at room temperature for 6 days. The test tubes with the emulsified samples were photographed 6 days after vortexing and the images of the samples were analyzed in ImageJ. The emulsification capacity of the starches and the stability of the emulsions were expressed as the volume of the cream emulsion layer in relation to the total volume of the sample. The emulsion volume fraction (E) was calculated as follows:
    Emulsion volume
    £ = __________ Total sample volume material, usually starch Ά quantity in remnant at the bottom of tube of test, too was calculated. See table 2-1.
    The drop size distribution of the emulsions was determined from microscopic images. The diameter of at least 250 drops was measured with ImageJ in samples that contained drops that were less than 1.4 mm in diameter. The average superficial droplet diameter (D 32 ) and the average volume diameter (D43) were calculated using the following equations:
    Where D is the measured diameter of a drop and n is the total number counted. The coefficient of variation (CV) as a percentage and the standard deviation were calculated according to the equations below to arrive at the distribution of the emulsion drops in each sample.
    34/79
    CF = - X 100 where σ = y £ C ^ -a 5S r] «-.ij
    Discussion as a result of Experiment 3
    Starches
    The starches selected for this study had different granule sizes, with quinoa as the smallest followed by rice, plus and barley, and these granules also had different shapes. The barley starch granules were smoothly shaped spheres and flattened spheroids with an average D32 of 17 pm, while quinoa, rice and more had more irregular polygonal shapes. The quinoa granules had a D 32 of approximately 2 pm and had smooth edges, while rice had granules with sharp edges with a D 32 of 4.5 and 5.4 pm for waxy and normal rice, respectively. The more waxy and the normal ones had smooth and sharp edges of their granules, while the more with high amylose content had smoother edges and also had some rod-shaped granules. The average size of the more granules was 9.3 pm for more with high amylose content and 15 pm for the other two more varieties. The shape of the granules was similar for all three quinoa granules: native, thermo-treated and modified by OSA. However, the size was increased for the granules that were subjected to heat treatment or modification by OSA, which occurred, in part, to a higher degree of aggregation caused by the increased hydrophobic capacity. The individual quinoa starch granules were between 0.7 and 2.2 µm in size.
    starch has a natural variation in the amylose / amylopectin composition and normal varieties have an amylose content of about 20 to 30%. Waxy starches have a very low content of amylose and, in the present study, waxy varieties of rice, barley and more were used. A variety
    35/79 too much with a high content of amylose (HylonVII) with 70% amylose was also used in order to observe the emulsification behavior in a broader spectrum of the amylose content. OSA binding has been shown to be non-uniform on a molecular scale and is affected by differences in the branching of starch molecules.
    Table 2-1 summarizes the test conditions used and the main results. The color after vortex is the measurement of the stability of the drops formed since the dye was added on top of the samples after emulsification and before the samples were mixed in a vortex. The stable drops had no exchange with any colored oil; therefore, the emulsion phase remained white. An emulsion phase with increased red color indicated that the drops were less stabilized by the absorption starch granules.
    The size of the drops is correlated to the color and stability of the emulsion. Starch granules, which had the ability to stabilize small drops, also created the most stable drops. This depends mainly on the size of the stabilizing granules, but also whether the shape of the granules had an impact. Quinoa, which had the smallest granule size, had the most satisfactory ability to stabilize an emulsion under the circumstances used in that study. The emulsions were produced regardless of the treatment and concentration of quinoa starch or the system used (sample No. 1-10 in table 2-1).
    The emulsification capacity of quinoa was definitely more satisfactory, followed by rice, which was only slightly larger granule size, but the granules were more irregularly shaped with sharp edges. The emulsification capacity was similar for the two rice varieties (sample no. 11 to 13 and 17 to 18, 20 in table 2-1). The most waxy and normal had granules
    36/79 irregularly conformed to, which may be a reason for slightly less stabilizing capacity compared to barley which had a larger granule size, but a smoother shape. A reduced surface contact of particles due to surface roughness or sharp edges has a negative impact on the emulsification powder, since the interfacial potential decreases. Another reason was probably the bimodal size distribution of the barley, where the smaller granules potentially increased drop stability and decreased drop size. Four samples were produced twice; No. 9 (quinoa), 20 (rice), 31 (more) and 42 (waxy barley) according to the classification in table 21. All with 200 mg of OSA starch and buffer with salt as the continuous phase. Quinoa and waxy barley, which produced stable emulsions, showed satisfactory reproducibility in relation to drop size, sediment fraction and emulsion volume fraction, while the reproducibility of results for rice and more was unsatisfactory.
    The stabilization capacity of more waxy and normal was similar (sample n ° 22 to 24 and 28 to 29, 31 in table 2-1), but the one with the highest amylose content (HylonVII) showed a different pattern. The three samples (n ç 33 to 35 in table 2-1) showed only small disparities in the emulsion fraction and drop size regardless of the treatment of starch granules. Stem-shaped granules have been shown to have a major impact on the stability ability and have shown that long particles with an aspect ratio greater than 4 are more effective emulsifiers than smaller particles of similar wettability.
    Treatments
    All starches in this study were used in their
    37/79 native form, thermo-treated and modified by OSA, respectively. Native starch granules are supposed to be ineffective as oil drop stabilizers due to low hydrophobic capacity, however, native quinoa (and, to some extent, HylonVII) has the ability to stabilize the droplets formed. All starch granules have proteins attached to the surface and, for small quinoa granules, the total large surface area of all granules can provide sufficient hydrophobic capacity to stabilize droplets, although the droplets stabilized by native quinoa starch are larger than when modified starches were used.
    Thermo-treated starches were, in some way, more satisfactory stabilizers than native starches since the hydrophobic capacity of surface proteins increased. Especially, the drops stabilized by quinoa, rice and waxy barley had a reduced drop size. The hydrophobic capacity of the starch granules apparently increased, but enough that the granules had the ability to act as less stabilizers when the granule size was as small as for quinoa.
    The OSA-modified starches all have the ability to stabilize oil droplets, but the use of the granules was not complete since the starch, to some extent, sedimented. The OSA content was between 2.6 and 3.6% for all starches and the quinoa was also modified to a lesser degree of 1.8%. No difference could be observed between the quinoa samples with the two degrees of OSA in relation to drop size, emulsion volume fraction or stability, which indicated that the 1.8% OSA bond provided hydrophobic granule surface capacity enough to stabilize an emulsion. Starch modified with 3% OSA
    38/79 is commercially available and approved as a food additive.
    Continuous phase
    Two different phosphate buffers, with and without 0.2M NaCl, were used as a continuous phase and the pH was 7 in both buffers. The difference in the pattern of drop formation was considerable between buffers with or without salt. The difference was apparent at both the macro- and microscopic level for hydrophobically modified starch granules, but not for native granules.
    When a continuous phase without salt was used, the emulsions had distinct conical shapes formed by the tip of test tubes, indicating a layer of reticulated emulsion with a yield stress, however, this shape was less obvious in the presence of salt. In addition, the volume fraction of the emulsion was higher and the starch sediment was lower in salt-free systems. The droplet size distribution also had a different nature where emulsions without salt showed bimodal droplet size distributions with a large CV (74 to 85%) and the droplets in emulsions containing salt show a more unimodal distribution with a CV of approximately 40 %. These observations can, to a large extent, be explained by the behavior of gout formation. In the absence of salt, the emulsion droplets formed a more rigid open network of droplet and granule clusters. While in salt systems, the droplets were less effectively stabilized and coalesced into a larger uniform size without significant aggregation. Emulsions stabilized by native starch were not affected by the presence of salt.
    Starch concentration emulsification starch concentration effect has been studied in four varieties and starch: quinoa, rice, mais and waxy barley, all of which have been modified by
    39/79
    OSA and in a 0.2 M NaCI phosphate buffer. These conditions were used since they presented the most satisfactory emulsification result in initial scanning studies, and salt emulsions showed a more uniform distribution of droplet sizes and were not gelatinized. Ά mass of the added starch was 100, 200 and 400 mg, which corresponds to approximately 3.2, 6.2, and 11.8% by volume of the oil (or 1.1, 2.2 and 3.9% by volume total system), respectively. The droplet size was decreased and the volume fraction of the emulsion phase was increased while the concentration of the amid granules was increased as can be seen for sample no. 8 a (quinoa), 19 to 21 (rice), 30 to 32 (more) and 41 to 43 (waxy barley) in table 2-1.
    It has previously been shown that the average droplet size of solid particle stabilized emulsions decreases with increasing particle concentration as more particles are available to stabilize smaller droplets. However, each system probably has a limiting droplet size, which depends on the physical and mechanical properties of the system (that is, the particle size and the emulsification method) and, when that droplet size is reached, any excess of particles will be in the continuous phase. In the current study, samples with the highest amount of starch produced emulsions with a density higher than the continuous phase. The droplet size decreased and the amount of starch left on the surface of the droplets increased as the starch concentration was increased, which resulted in a more stable emulsion. Another effect of the high starch concentration was that the amount of starch granules between the drops increased. This resulted in an increase in the total density of the drops and in the emulsion phase.
    It is interesting to note that even in low (100
    40/79 mg) starch concentrations, there was pellet pellet at the bottom of the test tubes. In reality, the starch sediment fraction decreased when the amount of starch was increased from 100 to 200 mg. The drops formed in a lower concentration of starch granules were less covered by the granules and more subjected to coalescence than granules desorbed from the surface of the larger drops. However, it was shown that Pickering emulsions were stabilized considerably even when silica (0.5 to 0.8 pm) or spore particles (-25 pm) were distributed highly unevenly on the surface of the droplets. The emulsion was also less dense at a low starch granule concentration, which means that the mobility of the droplets and granules promoted the sedimentation of the unsorbed granules in the continuous phase.
    Starch granule size
    To quantify the effects of the amount of added starch and granule size, the maximum possible surface coverage for starch concentration with a given particle size was estimated. The assumptions made were that all the drops would be identical in size and all the starch particles are identical, spherical and are attached to the oil and water interface at a 90 ° contact angle with a fraction of interfacial packaging φ 0.907 ie packaging hexagonal. The maximum theoretical coverage, TM, is estimated using the following equation:
    = 10 s where d sg is the average surface diameter of the starch granule, p sg is the starch density (1,550 kg / m 3 ) and φ is the packing density. Estimates of maximum surface coverage as well as average granule sizes of
    41/79 starch for the various starches are provided in Table 2-2.
    Since the surface coverage (mg / m 2 ) increases with starch granule size, it is not surprising that the drop diameter generated in the figure
    2-1 decrease with decreasing granule size as more area is covered by unit mass with smaller granules.
    The specific surface area of an emulsion, per volume of dispersed phase is defined by:
    and where is the average surface diameter d32 measured by light scattering. Based on the amount of added starch, theoretical C sg , Γ Μ , do (as mg per ml) the maximum coverage given the size of starch granules, a theoretical surface area could be calculated that could be generated by volume of dispersed branch, this is:
    A comparison of the calculated and measured drop surface areas is mapped in Figure 2-2 and illustrates very good agreement between these estimates and the measured starch stabilizes the drops despite the rather crude assumptions in the calculations. Starches above the line in Figure 2-2 have a larger drop area than expected and those below the line have a smaller drop area. A physical explanation of the larger drop areas is that the hexagonal closed-pack assumption overestimates the amount of starch on the surface and that it is possible to have less starch per unit area and still achieve drop stabilization.
    It could be argued by geometric analysis that as the starch granule size ratio to form the drop size increases, the minimum surface coverage
    42/79 required to stabilize the drops decreases, since larger spaces between the granules on the surface are possible while maintaining enough of a steric, making it difficult to prevent coalescence. For this reason, the larger starch granules, such as barley and more, have a larger than expected surface area and the trend increases with increasing area (i.e., smaller droplet sizes). Microscopic observations confirm this, showing larger spaces and gaps on the surface of droplets between adsorbed starch. In the case of rice, it has a smaller generated area than expected (data points are below the line in Figure 2-2). In the microscopic images of the rice emulsions, more free starch granules appeared in the continuous phase and a notable increase in the amount of sediment.
    Conclusions in view of Experiment 3
    This screening experiment, in the emulsifying capacity of a wide spectrum of starches in their granular form, revealed that starch granules effectively intact can stabilize oil droplets in an emulsion. Among the different starches that were examined, quinoa starch had the best prominent ability to act as a stabilizer, probably because of its small granule size. Quinoa starch was able to stabilize the droplets even in their native state, although the thermo-treated granules and, above all, those modified by OSA were more efficient, which was demonstrated by the smaller droplet size and increased droplet stability. All of the OSA-modified starches used in this study could stabilize the droplets and the droplet diameter decreased with the size of the granules. The droplet size was also decreased by increasing the concentration of the starch granules. The impact of salt concentration on the emulsifying capacity was studied in order to simulate the conditions of different food systems and
    43/79 other products based on emulsions. Salt-free systems produced very stable rigid emulsions with aggregate droplets with a bimodal droplet size distribution.
    Although the size of the starch granules stabilized with emulsion drops was relatively large, the drops may be suitable for encapsulating valuable ingredients in pharmaceutical and food products.
    Table 2-1. Summary of results and experimental conditions
    Master Or Trat Phase Amid Color Frac Sedi Drop size str IG amen cont O after to ment 6 days after The in to continuous postpone s in O vortexn ° in ofiona vor voluam amidof tic meid O and inO emul are Cone (mg) (0 6 (mm 3 / D (32) D (43) CV . inThe days mg) b (pm) (pm) (% salt4) 3 after vortex ) ice 1 Qu Nati Without 200 1 0.67 0.46 140 150 45in grandfather salt oa2 Qu Act Without 200 1 0.82 0.07 100 120 85in cido salt 5 oa3 Qu OSA Without 200 0 0.87 0 74 81 74in 1.8% salt oa4 Qu OSA 0.2 200 0 0.94 0 74 87 77in 2.9% M of oaNaCl 5 Qu Nati 0.2 200 1 0.60 0.35 320 370 46in grandfather M of oaNaCl 6 Qu How 0.2 200 1 0.68 0.31 160 170 41in cido M of oaNaCl 7 Qu OSA 0.2 200 0 0.78 0.01 76 79 40 1 in 1.8% M of 5
    44/79
    | oaNaCl 8 Quinoa OSA2.9% 0.2 M NaCl 100 1 0.58 0.32 270 290 32 9 Quinoa OSA2.9% 0.2 M NaCl 200 1 0.77 / 0.7 4 C 0.03 / 0, 0 2 C 100/110 c 110/120 c 37/3 7 C 10 Quinoa OSA 2.9% 0.2 M NaCl 400 0 1.00 n. v. 52 55 42 11 Ar ro zWx Natigrandfather 0.2M ofNaCl 200 4 0.40 2.3 > 1mm > 1 mm12 Ar ro zWx What I do 0.2 M NaCl 200 4 0.44 2.0 > 1 mm > 1 mm - 13 Ar ro zWx OSA3.8% 0.2 M NaCl 200 2 0.59 0.55 440 500 42 14 Ar ro z Natigrandfather Without salt 200 4 0.45 2, 1 > 1mm > 1 mm - 15 Ar ro z What I do Without salt 200 2 0.50 1.2 150 200 79 16 Ar ro z OSA2.8% Without salt 200 1 0.75 0, 12 100 170 70 17 Ar ro z Natigrandfather 0.2 M NaCl 200 4 0.42 1.7 > 1 mm > 1 mm - 18 Ar ro z What I do 0.2 M NaCl 200 3 0.46 1.7 530 590 71 19 Ar ro z OSA 2.8% 0.2 M NaCl 100 3 0.55 1.3 550 630 41 20 Airroz OSA2.8% 0.2 M NaCl 200 2 0.55 / 0.6 6 C 0.70 / 0.3 3 C 530/350 c 560/4 4 0 c 75/6 3 C 21 Ar ro z OSA 2.8% 0.2 M NaCl 400 2 0.85 n. v. 200 310 71 22 More Natigrandfather 0.2 M of 200 4 0.38 1.5 No drop No drop
    45/79
    Wx NaCIs s 23 MoreWx What I do 0.2 M NaCI 200 4 0.39 1, 9 No drop s No drop s - 24 MoreWx OSA3.3% 0.2 M NaCI 200 3 0.64 0.15 500 540 38 25 More Natigrandfather Without salt 200 4 0.34 1.5 No drop s No drop s26 More What I do Without salt 200 4 0.29 3, 7 No drop s No drop s - 27 More OSA2.6% Without salt 200 2 0.69 1.0 420 470 57 28 More Natigrandfather 0.2 M NaCI 200 4 0.38 1.2 No drop s No drop s - 29 More What I do 0.2 M NaCI 200 4 0.38 1.5 No drop s No drop s - 30 More OSA2.6% 0.2 M NaCI 100 3 0.53 0.27 1.300 1.400 26 31 More OSA2.6% 0.2 M NaCI 200 3 0.50 / 0.5 9 C 0.65 / 0, 1 4 C 1.30 0/72 0 c 1.40 0/75 0 c 30/2 9 C 32 More OSA2.6% 0.2 M NaCI 400 2 0.81 n. v. 290 300 34 33 More from AM to Natigrandfather 0.2 M NaCI 200 3 0.48 1.2 980 > 1 mm 51 34 More ofAM al to What I do 0.2 M NaCI 200 3 0.52 1, i 830 880 40 35 More than AM OSA3.1% 0.2 M NaCI 200 3 0.54 0.90 710 750 27
    46/79
    36 Wx ce va Natigrandfather Without salt 200 4 0.42 1.3 > 1 mm > 1mm37 Wx ce va What I do Without salt 200 3 0.51 1, 2 > 1 mm > 1 mm38 Wx ce va OSA3.6% Without salt 200 2 0.76 0.040 370 460 65 39 Cego fromWx Natigrandfather 0.2 M NaCl 200 4 0.38 1.3 > 1mm > 1mm40 BarleyWx What I do 0.2 M NaCl 200 3 0.50 0.90 890 930 41 41 Cego fromWx OSA3.6% 0.2 M NaCl 100 3 0.54 0.65 1.200 1.400 32 42 Wx ce va OSA3.6% 0.2 M NaCl 200 2 0.58 / 0.6 0 c 0.27 / 0.2 2 C 690/670 c 720/700 c 27/2 7 C 43 BarleyWx OSA3.6% 0.2 M NaCl 400 2 0.80 n.v. 270 300 34
    a Ο = white emulsion phase that was not colored by Red Solvent, 4 = red emulsion or oily phase that was completely colored by Red Solvent, 1 to 3 = increasing degree of red colored emulsion phase.
    b Sediment volume ratio to added starch.
    c Replicable results from two different samples.
    n.v. Not visible. The emulsion phase covers the bottom of the test tube and any sediment remaining on the bottom is not visible.
    47/79
    Table 2-2. Particle sizes and maximum surface coverage for starch granules.
    Shy Dio [m] D 32 [m] D 43 [m] r M [mg m 2 ] a Native Quinoa 1.14 1.7 2.51 1,590 Heated quinoa 1.33 2.23 3.38 2,090 Quinoa'sOSA 1.34 2.27 3.45 2,130 RiceOSA 3.45 4.46 5.25 4. 180 RiceWaxy ofOSA 3.57 5.38 7.46 5,040 OSA nylon VII 7.07 9, 32 11.1 8,740 OSA Waxier 9, 54 14.7 18, 0 13,800 More ofOSA 11.3 14.9 17, 1 14,000 BarleyWaxy fromOSA 7.49 17, 5 24.2 16,400
    Experiment 4
    In experiment 4, emulsions using a variety of oils and fats were made, as the physical properties of the dispersed phase vary depending on the type of oil. Oils that were used as the dispersed phase include: Miglyol 812, soy oil (natural and purified with AI2O3), rapeseed oil, paraffin oil, chestnut butter 10 (solid at room temperature), chestnut oil, Bassol C, glyceryl and hexadecane tributyrate. Small granular starch modified by OSA, as described in experiment 1, was used as droplet stabilizing particles. The emulsions were prepared as described in experiment 1 with the exception of 15 solid fats that were melted before high shear homogenization.
    Scattered phase effect
    Emulsions have been successfully created with all
    48/79 different oils used. However, the surface of the tributyrate oil droplets was sparsely occupied by the starch granules. This is probably due to the high solubility of the tributyrate in water.
    Conclusions in view of Experiment 4
    The stabilization of oil droplets with starch granules is effective over a wide range of oils. This is of practical impact as it indicated a robust system that is not particularly sensitive to the type of oil used, thus being applicable in a wide range of technical, pharmaceutical, food and cosmetic products.
    Experiment 5
    In experiment 5, emulsions using different processing techniques were made, the purpose being to demonstrate that starch granule stabilized emulsions can be made using a variety of methods.
    The oil phase in this experiment was from Bassol C (AAK, Sweden), the starch granules were isolated from quinoa and made more hydrophobic by modifying the OSA by 2.9% (as described in experiment 2), and the continuous phase was of 5 mM Phosphate buffer at pH 7 and 0.2 M NaCl. Four samples were weighed as follows: 3.50 g of starch granules were added to 59.5 g of phosphate buffer and then 7.00 g of Bassol C was added and stirred before homogenization. Each sample was made using a different homogenization method. Sample 1 was made using a 3,200 rpm Sorvall Omni Mixer (level 2) for 5 minutes. Sample 2 was made using a 12,800 rpm (level 8) Sorvall Omni Mixer for 5 minutes. Sample 3 was made in a 40 bar laboratory scale high pressure homogenizer (HPH) and the entire volume was passed through the HPH 10 times. Sample 4 was taken from a pump
    49/79 Masterflex peristaltic operated at 350 ml / min and the entire volume passed through the pump in the circulation loop a total of 300 times.
    The emulsions were diluted approximately 5 times with the same buffer solution as in the continuous phase before they were analyzed. Droplet Size distributions of the emulsions were determined using a laser diffraction particle analyzer (Mastersizer 2000, Malvern Instruments). The dispersion was diluted on the instrument to achieve an 8 to 12% glare. The size distribution was calculated based on Mie's theory using a refractory starch index of 1.54. Emulsions were also investigated using an optical microscope (Olympus BX50, Japan) equipped with a digital video camera.
    Experiment 5 Results
    Emulsions could be created using all four emulsification methods. Based on the amount of added starch (500 mg / mg of oil), a droplet size range of 26 to 33 pm (D 43 ) was expected. This was observed in the mixed samples of sorvall and in the one prepared in the peristaltic pump. The sample prepared in the high pressure homogenizer was subjected to a much larger mechanical treatment and, for this reason, the droplets were much smaller, but were also flocculated in structures of about 100 pm in size. This may be because there was not enough starch to cover the high surface area of oil generated in the homogenizer and the oil droplets shared starch particles, generating the observed microstructure. Average measured droplet sizes, micrograph of droplets, and emulsion appearance images in general are found in table 3-1.
    Table 3-1 - summarizes the conditions for Figure 3-1.
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    Figure 3-1 more topS sorvall level 2 with 10% Bassol C 500 mg / g OSA Q 2, 9% 300 (100X magnification) The second uppermost Figure 3-1S sorvall level 8 with 10% Bassol C 500 mg / g OSA Q 2.9% 300 (100X magnification) D32 = 7,873 pmD43 = 26.07 pmMode = 19.27 pm D32 = 10.08 pmD43 = 27.18 pmMode = 26.58 pm Smooth space fill emulsion Due to larger rpms, more air was swallowed, so the emulsion floated. Droplet size measured similar to level 2. The second lower Figure 3-1 Figure 3-1 lower HPH with 10% Bassol C 500 mg / g OSA Q 2.9% (100X magnification) Pump with 10% Bassol C 500 mg / g OSA Q 2.9% (magnification100X) D32 = 79.08 pm D 43 = 102.8 pm Mode = 96.15 pm D32 = 5.959 pmD43 = 31,104 pmMode = 53.93 pm Higher HPH intensity creates smaller droplets that exist as flakes seen in the image on the right, which measure about 100 pm in size in light scattering Lower intensity provides larger droplets, but smoother appearance. More free starch was also observed. Conclusions in view of Experiment 5
    This experiment showed that it is possible to use a variety of mechanical emulsification methods to generate starch granule-stabilized emulsions. This indicates a robust system that could be applied to a variety of different products and processes in a range of applications that have been provided (Figure 3).
    Experiment 6
    Food and other emulsion systems have a wide range in pH and salt concentration. Therefore, emulsification with continuous phases with a pH of 4 to 7 and
    51/79 salt concentrations of 0, 1 to 2 M NaCl and 0.2 M CaC12 were studied.
    The dispersed phase was Miglyol 812, small granular starch granules, as described in experiment 1, were used as drop stabilizing particles and the continuous phase was 5 mM phosphate buffer or milliQ water at varying pH and amounts of salts added. The emulsions were prepared as in experiment 1.
    Continuous phase effect
    The salt concentration was varied at pH 7 and the pH was varied at a salt concentration of 0.1 M NaCl. In another sample, 0.1 M CaC12 in MilliQ water was used as a continuous phase. Both pH and salt concentration had no significant effect on the volume fraction or the average drop size of the emulsion. However, the results of experiment 3 showed that there is a difference in the emulsion network between the systems with and without salt.
    Conclusions in view of Experiment 6
    A stabilization of oil droplets with starch granules is efficient regardless of the pH and salt concentration of the continuous phase. This indicates a very robust system that will have applications in a wide variety of products.
    Experiment 7
    In this experiment, emulsions with different oil phase contents were prepared to test their stability during storage and rheological properties, two properties that are important in a variety of emulsion applications. To determine the stability of the emulsions, neutral buoyancy emulsions, that is, the oil drops covered with starch had approximately the same density as the continuous phase, were prepared. The fractions of oil volume were 12.5, 16.6, 25.0 and 33.3%, the ratio of starch to oil was constant at 214 mg of
    52/79 starch / ml of oil and the total volume of the samples was 7 ml. Small granular starch isolated and modified by OSA to 1.8% as described in experiment 2.
    The continuous phase of the emulsions was a 5 mM phosphate buffer with pH 7 and 0.2 M NaCl (density of 1,009.6 kg / m 3 at 20 ° C), the dispersed phase was Miglyol 812 (density of 945 kg / m 3 at 20 ° C, Sasol GmbH, Germany). The emulsions were made by mixing high shear in a Ystral X10 mixer with a 6 mm dispersion tool (Ystral GmbH, Germany) at 22,000 rpm for 30 s.
    Storage Stability
    The samples were stored in 5 C sealed test tubes for 1 day, 1, 2, 4 and 8 weeks before the drop size measurements (using Coulter LS 130 laser diffraction, described in Method 2 experiment) and the determination of fractions of volume of photographs (method 2 experiment).
    Rheology measurements
    The elastic modulus and phase angle of samples stored for 8 weeks were measured using an oscillation stress scan, 20 s in each amplitude (Kinexus, Malvern, United Kingdom). The frequency was 1 Hz. A cone and plate system with a diameter of 40 mm and a cone angle of 4 degrees was used.
    Storage Stability Results
    The drop size was determined and the emulsion index was calculated at intervals of 5 times between 1 day and 8 weeks of storage. The drop size showed no significant difference, the oil concentration and storage time also showed no significant difference. The drop size (D 43 ) was between 34 and 39 pm for all samples. In this way, the drops were stable over time and were not susceptible to
    53/79 coalescence, irreversible flocculation or Ostwald maturation; the latter being unlikely in this system due to the relatively large drop sizes and low solubility of Miglyol in water.
    The emulsion index (EI, as defined in experiment 2) increased, as expected, with the oil concentration (Figure 4-1). The EI was close to 1 for samples with 33.3% oil, that is, the emulsion phase occupied almost the entire sample. The EI tended to increase with storage time, at least for the first four weeks, due to the corresponding densities for the drops and the continuous phase. During the 8 weeks of storage, the emulsion drops were stable for coalescence and the volume occluded by the emulsion phase was not affected or even increased. No significant difference in the average drop diameter over time or between concentrations, even after 8 weeks storage at 5 ° C.
    Rheology Results
    The rheology measurements confirmed the differences observed in the structure of the emulsions due to the variation in the volume fractions of the dispersed phase. In Figure 4-2 the elastic modulus is mapped as a function of complex shear stress. There is a short linear elastic region followed by a rapid decrease in stresses of ~ 1 Pa or less, which indicates that the samples have a weak gel structure. The elastic modulus G 'is a measure of the amount of stress energy per oscillation shear that can be stored in the sample structure, and is a function of the strength and the number of interactions between the components of the emulsions. As could be expected, the higher the oil concentration, the greater the elastic modulus, as there was more interaction material.
    As the shear stress was increased,
    54/79 the structure eventually broke, which was shown by the change in the phase angle. In low shear stresses, the samples had phase angles less than 45 °, that is, the samples exhibited more elastic behavior. As the shear stress was increased to the point that the weak gels started to flow, the phase angle increased to greater than 45 ° indicated a more liquid-like behavior in the samples. Table 4-2 shows that the higher the oil concentration, the more the shear stress could be increased before the gel structure in the emulsions was reduced to a liquid-like behavior.
    Conclusions in view of Experiment 7
    The resulting emulsions were found to be stable during storage (at least 8 weeks), despite their large droplet size. Rheological measurements showed a weak gel structure. This is important in many applications where an individual wants to be able to choose a final consistency based on the emulsion recipe. In addition, due to the partial double wettability of particles suitable for stabilizing emulsions, the stabilized particle droplet and free starch granules tend to form weak aggregates that give them a more gel-like consistency. This is important in many applications where thicker products, such as creams, are desirable; and in the present case, no additional viscosity modifier is required to achieve a gel-like consistency.
    Table 4-1: Average droplet diameter of starch granule stabilized emulsions before and after storage.
    Droplet diameter mean P 43 [μπι]
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    Oil content 1 day 8 weeks of storage at 5 ° C 12.5% 36.6 ± 1.98 37.2 + 0.735 16.6% 36.9 ± 0.240 37, l ± 0.219 25, 0% 35.9 ± 0.156 34.6 ± 0.014 33.3% 36.4 ± 2.16 35.2 ± 0.502
    Table 4-2. Shear stress values in the phase change from gel to liquid (phase angle 45 °).
    Concentrationof Oil Shear stress at 45 degrees (Pa) 12.5% 0.287 16.6% 0.334 25, 0% 0.480 33.3% 1.10
    Experiment 8
    The objective was to study the phase inversion of emulsions stabilized by starch granules and to identify relevant conditions for the formulation of topical creams.
    Methods
    Emulsions were produced using Miglyol 812, 5 mM phosphate buffer pH 7 and 0.2 M NaCl, Quinoa, OSA 1.8%. Samples were mixed at 22,000 rpm for 60 s. The total volume was 7 ml and each experiment was carried out in triplicates. The oil concentration and starch concentrations were varied as described in table 6-1. Samples L to M were also centrifuged in order to assess stability and to simulate 8 weeks of storage. Centrifugation was performed at 1,000 g for 81 min at room temperature (21 ° C).
    In addition to these experiments, two other oils, paraffin and shea oil, were used to produce emulsions in conditions that correspond to sample M. In a blind sensory classification test, 9 volunteers
    56/79 evaluated the parameters of consistency and applicability of these emulsions and two commercial products (Vaseline and a skin lotion).
    Phase inversion
    Samples containing 70% oil were water-in-oil emulsions at all concentrations of starch, while at lower oil concentrations, oil-in-water emulsions were formed (table 5-1).
    Relevant conditions for the formulation of creams
    At oil concentrations of 56% or 41%, the consistency in terms of thickness and homogeneity of the system was well suited for topical cream applications. After centrifugation, samples M and N had negligible phase separation, whereas sample L was slightly separated. The emulsion droplet size increased from 52.0 to 62.2 qm for sample L and from 33.0 to 37.3 qm for sample N, and was not affected for sample M (40.8 before and 40 , 5 qm after centrifugation). When different oils were tested, shea oil that had solid-like properties at room temperature produced an emulsion with a very thick consistency, while Miglyol and paraffin produced emulsions that were more slippery and slightly watery. Paraffin containing emulsion (higher rating by 1 test person) was better accepted than Miglyol emulsion, and shea oil emulsion was rated the best by 2 volunteers. Commercial products were rated as best by 2 (Vaseline) and 4 (skin lotion) volunteers, respectively · This is certainly not surprising as they contain other pleasant ingredients, such as perfume.
    Conclusions in view of Experiment 8
    Samples containing 70% oil or more were water-in-oil emulsions in all starch concentrations,
    57/79 while, in less oil concentrations, oil-in-water emulsions were formed (table 5-1). At oil concentrations of 56%, consistency in terms of thickness and homogeneity of the system was considered to be well suited 5 for topical cream applications. Under these conditions, the stability for conditions of forced storage and shear during centrifugation was negligible. Within the oils used in 56% and starch concentration of 214 mg / ml of oil, all produced very well accepted creams. The 10 emulsions containing Miglyol or paraffin were very similar, although paraffin was better accepted than Miglyol as an oil phase. The shea oil emulsion was more similar to solid and rated higher than commercial products by some testers.
    Table 5-1. Emulsion droplet size compositions and samples
    Sample Starch [mg] Oil [mg] Buffer [mg] Oil [%] Starch [mg / ml ofoil] Continuous phase Droplet size D 43 [m] THE 400 1,890 4,710 27 200.0 Water 35.7 B 400 2,890 3,710 41 130.8 Water 51.7 Ç 400 3,890 2,710 56 97.2 Water 61.1 D 400 4,890 1,710 70 77.3 Oil 64.1 AND 400 5,890 710 84 64.2 Oil 54.5 * F 200 2.8 90 3,910 41 65.4 Water 64, 6 G 200 3,890 2,910 56 48.6 Water 73, 9 H 200 4,890 1,910 70 38.7 Oil 55.5 I 600 2,890 3,510 41 196, 2 Water 31.7 J 600 3,890 2,510 56 145.8 Water 43.5 K 600 4,890 1,510 70 116.0 Oil 58.9 L 4 00 3,890 2,710 56 97.2 Water 52.0 M 856 3,890 2,254 56 214 Water 40, 8 N 642 2,890 3,468 41 214 Water 33, 0
    * Measured by micrographs (all other samples were measured using Coulter LS130)
    Experiment 9
    In experiment 9, the improved permeability of a
    58/79 lipophilic chemical in the skin by the use of starch granule stabilized emulsions was studied.
    Methods
    Emulsions were produced using 5 mM phosphate buffer pH 7 and 0.2 M NaCl, Quinoa, 1.8% OSA and Miglyol 812, paraffin or shea oil. Samples were mixed at 22,000 rpm for 60 s. The emulsions contained 56% oil and 214 mg of starch / ml of oil (corresponding to sample M in experiment 8). The total volume was 7 ml and each experiment was carried out in triplicates. Methyl salicylate, dissolved in the oily phase, was used as a control substance to study skin permeability.
    The measurement of skin diffusion was performed in a flow cell by monitoring the transport of methyl salicylate from the three different topical formulations through a pigskin membrane and silicone membrane under a phosphate buffer flow with pH 6.8 . The diffusion experiments were carried out in seven-chamber diffusion cells at 32 ° C and the donor and recipient phases were separated by a membrane with a diffusion area of 0.64 cm 2 (9 mm 0). About 1 g of the emulsions (donor phase) was spread evenly across the membranes. The cells were covered with parafilm to prevent evaporation. The buffer flowed through the pump (IsmatecIPN-16, L852) with a flow rate of 2 ml / h. Samples were collected every two hours for 12 hours and were analyzed using a spectrophotometer (Varian Carry 50Bio) at the detection wavelength for methyl salicylate (302 nm).
    In vitro skin penetration
    During in vitro skin penetration, the steady-state flow was about 8 qg / (cm2 * h) for all three formulations. This flow is almost twice as high as what was previously observed in a configuration
    59/79 similar experiment with the use of buffer solutions of the same concentration of methyl salicylate. This indicates that it was the presence of the emulsion system that increased penetration through the skin. Initially, the flow of penetration decreased over time (Figure 5-1), which could be due to the depletion of the oil droplets closest to the skin. In high viscosity systems like the one used, the diffusion of oil droplets is difficult and, thus, there will be a concentration gradient and a steady state region formed.
    Conclusions in view of Experiment 9
    There were no differences in skin penetration in vitro between the three used oils, which indicates that the system, as such, provided a very high penetration of 8 pg / (cm 2 * h). Therefore, similarities in terms of, for example, oil droplet size and particles used for stabilization were more important than the rheological properties and the individual properties of these rather unequal oils (see experiment 8) for the use of starch pickering emulsions as a topical drug delivery system.
    Experiment 10
    In experiment 10, the control of the flow and rheology properties of starch granule stabilized emulsions by changing the ratio of starch to oil is shown. Starch was isolated from Quinoa (Biofood, Sweden) by a wet grinding process and modified by OSA to 2.9% (as described in experiment 1). The continuous phase of the emulsions was 5 mM phosphate buffer with pH 7 with 0.2 M NaCl, and the dispersed phase was Miglyol 812. Emulsions were prepared using a Ystral high shear mixer at 22,000 rpm per 30 sec. Droplet size distributions were determined using laser diffraction, as described in experiment 1, and are shown in table 6-1 as the mean D 32 of surface and mode of distribution.
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    Emulsion samples for rheological characterization were prepared to contain the same total amount of dispersed phases (oil and starch together represent 40% of the emulsion) in ratios of three starches to oil: 143 mg / ml of oil (366 mg of starch and 2 , 56 ml of oil), 214 mg / ml of oil (526 mg of starch and 2.46 ml of oil) and 1,143 mg / ml of oil (1,841 mg of starch and 1.61 ml of oil), the entire emulsion had 4.2 ml of buffer, making a total of 7 ml. This amount was chosen to fill the space completely. All samples were prepared and measured in duplicates.
    Rheological Measurements
    Rheological measurements were performed on a rheometer (Malvern Kinexus, England) 24 h after preparation. The characteristics of the emulsions were analyzed at a temperature of 25 ± 0.1 ° C using a geometry from plate to sawn plate (upper plate 40 mm in diameter, lower plate 65 mm in diameter, span height of 1, 0 mm). All experiments were performed on duplicate samples. Oscillatory measurements were performed in order to determine the linear viscoelastic region of the sample (amplitude sweep). The phase angles, shear viscosity (η, Pa s), storage modules (G *, Pa) θ loss (G, Pa) were investigated. Oscillatory tests were performed in the shear stress range of 0.001 to 1,000 Pa at a frequency of 1 Hz.
    Rheology results
    All three samples exhibited a viscoelastic behavior that has a short linear elastic region over a resistance range from 0.0001 to 0.002 followed by a rapid decrease as the structure was divided. The shear dependence of the elastic modulus of the three emulsions tested is shown in Figure 6-1. These particular starch to oil ratios were chosen as
    61/79 below, at the neutral buoyancy starch concentration of 214 mg / ml of oil and well above it (as discussed in Experiment 1). The rheological properties in the linear region and the shear stress at the 45 ° phase angle (the point at which the structure divides) was measured and is shown in Table 6-1 for the 3 conditions tested. The elastic modulus G 'is a measure of the amount of energy from the oscillating shear stress that can be stored in the sample structure and is a function of the strength and the number of interactions between the components of the emulsions. As expected, the emulsion with the highest starch to oil ratio also has the largest elastic modulus since there was more surface interaction in the emulsion as well as there is both a small droplet size and excess starch. However, there may be several contributions to the higher modulus of the smaller droplet emulsion. With the increasing total surface of the dispersed phase, the attractive interactions seen in the aggregation of the starch granules would be more prominent. The higher Laplace pressure of smaller droplets leads to less droplet deformability and thus to a higher modulus. Furthermore, as the modules in constant sum of the dispersed phase volumes of oil and starch are
    compared, the system if become more rigid with the sharing growing From granules of starch completely not deformed.
    Conclusions in view of Experiment 10
    The emulsions produced by high shear homogenization had the droplet size 9 to 70 pm depending on the ratio of starch to oil. The rheological characterization indicated a gel structure with an elastic modulus in the range of 200 to 2,000 Pa depending on the droplet size. This is a useful attribute that allows you to adjust flow properties without adding viscosity modifiers.
    62/79
    Table 6-1: Rheological properties of starch-stabilized emulsions in various starch to oil ratios
    143 mg of starch / ml 214 mg of starch / ml 1143 mg of oil oil starch / ml of oil
    G ' u (Pa) in linear region 223 + 58.6 423 ± 12.7 2570 ± 69.4 Gt(Pa) in region 9.81 ± 3.24 20.4 ± 1.92 352 ± 38.6 linear (Pa s) in 35.6 ± 9.32 67.4 ± 2.04 415 ± 14.5 linear regionY * (resistance)at the phase angle 4.47 ± 1.01 2.55 ± 0.0667 0.761 ± 0.0263 45 °G '(Pa) at angle45 ° phase b, b ± U, / 5 b 81.1 ± 4.40 220 ± 26, 7 d 32 (pm) 13.8 ± 0.831 10.2 + 0.591 5.73 + 0.919 Mode (μπι) 33, 7 25, 9 9 , 6 Í5
    mean ± standard deviation, n = 2.
    Experiment 11
    In Experiment 11, the ability of starch granules to stabilize the external phase of double emulsions (W / O / W) was studied and the encapsulation efficiency of such double emulsions was demonstrated.
    An internal continuous emulsion of Ei oil was produced by emulsifying an aqueous phase consisting of 1.4 ml 0.1 M NaCI solution with 1.4 pL of household red food coloring (Ekstroms / Procordia, Eslov, Sweden) , in an oily phase consisting of 5.6 ml of Miglyol and 0.28 g of polyglycerol polyricinoleate surfactant (Grindstedt PGPR90, Danisco, Copenhagen Denmark) using a 6 mm Ystral Mixer with dispersant tool at 24,000 rpm for 10 minutes. The resulting Ei emulsion had a droplet size of 1.17 ± 0.13 pm (D 43 ± standard deviation), as measured by Malvern Mastersizer 2000S.
    Double Pickering emulsions were prepared with 20% internal Ei emulsion and 80% continuous phase (5 mM phosphate buffer with pH 7.0 0.2 M NaCI) containing 214 mg / ml of 1.8 % OSA modified quinoa starch
    63/79 in the Ystral X10 mixer at 22,000 rpm for 30 seconds.
    The resulting double emulsion had a droplet size of 48 ± 10 pm (D 43 ± standard deviation).
    The encapsulation stability of the double emulsion during storage was evaluated spectrophotometrically at 520 nm of the leakage of the dye in the external aqueous phase after different times as shown in Table 7-1.
    Table 7-1: Dye leakage in the external aqueous phase (5) after different storage times. (% leakage and standard deviation)
    Timestorage (days) in (%) Leak SD 0 0.14 0, 20 7 0.21 0, 19 14 0.37 0.16 21 0.49 0.17 30 1.00 0.23
    Conclusions in view of Experiment 11
    The successful use of starch granules to stabilize double emulsions has been demonstrated. The encapsulation efficiency of the emulsions was studied and remained excellent during storage. Such dual emulsions could be suitable for encapsulating water-soluble substances in food and pharmaceutical formulations.
    Experiment 12
    In Experiment 12, the excellent stability of starch-stabilized emulsions and double emulsions for freezing and thawing was studied.
    Experimental
    Small granular OSA modified starch prepared as in Experiment 1 was used. The continuous phase was a 5 mM phosphate buffer with pH 7 with 0.2 M NaCI, the dispersed phases were Miglyol 812 medium chain triglyceride oil (Sasol, Germany) or chestnut butter
    64/79 (solid at room temperature). The emulsions were prepared in glass tubes with a total volume of 6 ml based on 2 different recipes (7% and 33% oil) and 214 mg of starch per ml of oil. After adding starch to the tube plug, it was added and mixed for approximately 5 seconds using a vortex mixer (VM20, Chiltern Scientific Instrumentation Ltd, UK). Therefore, the oil was added and mixed with a Ystrol mixer (D-79282, BallrechtenDottingen, Germany) at 11,000 rpm for 30 seconds. Chestnut butter was melted in a water bath before emulsification. Some of the emulsions were then thermo-treated at 70 ° C for 1 minute in a water bath. The emulsions were stored at room temperature for 24 hours before further experiments. The emulsion samples were frozen in aluminum trays by stapling the trays in liquid nitrogen before storage in the freezer. The samples were produced in duplicates to study reproducibility. The samples were thawed the next day for additional particle size analysis and format analysis (microscopy) as described in Experiment 1. For unheated emulsions with 7% Miglyol samples, a second freezing method was evaluated using a rapid laboratory freezer (Frigoscandia, Sweden).
    The particle size distribution of emulsions before freezing and after thawing was analyzed as described in Experiment 2 and using microstructure imaging as described in Experiment 1.
    The double emulsions were prepared as described in Experiment 11 with the difference that Miglyol oil was replaced with chestnut butter. The freeze / thaw stability of emulsions
    65/79 pairs were analyzed as described above using the liquid nitrogen freezing method.
    Results
    The emulsions were stable for freezing and thawing, D 43 before freezing starch-stabilized emulsions with 7% Miglyol was 50.5 pm, after rapid freezing and thawing D 43 was 49.8 and after freezing in liquid nitrogen and thawing, 56.9 pm. The preserved drop shape was clearly seen under the microscope (see Figure 7-1). The heat treatment caused a slight increase in droplet size due to starch swelling and partial gelatinization and also increased droplet aggregation.
    The double untreated emulsions also showed excellent stability for freezing and thawing (Figure 7-2), although the drop aggregation was increased as seen from the particle size distribution curves (Figure 7-3). For thermo-treated double emulsions, the drop size distribution was undoubtedly unaffected by freezing and thawing although it indicated a collapse of the larger droplets (Figure 7-3).
    Freeze / thaw stability is important for product quality as products are exposed to a temperature range, etc.
    Conclusions in view of Experiment 12
    Starch-stabilized double emulsions and emulsions could be frozen and thawed with preserved emulsion droplet structure. The use of different oil phases, heat-treated or unheated emulsions or different freezing methods, all produced emulsions with highly acceptable freeze / thaw stability.
    66/79
    Experiment 13
    In Experiment 13, the emulsions were dried, producing an oil-laden powder.
    Experimental
    Freeze drying: The emulsions were prepared and frozen as described in Experiment 12. The sample trays were covered with perforated aluminum paper. The emulsions used contained Miglyol oil or chestnut butter as a dispersed phase at concentrations of 7% (untreated or thermotreated) or 33% of untreated. The frozen samples were transferred to a laboratory freeze dryer (Labconco Freeze Dryer, Ninolab USA). The freeze dryer was pre-cooled to -50 ° C and the samples were dried for 52 h.
    Spray drying: The emulsions were prepared by mixing small granular starch and buffer as in Experiment 1 with spiced chestnut butter using a Sorval Mixer at 1,800 rpm for 5min. The proportions used were 7% oil and 600 mg of starch / g of oil. The emulsions were thermo-treated at 70 ° C for 1 minute. The inlet temperature of the spray dryer was 130 ° C and the pump speed was set at 50.
    The particle size distribution of emulsions before freezing and after drying was analyzed as described in Experiment 2 and using microstructure imaging as described in Experiment 1. The dry powder was analyzed after rehydration in buffer. The dry powders were coated by cathodic sublimation with gold and the images recorded in a scanning electron microscopy (SEM, FegSEM, model JEOL JSM-6700F, Japan) operated at 5 kV and a working distance of 127 in 8 mm.
    Results
    67/79
    Dry emulsions, i.e. powders, were obtained by freeze drying and spray drying. The thermal treatment before drying resulted in the formation of a highly stable cohesive layer of partially gelatinized starch, which increased the stability of the drops during storage and processing. This layer was more important in the case of the dispersed liquid phase, since the physical state of the dispersed phase (liquid / solid) affected the stability of the emulsions through drying. The smaller drops of emulsion were better preserved after drying and rehydration while the larger drops were generally more susceptible to destabilization. The dried emulsion drops showed an increase in overall size distribution due to partial aggregation.
    The intact drops of thermo-treated emulsions containing liquid oil (Miglyol) were obtained after drying (see Figure 8-1). A cohesive starch layer was obtained by heat treatment that protects the oil droplets during freeze drying. The partial collapsed drops left empty starch pockets. There was a wide variation in drop size and some aggregation. Untreated emulsions containing liquid oil collapsed during drying. The intact drops of thermo-treated emulsions containing solid oil (chestnut butter) were obtained as seen in Figure 8-2 (unheated emulsion) and in Figure 8-3 (thermally treated before drying). After freeze drying non-thermo-treated emulsions, dry drops were obtained as well as free oil. Starch granules were seen on the surface of the drops. Heat treatment before freeze drying resulted in more drops intact after drying. The intact drops were also obtained by spray drying as seen in Figure 8-4. Spheres covered with oil-laden starch
    68/79 remained intact after spray drying although free starch is also present since starch was added in excess to 600 mg / g of oil.
    The aggregation of droplets, especially after rehydrating dried thermo-treated emulsions before drying was confirmed by the particle size distribution curves (see Figure 8-5). The particle size distribution curves showed similar results for freeze-dried emulsions and double freeze-dried emulsions (Figure 8-5) with chestnut butter as an oily phase (the emulsions were thermo-treated before drying).
    Conclusions in view of Experiment 13
    Starch-stabilized emulsions could be dried either by freeze drying or spray drying. The emulsions were more stable for drying when thermo-treated after emulsification causing partial gelatinization starch. This was particularly important when drying liquid oil. The resulting oil-laden powders had many attractive properties including the ability to be easily rubbed into the skin for a smooth feeling with no visible residue. This aspect can be considered useful in many products like cosmetics and topical delivery systems.
    Experiment 14
    In this experiment, the starch barrier was varied by swelling and gelatinization of starch granules after emulsification. The pH-stat method was used as a way to monitor the rate of lipolysis in order to use it as a means of comparing the relative barrier properties between the studied emulsions. Starch swelling and gelatinization occurred during heating in the presence of water.
    69/79
    The digestion of lipids is an interfacial process that involves the interaction of the enzyme lipase and its cofactors with the surface of the droplets so that the enzyme can come in close contact with its substrate. For this reason, the interfacial area, that is, the surface area of the specific emulsion is of importance and is given by:
    ^ 32 where S is the surface area per unit volume of emulsion (m 2 ), Φ is the fraction of volume of oil and D 32 is the average diameter of Sauter. S is used to scale the results of general activity to account for the different amount of surface area in the various samples. The pH-stat method for monitoring the release of free fatty acids (FFAs) to describe the rate of digestion is a well-known in vitro physiochemical method for screening the effects of compositions and structure of food and pharmaceutical products on the rate and extent of digestion lipid. The generation of FFAs is monitored in pH-stat by measuring the consumption of NaOH required to maintain a given pH (in this case, 7.0), the release rate (scaled by the oil's surface area) is the enzyme activity . The quantification of the barrier properties of the starch layer, an easily accessible oil interface (without barrier), is measured by setting the lipase activity to 100%. Next, the relative decrease in activity in starch granule stabilized emulsions is compared with the condition that the rate of NaOH consumption is proportional to the rate of FFA release if scaled by the S interface of the tested emulsion. The lower the lipolysis rate, the better protected the oil is by the partially gelatinized starch layer and the better the barrier properties.
    70/79
    Methods
    The small granular starch was isolated and modified by OSA as described in Experiment 1. The continuous phase was a pH 7 phosphate buffer with 0.2 M NaCI, the dispersed phase was Miglyol 812 medium chain triglyceride oil (Sasol , Germany).
    The assay used in lipolysis was a buffer with 4 mM NaTDC (bile salt), 1 mM Tris-Maleat, 1 mM CaCl2 and 150 mM NaCI. Lipase and colipase were used as enzymes for digestion of the oil phase.
    Emulsification
    The emulsions were prepared in glass test tubes with 2.7 ml of the continuous phase, 0.3 ml of the oil phase and 22.5 to 180 mg of starch by mixing with a Ystrol (D79282, Ballrechten-Dottingen, Germany) at 22,000 rpm for 30 seconds. A second set of emulsions was prepared in the same way using 7% oily phase and 214 mg of starch per ml of oil for heating at different temperatures.
    Emulsion heat treatments
    The first set of emulsions was thermo-treated in a 73 ° C water bath. The samples were kept above 70 ° C for 1 minute and the total warm-up time was approximately 3 minutes. After the samples had cooled to 40 ° C, the emulsions were stirred in a vortex mixer for 5 seconds. The second set of emulsions was thermo-treated as described at temperatures ranging from 45 to 100 ° C.
    Particle size measurements
    The particle size distribution of starch particles and emulsion droplets was measured as described in Experiment 1 for varying starch concentrations and as in Experiment 2 for varying temperatures. The drop size was measured both before and
    71/79 after lipolysis.
    PH-stat methods
    Lipase and colipase activity was determined by pH-stat titration using a TIM854 model radiometer (Analytical SAS, Cedex, France). The sample, emulsion or control was mixed with 15 ml assay buffer and 3 μΐ of each of the solutions containing lipase (1 mg / ml) and colipase (1 mg / ml). The pH was maintained at 7.0 by titrating 0.1 M NaOH and consumption (pmol / minutes) in 18 minutes was taken as the lipase and colipase activity. Lipase activity was determined as the amount of NaOH added to maintain the pH at 7 during lipolysis as the FFAs released by lipase lowered the pH. The average release of FFAs per minute between 15 and 18 minutes after adding the enzymes was used as the rate of lipolysis.
    Preparation of Controls
    The activity of the oil without the presence of starch was controlled with the use of Tween 20 stabilized emulsions. An appropriate amount of Tween 20 was used to produce drops of oil in the size range like the stabilized drops of starch, ie 10 to 20 pm. The effect of the heat treatment of the emulsions was controlled with the use of an unheated emulsion with the same composition as the corresponding heated emulsions. In addition, a buffer and starch control
    phase continuous, heated like the emulsions, was used for check the activity of the starch.Microscopy Inspection and imageamentc > da microstructure of emulsions were made before and after The microscopy in
    heat treatment as described in Experiment 1, with the modification that the light was also transmitted using a polarization filter (U-ANT, Olympus) and that the samples were placed on a microscopic slide and studied
    72/79 immediately without cover glass. The images were processed using the image processing program
    Java ImageJ (versionResults 1.42m). in view of Experiment 14 amount0 size decreased drop with a increased of starch added (see table 8-1) and the size of gout was not affected by lipolysis after 30 minutes. Table 8-1 . Drop size and activity of lipase
    for heat treated emulsions
    Starch (mg / ml ofoil) D32 (pm) Specific surface area Activity(pmol / minute) Activity / S Activity / S (% of max.) 75 47, 54 6.33E-03 5.31 E-05 8.39E-03 68% 150 39.00 7.69E-03 4.04E-05 5.25E-03 43% 225 33.49 8.96E-03 4.65E-05 5.19E-03 42% 300 28, 15 1.07E-02 4.86E-05 4,56E-03 37% 600 22.21 1.35E-02 5.73E-05 4.24E-03 34%
    The unheated emulsion had no effect on lipolysis compared to the emulsion with Tween20 as an emulsifier and drop stabilizer. The lipase activity decreased only in the heated emulsions due to the partial gelatinization of the starch granules as can be seen in Figure 9-1. The gelatinized granules created another impermeable layer around the droplets that differ from the distinct granules on the droplet surface in the unheated emulsions. However, the granules were not completely gelatinized during the heat treatment, which is shown by the polarized pattern of the starch closest to the drop interface in Figure 9-1 (bottom figure). Although the threshold between the individual granules becomes diffuse, a certain degree of particle remains intact at the oil interface. This could result in a particle stabilization mechanism maintained at the same time that it achieves a dense cohesive outer layer in the aqueous phase that gives rise to the enhanced barrier properties observed in the thermo-treated emulsions stabilized by starch. That
    3/79 intensification of the barrier increased with the temperature range studied as seen by decreased lipase activity (see Figure 9-2).
    Conclusions in view of Experiment 14
    The barrier of starch granules at the drop interface can be enhanced by heating the emulsion and thereby partially gelatinizing the granules. This intensified barrier prevents the lipase from reaching and digesting the oil. Lipase activity decreases by at least 60% compared to activity in an unheated emulsion, indicating that heating can achieve a cohesive starch layer that is useful for enhancing or adjusting barrier properties for encapsulation applications.
    Experiment 15
    In Experiment 15, the encapsulation of different substances in emulsions stabilized by starch and double emulsions is demonstrated.
    Experimental
    Small granular OSA modified starch prepared as in Experiment 1 was used. The continuous phase was a pH 7 5 mM phosphate buffer with 0.2 M NaCI. The oil phase and the emulsification method are described for each encapsulated substance below.
    Methyl salicylate (encapsulation in the oily phase) Methyl salicylate is used in pharmaceuticals as an analgesic, but it is also used in foods as a flavoring agent since it has a mint taste and smell. However, it is quite toxic, LD50 = 500 mg / kg for adult humans and is therefore used in very low concentrations. The aromatic nature of the substance makes it possible to detect by photo-spectroscopy at a wavelength of 302 nm.
    Methyl salicylate (CAS No. 1 19-36-8) was
    74/79 dissolved in chestnut butter during revolution at 50 ° C using a concentration of 50 L / g of oil. The starch (500 mg / g of oil), the buffer and the melted brown butter (33%) with methyl salicylate were then emulsified in batches of 50 g using a Sorvall mixer (level 8, 2 minutes) in a water bath at 40 ° C. The additional emulsions were freeze-dried as described in Experiment 13. Methyl salicylate was also encapsulated in the oil phase using three different oils and emulsified as described in Experiment 9. The encapsulated substance did not change the drop size distribution or appearance visual of the drops.
    Flavor (encapsulation in the oil phase)
    The starch (500 mg / g), the buffer and the brown butter (56%) with a few drops of a common almond flavoring agent for food use were emulsified using a Sorvall mixer as described above. The resulting emulsion had cream properties as described in Example 8 and had an almond flavor that was most apparent when the cream was applied to the skin. After 1 week of storage, the almond aroma was still detectable, although with reduced intensity.
    Penicillin (encapsulation in the internal aqueous phase of a double emulsion) active ingredient in Kâvepenin, phenoxymethylpenicillin (penicillin V), is a penicillin (antibacterial drug) that prevents bacteria from building a normal cell wall. The double emulsions were prepared as described in Experiment 11 with the modification that the starch concentration was 500 mg / ml of oil and that Kâvepenin was added to the internal aqueous phase at a concentration of 62.5 mg / ml. The emulsions were then centrifuged at 1,000 g for 5 minutes (Beckman Coulter,
    75/79
    Allegra (R) X-15R, L 284, England, the aqueous phase was removed and the emulsion washed with 5 ml of buffer. This procedure was repeated 5 times. As demonstrated earlier in Experiment 11, it is possible to produce double emulsions with a high degree of encapsulation efficiency and low leakage. For this reason, washing emulsions is useful to remove the small amount of internal aqueous phase that may have leaked during the initial emulsion step that causes unpleasant flavors or odors. This is particularly useful for bitter-tasting oral antibiotics, especially in liquid formulations for children where consent is a major problem. This aspect is further demonstrated in Experiment 16. The drop size was not changed by the washing procedure (D 43 was initially 30.4 pm, after washing 1: 30.4, washing 2: 42.5, washing 3: 34 , 7, wash 4: 42.9 and wash 5: 41.6 pm).
    The colorants (encapsulation in the internal aqueous phase of a double emulsion) Different colorants were encapsulated in the internal aqueous phase of double emulsions. A food coloring was encapsulated as described in Experiment 11 which shows excellent encapsulation efficiency and storage stability. These emulsions were further frozen in liquid nitrogen and thawed as described in Experiment 12 or freeze dried as described in Experiment 13 with an acceptable degree of encapsulation maintained. Coomassie Blue was encapsulated using the same method, but with a starch concentration of 500 mg / ml of oil. The encapsulated substance did not alter the drop size distribution or the visual appearance of double emulsion drops.
    Vitamin B12 was also encapsulated using the method described for Coomassie blue.
    Conclusions in view of Experiment 15
    76/79
    The substances could be effectively encapsulated in the oil phase of emulsions with good stability. Water-soluble substances could be encapsulated in double emulsions with starch particles that stabilize the external emulsion. These experiments show the suitability of emulsion drops stabilized by starch granules for encapsulation of active ingredients or substances in food and pharmaceutical products.
    Experiment 16
    In Experiment 16, a method to achieve a strange taste suppression was studied in double emulsions with encapsulated penicillin and in heated and unheated emulsions using fish oil as the dispersed phase. Fish oil contains omega 3 fatty acids and is generally considered to have health benefits although highly susceptible to oxidation which causes a strange taste. Penicillin is also known to cause a strange taste as well as a highly detectable bitter taste.
    Experimental
    Penicillin
    The double emulsions stabilized by starch with Penicillin (Kâvepenin) were prepared and washed as described in Experiment 15. A sensory analysis was performed before and after washing. Sensory parameters were assessed by a small amount of the double emulsion applied to the tongue and then swallowed. A standard sensorial curve was produced only with buffer and Kâvepenin in different concentrations to detect the sensory limit of the evaluator.
    OSA modified small granular fish oil prepared in Experiment 1 was used. The continuous phase was a pH 7 5 mM phosphate buffer with 0.2 M NaCI. THE
    77/79 oily phase was a commercial fish oil (Eskimo-3 Pure, Green Medicine ΆΜ, Malmo, Sweden). Emulsification using 500 mg of starch / ml of oil and 10% oily phase was performed as described in Experiment 1. Some of the emulsions were subsequently thermo-treated in a 70 ° C water bath for 1 minute. The emulsions were sealed and stored at 5 ° C for 1 week. The stability of the emulsion was observed immediately after sample preparation and one week later. Particle size distribution and microscopy analysis was performed as described in Experiment 2. A sensory analysis was performed by one person. Sensory parameters were evaluated from a small amount of the emulsion applied to the tongue and then swallowed.
    Penicillin Results
    The limit of voluntary detection of Kávepenin in buffer was below 10 mg / ml according to the standard curve. No flavor from Kávepenin was detected from double emulsions containing approximately 6 times that concentration. Washing did not result in any difference in the taste of the double emulsion.
    Fish oil
    Starch stabilized emulsions were formed (see Figure 10-1) and the emulsion drops were stable for heat treatment and storage. The untreated emulsions were white, whereas the thermo-treated emulsions were slightly yellow in color before and after storage. Storage for 1 week did not change the particle size distribution. The unheated emulsion tasted very strongly of fish oil. The heated emulsion had a moderate taste with respect to fish oil.
    Conclusions in view of Experiment 16
    78/79
    Strange taste suppression was demonstrated and highly effective when the penicillin was encapsulated. Starch-stabilized emulsions could also be produced with fish oil. The fish oil did not negatively affect the stability of emulsions. Starch-stabilized emulsions may be suitable for encapsulating ingredients or substances with undesirable taste in food and pharmaceutical products.
    Experiment 17
    In Experiment 17, starch granules to stabilize foam were used. The oily phase in this Experiment was brown fat (AAK, Sweden), the starch granules were isolated from quinoa and made more hydrophobic by modification by OSA by 2.9% (as described in Experiment 2) and the continuous phase was buffer of 5 mM phosphate at pH 7 and 0.2 M NaCI. The chestnut butter was melted at 60 ° C before homogenization in a Sorvall Omni mixer at level 8 for 5 minutes using a 300 ml dispersion unit. The larger container allowed air to be sucked into the liquid phases during mixing by a vortex on the liquid surface. In this way, both stabilized particle bubbles and droplets were formed.
    Result of Experiment 17
    A rigid foam-like structure was produced with a similar density of whipped cream. It was solid and could be cut into a piece shown in Figure 11-
    1. This foam was also unchanged after more than a month of storage.
    Conclusions in view of Experiment 17
    The successful use of starch granules to stabilize the foam has been demonstrated. The resulting structure could be attractive in a variety of applications
    79/79 food and cosmetics.
    权利要求:
    Claims (13)
    [1]
    1. EMULSION OR FOAM STABILIZED BY PARTICLE, characterized by comprising at least two phases and solid particles, said solid particles being granules of starch and said granules of starch or, a portion thereof, are located at the interface between the two phases that provide the particle-stabilized emulsion or foam, where the starch granules have a small granular size in the range of 0.2-8 microns and where the amount of added starch granules covers more than 10% of the surface of a emulsion droplet.
    [2]
    2. EMULSION OR FOAM STABILIZED BY PARTICLE, according to claim 1, characterized in that the starch granules have undergone a physical modification and / or chemical modification.
    [3]
    3. EMULSION OR FOAM STABILIZED BY PARTICLE, according to claim 2, characterized by the physical modification being carried out by dry heating or by other means that partially denature the surface proteins.
    [4]
    4. EMULSION OR FOAM STABILIZED BY PARTICLE, according to any one of claims 2 to 3, characterized in that the chemical modification is carried out by treatment of alkenyl succinyl anhydride or by grafting with other chemicals with a hydrophobic side chain.
    [5]
    5. EMULSION OR FOAM STABILIZED FOAM, according to any one of claims 1 to 4, characterized in that the starch granules preferably have a small granular size in the range of 0.2 to 4 microns, more preferably 0.2 to 1 micron.
    [6]
    6. EMULSION OR FOAM STABILIZED BY PARTICLE, according to any one of claims 1 to 5, characterized in that the starch granules are obtained from
    Petition 870190012161, of 05/02/2019, p. 13/18
    2/3 from any botanical source.
    [7]
    7. EMULSION OR FOAM STABILIZED BY PARTICLE, according to claim 6, characterized in that the starch granules are obtained from quinoa, rice, plus, amaranth, barley, unripened sweet corn, rye, triticale, wheat, buckwheat, tifa , filipendula, durian, teff, oats, parsnip, small millet, wild rice, birdseed,
    paw of cow, yam and taro including varieties waxy and with high content amylose From above. 8. EMULSION OR FOAM STABILIZED BY PARTICLE, in wake up with any an of claims 1 to 7, characterized by at any less two phases be chosen The
    from oil-based phase / water-based phase, and gas phase / water-based phase, such as an oil-in-water emulsion or a water-in-oil emulsion.
    [8]
    9. EMULSION OR FOAM STABILIZED BY PARTICLE, according to any one of claims 1 to 9, characterized in that said particle stabilized emulsion is subjected to a heat treatment.
    [9]
    10. DRY PARTICLE STABILIZED EMULSION OR FOAM, characterized in that a particle stabilized emulsion or foam as defined in any of claims 1 to 9 has been subjected to water removal as through drying.
    [10]
    11. USE OF A PARTICLE STABILIZED EMULSION, as defined in any one of claims 1 to 10, characterized in that it is for controlling the density of emulsion droplets.
    [11]
    12. USE OF A PARTICLE STABILIZED EMULSION, as defined in any one of claims 1 to 11, characterized in that it is for the encapsulation of substances chosen from biopharmaceuticals, proteins, probiotics, living cells, enzymes, antibodies, ingredients
    Petition 870190012161, of 05/02/2019, p. 14/18
    3/3 food, vitamins, and lipids.
    [12]
    13. USE OF A PARTICLE STABILIZED EMULSION, as defined in any one of claims 1 to 11, characterized by being in food products, products
    5 cosmetics, skin creams, lotions, pharmaceutical formulations.
    [13]
    14. FORMULATION, characterized in that it comprises a dry particle-stabilized emulsion as defined in claim 11 and a substance chosen from
    10 biopharmaceuticals, proteins, probiotics, living cells, enzymes, antibodies, food ingredients, vitamins and lipids.
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    同族专利:
    公开号 | 公开日
    CN103402370B|2015-07-15|
    DK2651243T3|2018-08-27|
    AU2011341738A1|2013-06-13|
    ZA201305229B|2014-09-25|
    CA2819581C|2019-06-18|
    EP2651243B1|2018-06-27|
    EP2651243A1|2013-10-23|
    US20150125498A1|2015-05-07|
    WO2012082065A1|2012-06-21|
    JP2014505673A|2014-03-06|
    BR112013014574A2|2017-09-26|
    CN103402370A|2013-11-20|
    CA2819581A1|2012-06-21|
    JP6046634B2|2016-12-21|
    ES2686145T3|2018-10-16|
    AU2011341738B2|2015-08-20|
    EP2651243A4|2014-08-20|
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    法律状态:
    2017-12-12| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-05-02| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-06-05| B15K| Others concerning applications: alteration of classification|Ipc: A23D 7/005 (2006.01), A23L 27/00 (2016.01), A23L 3 |
    2018-10-30| B07B| Technical examination (opinion): publication cancelled [chapter 7.2 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 7.4 NA RPI NO 2469 DE 02/05/2018 POR TER SIDO INDEVIDA. |
    2018-11-13| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2019-09-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2019-10-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/12/2011, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/12/2011, OBSERVADAS AS CONDICOES LEGAIS |
    优先权:
    申请号 | 申请日 | 专利标题
    SE1051328|2010-12-15|
    SE1051328-1|2010-12-15|
    PCT/SE2011/051522|WO2012082065A1|2010-12-15|2011-12-15|New particle stabilized emulsions and foams|
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