Clouding emulsion for a beverage

Abstract

A clouding emulsion for a beverage comprising from 80% to 99% w / w of an aqueous phase and from 1% to 20% w / w of acid stable oleosomes having an average particle size of from 0.1 μm to 5 μm, wherein the clouding emulsion is present in an amount of 0.00001% to 5% w / w, based on the total weight of the beverage. Methods for making the clouding emulsion and beverages containing the clouding emulsion are also disclosed.

Description

[0001] CLOUDING EMULSION FOR A BEVERAGE

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of beverage emulsions, more particularly to beverage clouding emulsions suitable for acidic beverages, beverages comprising such emulsions, particularly acidic beverages comprising such emulsions, and processes for the preparation of such emulsions.

[0004] BACKGROUND

[0005] A large proportion of soft drinks are “cloudy” beverages exhibiting a turbid appearance, which contain juice or exhibit a juice-like appearance. As juice is expensive, emulsion-based systems are often used, which contain oil droplets in a specific size range that scatter the light, thereby increasing the turbidity of the beverage and providing a juice-like visual impact.

[0006] Beverage emulsions consist of a continuous aqueous phase in which a discontinuous oil phase is dispersed in the form of microscopic droplets. They are thus characterised as oil- in-water (O / W) emulsions. Emulsions are thermodynamically unstable systems that tend to breakdown over time due to various physicochemical mechanisms, such as, creaming, sedimentation, coalescence, flocculation and Ostwald ripening. Common manifestations of beverage emulsion deterioration are “ringing” and “sedimentation”. Ringing is the formation of a ring around the neck of the container, whilst sedimentation is the precipitation of material at the bottom.

[0007] Due to demanding performance and stability requirements, beverage emulsions need to be carefully designed. They must be stable, both as a concentrate and when used in a fully formulated product, over a wide range of temperatures and storage conditions, whilst also being straightforward to formulate and incorporate into beverages. To achieve a stable system having the required oil droplet size and turbidity, conventional emulsifiers (e.g., gum acacia or modified starch) and weighting agents (e.g., sucrose acetate isobutyrate (SAIB), ester gum, damar gum, and brominated vegetable oil (BVO)) are needed. However, these ingredients are not clean-label and / or have poor consumer acceptance.

[0008] There remains a need to provide clean-label, acid-stable clouding emulsions and emulsifiers for beverages. Clean label generally refers to making a product using as few ingredients as possible, and making sure that consumers recognize these ingredients as being safe. Since one of the aims of the invention is to use a clean label cloudifier, conventional non-clean label cloudifiers, emulsifiers and weighting agents are avoided.

[0009] Beverages and beverage emulsions have an acidic pH due to the added acid (e.g., citric acid, lactic acid, etc.) to achieve the required taste impact. An acidic pH also provides microbiological stability to beverages and beverage emulsions. However, the use of oleosomes at acidic pH levels as a cloudifier and flavour emulsion for beverages has not been described so far.

[0010] SUMMARY OF ILLUSTRATIVE EMBODIMENTS

[0011] According to certain illustrative embodiments, disclosed is a clouding emulsion for a beverage comprising about 80% to about 99% w / w of an aqueous phase and about 1% to about 20% w / w of acid stable oleosomes having an average particle size of about 0.1 pm to about 5 pm.

[0012] According to certain illustrative embodiments, disclosed is a method of clouding a beverage, the method comprising i) preparing an emulsion comprising about 80% to about 99% w / w of an aqueous phase and about 1% to about 20% w / w of acid stable oleosomes having an average particle size of about 0.1 pm to about 5 pm; and ii) adding the emulsion to a beverage base in an amount of about 0.00001% to about 5% w / w, based on the total weight of the beverage.

[0013] According to certain illustrative embodiments, disclosed is a method for making a clouding emulsion for a beverage, the method comprising i) forming an aqueous mixture comprising plant material and water at a ratio of 1 :3 to 1 : 10, ii) adjusting the pH of the mixture to an alkaline pH; iii) comminuting the mixture; iv) removing insoluble components from the mixture and optionally separating an oleosome-containing top / cream layer from a protein-rich middle layer; v) forming a homogeneous slurry of the mixture; vi) reducing the pH of the mixture to an acidic pH; vii) optionally adding a flavor to the mixture and viii) adjusting the average size of oleosomes in the mixture to about 0.1 pm to about 5 pm.

[0014] According to certain illustrative embodiments, disclosed is a beverage product comprising a consumable beverage base and a clouding emulsion comprising about 80% to about 99% w / w of an aqueous phase and about 1% to about 20% w / w of acid stable oleosomes having an average particle size of about 0.1 pm to about 5 pm, wherein the emulsion is present in an amount of about 0.00001% to about 5% w / w, based on the total weight of the beverage.

[0015] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0016] The following text sets forth a broad description of numerous different, illustrative embodiments of the present disclosure. The description is to be construed as exemplary only and does not describe every possible embodiment since describing every possible embodiment would be impractical, if not impossible. It will be understood that any feature, characteristic, component, composition, ingredient, product, step or methodology described herein can be deleted, combined with or substituted for, in whole or part, any other feature, characteristic, component, composition, ingredient, product, step or methodology described herein. Numerous alternative embodiments could be implemented, using either current technology or technology developed after the filing date of this patent, which would still fall within the scope of the claims. All publications and patents cited herein are incorporated herein by reference.

[0017] The terms “comprises,” “comprising,” “has,” “having,” “includes,” “including,” “contains,” “containing,” or any other variation are open-ended and are intended to cover a non-exclusive inclusion of elements, such that an article, apparatus, compound, composition, combination, method, or process that “comprises,” “has,” or “includes,” or “contains” a recited list of elements does not include only those elements but may include other elements not expressly listed, recited or written in the specification or claims.

[0018] The terms “a” and “an” are defined as one or more unless expressly stated otherwise or constrained by other language herein. An element or feature proceeded by “a” or “an” may be interpreted as one of the recited element or feature, or more than one of the element or feature.

[0019] The terms “about,” “approximately,” “essentially,” “substantially,” any other version thereof, or any other similar relative term, or similar term of approximation, are defined as being close to as understood by one having ordinary skill in the art. By way of non-limiting, illustrative embodiments, these terms are defined to be within 10% of a recited value, or defined to be within 5% of a recited value, or defined to be within 4% of a recited value, or defined to be within 3% of a recited value, or defined to be within 2% of a recited value, or defined to be within 1% of a recited value, or defined to be within 0.5% of a recited value, or defined to be within 0.25% of a recited value, or defined to be within 0.1% of a recited value.

[0020] It should be understood that when an amount in weight percent is described in the present disclosure, it is intended that any and every amount within the range, including the end points, is to be considered as having been expressly disclosed. For example, the disclosure of "a range of from about 1 to about 10" is to be read as indicating each and every possible number along the continuum between about 1 and about 10. It is to be understood that the inventors appreciate and understand that any and all data points within the range are to be considered to have been specified, and that the inventors have possession of the entire range and all points within the range.

[0021] For the avoidance of doubt, preferences, options, particular features and the like indicated for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all other preferences, options, particular features and the like as indicated for the same or other aspects, features and parameters of the invention.

[0022] The present disclosure relates to the surprising finding that acid stable oleosomes having an average size of about 0.1 pm to about 5 pm can be utilized as clouding agents in acidic beverage products. As used herein, the terms "acid stable" and “acid-stabilized” refer to the disclosed modified oleosomes which exhibited increased stability against aggregation at the isoelectric point of the membrane proteins of the oleosomes.

[0023] The disclosed clouding emulsions can be used with or without flavor. An additional advantage of the use of emulsions containing flavor is the possibility to provide a high concentration of water insoluble flavor, e.g. terpenes, which significantly improves the overall flavor profile of a beverage compared to water-soluble flavors.

[0024] In certain aspects, the present disclosure relates to clouding emulsions for acidic beverages that are based on acid stable oleosomes having an average size of about 0.1 pm to about 5 pm, methods for making the clouding emulsions, compositions, formulations and products containing the clouding emulsions, and methods for preparing compositions, formulations and products containing the clouding emulsions.

[0025] In one or more embodiments, disclosed is an acid stable clouding emulsion for an acidic beverage comprising about 80% to about 99% w / w of an aqueous phase and about 1% to about 20% w / w of acid stable oleosomes having an average particle size of about 0.1 pm to about 5 pm. In certain embodiments, the clouding emulsion comprises about 1% to about 15% w / w of the acid stable oleosomes, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises about 1% to about 10% w / w of the acid stable oleosomes, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises about 3% to about 9% w / w of the acid stable oleosomes, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises about 5% to about 8% w / w of the acid stable oleosomes, based on the total weight of the emulsion.

[0026] In certain embodiments, the clouding emulsion comprises about 80% to about 95% w / w of an aqueous phase, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises about 80% to about 90% w / w of an aqueous phase, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises about 85% to about 95% w / w of an aqueous phase, based on the total weight of the emulsion.

[0027] According to certain embodiments, the oleosomes comprise an oily core that is surrounded by a phospholipid monolayer having hydrophobic proteins embedded in the phospholipid monolayer. According to certain illustrative embodiments, the oleosomes comprise an oil core containing about 94 weight percent to about 98% weight percent of a triglyceride that is surrounded by 0.6 weight percent to about 2 weight percent of a phospholipid monolayer, and from about 0.6 weight percent to about 4 weight percent of hydrophobic proteins embedded within the phospholipid monolayer surrounding the oil core of the oleosome.

[0028] In certain embodiments, the acid stable oleosomes are derived from one or more of: seeds, pollen, flowers, roots and stems of flowering plants, the spores and vegetative organs of non-flowering plants, algae, microalgae, animal cells, fungi and protists.

[0029] In certain embodiments, the acid stable oleosomes are derived from plant material. The plant material may be selected from the group consisting of a seed, a nut, a kernel, and combinations thereof. The seeds or nuts may be derived from one or more of the following plants, sunflower, soybean, oil palm, safflower, almond, macadamia, cotton seed, ground nut, coconut, rapeseed oil, echium, borage, linseed / flax / hemp, evening primrose, rice, wheat, oat, maize and barley. Particularly preferred are sunflower, soybean, rapeseed oil, flax and maize.

[0030] In certain embodiments, the plant material comprises seeds. By way of illustration, and not in limitation, suitable plant seeds from which oleosomes may be obtained include almond seeds, argan seeds, borage seeds, coconut, corn seeds, cotton seeds, chia seeds, cranberry seeds, flax seeds, grape seeds, hemp seeds, linseed, millet, mustard seeds, oil palm, pumpkin seeds, quinoa, rapeseeds, raspberry seeds, tomato seeds, mango seeds, safflower seeds, sesame seeds, sunflower seeds, soybeans, combinations thereof, and the like. In certain embodiments, the seeds comprise sunflower seeds. In certain embodiments, the clouding emulsion comprises acid stable oleosomes derived from sunflower seeds, wherein the oleosomes contain at least one flavour oil.

[0031] The material containing the oleosomes used in the present disclosure can be from the same source or it can be a mixture from different sources. For example, more than one type of seed could be used, or combinations of seeds and nuts.

[0032] In certain embodiments, the acid stable oleosomes utilized in the clouding emulsion act as emulsifiers. According to this embodiment, the clouding emulsion is free of, or substantially free of, further emulsifiers and / or weighting agents. In certain embodiments, the clouding emulsion is free of, or substantially free of, weighting agents. In certain embodiments, the clouding emulsion is free of, or substantially free of, gum acacia, modified starch, hydrocolloids, pectins, proteins, whey protein, sucrose acetate isobutyrate (SAIB), ester gum, damar gum, and / or brominated vegetable oil (BVO)).

[0033] In certain embodiments, the acid stable oleosomes contain at least one flavor oil. According to this embodiment, the clouding emulsion comprises about 1% to about 20% w / w of at least one flavor oil, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises from about 5% to about 15% w / w of at least one flavor oil, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises from about 5% to about 10% w / w of at least one flavor oil, based on the total weight of the emulsion.

[0034] Flavor oils include spearmint oil, cinnamon oil, oil of wintergreen (methyl salicylate), peppermint oil, Japanese mint oil, clove oil, bay oil, anise oil, eucalyptus oil, thyme oil, cedar leaf oil, oil of nutmeg, allspice, oil of sage, mace, oil of bitter almonds, and cassia oil; useful flavoring agents include artificial, natural and synthetic fruit flavors such as vanilla, and citrus oils including lemon, orange, lime, grapefruit, yuzu, sudachi, and fruit essences including apple, pear, peach, grape, raspberry, blackberry, gooseberry, blueberry, strawberry, cherry, plum, prune, raisin, cola, guarana, neroli, pineapple, apricot, banana, melon, apricot, cherry, tropical fruit, mango, mangosteen, pomegranate, papaya, and so forth.

[0035] Additional exemplary flavors imparted by a flavorant include a milk flavor, a butter flavor, a cheese flavor, a cream flavor, and a yogurt flavor, a vanilla flavor, tea or coffee flavors, such as a green tea flavor, an oolong tea flavor, a tea flavor, a cocoa flavor, a chocolate flavor, and a coffee flavor; mint flavors, such as a peppermint flavor, a spearmint flavor, and a Japanese mint flavor; spicy flavors, such as an asafetida flavor, an ajowan flavor, an anise flavor, an angelica flavor, a fennel flavor, an allspice flavor, a cinnamon flavor, a chamomile flavor, a mustard flavor, a cardamom flavor, a caraway flavor, a cumin flavor, a clove flavor, a pepper flavor, a coriander flavor, a sassafras flavor, a savory flavor, a Zanthoxyli Fructus flavor, a perilla flavor, a juniper berry flavor, a ginger flavor, a star anise flavor, a horseradish flavor, a thyme flavor, a tarragon flavor, a dill flavor, a capsicum flavor, a nutmeg flavor, a basil flavor, a marjoram flavor, a rosemary flavor, a bayleaf flavor, and a wasabi (Japanese horseradish) flavor; a nut flavor such as an almond flavor, a hazelnut flavor, a macadamia nut flavor, a peanut flavor, a pecan flavor, a pistachio flavor, and a walnut flavor; floral flavors; and vegetable flavors, such as an onion flavor, a garlic flavor, a cabbage flavor, a carrot flavor, a celery flavor, mushroom flavor, and a tomato flavor. According to some embodiments, flavorants may also include aldehydes and esters such as cinnamyl acetate ((E)-3-phenylprop-2-en-l-yl acetate); cinnamaldehyde ((2E)-3- phenylprop-2-enal); citral diethylacetal ((E)-l,l-dimethoxy-3,7-dimethylocta-2,6-diene), dihydrocarvyl acetate (2-methyl-5-prop-l-en-2-yl cyclohexyl acetate), eugenyl formate ((2S)- l,3,3-trimethylbicyclo[2.2.1]heptan-2-yl acetate), p-methylanisol (l-methoxy-4- methylbenzene), and so forth can be used. Further examples of aldehyde flavourings include acetaldehyde (apple), benzaldehyde (cherry, almond), anisic aldehyde (4- methoxybenzaldehyde) (licorice, anise), cinnamic aldehyde ((2E)-3-phenylprop-2-enal) (cinnamon), citral (E)-3,7-dimethylocta-2,6-dienal), i.e., alpha-citral ((EE)-3,7-dimethylocta- 2,6-dienal (lemon, lime), neral, i.e., beta-citral ((EZ)-3,7-dimethylocta-2,6-dienal (lemon, lime), decanal (orange, lemon), ethyl vanillin (vanilla, cream), heliotrope, i.e., piperonal (benzo[d][l,3]dioxole-5-carbaldehyde) (vanilla, cream), vanillin (vanilla, cream), alpha-amyl cinnamaldehyde ((E or Z)-2-benzylideneheptanal) (spicy fruity flavours), butyraldehyde (butanal) (butter, cheese), valeraldehyde (pentanal) (butter, cheese), citronellal (3,7- dimethyloct-6-enal) (modifies, many types), decanal (citrus fruits), aldehyde C-8 (octanal) (citrus fruits), aldehyde C-9 (nonanal) (citrus fruits), aldehyde C-12 (dodecanal) (citrus fruits), 2-ethyl butyraldehyde (2-ethyl. butanal) (berry fruits), hexenal, i.e., trans-2 hexenal (berry fruits), tolyl aldehyde (4-methylbenzaldehyde) (cherry, almond), veratraldehyde (3,4- dimethoxybenzaldehyde) (vanilla), 2,6-dimethyl-5-heptenal, i.e., melonal (melon), 2,6- dimethyloctanal (green fruit), and 2-dodecenal (citrus, mandarin), and the like.

[0036] The clouding emulsion may have an acidic pH. In certain embodiments, the clouding emulsion has a pH of about 6 or less. In certain embodiments, the clouding emulsion has a pH of about 5 or less. In certain embodiments, the clouding emulsion has a pH of about 4 or less. In certain embodiments, the clouding emulsion has a pH of about 3.5 or less. In certain embodiments, the clouding emulsion has a pH of about 3 or less. The clouding emulsion may further comprise a phospholipid and / or a free phospholipid which is not derived from the oleosome phospholipid monolayer. According to this embodiment, the clouding emulsion may comprise about 0.1% to about 3% w / w phospholipids and / or free phospholipids, based on the total weight of the emulsion. In certain embodiments, the clouding emulsion comprises about 0.5% to about 1.5% w / w phospholipids and / or free phospholipids, based on the total weight of the emulsion.

[0037] Also disclosed is a method for making a clouding emulsion for a beverage, the method comprising i) forming an aqueous mixture comprising plant material and water at a ratio of 1 :3 to 1 : 10, ii) adjusting the pH of the mixture to an alkaline pH; iii) comminuting the mixture; iv) removing insoluble components from the mixture; v) stabilizing oleosomes in the mixture against aggregation of oleosomes when they are close to the isoelectric point by forming a homogeneous slurry of the mixture comprising free protein and / or free phospholipids; vi) reducing the pH of the mixture to an acidic pH; and vii) adjusting the average size of oleosomes in the mixture to about 0.1 pm to about 5 pm.

[0038] According to certain embodiments, plant material is mixed with water in a ratio between about 1 :4 to about 1 :8 at room temperature. In certain embodiments, plant material is mixed with water in a ratio between about 1 : 5 to about 1:7. In certain embodiments, the method utilizes deionized water. Any food grade base can be used in order to form or maintain alkaline conditions of the aqueous mixture. Examples of bases include sodium hydroxide, sodium carbonate, potassium bicarbonate, and sodium acetate, the like, and combinations thereof. The mixture is then soaked for about one to ten hours at room temperature or overnight (16 hours) at 4°C. Thereafter, the pH of the mixture may be further adapted to an alkaline pH for approximately one hour.

[0039] In certain embodiments the plant material comprises one or more plant seeds that are mixed with water in a ratio of about 1 :7 while stirring with 1 M sodium hydroxide. The plant seeds in the aqueous mixture are soaked for about 4 hours at room temperature. The pH of the mixture is adjusted every hour to maintain alkaline conditions. Upon pH adjustment, a high pH should be avoided, as long exposure to alkaline conditions may lead to undesired oxidating effects. For example, in the regard to sunflower seeds, a pH of higher than 7.8 should be avoided; and in regard to rape seeds, a pH of higher than 9 should be avoided.

[0040] According to certain illustrative embodiments, the plant material in the aqueous mixture is comminuted. This is accomplished by any known process for comminuting a material. For example, and without limitation, the plant material may be comminuted by granulating, mixing, grinding, grating, shredding, mincing, crushing, crumbling, comminuting, pulverizing, stirring, milling, macerating, rolling, blending and like processes. In certain embodiments, the plant material is blended for about 1 to about 5 minutes using a laboratory blender (e.g., Waring Commercial, 400 W, USA). The slurry is recovered and stirred for about one hour while the pH is adjusted to about 7.8 using 1 M NaOH (adjusted every 15 min).

[0041] According to certain illustrative embodiments, the method comprises removing insoluble components from the mixture by, for example, filtration and / or centrifugation. The filtration may be performed by any suitable filtration method, for example, straining through a cheesecloth to remove the solid part of the mixture. According to this embodiment, the remaining liquid in the retentate can be manually squeezed to obtain the final filtrate.

[0042] In one or more embodiments, the method comprises centrifuging the mixture to further remove insoluble components. According to certain illustrative embodiments, the filtrate containing oleosomes and other solutes (e.g., proteins, phenols, etc.) is subjected to centrifugation. The filtrate can be centrifuged at approximately 10,000xg, however different speeds can be used, for 30 min (4 °C) in a suitable centrifugation apparatus, such as Avanti J- 26XP (Beckman Coulters, USA). The centrifugation step results in a three-layer system: i) the oleosome-containing top / cream layer, ii) a protein-rich middle layer, and iii) the pellet bottom layer with insoluble material, for example, remaining plant fibers. In certain embodiments, the top / cream layer is further purified and washed by separating the top / cream layer and mixing it with water and repeating centrifugation. If only the top / cream layer is used, the risk of flocculation and aggregation during the pH reduction step is high. However, it was surprisingly found that if the top / cream layer together with the middle layer containing the protein fraction is used, the emulsion remains stable during the pH reduction step. Without being bound by theory, it is believed that the proteins form a protection layer around the oleosomes preventing oleosome aggregation at the isoelectric point of the membrane proteins of the oleosomes.

[0043] The oleosome-containing cream layer and the protein-rich middle layer can be collected by pouring out the content from centrifuge tubes. The recovered sample is referred to as the “oleosome milk”. The oleosome milk can also be obtained without centrifugation. The disclosed aqueous extraction method can be used to obtain oleosomes at different purities. For instance, oleosome milk (10% w / w dry matter) can be obtained which is high in proteins (18% of dry matter) and oleosomes (69% of dry matter). In addition, oleosome creams can be obtained having high oleosome purity (88% of dry matter) and low protein content (8% of dry matter). It has been found that the presence of free proteins in the clouding emulsion increases the stability of the emulsion and the beverage to which it is added. As used herein, the term “free protein” refers to a protein that is present in the emulsion but is not an oleosome membrane protein, such as oleosins. Fur purposes of illustration, and not by way of limitation, a free protein may comprise storage protein.

[0044] According to certain illustrative embodiments, the method comprises stabilizing oleosomes in the mixture against aggregation of oleosomes when they are close to the isoelectric point by forming a homogeneous slurry of the mixture with free proteins and / or free phospholipids. In certain embodiments, the homogeneous slurry comprises the oleosome- containing cream layer and the protein-rich middle layer, i.e., the oleosome milk. In certain embodiments, phospholipids and / or free phospholipids are added to the oleosome milk. As disclosed herein, it is believed that the proteins and / or phospholipids form a protection layer around the oleosomes thereby preventing their aggregation at the isoelectric point. In certain embodiments, the homogeneous slurry comprises the oleosome-containing cream layer and free phospholipids. As used herein, the term “free phospholipids” refers to phospholipids that are present in the emulsion but are not derived from the oleosome. Without wishing to be bound by theory, it is believed that phospholipids can interact with the proteins, thereby strengthening the oleosomes and reducing the interaction of proteins which reduces aggregation between oleosomes. It has also been found that the addition of phospholipids can increase the membrane density of the oleosomes, leading to more stable droplets. In certain embodiments, the emulsion comprises about 0.1% to about 5% w / w phospholipids, based on the total weight of the emulsion. In certain embodiments, the emulsion comprises about 0.25% to about 4% w / w phospholipids, based on the total weight of the emulsion. In certain embodiments, the emulsion comprises about 0.5% to about 3% w / w phospholipids, based on the total weight of the emulsion. In certain embodiments, the emulsion comprises about 0.75% to about 2% w / w phospholipids, based on the total weight of the emulsion. In certain embodiments, the emulsion comprises about 1% w / w phospholipids, based on the total weight of the emulsion.

[0045] According to further embodiments of the present invention, the phospholipids are selected from the group consisting of phosphatidylethanolamine, phosphatidylcholine, phosphatidyl serine, phosphatidylglycerol, phosphatidylinositol, and combinations thereof. In certain embodiments, the phospholipid comprises phosphatidylcholine. In certain embodiments lecithin comprising a mixture of phospholipids is used.

[0046] In one or more embodiments, the phospholipids are first dispersed in demineralized water at a concentration of about 5% (w / w). The phospholipid dispersion is stirred for approximately 4 hours, followed by overnight (16 hours) hydration. The phospholipid dispersion is mixed with the oleosome-containing cream layer alone, or in combination with the protein-rich middle layer to obtain the desired phospholipid concentration. In certain embodiments, the phospholipid dispersion is mixed with the oleosome-containing cream layer alone.

[0047] According to certain illustrative embodiments, the method comprises reducing the pH of the mixture to an acidic pH, for example, a pH of about 6 or less, or a pH of about 5 or less, or a pH of about 4 or less, or a pH of about 3.5 or less, or a pH of about 3 or less. It was found that reducing the pH of the mixture from between about 3 to about 3.5 achieved the most stable emulsions upon storage.

[0048] Any food grade acid can be used in order to reduce the pH of mixture to the desired acidic conditions. Examples of acids include hydrochloric acid, lactic acid, phosphoric acid, acetic acid, citric acid, malic acid, tartaric acid, oxalic acid, tannic acid, caffeic acid, benzoic acid, butyric acid, and combinations thereof. In general, the amount of acid required depends on the type of acid.

[0049] According to further embodiments of the present invention, the pH is adjusted using about 1 M hydrochloric acid. Approximately 100 mL of the mixture is stirred on a magnetic stirrer plate. First, the pH is adjusted to about 6.5 to about 6.8. Then, several mL of hydrochloric acid is added to pass the isoelectric point of the proteins (about pH 4.5) with a target pH of about 3.5. Afterwards, the pH was reduced to pH 3.0.

[0050] In certain embodiments, the method comprises adding one or more flavor oils during mixing. The emulsion can be mixed with a high shear mixer, and subsequently homogenized and pasteurized. The resulting emulsion may comprise about 1% to about 20% w / w of oleosomes and about 1% to about 20% w / w of flavor oil. An additional advantage of the clouding emulsions containing flavor is the possibility to provide a high concentration of water insoluble flavor, e.g. terpenes, which significantly improves the overall flavor profile of a beverage compared to water-soluble flavors. Applicant has found that the disclosed oleosomes can absorb and stabilize oil. This allows the incorporation of oil, for example flavor oils, and thereby overcoming the need for emulsifiers to stabilize such oils.

[0051] In certain embodiments, the method comprises adjusting the average size of oleosomes in the mixture to about 0.1 pm to about 5 pm, or about 0.5 pm to about 4 pm, or about 0.1 pm to about 3 pm. This is accomplished by any known process, for example, high-speed shearing and homogenization. The high-speed shearing may be carried out at about 6,000 rpm for approximately 1 minute. A suitable high-speed mixer is commercially available under the designation Ultra-Turrax (IKA, Germany). Homogenization may be carried out at 500 / 50 bar. A suitable homogenizer is a Niro-Soavi type NS 1001 L Panda high-pressure homogenizer.

[0052] In one or more embodiments, flavor oil, for example, orange oil, is slowly dripped into the sample while being sheared. It has been found that stepwise oil loading avoids destabilisation and aggregation of the oleosomes. In certain embodiments, flavor oil is added using a 2.5 mL pipette, and a full pipette is added approximately every 10 seconds. In certain embodiments, after the full addition of flavor oil, the mixing speed is increased to 12,000 rpm for approximately 1 minute. Afterwards, the sample is stirred on a stirrer plate for approximately 10 minutes to allow the formed foam to collapse.

[0053] In certain embodiments, the sample is then homogenized, for example, at 500 / 50 bar. According to further embodiments of the present invention, the sample is homogenized for 2 or more passes at 500 / 50 bar, or 3 or more passes at 500 / 50 bar, or 4 or more passes at 500 / 50 bar, or 5 or more passes at 500 / 50 bar.

[0054] Also disclosed is a finished beverage comprising an acid stable clouding emulsion for a beverage comprising from about 80% to about 99% w / w of an aqueous phase and from about 1% to about 20% w / w of acid stable oleosomes having an average particle size of from about 0.1 pm to about 5 pm.

[0055] The finished beverage may have an acidic pH. In certain embodiments, the finished beverage has a pH of about 6 or less. In certain embodiments, the finished beverage has a pH of about 5 or less. In certain embodiments, the finished beverage has a pH of about 4 or less. In certain embodiments, the finished beverage has a pH of about 3.5 or less. In certain embodiments, the finished beverage has a pH of about 3 or less.

[0056] The clouding emulsion may be present in the beverage in an amount of about 0.00001% to about 5% w / w, or about 0.0001% to about 4% w / w, or 0.001% to about 3% w / w, 0.01% to about 2% w / w, 0.1% to about 5% w / w, based on the total weight of the beverage.

[0057] Also disclosed is a method of clouding a beverage comprising: preparing an emulsion comprising from about 80% to about 99% w / w of an aqueous phase and from about 1% to about 20% w / w of acid stable oleosomes having an average particle size of from about 0.1 pm to about 5 pm; and adding the emulsion to a beverage base in an amount of about 0.00001% to about 5% w / w, based on the total weight of the beverage.

[0058] EXAMPLES

[0059] The disclosure is further described with reference to the following non-limiting examples. The following examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations of the invention are possible without departing from the spirit and scope of the present disclosure.

[0060] Preparation of Compositions Comprising Oleosomes

[0061] Sunflower seeds (Notenstore, The Netherlands) were soaked for 4 hours with water in a 1 :7 (w:w) seed-to-water ratio at room temperature. During the soaking time, the pH was adjusted to 7.8 using 1 M NaOH. The pH was adjusted every 1 hour. The composition of sunflower seeds varies based on breed and agricultural conditions. A typical composition of sunflower seeds, on a dry basis, is shown below in Table 1. Table 1

[0062] After 4 hours of soaking, the seeds were blended in a kitchen blender for 1 minute at max speed using a laboratory blender (Waring Commercial, 400 W, USA). The slurry was recovered and stirred for 1 hour, while the pH was adjusted to 7.8 using 1 M NaOH (adjusted every 15 min). The slurry was passed through a cheesecloth, and the remaining liquid in the retentate was manually squeezed to obtain the final filtrate. The filtrate containing oleosomes and other solutes (e.g. proteins, phenols, mineral, sugars, etc.) was centrifuged at 10,000xg for 30 minutes at 4 °C. This resulted in a three-layer system: i) the oleosome-containing cream / top layer, called

[0063] “first cream” ii) a protein-rich middle layer, and iii) the pellet with insoluble seed material. The cream / top layer (“first cream”) and middle layer were combined and referred to as “oleosome milk”. “Washed cream” was produced by removing the free proteins from the “first cream”. The washing was performed by re-dispersing the cream in water (1 :4 w:w ratio), followed by centrifugation and collection of the formed cream layer. This step was repeated once more, leading to “washed cream”. The “first cream” and “washed cream” were often dispersed in a

[0064] 1 :2 (w:w) ratio.

[0065] Chemical Analysis of Samples

[0066] To define the dry matter of all samples, they were first stirred on a magnetic stirrer plate for 1 hour, until all large flocs of creams were not visible anymore. Sometimes, the cream was high speed stirred for 1 minute at 6,000 rpm (lowest speed) using an Ultra-Turrax (IK A, Germany) to break up the remaining flocculates. Afterwards, dry matter was determined by drying aliquots of the sample at 105 °C for 1 hour.

[0067] The oil content of the blended sunflower seeds and the obtained oleosomes were studied using Soxhlet extraction. All samples were dried overnight at 60 °C. About 2 - 5 g of material was used for oil extraction, which was performed for 16 hrs using petroleum ether as a solvent.

[0068] The oil content of each sample was determined in duplicate. The oil content was calculated using the following equation:

[0069] Mass of extracted oil (a

[0070] Oil content (%) = — - , . — ; - - — — — x 100

[0071] Mass of dried sample g)

[0072] The protein content was measured in a Flash EA 1112 Series Dumas (Interscience, The

[0073] Netherlands). The nitrogen content of the samples was obtained and converted into protein content with a conversion factor of 5.7. The protein content of each sample was determined in triplicate.

[0074] The dry matter, oil and protein extraction yield was determined using the following equation:

[0075] Dry matter, oil or protein extracted from 150$ seed

[0076] Yield (%) = — - - - - — — — - ,z100

[0077] Dry matter, oil or protein content in 150 g seeds (

[0078] The oil and protein content of the sunflower seed and various preparations obtained therefrom are shown below in Table 2. Table 2

[0079] Emulsion Production

[0080] In case a cloudifier emulsion was produced, the extract was optionally diluted with water to the desired dry matter concentration. The pH was adjusted to the desired pH using IM HC1 solution while stirring the emulsion on a magnetic stirrer plate. First, the pH was adjusted to

[0081] 6.5-6.8. Then, high amounts of HC1 (several mL) were added to pass the isoelectric point of the proteins (pH 4.5). Afterwards, the pH was slowly reduced to the desired pH. After reaching the desired pH, the sample was pre-homogenized using a high-speed mixer (Ultra-Turrax, IKA, Germany) at 6,000 rpm for 1 min, if not mentioned differently. If not mentioned differently, the sample was homogenised for 5 passes at 500 / 50 bar in a Panda high-pressure homogeniser (GEA, Niro Soavi NS 1001 L, Italy). In case a flavour loaded emulsion was produced, the dry matter concentration was adapted to the desired concentration by adding water and the pH reduced as described before. The sample was pre-homogenized at 6,000 rpm using an Ultra- Turrax high-speed mixer (IKA, Germany). The desired amount of orange oil was slowly dripped into the sample while being sheared. After the addition of the oil, the mixing speed was increased to 12,000 rpm for 1 min. Afterwards, the sample was stirred on a stirrer plate for 10 min to allow the formed foam to collapse. If not mentioned differently, the sample was homogenised for 5 passes at 500 / 50 bar in a Panda high-pressure homogeniser (GEA, Niro Soavi NS 1001 L, Italy). Emulsion analysis - Droplet size distribution

[0082] The droplet size was determined using static light scattering in a Bettersizer S3 Plus (3P Instruments, Germany). The refractive indices of the continuous and dispersed phase were set at 1.330 and 1.469, respectively. The Bettersizer water basin was filled with water at pH 3 to prevent flocculation in the device. All measurements were performed in triplicate. In some cases, sodium dodecyl sulfate (SDS) was added to reduce aggregation of the oleosomes during the measurement.

[0083] Beverage Preparation

[0084] A beverage syrup with 55°Brix was prepared consisting of 45.25% water, 0.07% sodium benzoate, 0.16% trisodium citrate, 53.33% of sugar and 1.19% of citric acid. To prepare a beverage, the syrup was diluted with water to obtain a beverage with 7.5° Brix. The necessary amount of flavour emulsions was added to achieve a beverage containing 100 ppm flavour. Different concentrations of the cloudifier emulsion were added to create a cloudified beverage.

[0085] Beverage Analysis

[0086] Beverages were filled into clear bottles, which were stored at 20°C where the samples were not moved or shaken and the formation of a ring was visually evaluated over time applying a scale representing the ring formation (table 3). The cloudiness of beverages was analysed by defining the average transmission at 20°C using a Turbiscan Tower (Formul action, Toulouse). Table 3: Ring formation evaluation in beverages

[0087] Example 1

[0088] A “first cream” was produced according to the above-described procedure.

[0089] Cloudifier emulsion: The “first cream” was dispersed in water to achieve a solution with 10% dry matter (w / w). The pH was adjusted to pH=3.0 with IM HC1 solution while stirring. The sample was homogenized (step 1 : pre-homogenization step with Ultra-Turrax for 30 sec at 10,000 rpm, step 2: high pressure homogenization at 50 / 500 bar for 5 passes). It was observed that the sample was very unstable when the “first cream” was used. In several cases, the emulsion already flocculated before the high-pressure homogenization. In case the emulsion was stable after the pH reduction, the emulsion flocculated after the high-pressure homogenization step.

[0090] Flavour emulsion: The “first cream” was dispersed in water to achieve a solution with 7.53% dry matter (w / w). The pH was adjusted to pH=3.0 with IM HC1 solution while stirring. 7% orange oil was added while stirring (Ultra-Turrax, 6000 rpm). After the oil addition, the sample was homogenized (step 1 : pre-homogenization step with Ultra-Turrax for 60 sec at 12,000 rpm, step 2: high pressure homogenization at 50 / 500 bar for 5 passes). It was observed that the sample was very unstable when the “first cream” was used. The emulsion flocculated during the high-pressure homogenization step.

[0091] Based on these results, the “first cream” is not suitable to produce stable cloudifier or flavour emulsions with a low pH for beverages.

[0092] Example 2

[0093] An oleosome milk was prepared according to the above-described procedure and separated into two different batches.

[0094] First Batch: The oleosome milk was further processed to obtain a “washed cream” as described above. The “washed cream” was re-dispersed in water and the pH adjusted to pH=6.6 for Ih while stirring. Afterwards, it was diluted to 1% dry matter concentration (w / w) and the pH was adjusted to pH=3.5 with IM HC1 solution. The sample was homogenized (step 1 : prehomogenization step with Ultra-Turrax for 30 sec at 10,000 rpm, step 2: high-pressure homogenization at 500 bar for 5 passes). When the pH was adjusted, strong flocculation was observed, which was formed as soon as the stirring stopped. It was not possible to homogenize this sample due to the strong flocculation. Droplet size analysis also shows the formation of large aggregates (see table 4).

[0095] Second Batch: The “oleosome milk” was used and the pH was adjusted to pH=3.5 with IM HC1 solution while stirring for Ih. The dry matter concentration was adjusted to 1% (w / w) by adding water and the sample was homogenized (step 1 : pre-homogenization step with Ultra- Turrax for 30 sec at 10,000 rpm, step 2: high-pressure homogenization at 500 bar for 5 passes). No flocculation was observed in this case. The measured droplet size distribution after production (0 days) and after 4-day storage is in the desired submicron range with a D50 < 0.5 pm (see table 4).

[0096] Table 4: Droplet size distribution parameters of emulsions after production (day 0) and 4-day storage

[0097] In contrast to the “washed cream” of the First Batch, the “oleosome milk” of the

[0098] Second Batch is suitable to produce an acid stable emulsion according to the invention.

[0099] Example 3

[0100] To evaluate the suitability of the “oleosome milk” to produce stable emulsions at low pH, a cloudifier emulsion and a flavour emulsion were prepared using an oleosome milk according to the above-described procedure. The oleosome milk had a dry matter concentration of 9.8% (w / w).

[0101] Cloudifier emulsion: A portion of the oleosome milk was taken and the pH was adjusted to 3.0 with IM HC1 while stirring. The emulsion was homogenized with a high-pressure homogenizer (5 passes, 50 / 500 bar).

[0102] Flavour loaded emulsion: A portion of the oleosome milk was diluted to a dry matter concentration of 7.53% (w / w) by adding water. A flavour loaded emulsion with 7% orange oil was prepared according to the above-described procedure. No aggregation or further destabilization of the emulsions was observed. The droplet size distribution was analysed and was found to be in the desired submicron range with a D50 < 0.5 pm (see table 5).

[0103] Table 5: Droplet size distribution parameters of emulsions after emulsion production

[0104] As shown in example 2, the oleosome milk is capable of producing cloudifier and flavour emulsions for beverages in the desired droplet size range at low pH levels.

[0105] Example 4

[0106] Extraction according to the above-described procedure was performed. The obtained oleosome milk had a dry matter concentration of 9.5%, which was used to produce the cloudifier emulsion. To create a flavour emulsion, the oleosome milk was diluted with water to achieve 7% dry matter. 5% oil was added during high shear mixing to obtain a flavour emulsion, as described above. Both emulsions were homogenized at 50 / 500 bar for 5 passes. The droplet size distributions of both emulsions were analysed (see Table 6). The droplet size distribution was in the desired submicron range with a D50 < 0.5 pm.

[0107] Table 6: Droplet size distribution parameters of emulsions after emulsion production

[0108] Both emulsions were used to prepare beverages as described above with 4 g / kg of cloudifier emulsion and 2 g / kg of flavour emulsion. The turbidity of the beverages was measured after production (0 days) and after 5 days (see table 7). Ring formation of beverages was evaluated over time (see table 8).

[0109] Table 7: Beverage turbidity expressed as transmission

[0110] Table 8: Ring formation evaluation of beverages over time

[0111] The results of the above experimental tests show that beverages produced with the cloudifier and flavour emulsion are cloudy and only show minor ringing over time.

[0112] Example 5

[0113] An oleosome milk was prepared as described above. A 5% (w / w) phospholipid solution (phosphatidylcholine (referred to as “PC”), Lipoid H90, Lipoid, Steinhausen, Switzerland) was prepared in demineralised water. The oleosome milk was mixed with the phospholipid solution in a 4: 1 (v:v) ratio, which yielded in a solution with 8% (w / w) dry matter concentration and 1% (w / w) phospholipids.

[0114] Cloudifier emulsion: A portion of the above-described solution was utilized and the pH was adjusted to pH=3.0 with IM HC1 while stirring. The emulsion was homogenized with a high- pressure homogenizer (5 passes, 50 / 500 bar) to produce a cloudifier emulsion. Flavour emulsion: The dry matter of the oleosome milk containing phospholipids was adjusted to 7.37% (w / w) by adding water and the pH was adjusted to pH=3.0 with IM HC1 while stirring. 7% orange oil was slowly added while shearing (Ultra-Turrax 6000 rpm). After the addition of the oil, the emulsion was pre-homogenized at 12,000 rpm and afterwards high pressure homogenized (5 passes, 50 / 500 bar) to obtain a flavour emulsion.

[0115] Droplet size distribution parameters were measured of freshly produced emulsions (0 days) and after 14 days of storage (see table 9).

[0116] Table 9: Droplet size distribution parameters after production (day 0) and after storage (day

[0117] 14) Beverages were produced with the cloudifier (dosage: 4 g / kg) and the flavour emulsion

[0118] (dosage 2 g / kg) and the ring formation was evaluated over time (see table 10).

[0119] Table 10: Ring formation evaluation in beverages over time

[0120] Surprisingly, beverages containing phospholipids were found to be even more stable compared to beverages without phospholipids. The beverages containing the cloudifier emulsion only showed minor ringing. The beverage made with the flavour-loaded emulsion also showed no ringing.

[0121] Example 6

[0122] A “first cream” was produced as described above as well as a 5% (w / w) phospholipid solution (phosphatidylcholine, Lipoid H90, Lipoid, Steinhausen, Switzerland) was prepared in demineralised water.

[0123] The “first cream” was diluted with water and the prepared phospholipid solution to obtain a solution containing 8% oleosomes on dry matter (w / w) and 1% phospholipids. The pH was reduced to pH=3.0 and the emulsion homogenized as described above to obtain a cloudifier emulsion. 10% sodium benzoate was added to obtain 0.02% sodium benzoate in the final solution.

[0124] No flocculation was observed during the production of the emulsion. The droplet size distribution was analysed after production and after storage (see table 11). The droplet size distribution shows very good stability of the emulsion.

[0125] Table 11 : Droplet size distribution parameters after production (day 0) and after storage (day 28) Beverages were produced containing 4 g / kg of the above described cloudifier emulsions and stored over 8 weeks (see table 12). No ring formation was observed.

[0126] Table 12: Ring formation evaluation in beverages

[0127] While the clouding emulsion, the compositions, formulations and products containing the clouding emulsion, and methods of making the clouding emulsion and compositions, formulations and products containing the clouding emulsion, have been described in connection with various embodiments, it is to be understood that other similar embodiments may be used or modifications and additions may be made to the described embodiments for performing the same function. Furthermore, the various illustrative embodiments may be combined to produce the desired results.

Claims

CLAIMS:

1. A clouding emulsion for a beverage comprising from 80% to 99% w / w of an aqueous phase and from 1% to 20% w / w of acid stable oleosomes having an average particle size of from 0.1 pm to 5 pm, wherein the clouding emulsion is present in an amount of 0.00001% to 5% w / w, based on the total weight of the beverage2. The clouding emulsion of claim 1, wherein the acid stable oleosomes are derived from plant material.

3. The clouding emulsion of claim 1 , wherein the plant material is selected from the group consisting of a seed, a nut, a kernel, and combinations thereof.

4. The clouding emulsion of claim 3, wherein the plant material comprises a seed.

5. The clouding emulsion of claim 4, wherein the seed is selected from the group consisting of almond seeds, argan seeds, borage seeds, coconut, com seeds, cotton seeds, chia seeds, cranberry seeds, flax seeds, grape seeds, hemp seeds, linseed, millet, mustard seeds, oil palm, pumpkin seeds, quinoa, rapeseeds, tomato seeds, mango seeds, raspberry seeds, safflower seeds, sesame seeds, sunflower seeds, soybeans, and combinations thereof.

6. The clouding emulsion of claim 5, wherein the seed comprises sunflower seeds.

7. The clouding emulsion of claim 1, wherein the emulsion is free of pectin, whey protein, hydrocolloids, gum acacia and modified starch.

8. The clouding emulsion of claim 1, wherein the emulsion is free of weighting agents.

9. The clouding emulsion of claim 1, wherein the acid stable oleosomes encapsulate at least one flavor oil.

10. The clouding emulsion of claim 9 comprising from 1% to 15% w / w of the at least one flavor oil, based on the total weight of the emulsion.

11. The clouding emulsion of claim 1, wherein the emulsion has a pH of 3.5 or less.

12. The clouding emulsion of claim 1, wherein the acid stable oleosomes are at least partially encapsulated by free proteins and / or free phospholipids.

13. The clouding emulsion of claim 12 comprising 0.5% to 3% w / w free protein and / or free phospholipids, based on the total weight of the emulsion.

14. A finished beverage comprising the clouding emulsion of claim 1.

15. The finished beverage of claim 14 having an acidic pH.

16. The finished beverage of claim 14, wherein the emulsion is present in an amount of 0.00001% to 5% w / w, based on the total weight of the beverage.

17. A method of clouding a beverage comprising: preparing an emulsion comprising from 80% to 99% w / w of an aqueous phase and from 1% to 20% w / w of acid stable oleosomes having an average particle size of from 0.1 pm to 5 pm; and adding said emulsion to a beverage base in an amount of 0.00001% to 5% w / w, based on the total weight of the beverage.

18. A method for making a clouding emulsion for a beverage, the method comprising: forming an aqueous mixture comprising plant material and water at a ratio of 1 :3 to1 : 10; adjusting the pH of the mixture to an alkaline pH; comminuting the mixture; removing insoluble components from the mixture; stabilizing oleosomes in the mixture against aggregation by adding free proteins and / or free phospholipids to the mixture; reducing the pH of the mixture to an acidic pH; adjusting the average size of the oleosomes in the mixture to 0.1 pm to 5 pm.

19. The method of preparing a clouding emulsion for a beverage according to claim 18 comprising centrifuging the mixture to further remove insoluble components.

20. The method of preparing a clouding emulsion for a beverage according to claim 19 comprising adding a flavor oil after reducing the pH of the mixture to an acidic pH.

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