阅读说明：本技术 提取感兴趣的物质的方法 (Method for extracting substance of interest ) 是由 C·孔蒂诺-佩平 S·德格郎热 A·达尔阿尔梅利娜 C·杜瓦尔 M·莱坦-马丁 于 2019-11-27 设计创作，主要内容包括：本说明书涉及一种用于提取至少一种感兴趣的物质的方法,该方法包括以下步骤：-提供包含水、包含至少一种感兴趣的物质或其至少一种前体的来源、至少一种表面活性剂以及至少一种亲脂性化合物的混合物；-混合物的乳化,导致乳液的形成；其中乳液包含：-连续水相；以及不连续脂相,该不连续脂相包含至少一种亲脂性化合物、至少一种表面活性剂以及在乳化过程中从来源中提取的至少一种感兴趣的物质；其中,该方法还包括向混合物添加至少一种冷冻保护剂并使乳液冻干。(The present description relates to a method for extracting at least one substance of interest, comprising the steps of: -providing a mixture comprising water, a source comprising at least one substance of interest or at least one precursor thereof, at least one surfactant and at least one lipophilic compound; -emulsification of the mixture, resulting in the formation of an emulsion; wherein the emulsion comprises: -a continuous aqueous phase; and a discontinuous lipid phase comprising at least one lipophilic compound, at least one surfactant and at least one substance of interest extracted from the source during emulsification; wherein the method further comprises adding at least one cryoprotectant to the mixture and lyophilizing the emulsion.)

1. A method for extracting at least one substance of interest from a source, the method comprising the steps of:

-providing a mixture comprising water, a source comprising the at least one substance of interest or at least one precursor thereof, at least one surfactant and at least one lipophilic compound;

-emulsifying the mixture, thereby forming an emulsion;

wherein the emulsion comprises:

-a continuous aqueous phase; and

-a discontinuous lipid phase comprising said at least one lipophilic compound, said at least one surfactant and said at least one substance of interest extracted from said source during emulsification;

wherein the method further comprises adding at least one cryoprotectant to the mixture and freeze drying the emulsion.

2. The method of claim 1, wherein said emulsifying said mixture comprises sonicating said mixture.

3. The method according to claim 1 or 2, wherein the source is a plant, animal, mineral, prokaryotic and/or unicellular eukaryotic source.

4. The method of claim 3, wherein the source is a plant source.

5. A process according to any one of the preceding claims, wherein the at least one surfactant is selected from the group comprising Dendri-TAC type dendrimers, F_iTAC_nOr H_iTAC_nType oligomers, TPGS1000, TPGS 750M, surfactants derived from sugars and/or amino acids, and combinations thereof.

6. The method of any one of the preceding claims, wherein emulsifying is performed by maintaining the temperature of the mixture between about 4 ℃ and about 60 ℃.

7. The method of any one of the preceding claims, wherein the emulsification duration is between about 0.5 seconds and about 5 hours.

8. A method according to any preceding claim, wherein the discontinuous lipid phase is in the form of droplets having a predetermined mean diameter D.

9. The method according to any one of the preceding claims, wherein at least one cryoprotectant is added to the mixture after emulsifying the mixture.

10. The method according to any of the preceding claims, wherein the at least one cryoprotectant is selected from the group comprising polymers, amino acids, saccharide compounds such as mono-, di-and polysaccharides, and combinations thereof.

11. The method according to any one of the preceding claims, wherein the at least one lipophilic compound, the at least one surfactant and the at least one cryoprotectant are biocompatible.

12. The method of any one of the preceding claims, further comprising a pre-treatment step prior to emulsifying the mixture, wherein the pre-treatment step comprises soaking the mixture at a temperature between about 20 ℃ and about 60 ℃.

13. A dry formulation obtained by the method of any one of claims 1-12.

14. Use of a dry formulation according to claim 13, in the manufacture of a product comprising at least one substance of interest extracted from a source comprising the at least one substance of interest or at least one precursor thereof, by adding to the dry formulation a compound selected from the group comprising water or oil.

15. Use of the dry formulation according to claim 13 in the field of cosmetics, food processing, pharmaceuticals, neutraceuticals and/or probiotics.

Technical Field

The present invention relates to a process for extracting at least one substance of interest, to an emulsion produced by the process and to the use of the emulsion. The present description finds particular, but not exclusive, application in the food processing, cosmetic and pharmaceutical fields, and in the perfume field.

Background

Natural substances, such as natural substances of plant, animal or mineral origin, are of increasing interest in many fields. In fact, for example, in view of the rich chemical diversity of molecules offered by the plant world, and the intense public enthusiasm for natural products, a certain number of industrial sectors (cosmetics, pharmaceuticals, food processing, nutraceuticals, probiotics) are becoming transformed to incorporate into their formulations molecules of plant origin. Adding value to these natural active ingredients, it has become a great economic potential for the development of the food processing, cosmetic and pharmaceutical sectors.

To add value, natural compounds (e.g., biologically active compounds, i.e., compounds having biological activity) can be first isolated from their plant matrix during an extraction step and then used in the form of an extract or in the form of isolated molecules after purification. The extraction sector is particularly concerned with environmental and economic problems and nowadays is also striving to find solutions to reduce energy costs and waste of the whole extraction process chain.

In the field of extraction of high value-added natural products, solvent extraction remains the most common method. However, this extraction method relies on the use of organic solvents (e.g., hexane, cyclohexane, petroleum ether, methanol, ethanol or hydroalcoholic mixtures). For example, if the natural product to be extracted (i.e. the substance of interest) is hydrophilic, the extraction solvent used may be a polar aprotic solvent, such as acetone, or a polar protic solvent, such as water, methanol or ethanol. On the other hand, if the natural product to be extracted (i.e. the substance of interest) is lipophilic, the extraction solvent used may be non-polar (i.e. hydrophobic), such as hexane, or a non-polar less than hexane solvent, such as chloroform or dichloromethane. Many of these solvents are flammable, volatile, and toxic. Furthermore, the gradual depletion of oil resources, especially the tightening of regulations, is forcing manufacturers to move to more environmentally friendly processes. Finding an alternative to the use of organic solvents and/or energy-consuming extraction methods (which sometimes denature biomolecules) is a major economic and scientific challenge to which the present invention is directed.

Although water is ubiquitous in plants, many bioactive molecules of plant origin (e.g., vitamins, antioxidants, natural anti-inflammatory agents, etc.) are poorly soluble or insoluble in water, thereby limiting extraction yield and the quality (e.g., in terms of integrity and stability) of the resulting extract.

The subject matter of the present description relates in particular to a process which, in certain embodiments, makes it possible to extract, without modifying it, substances of interest of vegetable, animal or mineral origin (for example fat-soluble substances) in good yields, for example by emulsifying (for example by sonication) an aqueous medium to which lipophilic compounds and natural or synthetic surfactants are added, and which makes it possible to obtain, in the same step ("one-pot"), formulations which are stable for a long time and which are easy to store and transport.

Disclosure of Invention

In this specification and the appended claims, the term "comprising" is synonymous (meaning the same) with "including" and "containing", is inclusive or open-ended and does not exclude additional elements not described or shown. Further, in this specification, the term "about" is synonymous with (has the same meaning as) a lower margin and/or an upper margin of 10% (e.g., 5%) of the corresponding value.

In the present description and claims, D is the mean droplet diameter, measured on a granulometer based on light diffraction for droplets with a diameter D greater than 1000nm (De Brouckere volume weighted mean diameter of the emulsion particles), and with a Dynamic Light Scattering (DLS) device for droplets with a diameter D less than 1000 nm. When D is measured using a Dynamic Light Scattering (DLS) device, the polydispersity index (PdI) of the emulsion is defined as the ratio of the square of the standard deviation to the square of the mean droplet diameter. When using a particle size meter, the polydispersity index (PdI) of the emulsion refers to the ratio D90/D10, where droplet diameter D10 refers to the diameter of droplets having a diameter greater than or equal to 90% of the total number of droplets in the emulsion, and droplet diameter D90 refers to the diameter of droplets having a diameter greater than this 10% of the total number of droplets in the emulsion.

It is an object of the present specification to provide a method of extracting at least one substance of interest from a source. The at least one substance of interest may be contained in the source or generated by the extraction process, in the latter case, the at least one substance of interest is derived from at least one molecule previously contained in the source, which is chemically converted into the at least one substance of interest during the extraction process.

According to a first aspect, the present description relates to a method for extracting at least one substance of interest from a source, comprising the steps of:

-providing a mixture comprising water, a source comprising the at least one substance of interest or at least one precursor thereof, at least one surfactant and at least one lipophilic compound;

-emulsifying the mixture, thereby forming an emulsion;

and wherein the emulsion comprises:

-a continuous aqueous phase; and

-a discontinuous lipid phase comprising said at least one lipophilic compound, said at least one surfactant and said at least one substance of interest extracted from said source during emulsification;

wherein the method further comprises adding at least one cryoprotectant to the mixture and freeze drying the emulsion.

The method according to embodiments herein may advantageously be a "one-pot" process, without changing the at least one substance of interest and with a good yield of the substance of interest. The extraction of molecules by emulsification in an aqueous medium to which lipophilic compounds and surfactants are added is a "green chemistry" and environmentally friendly process.

According to one or more embodiments, emulsifying the mixture includes applying an energy source, which may be ultrasonic energy and/or purely mechanical energy, to the mixture. According to one or more embodiments, emulsifying the mixture comprises sonicating the mixture. Sonicating the mixture includes applying an ultrasonic energy source to the mixture, resulting in the formation of an emulsion.

Thus, the method comprises emulsifying a mixture comprising water, a source comprising at least one substance of interest or at least one precursor thereof, at least one surfactant and at least one lipophilic compound. Emulsifying the mixture results in the formation of an emulsion comprising a continuous aqueous phase and a discontinuous lipid phase comprising the at least one lipophilic compound, the at least one surfactant and at least one substance of interest extracted from the source during emulsification. Emulsification induces the formation of an emulsion having the effect of increasing the contact surface between both the aqueous phase and the lipid phase, thereby increasing the transfer of the at least one substance of interest from one phase to the other. In accordance with one or more embodiments, a residual portion of the surfactant may be included in the aqueous phase after emulsifying the mixture resulting in the formation of the emulsion.

According to one aspect, the present description relates to a method for extracting at least one substance of interest from a source, the method comprising the steps of:

-providing a mixture comprising water, a source comprising at least one substance of interest or at least one precursor thereof, at least one surfactant and at least one lipophilic compound;

-sonicating the mixture, thereby forming an emulsion;

and wherein the emulsion comprises:

-a continuous aqueous phase; and

-a discontinuous lipid phase comprising said at least one lipophilic compound, said at least one surfactant and said at least one substance of interest extracted from said source during sonication;

wherein the method further comprises adding at least one cryoprotectant to the mixture and freeze drying the emulsion.

By exploiting the lipophilicity of certain molecules that are difficult to extract in pure water (e.g., without the addition of alcohol), the present inventors have been able to implement an extraction process in water to which at least one lipophilic compound is added, together with a surfactant of natural or synthetic origin, so that extracts of the "oil and/or wax in water" type, enriched with the potential bioactive molecule of interest, can be obtained in a limited number of steps.

According to one or more embodiments, the source can be, for example, a plant (e.g., olive pomace, distillers grains, brewery residue, etc.), an animal (e.g., a carcass, skin, or other portion of an animal), a mineral, a prokaryote, and/or a unicellular eukaryotic source.

According to one or more embodiments, the source may be derived from a by-product of the food and/or agricultural industry, whether plant, animal, mineral, prokaryotic, and/or unicellular eukaryotic or otherwise. The source may be comprised of any material that may contain or produce at least one substance of interest. The substances of interest may for example comprise lipophilic molecules which are poorly soluble in water (fat-soluble) and which are of potential interest for application in the field of cosmetics, pharmaceuticals and/or food processing. The molecules may, for example, have biological activities beneficial to human health. For example, antioxidants are molecules that may play a major role in protecting the body from the development of cardiovascular diseases and certain cancers, particularly because antioxidants are able to rapidly capture reactive oxygen species involved in the development of these diseases. The lipophilic molecule may for example comprise a polyphenol or a terpene.

According to one or more embodiments, the source is a plant source. The plant source may include at least a portion of at least one plant (seed, leaf, root, stem, fruit, flower, etc.). The plant sources may be cited, for example, from the book by Albert Y.Leung and Steven Foster ("Encyclopedia of common natural ingredients used in food, drugs and cosmetics", 2nd edition, Wiley-Interscience, 1996). The plant source may for example be selected from soothing properties, anti-inflammatory properties, antiseptic properties, antiperspirant properties, calming properties, healing properties, tonic properties, properties promoting control of blood and lymph microcirculation, texturizing properties, antioxidant properties, foaming or emulsifying properties, photoprotective properties, thickening, absorbing and/or odorous properties.

As examples of plant sources classified according to their different characteristics, mention may be made in particular of:

for its soothing properties: apricot, blueberry, mullein, roman chamomile, fenugreek, marshmallow, flax, lily, mallow, marigold, elderberry, basswood, coltsfoot, plantain seed, plantain, papaya, peach, orange, cactus, apple;

for its anti-inflammatory properties: herba et Gemma Agrimoniae, fructus crataegi, flos Photiniae, creeping wheatgrass, juniper, radix Althaeae Roseae, ramulus Sambuci Williamsii, Tilia Miqueliana Maxim, semen Trigonellae, radix Gentianae, caulis et folium Lactucae Sativae, herba Violae Hortensis, herba plantaginis, fructus Rubi Corchorifolii Immaturus, herba Rosmarini officinalis, herba Salvia officinalis, rhizoma Dioscoreae, coltsfoot, Helichrysum arenarium L, and Bellis perennis;

for its anti-corrosion properties: garlic, hairyvein agrimony, cowberry fruit, burdock, oak, lithospermum, eucalyptus, juniper, geranium, bay, lavender, marjoram, mint, pine, rosemary, sandalwood, thyme, sage, honeysuckle, helichrysum, daisy, chrysanthemum indicum, pepper and pepper;

for their antiperspirant properties: sage, oak, walnut, pine, horsetail, coltsfoot;

for its convergence properties: acacia, yarrow, agrimony, collard, strawberry, mugwort, gromwell, cypress, oak, rose hip, witch hazel, black mulberry, blueberry, hazelnut, walnut, nettle, poplar, plantain, raspberry, ratanhia (ratanhia), rose, loosestrife, willow, Potentilla tormentosa, sarmentia, sargentgloryvine, sweet clover;

for its sedative properties: carrot, pansy, elderberry, basswood, passion flower, basil, camphor tree, pear, apple, grape, lettuce, rose and ginger;

for its healing properties: herba Hyperici perforati, radix Potentillae Anserinae, flos Tagetis Erectae, flos Matricariae Chamomillae, radix Arnebiae, milfoil, folium Artemisiae Argyi, herba et Gemma Agrimoniae, folium Artemisiae Argyi, Arnica Montana, radix Raphani, myrtle, herba Catharanthi rosei, herba plantaginis, poplar, flos Primulae, herba Salvia officinalis, herba Senecionis Scandentis, herba Verbenae, radix Angelicae sinensis, fructus Aristolochiae, alder, aurone (aurone), Polygonum hydropiper, birch, hydrocanthus, folium et cacumen Najaponici, fructus Lycii, Tasmani, flos Abutili, herba Gei, herba Sedi indicae, herba Selaginellae Doederleinii, herba Potentillae Anserinae, strawberry, herba Equiseti hiemalis, spiraea, flos Farfarae Japonicae, herba Violae, burdock, Bellis perennial, and Bulbus Lilii;

for its tonic properties: hypericum perforatum, rosehip, mistletoe, mate, blackcurrant, mugwort, arnica, dragon fruit, cinnamon, geranium, hyssop, marjoram, lemon balm, parsley, red pine, rosemary, savory, thyme, basil, rosehip, gentian, hops, bay, mint, sage, Potentilla chinensis, yarrow, Agrimonia, myrica rubra, polygonum, oak, quince, cypress, horse chestnut, matrimony, walnut, nettle, plantain, water pear, potentilla chinensis, polygonum cuspidatum, loosestrife, speedwell, angelica, mugwort, clover, juniper, mustard, cinchona, viola, nasturtium, watercress, sage, ruscus, butea frondosa, and wormwood;

for its properties that promote the control of the blood and lymphatic microcirculation: blackcurrants, bilberries and grape seeds;

for its hardening and tempering properties: wheat, fucoidin;

for its antioxidant properties: rice, rosemary, sage, thyme, green tea, licorice, pepper;

for its foaming or emulsifying properties: soapwort, ivy, butcher's broom, panama, soapbark tree, smilax china mexicana, quinoa and soybean;

for its photoprotective properties: aloe, sunflower, licorice, magnolia, kaempferia galanga;

for its thickening or absorbing properties: pea, wheat, potato, corn; and

for its aromatic character: rosemary, violet, lavender, jasmine, lily of the valley, vanilla, rose, citrus fruit (e.g. lemon, grapefruit). According to one or more embodiments, the plant source is a curcuma species plant.

According to one or more embodiments, the source comprises propolis and/or honey.

The at least one lipophilic compound may be comprised in the source and/or be produced by a previous addition of an external source. Depending on the source considered, the addition of an external source of the at least one lipophilic compound is not always necessary to obtain a stable emulsion. For example, certain plant substrates can release essential oils, phospholipids, and amphiphilic proteins under the action of ultrasound. For example, citrus peel may release variable amounts of essential oils under the action of ultrasound, which may be sufficient to obtain an emulsion for which the method according to embodiments of the present description is directed. According to one or more embodiments, the at least one lipophilic compound is selected from the group consisting of oils, waxes, and combinations thereof. The at least one lipophilic compound may for example comprise a mixture between oils, a mixture between waxes or an oil/wax mixture. The inventors have found that for some sources, in the presence of at least two lipophilic compounds in the mixture provided by the process of the invention (for example in the presence of two different oils or in the presence of an oil and a wax) it is possible to reduce the polydispersity index of the emulsion and/or to reduce the mean diameter D of the emulsion droplets and/or to increase the extraction rate of the at least one active substance, compared to the case where only one oil is present in the mixture. According to one or more embodiments, the lipophilic compound is selected from the group consisting of mono-, di-, or tri-or glycerol derivatives of glycerol, mono-, di-, or tri-, or tetra-or citric acid derivatives of citric acid, fatty acids, mono-esters of fatty acids, essential oils, fat substitutes, waxes, and combinations thereof. Advantageously, the process according to the invention does not require the use of typical organic extraction solvents, such as hexane, cyclohexane, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, petroleum ether, methanol, ethanol or hydroalcoholic mixtures. According to one or more embodiments, the at least one lipophilic compound is present in the mixture in a weight to volume ratio of between 0.01% w/v and 70% w/v, such as between 3% w/v and 70% w/v, based on the total volume of the mixture (excluding the source) comprising water, lipophilic compound and surfactant. The weight-to-volume ratio of the at least one lipophilic compound in the same mixture, excluding the source, is for example between 0.1% w/v and 20% w/v, for example between 5% w/v and 20% w/v.

The at least one surfactant may be anionic, cationic, zwitterionic, amphoteric or non-ionic. The at least one surfactant may be natural or synthetic. According to one or more embodiments, its hydrophilic-lipophilic balance (HLB) is between 4 and 19. The at least one surfactant may, for example, be chosen from those comprising, for example, alcohols, aminoalcohols, esters, amine oxidesNonionic surfactants and combinations thereof. The surfactant may be, for example, Lauroglycol^TMFCC (propylene glycol monolaurate (type I)), Lauroglicol^TM 90、Capryol^TM 90、Oleique CC 497, (polyglycerol-3 dioleate),Caprylic/capric polyethylene glycol glycerides (Caproloyl Polyoxyl-8glycerides),M 1944CS、ALF、M 2125CS、50/13、M 2130CS、48/16、44/14、HP、CBG MB. The surfactant may be selected from the group consisting of Axol C62(ADARA), Cutina CP (BASF), Cutina GMS (BASF), Emulium 22(GATTEFOSSE), Emulium Delta (GATTEFOSSE), Emulium melifera (GATTEFOSSE), Eumulgin SG (BASF), Montanov 202(Seppic), Montanov 68(Seppic), Montanov 82(Seppic), Montanov L (Seppic), Olivem 1000(Univar), Olivoil acetate (ACS Phyto), Xylance (MASSO). The surfactant may be derived from a sugar, such as an alkyl glycoside, sorbitan ester, sucrose ester (or ester of sucrose), or alkyl methyl glucamide. For example, the surfactant may be an alkyl glycoside, such as an alkyl glucoside. Alkyl glycosides are a class of nonionic surfactants that are widely used in a variety of cosmetic, household, and industrial applications. These surfactants are biodegradable, typically derived from glucose and fatty alcohols. The sugar-derived surfactant may also be a sucrose ester of a fatty acid or a sucrose ester of a fatty acid, e.g. E473. Sucrose esters of fatty acids are listed as emulsifiers and stabilizers in the International food codex. In other uses, sucrose esters are added to the surface treatment of ice cream, candies, chewing gum, spirits, food supplements, infant formula and fresh fruit. In addition to processed foods, sucrose esters are also used in cosmetics and pharmaceuticals, for example as emulsifiers for creams, skin lotions, lotions and ointments. The saccharide surfactant may also be a sorbitan ester, such as E491 (sorbitan monostearate, Span 60), E492 (sorbitan tristearate, Span 65), E493 (sorbitan monolaurate, Span 20), E494 (sorbitan monooleate, Span 80), E495 (sorbitan monopalmitate, Span 40), E496 (sorbitan trioleate, Span 85), E432 (polyoxyethylene sorbitan monolaurate, polysorbate 20), E433 (polyoxyethylene sorbitan monooleate, polysorbate 80), E434 (polyoxyethylene sorbitan monopalmitate, polysorbate 40), E435 (polyoxyethylene sorbitan monostearate, polysorbate 60), E436 (polyoxyethylene sorbitan tristearate, polysorbate 65). The sugar-derived surfactant may be selected from the group consisting of Simulsol AS 48, Simulsol SL 7G, coco glucoside, decyl glucoside, lauryl glucoside, sodium lauryl glucarate.

The surfactants may also be derived from amino acids such as sodium cocoyl glutamate, disodium cocoyl glutamate, sodium lauroyl glutamate, and also from peptides such as sodium cocoyl hydrolysed wheat protein, sodium cocoyl hydrolysed collagen. The surfactant can be alkyl PEG sulfosuccinates (e.g., disodium laureth sulfosuccinate, disodium capeth sulfosuccinate), acyl sarcosines (e.g., sodium lauroyl sarcosinate, sodium cocoyl sarcosinate), acyl isethionates (e.g., sodium cocoyl isethionate). The surfactant may be disodium laureth sulfosuccinate, cocamidopropyl betaine, cocoyl betaine, sodium cocoyl sulfate, sodium laureth sulfoacetate. The surfactant may be a glycolipid, such as Rhamnolipid (RL), mannosylerythritol lipid (MEL), trehalose glycolipid (TL), xyloglycolipid, sophorolipid, lipopeptide, such as fengycin (fengycin), iturin (iturin), surfactin (surfactin).

According to one or more embodiments, the at least one surfactant is chosen from the group comprising dendrimers (such as those of the Dendri-TAC type described in patent application PCT/IB 2016/052952), F_iTAC_nOr H_iTAC_nOligomers of type (la), TPGS1000, TPGS 750M, and combinations thereof. For each surfactant, the concentration of the surfactant may, for example, be adapted to the concentration of the lipophilic compound, in order to obtain an emulsion with a low polydispersity index and thus to influence the extraction yield and/or the purity of the substance of interest. According to one or more embodiments, the concentration of surfactant in the mixture, excluding the source, is between 0.5mg/mL and 250mg/mL, such as between 5mg/mL and 250 mg/mL. Above the upper limit of this range, the extraction rate of the substance of interest may decrease. This reduction may be due to the increased viscosity making it difficult to transfer the substance of interest from the source to the discontinuous lipid phase of the emulsion.

According to one or more embodiments, the emulsification is performed by maintaining the temperature of the mixture between about 0 ℃ and about 60 ℃ (e.g., between 4 ℃ and 60 ℃).

According to one or more embodiments, the temperature of the emulsion or formulation does not exceed 60 ℃ during the process. The method according to these examples therefore provides a considerable added value to the cosmetics, food and/or pharmaceutical sector, in particular for substances of interest which are susceptible to rapid degradation in the presence of air and/or when subjected to thermal treatment. According to one or more embodiments, the temperature of the mixture does not exceed 25 ℃ during emulsification and is maintained, for example, between 0 ℃ and 20 ℃, for example between 5 ℃ and 15 ℃.

According to one or more embodiments, the slurry may be passed through an ultrasonic bath and/or ultrasonic rod (sonotrode) and/or ultrasonic cup-horn reactor and/or disperser-homogenizer (e.g., a homogenizerType) is emulsified. According to one or more embodiments, the mixture is stirred during emulsification. According to one or more embodiments, the duration of the emulsification is between about 0.5 seconds and about 5 hours. According to one or more embodiments, the duration of the emulsification does not exceed 2 hours, and is for example between 0.5 and 1.5 hours.

According to one or more embodiments of the method wherein emulsifying the mixture comprises sonicating the mixture, the sonication is performed at a power of between about 100W and about 800W, at a frequency of between about 20kHz and about 2000kHz, in a pulsed or continuous mode.

According to one or more embodiments, the discontinuous lipid phase is in the form of droplets having a predetermined average diameter (D). According to one or more embodiments, the predetermined average diameter (D) of the droplets is between about 30nm and about 6000 nm. For some applications of the emulsion obtained by the process according to the invention, the mean diameter (D) of the droplets of the lipid phase is preferably within a predetermined range of values. For example, emulsions used in connection with oral bioavailability may comprise a lipid phase in the form of droplets having a mean diameter (D) preferably less than 200 nm.

Furthermore, according to one or more embodiments of the present invention, the polydispersity index (PdI) of the emulsion may preferably be less than 0.5, such as less than 0.3, or even less than 0.2. For certain emulsions obtained by a method according to one or more embodiments of the present description, a polydispersity index of greater than 0.3 may reflect poor stability of the emulsion, which may be reflected, for example, by poor freeze-drying resistance.

The method according to the invention further comprises adding at least one cryoprotectant to the mixture. This addition can be carried out before and/or during and/or after emulsification of the mixture. According to one or more embodiments, the concentration of cryoprotectant in the mixture, excluding the source, is between 5mg/mL (or 0.5% w/v when expressed as a weight to volume ratio based on the total volume of the mixture) and 250mg/mL (or 25% w/v). In particular, the addition of a cryoprotectant may provide additional protection to the emulsion obtained after emulsification, for example in a freeze-drying step. The cryoprotectant can replace hydrogen bonds between water and the surfactant so as to maintain the spatial organization of the emulsion, and can reduce the interaction between the surfactants of different liquid drops so as to generate aggregates so as to avoid the instability of the emulsion liquid drops. Furthermore, cryoprotectants can form an amorphous matrix and avoid the formation of ice crystals, which may be responsible for the droplet instability mechanism.

According to one or more embodiments, the at least one cryoprotectant is selected from the group consisting of polymers, amino acids, saccharide compounds (e.g., monosaccharides, disaccharides, and polysaccharides), and combinations thereof. According to one or more embodiments, the at least one cryoprotectant is selected from the group consisting of trehalose, sucrose, maltose, glucose, mannitol, hydroxypropyl- β -cyclodextrin and combinations thereof. In other exemplary embodiments, the at least one cryoprotectant is a polymer, such as polyvinylpyrrolidone or polyvinyl alcohol. The at least one cryoprotectant may also be an amino acid, such as glycine.

According to one or more embodiments, the concentration of the at least one cryoprotectant in the mixture excluding the source is between 5mg/mL (or 0.5% w/v) and 250mg/mL (or 25% w/v) and is selected based on factors such as system composition, cooling rate, and freezing temperature. According to one or more embodiments, said addition of at least one cryoprotectant is carried out after the mixture has been emulsified, this addition being carried out in a weight to volume ratio of at least 5% w/v (based on the total volume of the mixture excluding the source), for example comprised between 5% w/v and 10% w/v, for example comprised between 6% w/v and 8% w/v. The addition of such a concentration of cryoprotectant may, for example, increase the homogeneity of the emulsion obtained by the extraction method according to the present description, which comprises a step of freeze-drying the emulsion before the rehydration step.

According to one or more embodiments, the at least one lipophilic compound, the at least one surfactant, and the at least one cryoprotectant are biocompatible. Biocompatible substances are defined as substances which are non-toxic and have no harmful effects on health and which can be used in cosmetic and/or pharmaceutical and/or food applications. For example, in accordance with one or more embodiments, the at least one surfactant and the at least one cryoprotectant may be considered harmless according to standards approved by the United States Food and Drug Administration (USFDA) and benefit from Generally Recognized As Safe (GRAS) certification.

According to one or more embodiments, the method further comprises a "post-extraction" treatment step, i.e. a step after emulsifying the mixture, thereby forming an emulsion. This treatment step may for example comprise the subsequent step of ultrasonicating the emulsion obtained after emulsification of the mixture: this processing step may be performed, for example, by a sonotrode. The "post-extraction" processing step may also, for example, comprise one or more ultracentrifugation steps at speeds between 1000G and 50000G. According to one or more embodiments, this treatment step may reduce the average diameter D of the droplets and the polydispersity index of the emulsion.

According to one or more embodiments, the method further comprises a pre-treatment step prior to emulsifying the mixture, the pre-treatment step comprising heat-soaking a mixture comprising water, a source, at least one surfactant and at least one lipophilic compound at a temperature between about 20 ℃ and about 60 ℃. According to one or more embodiments, the pre-treatment step may also include grinding, passing through a disperser-homogenizer (e.g.,) Ultrasonic bath or microwave oven.

The method according to the invention further comprises freeze-drying the emulsion. For example, a freeze-drying step may be performed after emulsification and addition of at least one cryoprotectant. The emulsion freeze-drying step may result in the formation of an emulsion in the form of a dry formulation. Thus, a dry formulation rich in potentially bioactive and lipid soluble substances can be obtained in a minimum of steps. The corresponding emulsion is easily reconstituted by simple rehydration, i.e. by adding a certain amount of water or an aqueous solution to the dry formulation to re-form the emulsion. The reconstituted emulsion does not necessarily have the same characteristics as the starting emulsion (i.e. the emulsion before freeze-drying). For example, the average droplet diameter D and/or the polydispersity index PdI of the reconstituted emulsion may be significantly different from the values in the emulsion before freeze-drying and after freeze-drying/rehydration (i.e., post-freeze-drying rehydration). According to one or more embodiments, the method comprising freeze-drying the emulsion before the rehydration step further comprises a "post-rehydration" treatment step, i.e. a step after rehydration of the dried formulation obtained by freeze-drying. This step may consist, for example, of sonicating the emulsion obtained after rehydration, for example by a sonotrode. According to one or more embodiments, this step may reduce the average diameter D of the droplets and the polydispersity index of the emulsion obtained after rehydration.

The freeze-drying step can be adapted to the nature of the substance of interest and its inherent sensitivity as well as to the volume of the mixture after emulsification. The duration of the freeze-drying step may vary from a few hours to a few days. According to one or more embodiments, the freeze-drying step comprises at least three sub-steps: freezing, wherein the temperature of the emulsified mixture is less than about-20 ℃; sublimating, which occurs at a pressure below about 500 μ bar, to sublimate water in the mixture; drying, wherein the resulting mixture is heated to a temperature above about 20 ℃ to reduce residual moisture content.

In the freeze-drying step, at least one cryoprotectant may provide stability to the emulsion during the heat treatment to which the emulsion is subjected.

According to a second aspect, the present description relates to a dry formulation obtained by the method according to the first aspect.

In order to ensure the stability, preservation and storage of the emulsion produced by the method according to the first aspect, the present invention comprises converting the emulsion into a dry formulation by subjecting it to lyophilization, in order to facilitate and expand its field of application to various industrial sectors. Among other advantages, the dry formulation obtained by freeze-drying the emulsion makes it possible to limit any oxidative degradation of the substance of interest it contains. Thus, the present description may relate to a dry formulation.

The present description makes it possible to obtain, by a "one-pot process", a dry formulation enriched in potentially bioactive lipid-soluble substances and easily reconstituted by simple rehydration, in a minimum of steps. In addition to the benefits in storage, preservation and use, these formulations may also improve the stability and bioavailability of bioactive molecules, which opens up prospects for applications in various fields, some of which are high value-added, such as the cosmetics, food processing or pharmaceutical industries. In case the extract is used for food (e.g. food supplement or additive) or pharmaceutical purposes, such a formulation may e.g. improve the oral bioavailability of the low water soluble molecules it comprises.

According to another aspect, the present description relates to the use of a dry formulation according to the second aspect, extracted from a source comprising said at least one substance of interest or at least one precursor thereof, for the manufacture of a product comprising at least one substance of interest, by adding to said dry formulation a compound selected from the group comprising water or oil.

According to another aspect, the present description relates to the use of the dry formulation according to the second aspect in the field of cosmetics, food processing, pharmaceuticals, probiotics and/or neutraceuticals.

The above embodiments are not exhaustive. In particular, it is to be understood that other embodiments may be envisaged based on different combinations of embodiments that have been explicitly described. Unless otherwise stated in the present specification, it is obvious to those skilled in the art that the above embodiments may be combined with each other. For example, all features of the above-described embodiments, regardless of which embodiment they relate to the method, may be combined with or replaced by other features of other embodiments, unless stated otherwise.

Embodiments in accordance with the above aspects and additional advantages will become apparent from the following detailed description and the appended claims.

Drawings

Fig. 1 is a schematic diagram illustrating an example of a method according to an embodiment of the present invention.

Detailed Description

In the following detailed description of embodiments of the invention, numerous specific details are set forth in order to provide a more thorough understanding of the present description. It will be apparent, however, to one skilled in the art that the present description may be practiced without these specific details. In other instances, well-known features have not been described in detail to avoid unnecessarily complicating the description.

Fig. 1 shows an embodiment of the extraction method according to the present description, wherein an additional step of freeze-drying the emulsion obtained by sonication is included. The mixture to be emulsified comprises water (1), plant origin (2) (i.e. turmeric root), surfactant (3), lipophilic compound (4). The mixture is contained in a container (5) and the emulsification of the mixture comprises a sonication step carried out by an ultrasonic rod (6) capable of emitting ultrasonic waves (7). Sonication of the mixture results in the formation of an emulsion comprising a continuous aqueous phase (10) and a discontinuous lipid phase (40) in the form of droplets having a content of the substance of interest (21), which in this case is curcumin. A cryoprotectant is then added to the mixture and the freeze drying step results in the formation of a dried formulation (400). The addition of water to the dried formulation (i.e., rehydration) can rehydrate the dried formulation and produce an emulsion having the same characteristics as before drying. However, in other embodiments of the extraction method according to the invention, different from the embodiment shown in fig. 1, rehydration of the emulsion may result in an emulsion having different characteristics than the emulsion before drying, for example in terms of average diameter D and polydispersity index.

An example of an extraction process according to the present description is detailed below, wherein the source chosen is turmeric root, in order to extract the substance of interest curcumin. Curcumin is a polyphenol, and various pharmacological properties (anti-inflammatory, anti-oxidation, anti-cancer, treatment of certain neurodegenerative diseases and the like) of curcumin are only partially researched so far. Furthermore, curcumin exhibits low solubility in water (11 ng/mL in a buffer solution at pH-5), which makes the process according to the present specification particularly attractive for the extraction of curcumin from turmeric. Before attempting to extract curcumin, the inventors tested the solubility of this molecule in various lipophilic compounds by HPLC assay. Among the lipophilic compounds tested, tributyrin has proved to be a good candidate in particular. Interestingly, such lipophilic compounds are biocompatible oils, which may make the method of the invention attractive for certain applications in the field of cosmetics, food processing, pharmaceuticals, neutraceuticals and/or probiotics.

The emulsions numbered 12 to 18 are emulsions obtained by the process of the invention according to many different examples of the process of the invention, some characteristic conditions of the process for obtaining these emulsions being gathered in table 1 (otherwise identical). These examples of methods of producing the emulsions 12 to 18 all involve the use of the surfactant TPGS1000 (alpha-tocopherol polypropylene glycol 1000 succinate), a commercial surfactant derived from vitamin E (tocopherol), and the use of tributyrin as at least one lipophilic compound in the mixture. In emulsions 13 to 18, a second lipophilic compound is added to the mixture provided by the process, in the form of a wax (i.e. jasmine wax in emulsion 13, Suppocire NB in emulsion 14) or another oil (i.e. C8-C10 triglycerides in emulsion 18).

It is evident from the emulsion 14 that the inventors succeeded in obtaining an emulsion comprising a curcumin content corresponding to 100% extraction by the process of the invention. This extraction rate was obtained by sonicating the mixture for only 2 minutes, under the conditions of the present inventors, resulting in the formation of emulsion 14, which was fixed at 100% in acetone for up to 8 hours for the soxhlet extraction apparatus.

TABLE 1

Emulsion and method of making	12	13	14	18
					mTributyrin(mg)	206	181	175	85
mC8-C10 Triglycerides(mg)	0	0	0	15
					mSuppocireNB(mg)	0	0	25	0
mJasmine flowerWax(mg)	0	25	0	0
					m(Curcuma longa)(mg)	100	100	100	100
mTPGS 1000(mg)	60.8	60.8	60.8	60.8
					mH2O(mg)	2027.6	2027.6	2027.6	2027.6
D(nm)	286.5	168.3	194.7	113
					PdI	0.296	0.232	0.254	0.257
Extraction ratio (%)	67	88	100	93

Furthermore, it appears that the substitution of a certain amount of tributyrin with a jasmine wax (emulsion 13) or suppocireNB (emulsion 14) approximately equal to its mass equivalent increases the extraction yield and reduces the polydispersity index PdI, compared to the emulsion 12 obtained with the first compound only, i.e. tributyrin.

The following paragraphs provide some protocol details for the production and analysis of emulsions 12-18.

Protocol for the preparation of the emulsions 12 to 18:

a single lipophilic compound (tributyrin) or two lipophilic compounds (tributyrin/C8-C10 triglyceride or tributyrin/jasmine wax or tributyrin/Suppocire NB) were weighed, in the case of a mixture of the two lipophilic compounds, in a certain ratio. Furthermore, in the case of an oil/wax mixture, the oil/wax mixture is placed in a bath at 40 ℃ until a homogeneous mixture is obtained.

By dispersing surfactant (TPGS 1000)Water to prepare an aqueous phase. The aqueous phase was then added to the at least one lipophilic compound and stirred in its entirety using a vortex stirrer for 20 seconds. The solution was then poured into a 50mL conical centrifuge tube which had been pre-weighed with a certain mass of turmeric (100 mg).

The macroemulsion was prepared by first emulsifying the at least one lipophilic compound, the aqueous phase and the plant source by vortexing for 10 seconds and then placing the centrifuge tube in an ultrasonic bath for 5 minutes at room temperature.

A finer emulsion was then prepared from this crude emulsion using an ultrasonic probe (biolock SCIENTIFIC, Vibracell 7504). The ultrasound probe (Φ ═ 13mm) was placed in a centrifuge tube in an ice bath for 16.75 minutes (in pulsed mode, corresponding to a total of 2 minutes of sonication). The temperature of the mixture during sonication of the mixture is measured, which is in the range of about 4 ℃ to 20 ℃. The applied duty cycle was 11.94% and the ultrasound intensity was 60% (450W).

After removing the ultrasonic probe from the emulsion, the same probe was immersed in a solution containing 2mLIn the pipe of water. Rinse water was then added to the emulsion and the whole was centrifuged at 1100G for 1 minute.

The resulting emulsion was then diluted and divided into 2mL batches. Then 300. mu.L of an aqueous solution of a cryoprotectant (concentration 500mg/mL), such as trehalose or maltose, may be added. These aliquots were then placed in a freezer overnight and freeze-dried for one day.

Analytical protocol for emulsions 12 to 18:

the supernatant obtained after centrifugation was recovered and the droplet size distribution was analyzed by Dynamic Light Scattering (DLS) using a Nano-S Nanosizer (Malvern Instrument). Will useThe supernatant diluted 1:10 in water was placed in a 45. mu.L quartz cell and 10 measurements were made for 10 seconds each. The hydrodynamic diameter or average diameter D is obtained by averaging the results of 10 measurements. The measurement was carried out at an angle of 173 ℃ using a laser having a wavelength of 633 nm. DLS data is calculated based on intensity.

The emulsion was diluted with acetonitrile and then filtered through a 0.2 μm nylon syringe filter, after which the curcumin concentration of the supernatant was determined by high performance liquid chromatography.

The Shimadzu system with diode array detector (SPD-M20A) was equipped with a pumping system (LC-20AD), a degasser (DGU-20A3), a communication module (CBM-20A) and a column oven (Waters). In a Phenomenex Kinetex Biphenyl column (C)4.6 x 100mm,2.6 μm) and the column temperature set at 30 ℃. And carrying out data analysis by adopting Labsolution software. Using a linear gradient of a (water containing 0.1% trifluoroacetic acid) and B (acetonitrile containing 0.1% trifluoroacetic acid), the elution gradient was as follows (v/v): 0min, B37%; 10min, B50%; 15min, B100%, hold for 10 min. The flow rate was 1.3mL/min, and the sample size was 5. mu.L. Detection was performed at 420 nm.

Tables 2 to 9 below collect certain characteristics of the emulsions (i.e., emulsions 19 to 65) prepared according to one or more examples of the method of the present invention. These emulsions are prepared according to non-detailed example protocols (but similar to those described above for preparing emulsions 12 to 18). On the other hand, the analytical protocol for emulsions 19 to 65 was the same as that for emulsions 12 to 18.

Tables 2 to 9 are intended to provide comparable results obtained under otherwise identical conditions.

TABLE 2

Emulsion and method of making	19	18	20	22
					mTributyrin(mg)	100	85	85	42,5
mC8-C10 Triglycerides(mg)	0	15	0	0
					mSuppocireNB(mg)	0	0	15	7.5
m(Curcuma longa)(mg)	100	100	100	100
					mTPGS 1000(mg)	60.8	60.8	60.8	60.8
mH2O(mg)	2027.6	2027.6	2027.6	2027.6
					D(nm)	-	113	109	-
PdI	>0.3	0.257	0.251	>0.3
					Extraction ratio (%)	74	93	87	82

Table 2 shows emulsions 19 and 20 prepared by weighing the same total mass (i.e. 100mg) of lipophilic compounds as emulsion 18 in table 1. The results particularly show the benefit of adding a second lipophilic compound to reduce PdI. Emulsion 22 corresponds to emulsion 20, in which the mass of the lipophilic compound is reduced by half (i.e. 50mg), thereby compromising the homogeneity of the emulsion, emulsion 22 having a PdI higher than 0.3.

The inventors also showed that in the case of freeze-drying followed by rehydration in the presence of 6.5% (w/v) trehalose (used as cryoprotectant), the D of emulsion 18 was 147nm and the PdI was 0.26 (results not shown in Table 2).

TABLE 3

Emulsion and method of making	23	24	25	26	27
						mTributyrin(mg)	100	100	100	100	100
m(Curcuma longa)(mg)	100	150	200	250	300
						mTPGS 1000(mg)	60.8	60.8	60.8	60.8	60.8
VH2O(mL)	4000	4000	4000	4000	4000
						D(nm)	-	134	112	111	119
PdI	>0.3	0.278	0.234	0.209	0.182
						Extraction ratio (%)	87	90	87	94	97

Table 3 shows that the extraction rate increases with the amount of source (turmeric) included in the mixture, as does the homogeneity of the emulsion, which results in a decrease in PdI.

TABLE 4

Unlike opaque emulsion 27, emulsions 28 to 31 shown in Table 4 are translucent due to the inclusion of a "post-extraction" treatment step comprising several ultracentrifugation steps during the extraction process.

The addition of a second ultracentrifugation step in this post-treatment step resulted in a slight decrease in extraction yield, but also in an increase in emulsion homogeneity, which can be reflected by a decrease in PdI.

TABLE 5

Table 5 shows the effect of the properties of the lipophilic compounds in the context of emulsions comprising a single lipophilic compound (or fat 1, F1).

The series of emulsions 32 to 36 shown in table 5 shows, in particular, a good homogeneity of emulsions comprising tocopherol (also referred to as vitamin E) or squalene as lipophilic compound.

TABLE 6

Table 6 shows the effect of a specific step of the pre-treatment and the effect of adding a second lipophilic compound in case the emulsion contains tocopherol as the lipophilic compound (or fat 1, F1).

The results show that the mixture is passed through a disperser-homogenizer (Type) reduces the PdI of the emulsion. The same effect is observed when a second lipophilic compound is used, compared to the case of a single lipophilic compound.

TABLE 7

Table 7 comparison of an emulsion (emulsion 40) obtained by a method comprising a step of emulsifying a mixture by ultrasonication with a homogenizer (Type) was compared two emulsions obtained by the process of the step of performing emulsification.

As shown in table 7, the total sonication time for emulsion 40 was 2 minutes, which corresponds to the total time for applying ultrasound to the mixture. Similarly, for emulsions 41 and 42, the time shown is the total time the disperser-homogenizer was applied to the mixture.

The extraction yield and homogeneity of emulsions 41 and 42 (obtained with a disperser-homogenizer) are similar and slightly higher than the corresponding values characterizing emulsion 40 (obtained by sonication).

The inventors also show that when the emulsions were subjected to a "post-extraction" treatment step consisting of 5000G ultracentrifugation for 5 minutes, the (D; PdI) values of emulsions 40 and 42 were (81; 0.200) and (111; 0.222), respectively (results not shown in Table 7).

Tables 8 and 9 below show the characteristics of other emulsions obtained by applying the extraction method according to the present description to sources other than turmeric root, such as lavender flowers (table 8) or orange peel (table 9). In both cases, the source is of plant origin and contains essential oils representative of the substance of interest.

Two different surfactants were used to extract the lavender essential oil, i.e. TPGS1000 for lotions 43 to 52 and H for lotions 53 to 55₁₂TAC₅. As contemplated in this specification according to one or more embodiments of the process, the emulsions 43 to 47 not comprising sunflower oil also show the possibility of including at least one lipophilic compound only in the source. Table 8 also shows the use of different amounts of cryoprotectant (trehalose) to reduce the polydispersity index of the emulsion after freeze drying/rehydration.

As contemplated in this specification according to one or more embodiments of the method, the emulsions 56 to 60 not comprising sunflower oil show the possibility of including at least one lipophilic compound only in the source. Table 9 also shows the use of different amounts of cryoprotectant (trehalose) to reduce the polydispersity index of the emulsion after freeze drying/rehydration.

TABLE 8

TABLE 9

Various amounts of cryoprotectants (trehalose) were further used to reduce the polydispersity index of the emulsion after freeze-drying/rehydration.