Liquid crystalline phase compositions and methods for their preparation

Abstract

The present invention relates to an enzymatic method for producing stable lipid-based liquid crystal phases of the Fd3m or HII space group, compositions comprising such phases and uses thereof, wherein the starting mixture comprises 30 to 65 wt% of polar lipids and 35 to 70 wt% of oil; which is well dispersed; and triglyceride lipase is added to produce an oil hydrolysate which leads to liquid crystal formation in the presence of an aqueous solvent.

Description

Technical Field

[0001] This invention relates to an enzymatic method for producing stable lipid-based liquid crystal phases, compositions comprising such phases, and their uses. Background of the Invention

[0003] Liquid crystal phases are structures formed by the self-assembly of amphiphilic molecules (such as lipids) in an aqueous environment. These phases are known for their highly organized and unique molecular arrangements. Unlike emulsions (which are mixtures of immiscible liquids stabilized by emulsifiers), liquid crystal phases exhibit specific geometric patterns and are characterized by their unique physical properties.

[0004] Fd3m and HII liquid crystal phases represent two different arrangements. The Fd3m phase is a discontinuous cubic structure, characterized by its complex three-dimensional network of two different-sized antiphase micelles. The HII phase, also known as the antiphase hexagonal phase, is characterized by its tubular structure of hexagonal stacks. These phases hold significant potential in a variety of applications, including drug delivery systems, food technology, and cosmetic formulations.

[0005] However, producing these specific liquid crystal phases can be extremely challenging, especially when the goals are cost-effectiveness and compliance with clean label standards. Traditional methods often involve complex processing and may require the use of synthetic or potentially health-impacting additives.

[0006] Therefore, innovative, low-cost, and efficient methods are needed to produce these specialized liquid crystal phases to meet the growing consumer demand for products that are both effective and composed of natural, healthy ingredients. Summary of the Invention

[0007] This invention relates to compositions comprising a stable liquid crystal phase containing lipids organized in the Fd3m or HII space group, and methods for producing them.

[0008] In a first aspect, the present invention relates to a method for producing a stable liquid crystal phase comprising lipids of tissue in the Fd3m and / or HII space groups, the method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%; and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add an aqueous solvent to the starting mixture and mix; and c. Add at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase, and then mix; This results in a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

[0009] In a second aspect, the present invention relates to a liquid crystal phase of lipids in the Fd3m or HII space group, said liquid crystal phase being obtained or obtainable by the method of the present invention.

[0010] In another aspect, the present invention relates to a liquid crystal phase comprising lipids organized in the Fd3m and / or HII space groups, the liquid crystal phase comprising a phase mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, ii. Oils in the range of 35 to 70 wt%; and iii. Aqueous solvents; The liquid crystal phase described herein has a lipid structure characterized by small-angle X-ray scattering (SAXS) spectra, which are defined by peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44 for Fd3m space group, and / or 1, √3, 2, √7, 3, √12 for HII space group.

[0011] In a third aspect, the present invention relates to the use of liquid crystal phases comprising lipids in Fd3m and / or HII space groups, which are obtained or obtainable by the method of the present invention.

[0012] In another aspect, the present invention relates to a hydrolysate mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and iii. Other components less than 15 wt%.

[0013] In a fourth aspect, the present invention relates to the use of triglyceride lipase to generate a liquid crystal phase of tissue according to the invention in space group Fd3m or HII.

[0014] In a further aspect, the present invention relates to a product selected from food, feed, cosmetics and nutritional supplements, wherein the product comprises a liquid crystal phase as described herein.

[0015] Another aspect of the invention relates to a liquid crystal phase as described herein, which is used as a pharmaceutical.

[0016] Brief description of the attached figures

[0017] Figure 1- SAXS diagram of the liquid crystal phase formed in Example 2. The diagram shows a well-defined liquid crystal phase with Fd3m having a mode with peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44.

[0018] Figure 2 - SAXS diagram of the liquid crystal phase formed in Example 7. This diagram shows a SAXS diagram of a liquid crystal phase characterized by a less defined phase. It exhibits peak patterns that may be partially or completely overlapping, and in SAXS measurements, Fd3m is defined by the peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44.

[0019] Figure 3 - Optical microscope images of the hydrolysate mixture before (a) and 5–20 seconds after (b) the addition of water. Figure 3 In diagram a, a boundary is visible that separates the air (to the left) from the hydrolysate mixture (to the right). Figure 3 In b, the LCP structure is visible in the middle. Invention Details

[0021] This invention is based on a surprising discovery: when oil hydrolysates are mixed with polar lipids in the presence of water, the components present in the mixture assemble into a liquid crystal phase containing lipids organized in the Fd3m and / or HII space groups. These liquid crystal phases (LCPs) are useful in many applications, such as in food, animal feed, cosmetics, nutritional supplements, and pharmaceuticals.

[0022] The presence of the Fd3m and / or HII space groups endows it with exceptional stability.

[0023] In this invention, the liquid crystal phase formation can be achieved using oils derived entirely from natural fats and oils, without any synthetic components (e.g., synthetic lipids or emulsifiers). The method of this invention provides a means to avoid the use of chemicals or compounds that may be considered undesirable or unhealthy by consumers, such as organic solvents.

[0024] Furthermore, the methods of this invention provide robust approaches for the large-scale production of LCPs of tissues in the Fd3m and / or HII space groups. This method is particularly effective in converting the starting mixture into a liquid crystal phase. Previously, liquid crystal phases of tissues in the Fd3m space group were only known to form under a narrow range of conditions. In contrast, this invention allows the use of several types of lipids, and the LCPs of this invention are formed under a wide range of conditions.

[0025] These methods can be implemented using plant-based oils, thus avoiding less desirable components due to their effects on plant, animal, or human health.

[0026] The method of the present invention further provides a method for producing a stable composition containing bioactive molecules.

[0027] definition

[0028] MAG: Monoglyceride; DAG: Diglyceride; TAG: Triglyceride; FFA: Free fatty acid

[0029] In the context of triglyceride hydrolysis, the term "hydrolysis" refers to the removal of at least one fatty acid chain from the glycerol backbone by a lipase.

[0030] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural references unless the context clearly indicates otherwise.

[0031] It should be understood that the embodiments described herein include those that are “composed of” and / or “substantially composed of”. As used herein, unless the context requires otherwise due to the language of expression or necessary implication, the word “comprising” or variations such as “including” or “containing” are used in an inclusive sense, that is, specifying the presence of the described features but not excluding the presence or addition of further features in various embodiments.

[0032] Overview of the method of the present invention

[0033] The hydrolysis products of oils bind with aqueous solvents (e.g., water) and polar lipids to assemble into liquid crystal phases organized in space groups Fd3m and / or HII. The inventors have remarkably determined the ratio of hydrolysis products to polar lipids required for the formation of these liquid crystal phases, as well as the necessary parameters. Hydration of the components is crucial. Therefore, a certain amount of water must be present, and the components must be allowed to hydrate to form LCPs.

[0034] It should be understood that there are many methods for generating hydrolysis products, providing polar lipids, ensuring the hydration of these components, and mixing these components. This invention covers all suitable methods, provided that a defined ratio of hydrolysis products to polar lipids is achieved, resulting in the formation of a liquid crystal phase.

[0035] Therefore, this invention relates in one aspect to a method for producing LCPs comprising lipids from tissues in the Fd3m and / or HII space groups. Note that the disclosure of the method parameters of this invention is also relevant to all other aspects of this invention.

[0036] Liquid Crystal Phase (LCP)

[0037] Liquid crystals possess properties intermediate between liquids and solid crystals. They are classified into different types, including lyotropic liquid crystals, thermotropic liquid crystals, and metallotropic liquid crystals. This invention relates to lyotropic liquid crystals, meaning they are composed of amphiphilic molecules and a solvent (such as water).

[0038] This invention relates to a method for producing a liquid crystal phase comprising lipids organized in Fd3m and / or HII space groups. As used herein, the term "liquid crystal phase" refers to a lipid-containing phase formed when oil hydrolysis products and polar lipids are organized into specific space groups. This LCP is organized and separated from the residual aqueous phase. The LCP according to the invention organizes lipid components into specific space groups, which are identifiable using SAXS, as further described below. Figure 1 and Figure 2 The Fd3m and HII space groups are particularly stable molecular arrangements.

[0039] The inventors have determined the conditions under which the Fd3m or HII phases form, either in large quantities (well-defined) or in smaller quantities and sometimes together with other phases (less well-defined). The classification is based on the very typical peak patterns of these phases observed in SAXS, as detailed below.

[0040] When no significant amounts of Fd3m or HII are formed, this structure is referred to in this paper as disordered (when we cannot identify any typical LC phase using SAXS) or “layered.” For example, layered phases can be observed at low levels of polar lipids (PL4–PL25). This space group is not related to stability.

[0041] The viscoelastic properties of liquid crystal phases (such as Fd3m) with storage moduli typically exceeding 10,000 Pa further demonstrate their stability.

[0042] A well-defined liquid crystal phase: a product comprising a polar lipid-based liquid crystal phase having a non-layered intermediate phase space group of Fd3m or HII. This is characterized by a characteristic pattern of peak ratios in SAXS measurements, where Fd3m is defined by the peak positions at q values ​​of the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44, and HII by the peak positions at q values ​​of the following ratios: 1, √3, 2, √7, 3, √12. See also Figure 1 An example SAXS diagram is shown.

[0043] Less clearly defined liquid crystal phase: Products containing polar lipid-based liquid crystal phases having Fd3m and / or HII space groups. This manifests as potentially partially or completely overlapping peak patterns, in SAXS measurements, where Fd3m is defined by the peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, and √44, and HII is defined by the peak positions at q values ​​at the following ratios: 1, √3, 2, √7, 3, and √12. See also Figure 2 An example SAXS diagram is shown.

[0044] Laminar or lipid micelle aggregates: especially formed at 25% or lower polar lipid content. In SAXS measurements, the laminar phase is defined by the peak positions at q values ​​at the following ratios: 1, 2, 3, 4, 5, 6.

[0045] Furthermore, some embodiments relate to the method according to the invention, wherein at least 50 wt% of lipids from the starting mixture are incorporated into the lipid-based liquid crystal phase; for example, at least 60%, 70%, 80%, 90%, 95%, 96%, 97%; or for example, in the range of 50 to 98 wt%.

[0046] Therefore, some embodiments relate to the method according to the invention, wherein at least 50 wt% of the lipids present in the stable liquid crystal phase are organized in the Fd3m or HII space group, for example at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 96 wt%, 97 wt%; or for example 50 wt% to 98 wt%.

[0047] Methods for preparing LCP

[0048] The invention relates, in a first aspect, to a method for producing a stable liquid crystal phase comprising lipids of tissue in the Fd3m and / or HII space groups.

[0049] Therefore, one embodiment of the present invention relates to a method for generating a stable liquid crystal phase comprising lipids of tissue in the Fd3m and / or HII space groups, the method comprising the following steps: A. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%; and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; B. Perform steps (B1) and (B2) in any order or simultaneously: B1. Add an aqueous solvent to the starting mixture and mix; and B2. Contacting the mixture from step (A) or (B1) under conditions allowing lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; and C. Further mix the mixture from step (B); This results in a liquid crystal phase containing lipids organized in the Fd3m and / or HII space groups.

[0050] In one embodiment of the invention, step (B1) precedes step (B2).

[0051] In another embodiment of the invention, step (B2) precedes step (B1).

[0052] Optionally, the lipase is removed before step (C). For example, if step (B2) precedes step (B1), the lipase can be removed between steps (B2) and (B1).

[0053] In some embodiments, an aqueous solvent is added at a ratio of at least 0.01:1, for example at least 0.2:1, relative to the volume of the starting mixture.

[0054] In one related embodiment, the present invention relates to a method for generating a stable liquid crystal phase comprising lipids of tissue in the Fd3m and / or HII space groups, the method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%; and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add an aqueous solvent to the starting mixture and mix; and c. Add at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase, and then mix; This results in a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

[0055] In some embodiments, an aqueous solvent is added at a ratio of at least 0.01:1, for example at least 0.2:1, relative to the volume of the starting mixture.

[0056] One embodiment of the invention relates to the method described herein, wherein step a precedes steps (b) and (c), and steps (b) and (c) may be performed in any order or simultaneously.

[0057] Another embodiment of the invention relates to the method described herein, wherein step (b) precedes step (c).

[0058] Further embodiments of the invention relate to a method as described herein, wherein step (c) precedes step (b). In a further embodiment, the invention relates to a method for producing a stable liquid crystal phase comprising lipids of tissue in the Fd3m and / or HII space groups, the method comprising the following steps: a. Mix the oil and polar lipids to form a starting mixture consisting of the following: i. Polar lipids in the range of 30 to 65 wt%, and; ii. Oils in the range of 35 to 70 wt%; and iii. Other components less than 15 wt%, The oil and polar lipids are well dispersed in each other; b. Add water at a ratio of at least 0.2:1 relative to the volume of the initial mixture, and mix; c. Contact the mixture from step (b) with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; and d. Further mix the mixture from step (c); This results in a liquid crystal phase containing lipids organized in the Fd3m and / or HII space groups.

[0059] Without being bound by theory, it appears that hydrolysis products from lipase reactions are organized together with polar lipids to form a liquid crystal phase comprising lipids organized in space groups Fd3m and / or HII. Preferably, a relatively large proportion of the lipids are incorporated into the LCP phase.

[0060] Therefore, one embodiment of the present invention relates to a method as described herein, wherein at least 50 wt% of lipids from the starting mixture are incorporated into the liquid crystal phase.

[0061] Another embodiment of the invention relates to the method described herein, wherein at least 50 wt% of the lipids in the liquid crystal phase are organized in the Fd3m or HII space group.

[0062] Oil

[0063] In some embodiments of the method of the present invention, oil and polar lipids are mixed to form a starting mixture. This starting mixture may also be provided as a premixed mixture.

[0064] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the oil and the polar lipid in the starting mixture have been premixed.

[0065] As used herein, the term "oil" refers to a lipid composition that primarily comprises triglycerides and is liquid at 40°C. Typically, oil contains triglycerides in the range of 94 to 99 wt%. In certain embodiments, oil may contain 95 to 99 wt% TAG, or, for example, at least 97 wt% TAG, or at least 98 wt% TAG, or at least 99.5 wt% TAG, or consist substantially of TAG.

[0066] The oil according to the invention may contain oil from several sources, or may consist of oil from a single source.

[0067] The specific implementation involves the oil being selected from the group consisting of vegetable oils, microbial oils, marine oils, animal oils, and any combination thereof.

[0068] As used in this article, vegetable oil refers to any oil derived from a plant, such as from seeds, leaves, or any other part of a plant. Microbial oil refers to any oil derived from microorganisms. An example of microbial oil is algal oil. Marine oil refers to any oil derived from any marine organism, such as one or more of algae, seaweed, fish, or plankton. Fish oil refers to any oil derived from any fish. Fish oil is an example of animal oil.

[0069] The choice of oil used in the methods of this invention can be based on specific diets or customer needs. For example, vegetarian products can be manufactured using one or more vegetable oils and / or microbial oils. Compositions high in omega-3 fatty acids can be manufactured using one or more marine oils.

[0070] Specific embodiments of the present invention relate to the oil comprising or consisting of vegetable oil and / or marine oil.

[0071] In certain embodiments, such as when relating to food, feed, and cosmetic applications, the oil may be one or more vegetable oils, such as one or more oils selected from the group consisting of oils derived from soybean, grapeseed, cocoa, olive, palm, rice bran, rapeseed, canola, sunflower seed, safflower seed, peanut, cottonseed, almond, coconut, palm kernel, shea butter, corn, hazelnut, flaxseed, wheat germ, and sesame; or one or more marine oils, such as one or more selected from the group consisting of fish oil, algae oil, and seaweed oil.

[0072] In a particular embodiment, the present invention relates to wherein the oil is a food-grade oil, particularly a food-grade vegetable oil.

[0073] Although animal fats are typically solid at 40°C, they can be processed to become liquid at room temperature, such as ghee. Alternatively, the method of the present invention can be carried out at a temperature where the animal fats are liquid.

[0074] The method of the present invention relates to cases where the lipids are in liquid form. Therefore, specific embodiments relate to oils and polar lipids that are liquid at 40°C. However, in principle, any fat can be used as a source of oil and / or polar lipids, provided the method is carried out at parameters where the lipids are in liquid form. For example, the method can be carried out at high temperatures that melt the lipids, or at a combination of temperature and pressure where the lipids are in liquid form.

[0075] polar lipids

[0076] The term "polar lipid" as used in this article refers to lipids that have both hydrophobic and hydrophilic portions.

[0077] In one embodiment, the polar lipid comprises one or more phospholipids.

[0078] In a further embodiment, the polar lipid comprises one or more glycolipids.

[0079] Further embodiments of the invention relate to polar lipids comprising one or more phospholipids and / or one or more glycolipids.

[0080] Phospholipids may be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and combinations thereof. Glycolipids may be selected from the group consisting of digalactosyl diglyceride (DGDG), monogalactosyl diglyceride (MGDG), digalactosyl monoacylglyceride (DGMG), monogalactosyl monoacylglyceride (MGMG), and combinations thereof.

[0081] Polar lipids can come from any suitable source, such as those endogenous to the oil, those exogenously added to the oil, or any combination thereof.

[0082] A particular implementation involves the polar lipid comprising an exogenously added polar lipid.

[0083] As used in this article, lecithin refers to an oil component with a high content of polar lipids. Lecithin is an readily available source of polar lipids. In addition to polar lipids, lecithin may contain neutral fats, particularly TAGs.

[0084] In some embodiments of the present invention, the polar lipids comprise or consist of one or more lecithins, such as one or more lecithins selected from the group consisting of plant lecithins, microbial lecithins, marine lecithins, animal lecithins, and combinations thereof.

[0085] Specific embodiments of the present invention relate to polar lipids comprising or consisting of one or more plant lecithins. Any plant lecithin can be used in the methods of the present invention. Examples of plant lecithins include lecithin derived from sunflower seeds, soybeans, oats, rapeseed, and cottonseed.

[0086] Some embodiments of the present invention involve polar lipids comprising or consisting of one or more animal lecithins, such as, for example, egg yolks and milk.

[0087] Oat oil has a particularly high content of polar lipids, ranging from 20-40 wt%, with digalactosyl diglyceride (DGDG) and phosphatidylcholine being the most abundant. In a specific embodiment of the method of the present invention, the oil provided is oat oil, and the polar lipids are provided by oat oil lecithin.

[0088] Other components

[0089] The starting mixture contains less than 15 wt% of components other than polar lipids and oils. These components may, for example, consist of or be composed of sterols, carbohydrates, or proteins present in oils and / or polar lipids. Other components may also include one or more additives as described below.

[0090] Therefore, one embodiment of the present invention relates to a method as described herein, wherein the starting mixture contains less than 15 wt% of other components.

[0091] In some embodiments, the starting mixture contains less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1% of other components (all percentages are in wt).

[0092] In other embodiments, the starting mixture contains other components in the range of 0.5% to 15%, 0.5% to 10%, 0.5% to 8%, 0.5% to 6%, 0.5% to 5%, 0.5% to 4%, 0.6% to 3%, or other components (by weight) in the range of 0.05% to 6%, 0.05% to 4%.

[0093] Starting Mixture

[0094] In one method of the present invention, oil and polar lipids are mixed to form a starting mixture comprising polar lipids in the range of 30 to 65 wt% and oil in the range of 35 to 70 wt%.

[0095] In one embodiment, the starting mixture consists of the following: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oils in the range of 35 to 70 wt%; and iii. Other components less than 15 wt%.

[0096] This ratio of polar lipids to oil is important for the formation of the crystalline phase, see Example 5.

[0097] The starting mixture can be formed by combining one or more oils with a further composition comprising one or more polar lipids. For example, oils can be combined with polar lipids (e.g., lecithin) to achieve mixtures within the range specified by the method of the present invention. Any oil can be combined with any polar lipid.

[0098] In one embodiment, an oil that already naturally possesses the polar lipid content according to the invention can be selected, and no further polar lipids need to be added. Therefore, one embodiment of the method of the invention relates to a case where no exogenous polar lipids are added.

[0099] In an alternative embodiment, the method of the present invention includes step (a) of mixing at least one lecithin and at least one oil to form a starting mixture consisting of lecithin in the range of 30 to 65 wt% and oil in the range of 35 to 70 wt%; wherein the oil and polar lipids are well dispersed in each other.

[0100] mix

[0101] The method of the present invention includes mixing a starting mixture. This ensures that polar lipids are dispersed in the oil. This mixing can be carried out in any suitable manner, and those skilled in the art can select such a manner. In some embodiments, the mixing occurs prior to contact with a lipase.

[0102] In some cases, the oil may already inherently possess the polar lipid qualities specified in this invention. In such cases, the oil may still be blended to ensure that the polar lipids are dispersed throughout the oil.

[0103] In one implementation, the mixing can take place for at least 20 minutes, for example, at least 30, 40, 60, 90, or 120 minutes. In other examples, the mixing can take place in the range of 20 minutes to 10 hours, or for example, 1 hour to 10 hours.

[0104] water

[0105] Without wishing to be bound by theory, water is considered to have two functions in the method of the present invention. The enzymatic activity in step (c) requires water. The addition of water also initiates the formation of LCPs, and some of the water becomes a component of the LCPs formed in the method of the present invention, which contain lipids organized in the Fd3m and / or HII space groups.

[0106] Therefore, in some embodiments, the method of the present invention includes the step of adding an aqueous solvent (e.g., water) to the starting mixture to achieve an amount of at least 0.2 parts aqueous solvent to 1 part starting mixture based on the volume of each portion. The upper limit is not critical, as excess water is excluded from the formed LCP into the aqueous phase. Thus, the volume ratio can be, for example, in the range of 0.2:1 to 10:1, e.g., from 0.3:1 to 6:1; or, for example, from 0.2:1 to 5:1, or, for example, from 1.2:1 to 4:1.

[0107] Therefore, one embodiment of the invention relates to a method as described herein, wherein the volume ratio of the aqueous solvent to the starting mixture is in the range of 0.2:1 to 10:1, for example from 0.3:1 to 6:1. In some variations of this method, only a very small amount of aqueous solvent is required to initiate the formation of LCP, for example when the aqueous solvent is added to the hydrolysate mixture. Therefore, one embodiment of the invention relates to a method as described herein, wherein the aqueous solvent is added to achieve a ratio of at least 0.01:1 relative to the volume of the hydrolysate mixture, for example at least 0.02:1, at least 0.05:1, at least 0.1:1, or at least 0.2:1 relative to the volume of the hydrolysate mixture.

[0108] Therefore, in some embodiments, the aqueous solvent is added at a ratio of at least 0.01:1 (e.g., at least 0.2:1) relative to the volume of the starting mixture.

[0109] Another embodiment of the invention relates to the method as described herein, wherein the aqueous solvent comprises at least 70 vol% water, such as at least 80 vol% water, such as at least 90 vol% water, such as at least 95 vol% water, such as at least 99 vol% water.

[0110] Further embodiments of the present invention relate to the method as described herein, wherein the aqueous solvent is composed of water.

[0111] Mixing in the presence of water ensures that the components of LCP can be assembled efficiently. The transport of water to the reverse micelles of the Fd3m structure is a potentially slow step, which is facilitated by mixing. Furthermore, this mixing contributes to efficient enzymatic hydrolysis.

[0112] Water can be in any suitable form. For example, the at least one lipase in step (c) can be provided in aqueous solution in step (b). The aqueous lipase solution can form some or all of the added water. Therefore, specific embodiments relate to cases where the lipase is provided in aqueous solution.

[0113] Therefore, one embodiment of the present invention relates to the method as described herein, wherein the at least one lipase is contained in an aqueous solvent.

[0114] Aqueous solvents

[0115] Water can be provided as an aqueous solvent that mainly comprises water or is composed entirely of water. Therefore, the aqueous solvent may contain additional polar solvents. These additional polar solvents may be, but are not limited to, methanol, ethanol, acetone, acetonitrile, and ethylene glycol, preferably methanol or ethanol.

[0116] The volume ratio between water and the additional polar solvent can be from 100:0 to 80:20, for example 100:00 to 90:10, for example 100:00 to 95:5.

[0117] Excessive concentrations of the additional polar solvent can interfere with enzyme functionality and are undesirable.

[0118] Lipase activity

[0119] The inventors have determined that diglycerides, monoglycerides, and free fatty acids produced during the hydrolysis of triglycerides contribute to the formation of liquid crystal phases containing lipids in the Fd3m and / or HII space groups.

[0120] The at least one lipase according to the present invention can be any lipase capable of hydrolyzing triglycerides.

[0121] Therefore, one embodiment relates to the method according to the invention, wherein, under the conditions of step (c) or step (a1), the primary activity of the at least one lipase is the hydrolysis of triglycerides.

[0122] Some embodiments of the present invention involve a mixture of oil, polar lipids and an aqueous solvent (e.g., water) being contacted with at least one lipase under conditions that allow lipase activity, such that at least 1% of the triglycerides in the oil are hydrolyzed by said at least one lipase.

[0123] In a particular embodiment, the at least one lipase comprises or consists of triglyceride lipase [EC 3.1.1.3].

[0124] In a further specific embodiment, the at least one lipase comprises or consists of lipases having sn1,3-position specificity, such lipases preferentially cleaving at positions 1 and 3 of the glycerol backbone.

[0125] The sn 1,3 lipase can be any lipase having this position specificity. For example, the sn 1,3 lipase can be selected from Rhizopus oryzae (…). Rhizomucor miehie Lipases (e.g., RM lipase from Novozymes), *Thermophilus sparsely cottony* ( Thermomyces lanuginosus Lipases (e.g., Lipozyme TL lipase) and Rhizopus oligosporus ( Rhizomucor Arrhizus The group consisting of lipases (such as Palatase from Novozymes), and any variants or modifications of these enzymes, such as thermostable variants, etc.

[0126] Some embodiments of the method of the present invention involve the lipase having little or no phospholipase activity under the conditions of step (c) or (a1). In some embodiments, the at least one lipase is not classified as a phospholipase.

[0127] The conditions for enzyme activity can be selected by those skilled in the art. These parameters include temperature, water volume, mixing rate, incubation time, and enzyme quantity. Those skilled in the art will be able to select the conditions under which the enzyme will achieve triglyceride hydrolysis.

[0128] The lipase can be provided in any formulation compatible with the hydrolysis of oil, and those skilled in the art will be able to select such formulations. Typically, in the method of the present invention, the lipase is provided in the form of an aqueous suspension and incubated with oil under mixed conditions. Alternatively, the lipase can be provided as an immobilized lipase, as detailed below. Any combination of various formulations (e.g., aqueous formulations and / or immobilized formulations) can be used; in any order, including parallel use, simultaneous use, and / or sequential use.

[0129] Lipase Immobilization

[0130] Some embodiments of the present invention involve the at least one lipase comprising or consisting of an immobilized lipase.

[0131] In some embodiments of the invention, at least one lipase may be provided in the form of an immobilized lipase. For example, the lipase may be any of the immobilized lipases described above. An immobilized lipase refers to the at least one lipase covalently or non-covalently attached to solid particles (e.g., beads). Immobilized enzyme particles can be used in various types of reactors, including stirred tanks, rotating bed reactors, and packed bed reactors. Immobilized lipases have the advantage of being reusable. Furthermore, it provides a means of controlling the degree of hydrolysis of triglycerides. The reaction can be stopped by separating the immobilized lipase from the reaction mixture (hydrolysis product mixture). A further advantage is that removing the immobilized lipase ensures an enzyme-free product.

[0132] In one embodiment, the at least one lipase comprises or is composed of a triglyceride lipase (e.g., sn-1,3-specific lipase), said triglyceride lipase being immobilized, such as immobilized TL lipase.

[0133] Degree of hydrolysis

[0134] The reaction products of lipase hydrolysis of triglycerides are important for the formation of stable liquid crystal phases of lipids containing tissues in space groups Fd3m and / or HII of the present invention, see Example 7.

[0135] Therefore, some embodiments of the method of the present invention include step (c) contacting the mixture of step (b) with at least one lipase under conditions allowing lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase. Here, "hydrolyzed" means the removal of at least one fatty acid chain from the glycerol backbone.

[0136] In other embodiments, the amount of hydrolyzed triglycerides is at least 5 wt%, for example at least 30, at least 40, at least 50, at least 55, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 92, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 99% wt.

[0137] Alternatively, in some embodiments of the invention, the invention relates to a residual triglyceride content of no more than 60 wt%, such as no more than 50, 45, 40, 38, 35, 32, 30, 27, 25, 20, 18, 15, 12, 10, 8, 5, 4% or no more than 3 wt%, calculated as wt% based on the amount of triglycerides in the starting mixture (see step (a)) containing oil, polar lipids and other components that may be present.

[0138] In a particular implementation, the LCP does not contain triglycerides, meaning that all triglycerides affected by lipases have been hydrolyzed.

[0139] In some embodiments, the LCP contains components within the following ranges: at least 35% FFA, no more than 20% DAG, and no more than 10% TAG; or, for example, at least 40% FFA, no more than 15% DAG, and no more than 10% TAG. This content can be calculated as a percentage of peak area in TLC data (see Example 6).

[0140] Without being bound by assumptions, it appears that the levels of TAG and FFA are more important for LCP formation than the levels of DAG and MAG. Therefore, specific embodiments relate to LCPs of the present invention comprising components within the following ranges: at least 35% FFA and no more than 10% TAG; for example, at least 40% FFA and no more than 10% TAG; for example, at least 50% FFA and no more than 10% TAG; or for example, at least 60% FFA and no more than 5% TAG, or at least 60% FFA and no more than 3% TAG, or at least 60% FFA and substantially TAG-free.

[0141] Low amounts (e.g., less than 10%) of TAG appear to be particularly useful when the goal is to form LCPs containing lipids organized in the Fd3m space group.

[0142] Typically, LCPs containing lipids in the HII space group of tissue contain higher amounts of TAG compared to LCPs containing lipids in the Fd3m space group.

[0143] For example, LCPs containing lipids in the HII space can be contained in TAGs ranging from about 15% to 60%, such as from 25% to 58%, or from 35% to 55%.

[0144] Further mixing

[0145] The method of the present invention includes a first step of mixing, which is performed to ensure the dispersion of polar lipids in the oil, as described above. The method of the present invention may further include mixing in which a lipase is contacted with the mixture. Mixing aimed at ensuring contact between the lipase and the substrate and / or water can be carried out by any suitable means.

[0146] Some embodiments of the invention involve allowing the formation of LCP to last for at least 12 hours, such as at least 14 hours, at least 20 hours, or in the range of 12 to 100 hours, or in the range of 16 to 100 hours.

[0147] Another embodiment of the invention relates to the method as described herein, wherein the formation of the liquid crystal phase is allowed to continue for at least 12 hours, for example at least 14 hours, preferably at least 16 hours.

[0148] A relatively long formation time is required to ensure sufficient lipase activity. Once sufficient hydrolysis has occurred, LCP formation proceeds rapidly. Sufficient hydrolysis is considered to have occurred when TAG levels are within the ranges described herein.

[0149] Therefore, in the embodiment of forming the hydrolysate mixture, the time-consuming part is the formation of the hydrolysate mixture. Thus, if the hydrolysate mixture is stored for at least the aforementioned time period, then LCP formation upon addition of water is instantaneous. Instantaneous means no more than 1 minute, for example, no more than 50 seconds, for example, no more than 40 seconds, for example, no more than 30 seconds, for example, no more than 20 seconds, for example, no more than 10 seconds, for example, no more than 5 seconds.

[0150] additive

[0151] The inventors have demonstrated that the liquid crystal phase of this invention can be formulated to contain additives. Additives may also be referred to as ingredients.

[0152] Therefore, some embodiments involve a method according to the invention, wherein at least one additive is added to produce a stable liquid crystal phase comprising said additive.

[0153] If water-soluble, these one or more further components can be added to the aqueous composition; or if lipophilic, they can be mixed with process oils and / or polar lipids and incorporated into the LCP. Therefore, one embodiment of the invention relates to the method as described herein, wherein if the additive is lipophilic, the at least one additive is added to the starting mixture or hydrolysate mixture; or if the additive is water-soluble, the at least one additive is added together with the aqueous solvent.

[0154] Further ingredients can be any ingredients. They can be, for example, bioactive molecules such as vitamins, pharmaceutically active ingredients, etc. In other embodiments, as an alternative or supplement, the additive may include one or more of colorants, flavorings, and taste agents. The additives can be selected based on the product's needs and objectives, and there are no particular limitations.

[0155] One embodiment of the present invention relates to the method as described herein, wherein the at least one additive is selected from the group consisting of bioactive molecules, pharmaceutically active ingredients, colorants, flavorings, and tasters.

[0156] Further steps

[0157] The method of the present invention may include one or more further steps to further process the LCP. For example, the method of the present invention may include a step of spray drying the LCP. Spray drying may be performed, for example, by dispersing the LCP and then subjecting it to, for example, ultrasonic treatment to obtain particles dispersed in a solution. The solution is then spray dried to obtain a final product in powder form. Therefore, one embodiment of the present invention relates to the spray-dried LCP of the present invention.

[0158] Further methods for generating LCP

[0159] When oil hydrolysate, polar lipids, and aqueous solvents (e.g., water) are combined in the amounts according to the invention, an LCP is formed.

[0160] In one embodiment, the oil is hydrolyzed after mixing with the polar lipids in the presence of water. In these embodiments, as hydrolysis proceeds, the resulting hydrolysis products (e.g., FFA, MAG, and / or DAG) and polar lipids assemble into LCPs. This principle may be referred to as "one-pot production".

[0161] In some embodiments, LCPs are produced in a two-step process, wherein the hydrolysis of the oil and the formation of the LCP are carried out in separate reactions. Thus, in the first step, the oil is hydrolyzed to produce a hydrolysate (hydrolysate). In a separate second step, the hydrolysate is combined with water and polar lipids in amounts according to the invention to form an LCP. These two steps can be separated temporally or physically. An example of temporal separation is, for example, where the oil is first hydrolyzed; thereafter, at least one enzyme is inactivated or removed; and subsequently, the hydrolysate is combined with polar lipids and water in amounts according to the invention to form an LCP. An example of physical separation is, for example, where the oil is hydrolyzed using an immobilized lipase in a reactor bed; thereafter, the hydrolysate reaction mixture containing the hydrolysate is separated from the immobilized lipase, for example, by transferring the hydrolysis reaction mixture containing the hydrolysate to another container, leaving the immobilized lipase in place; and subsequently, in said container, the hydrolysate is combined with water and polar lipids in amounts according to the invention to form an LCP.

[0162] Therefore, the enzymatic hydrolysis of oils to produce hydrolysis products can be carried out before, during, or after mixing with polar lipids.

[0163] Therefore, in some embodiments of the present invention, therein relates to a method for generating a stable liquid crystal phase comprising lipids of tissue in the Fd3m and / or HII space groups, the method comprising the following steps: a1. Contacting an oil with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by the at least one lipase to obtain an oil hydrolysate; b1. Optionally, remove at least one lipase from the oil hydrolysate. c1. The oil hydrolysate from step a1 or b1 is mixed with a polar lipid to form a hydrolysate mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and The oil hydrolysate and polar lipids are well dispersed in each other; d1. Add an aqueous solvent to the hydrolysate mixture and mix further; This results in a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

[0164] In one embodiment, an aqueous solvent is added to achieve a ratio of at least 0.01:1 relative to the volume of the hydrolysate mixture, for example, at least 0.2:1.

[0165] Another embodiment of the present invention relates to the method as described herein, wherein the method includes the following steps: a1. Contacting an oil with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; b1. Optionally, remove the lipase from the reaction mixture. c1. The oil hydrolysate from step a1 or b1 is mixed with polar lipids to form a mixture consisting of: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and iii. Other components less than 15 wt%; The oil hydrolysate and polar lipids are well dispersed in each other; d1. Add water to achieve a ratio of at least 0.2:1 relative to the starting mixture and mix. This results in the formation of a stable lipid-based liquid crystal phase.

[0166] The above method can be stopped after step c1 to obtain a hydrolysate mixture. The hydrolysate mixture is storage stable.

[0167] Then, a method for forming a liquid crystal phase includes the following steps: i. Provide a mixture of hydrolysates; ii. Add an aqueous solvent to the volume of the hydrolysate mixture and mix; This results in the formation of a stable lipid-based liquid crystal phase.

[0168] In some embodiments, an aqueous solvent is added to achieve a volume ratio of at least 0.01:1 relative to the hydrolysate mixture, for example at least 0.2:1, and then further mixed.

[0169] When an aqueous solvent (such as water) is added to a mixture of hydrolysates, LCP formation is typically instantaneous. Instantaneous means no more than 1 minute, for example, no more than 50 seconds, for example, no more than 40 seconds, for example, no more than 30 seconds, for example, no more than 20 seconds, for example, no more than 10 seconds, for example, no more than 5 seconds.

[0170] Hydrolysate mixture

[0171] This disclosure also relates to the hydrolysate mixture itself. It has been found that the hydrolysate mixture is unexpectedly stable. After prolonged storage, additives can be added to the hydrolysate mixture and then mixed with an aqueous solvent to form the desired LCP. If the additive is fragile, it may be preferable to delay its inclusion in the system; and therefore, for some applications, delaying the addition of the additive and the aqueous solvent may be advantageous.

[0172] Therefore, one aspect of the present invention relates to a hydrolysate mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and iii. Other components in the range of less than 15 wt%.

[0173] One embodiment of the present invention relates to a method for forming a mixture of hydrolysates, the method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase, and then mix.

[0174] Another embodiment of the present invention relates to a method for forming a mixture of hydrolysates, the method comprising the following steps: a1. Contacting an oil with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; b1. Optionally, remove the lipase from the reaction mixture. c1. The oil hydrolysate from step a1 or b1 is mixed with a polar lipid to form a hydrolysate mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and iii. Other components less than 15 wt%; The oil hydrolysate and polar lipids are well dispersed in each other.

[0175] One related embodiment of the present invention relates to a method for forming a mixture of hydrolysates, the method comprising the following steps: a1. Contacting an oil with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by the at least one lipase to obtain an oil hydrolysate; b1. Optionally, remove at least one lipase from the oil hydrolysate. c1. The oil hydrolysate from step a1 or b1 is mixed with a polar lipid to form a hydrolysate mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and The oil hydrolysate and polar lipids are well dispersed in each other.

[0176] Hydrolysate mixtures can be produced with or without additives and stored for later use. Therefore, at a later time, an aqueous solvent can be added to the hydrolysate mixture to form the desired LCP.

[0177] The hydrolysate mixture may contain lipase. Alternatively, if the lipase is removed in step b1, then the hydrolysate mixture contains little or no lipase.

[0178] LCP achieved by the method of the present invention

[0179] In a second aspect, the present invention relates to a liquid crystal phase comprising lipids organized in the Fd3m and / or HII space groups, said liquid crystal phase being obtained or obtainable by the method of the present invention.

[0180] In the final mixing step of the method of the present invention, the oil hydrolysate, polar lipids, and a portion of water combine to form a liquid crystal phase in which the lipids are organized in a specific spatial group. As the LCP forms, it separates from the residual aqueous phase.

[0181] The liquid crystal phase comprises or consists of oil hydrolysis products, polar lipids, and an aqueous solvent (e.g., water), as well as any further components (e.g., additives). Additives may be described as being encapsulated or enclosed within the LCP of this invention.

[0182] Conventional methods for manufacturing liquid crystal phases only produce transient LCPs with Fd3m. In contrast, the present invention relates to the formation of stable liquid crystal phases comprising lipids of tissue in the Fd3m and / or HII space groups.

[0183] The LCPs containing lipids in the Fd3m and / or HII space groups according to the present invention are stable. Stability is related to the degree of formation of Fd3m and / or HII. Therefore, in some embodiments, the present invention relates to cases where the LCP has a well-defined crystalline phase. This can be determined by SAXS mapping, but can also be determined by visual inspection and stability testing (see Example 4).

[0184] Therefore, in some implementations, the LCP is stable at room temperature (25°C) for at least 4 days, such as 5 days, 6 days, or 7 days; or, for example, stable at 25°C for 1 week, 2 weeks, 3 weeks, or 4 weeks; or, for example, stable at 25°C for 1 month, 2 months, or 3 months.

[0185] Specific implementation schemes involve the LCP being stable at 25°C for at least 2 months, such as at least 3 months or at least 4 months.

[0186] In addition, or alternatively, in some embodiments, the LCP according to the invention is stable at 4°C for at least 4 weeks, for example at least 5 weeks, at least 6 weeks or at least 7 weeks; or for example at 4°C for at least 1 month, 2 months, 3 months or 4 months, or for example at 4°C for at least 1 year, 2 years or 3 years.

[0187] A specific implementation involves that the LCP is stable at 4°C for at least 6 months, such as at least 12 months or at least 18 months.

[0188] In one embodiment, a liquid crystal phase is obtained or is obtainable by the method of this disclosure, the liquid crystal phase comprising 30 to 65 wt% of polar lipids and having a lipid structure characterized by small-angle X-ray scattering (SAXS) spectra defined by peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44 for Fd3m space group; and 1, √3, 2, √7, 3, √12 for HII space group.

[0189] One aspect of the present invention relates to a liquid crystal phase comprising lipids organized in space groups Fd3m and / or HII, said liquid crystal phase comprising a phase mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, ii. Oils in the range of 35 to 70 wt%; and iii. Aqueous solvents; The liquid crystal phase has a lipid structure characterized by small-angle X-ray scattering (SAXS) spectra, which are defined by peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44 for Fd3m space group, and / or 1, √3, 2, √7, 3, √12 for HII space group.

[0190] In the context of this application, a phase mixture is a composition comprising polar lipids, oils and an aqueous solvent. For example, the phase mixture may comprise 30 to 65 wt% of polar lipids; 35 to 70 wt% of oils; and an aqueous solvent.

[0191] One embodiment of the present invention relates to a liquid crystal phase as described herein, wherein the volume ratio between the aqueous solvent and the phase mixture is at least 0.01:1, for example at least 0.2:1.

[0192] It has been found that specific amounts of triglycerides (TAG) and free fatty acids (FFA) result in well-defined and highly stable liquid crystal phases with lipids organized in the Fd3m space group.

[0193] Therefore, one embodiment of the present invention relates to a liquid crystal phase as described herein, wherein the lipid structure is in the Fd3m space group.

[0194] The ratio between TAG and FFA can be given by the peak intensities corresponding to TAG and FFA, respectively, as determined by TLC. In short, the area under each peak can be measured, and the ratio can be calculated as the peak area of ​​TAG versus the peak area of ​​FFA.

[0195] Alternatively, the ratio can be given as wt% of TAG to wt% of FFA.

[0196] One embodiment of the present invention relates to a liquid crystal phase as described herein, wherein the ratio between triglycerides (TAG) and free fatty acids (FFA) is equal to or less than 1:5.

[0197] Another embodiment of the invention relates to a liquid crystal phase as described herein, wherein the ratio between TAG and FFA is equal to or less than 1:10, for example equal to or less than 1:20, for example equal to or less than 1:30, for example equal to or less than 1:40, for example equal to or less than 1:50, for example equal to or less than 1:60, for example equal to or less than 1:70, for example equal to or less than 1:80, for example equal to or less than 1:90, for example equal to or less than 1:95, for example equal to or less than 1:99.

[0198] Further embodiments of the invention relate to liquid crystal phases as described herein, wherein the liquid crystal phase is substantially free of TAG.

[0199] For certain applications, it may be preferable to produce liquid crystal phases as described herein, in which the lipid structure is located in space group HII. As demonstrated herein, these LCPs are formed with higher amounts of TAG, i.e., when less hydrolysis occurs. In particular, the TAG content can be higher than that of FFA.

[0200] Therefore, one embodiment of the present invention relates to a liquid crystal phase as described herein, wherein the lipid structure is organized in the HII space group.

[0201] Another embodiment of the invention relates to a liquid crystal phase as described herein, wherein the content of TAG is greater than or equal to the content of FFA. Such LCPs can be in the HII phase.

[0202] Further embodiments of the invention relate to a liquid crystal phase as described herein, wherein the ratio between triglycerides (TAG) and free fatty acids (FFA) is in the range of 1:1 to 50:1, for example 2:1 to 25:1, for example 3:1 to 20:1, for example 5:1 to 15:1.

[0203] Another embodiment of the invention relates to a liquid crystal phase as described herein, wherein the ratio between TAG and FFA is in the range of 12:1 to 6:1.

[0204] Further embodiments of the present invention relate to a liquid crystal phase as described herein, wherein the liquid crystal phase contains less than 15 wt% of other components.

[0205] Liquid crystal phases can be loaded with various additives to produce beneficial functions in a wide variety of products. These additives can be components that are important for the development and preparation of food compositions or nutritional supplements for which high stability is an excellent property, or they can be active pharmaceutical ingredients (APIs) that require sustained release and / or stability.

[0206] Therefore, one embodiment of the present invention relates to a liquid crystal phase as described herein, which further comprises one or more additives.

[0207] Another embodiment of the invention relates to a liquid crystal phase as described herein, wherein the one or more additives are selected from the group consisting of: bioactive molecules, pharmaceutically active ingredients, colorants, flavorings, and tasters.

[0208] The LCP disclosed herein typically contains 10 to 35 wt% water, for example, 15 to 25 wt% water.

[0209] One aspect of the present invention relates to a product selected from the group consisting of food, animal feed, cosmetics and nutritional supplements, wherein the product comprises a liquid crystal phase as described herein.

[0210] use

[0211] In a third aspect, the present invention relates to the use of compositions obtained or available by the method of the present invention.

[0212] One aspect of the present invention relates to the use of liquid crystal phases as described herein in the preparation of food, feed, cosmetics or nutritional supplements.

[0213] Another aspect of the invention relates to liquid crystal phases as described herein, which are used as food, feed, ingredients, cosmetics, nutritional supplements or excipients.

[0214] Alternatively, the liquid crystal phase can contain the active pharmaceutical ingredient (API). In this case, the liquid crystal phase can be used in pharmaceuticals for API delivery.

[0215] Therefore, one aspect of the present invention relates to a liquid crystal phase as described herein, which is used as a pharmaceutical.

[0216] One embodiment of the present invention relates to a liquid crystal phase as described herein, which is used in medicine, for example as a nanocarrier for an active pharmaceutical ingredient.

[0217] Lipase used to produce LCP

[0218] In a fourth aspect, the present invention relates to the use of triglyceride lipase in the method of the present invention.

[0219] Therefore, the present invention relates to a triglyceride lipase with particular sn 1,3 position specificity for producing the LCP of the present invention. In a particular embodiment, this use is in any method according to the present invention.

[0220] Detailed Implementation Plan

[0221] In one specific embodiment, the present invention relates to a method for generating a stable liquid crystal phase comprising lipids organized in the Fd3m and / or HII space groups, the method comprising the following steps: a. Mix oils and polar lipids to form a starting mixture consisting of: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and iii. Other components less than 5 wt% The oil and polar lipids are well dispersed in each other; b. Add water to a ratio of at least 0.2:1 relative to the starting mixture and mix. c. Contact the mixture from step (b) with at least one triglyceride lipase having sn 1,3-position specificity under conditions allowing lipase activity, such that at least 50 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; and d. Further mix the mixture from step (c); This results in the formation of a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

[0222] In one related embodiment, the present invention relates to a method for generating a stable liquid crystal phase comprising lipids organized in the Fd3m and / or HII space groups, the method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%; and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add an aqueous solvent to the starting mixture and mix. c. Contact the mixture from step (b) with at least one triglyceride lipase having sn 1,3-position specificity under conditions allowing lipase activity, such that at least 50 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; and d. Further mix the mixture from step (c); This results in the formation of a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

[0223] In some embodiments, an aqueous solvent is added to achieve a volume ratio of at least 0.01:1 relative to the starting mixture, for example at least 0.2:1, and then further mixed.

[0224] In one related embodiment, the present invention relates to a method for generating a stable liquid crystal phase comprising lipids organized in the Fd3m and / or HII space groups, the method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%; and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Contact the mixture of step (a) with at least one triglyceride lipase having sn 1,3-position specificity under conditions that allow lipase activity, such that at least 50 wt% of the triglycerides in the oil are hydrolyzed by the at least one lipase. c. Add an aqueous solvent to the starting mixture; and d. Mix the mixture from step (c); This results in the formation of a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

[0225] In some embodiments, an aqueous solvent is added to achieve a volume ratio of at least 0.01:1 relative to the starting mixture, for example, at least 0.2:1, and then further mixed.

[0226] The enumeration or discussion of documents that appear to be prior art in this specification should not necessarily be construed as an admission that such documents are part of the prior art or are common general knowledge.

[0227] Unless the context otherwise indicates, the preferences, choices, and embodiments of the aspects, features, or parameters given in this invention should be considered as disclosed in combination with any and all preferences, choices, and embodiments of all other aspects, features, and parameters of this invention. This is especially true for the description of methods for producing stable crystalline phases and all their features, which may readily be part of the stable crystalline phase itself or obtained by means of methods as described herein or their uses. Embodiments and features of the invention are also outlined in the following sections.

[0228] The invention will now be described in further detail in the following non-limiting embodiments.

[0229] Example

[0230] Example 1 - Lipase

[0231] The usefulness of various lipases in LCP production was tested. The tested lipases are described in Table 1 below. They are commercially available enzymes from Novozymes, and their activity is defined by Novozymes as: 1 LU is the amount of enzyme activity that releases 1 μmol of titratable butyrate per minute from the substrate glycerol tributyrate under defined standard conditions. 1 LU equals 1 IUN. 1 PLU is the amount of enzyme activity that produces 1 μmol of propyl laurate per minute under defined standard conditions.

[0232] The experiment was conducted using oat oil enriched with polar lipid fractions from oats. Therefore, 300 mg of polar lipid-rich oat oil containing 40 wt% polar lipids and 1.2 ml of aqueous buffer were added to a 4.5 ml glass sample tube.

[0233] Add 10 µl of lipase solution and incubate the sample at 40°C for 16 hours on a constant temperature mixer (700 RPM).

[0234] After incubation, the samples were removed from the incubator, and the formation of the liquid crystal phase was assessed by small-angle X-ray scattering (SAXS), as described below, with results presented in Table 1 and [other tables not provided]. Figure 1 middle.

[0235] Phase identification was performed using SAXS.

[0236] Most SAXS experiments were conducted in the SAXS laboratory of the Department of Physical Chemistry, Department of Chemistry, Lund University. A few experiments were conducted in the CoSAXS beamline of the MAX IV laboratory at Lund University. Phases were identified based on peak modes observed in the SAXS experiments. Fd3m was characterized by peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44, and HII by peak positions at q values ​​at the following ratios: 1, √3, 2, √7, 3, √12.

[0237] surface 1 K = kilo, LU = lipase unit, PLU = propyl lauryl ester unit, IUN = transesterification unit. 1 LU is the amount of enzyme activity that releases 1 μmol of titratable butyrate per minute under defined standard conditions. 1 LU equals 1 IUN. 1 PLU, as defined by Novozymes, is the amount of enzyme activity that produces 1 μmol of propyl lauryl ester per minute under defined standard conditions.

[0238] Therefore, the formation of the LCP according to the present invention requires the hydrolysis of triglycerides of oil. Buffer solution and tap water work equally well as aqueous phases.

[0239] Example 2 - Time Process

[0240] This method is performed according to the method in Example 1. The lipase used is Lipozyme TL. Different durations of LCP are allowed.

[0241] Table 2

[0242] The results showed that the appearance of LCPs occurred over time. LCP formation led to phase separation, with virtually all lipids forming the LCP phase and excess water forming a separate phase. It was observed that LCPs that appeared even after 96 hours were still present, indicating that LCPs possess good stability.

[0243] Example 3 – Temperature

[0244] The method was performed according to that of Example 1, but with an incubation time of 24 hours. The samples were then transferred to various temperatures and their structures were determined using SAXS.

[0245] Therefore, the Fd3m phase can exist over a wide temperature range.

[0246] Table 3

[0247] Example 4 - Stability

[0248] LCP was prepared using oat oil with 40% polar lipids and Lipozyme TL, according to the method in Example 1.

[0249] The LCP was stored as described, and its stability was checked visually and using a SAXS device.

[0250] In this experiment, phase separation refers to the decomposition of LCP into an oil phase and an aqueous phase, with the oil phase no longer exhibiting any identifiable structure. For example, the SAXS spectrum associated with the LCP can no longer be identified in the LCP (see definition). This table indicates the time it takes for an LCP to remain intact until its decomposition (phase separation).

[0251] Therefore, the lamellar mesophase structure develops within several days. LCPs containing lipids from tissues in the Fd3m and / or HII space groups exhibit greater stability.

[0252] Table 4

[0253] Example 5 - Content of polar lipids

[0254] The method described in Example 1 used oat oil with varying amounts of polar lipids from oats (designated PL4 to PL60, having polar lipids from 4% to 60 wt% of total weight). 10 µl of Lipozyme TL (Novozymes) lipase was used.

[0255] The results are shown in Table 5.

[0256] surface 5

[0257] Therefore, in the case of oat oil, the formation of the Fd3m phase requires the presence of at least 30% polar lipids. At a polar lipid content of 60%, an anti-hexagonal phase is formed instead.

[0258] Example 6 – Effects of Dispersion

[0259] The method was performed according to the general procedure in Example 1, using 300 mg of oat oil containing 40 wt% polar lipids (PL40), 1.2 ml of water, and 10 µl of lipase (Lipozyme TL, Novozymes). After incubation for 16 hours, the LCPs were collected and dispersed in water by sonication. This allowed for pipetting of the dispersion, which was analyzed by SAXS at the CoSAXS beamline of Lund University's MAX IV facility. The analysis revealed that the dispersion contained the HII phase.

[0260] Therefore, under the conditions used, dispersion via ultrasonic treatment leads to the transformation of the Fd3m phase to the HII phase. The HII phase may be significant because its water bodies are more interconnected than those of Fd3m, thus facilitating the diffusion of hydrophilic molecules. Furthermore, the liquid dispersion can be manipulated by pumping, pipetting, etc., which is impossible with rigid bulk LCPs.

[0261] Example 7 - Formation of liquid crystal phase from oil hydrolysis products

[0262] Sunflower seed oil was hydrolyzed using an immobilized TL IM lipase from Novozymes, following the general method described in Example 12. Samples were taken at different time points and analyzed by TLC, which is sensitive to TAG, MAG, DAG, and FFA.

[0263] The sample containing the hydrolysis products was combined with lecithin and water (two-step method, see Example 12) to generate a liquid crystal phase. The LCP was analyzed by SAXS, and the results are shown in Table 6.

[0264] Two control samples were run using the general method described in Example 1. These samples were incubated for 16 hours to ensure complete hydrolysis of TAG. The resulting LCPs were analyzed by TLC, as shown in Table 6.

[0265] TLC Analysis: Lipid hydrolysis products were extracted according to Bligh and Dyer (Canadian Journal of Biochemistry and Physiology, 1959. 37(8): p. 911-917) and analyzed by thin-layer chromatography (TLC). Lipids were separated on TLC silica gel 60 plates (Merck) using heptane:ethyl ether:acetic acid (70:30:1) as the mobile phase and imaged under UV light after spraying with primulin solution. The TLC plates were analyzed using a CAMAG scanner, and peak intensities were recorded. The sensitivity of this method was not equal for all sample components; however, the results are still useful for demonstrating the continuous conversion of TAG to DAG, MAG, and FFA. Data were generated using the software visionCATS, which produces approximate peak areas. The results of TLC and SAXS analyses are shown in Table 6. The values ​​for each of “PL, MAG”, “FFA”, “DAG”, and “TAG” correspond to the peak intensities measured in the TLC analysis.

[0266] It can be seen that the contents of free fatty acids and diglycerides increase significantly after enzymatic hydrolysis. This increase is even greater with longer incubation times, accompanied by a decrease in triglycerides.

[0267] The formation of the Fd3m structure is associated with the removal (hydrolysis) of TAG and the generation of FFA. In contrast, the concentrations of MAG and DAG do not vary as drastically as those of FFA and TAG, respectively. In the well-defined Fd3m phase, the majority of the components are FFA (peak area approximately 60%). TAG and PL together account for approximately 40% of the peak area. It can be seen that there is no TAG in the well-defined Fd3m phase.

[0268] Table 6

[0269] PL = polar lipids; MAG = monoglyceride; DAG = diglyceride; TAG = triglyceride; FFA = free fatty acids.

[0270] Example 8 - The dispersion of polar lipids in oil is important for the formation of LCPs.

[0271] Samples were prepared according to the method in Example 1.

[0272] Peanut oil was mixed with a polar lipid in the form of sunflower seed lecithin (see Example 9). In one case, the mixture was thoroughly mixed for 2 hours before adding water and enzyme to obtain a visually homogeneous dispersion. In another case, water and enzyme were added directly and incubation was initiated. Both mixtures were incubated at 700 rpm for 16 hours, and the products were analyzed (see Table 7).

[0273] Therefore, the dispersion of polar lipids in oil is important for the subsequent formation of the Fd3m phase.

[0274] Table 7

[0275] Example 9 - LCP can be formed using various oils.

[0276] The sample was prepared according to the method of Example 1, using the following oils: peanut oil, flaxseed oil, rapeseed oil, and coconut oil. These oils were all food-grade oils for cooking and were commercially available in grocery stores.

[0277] Peanut oil has a high smoking temperature. Flaxseed oil has a high content of omega-3 fatty acids, especially ALA. The beneficial qualities of flaxseed oil may decrease if used at high temperatures. Coconut oil is solid below 25°C. See Table 8 for the composition of the oils.

[0278] The sunflower seed lecithin was purchased from a health product store and contains the following ingredients per 100g: 99g phospholipids; 12g saturated fat; 23g monounsaturated fat; and 64g polyunsaturated fat. This product contains 0g carbohydrates, 0g protein, and 0g salt.

[0279] Table 8

[0280] 300 mg of oil was thoroughly mixed with various amounts of sunflower seed lecithin (see above) to obtain a visually uniform dispersion. 1.2 ml of water and 10 µl of lipase (TL) solution were added, followed by incubation at 700 rpm for 16 hours at 40°C. The results are shown in Table 9.

[0281] Therefore, the Fd3m phase can be produced by combining a vegetable oil from one source with polar lipids from other plant species. Furthermore, examples show that this method is suitable for producing the Fd3m phase using various oils.

[0282] Table 9

[0283] Example 10 - LCP can be manufactured to contain additives

[0284] LCP was prepared according to the method in Example 1. 300 mg of oat oil containing 40% polar lipids (PL40) was mixed with 1.2 ml of water and 10 µl of lipase solution (TL lipase) and incubated at 40 °C and 700 RPM using a thermostatic mixer for 16 hours.

[0285] The following additives were tested as inclusions: curcumin from turmeric (Curcuma longa (Turmeric)), powder (CAS: 458-37-7), Sigma-Aldrich; vitamin D (ergocalciferol, CAS: 50-14-6), >98%, Sigma-Aldrich; ferrous sulfate heptahydrate: ACS reagent, ≥99.0%, (CAS: 7782-63-0), Sigma-Aldrich; fish oil: capsules, ICA AB (the capsules were cut open, the fish oil was recovered and used in the experiment).

[0286] Water-soluble additives (curcumin, ferrous sulfate) are dissolved in water before being mixed with oil. Fat-soluble additives (fish oil, vitamin D) are dissolved in oil before being mixed with water. For tests containing 5%–40% fish oil, the amount of PL40 is reduced accordingly.

[0287] The results are shown in Table 10. The results show that additives can be included and encapsulated within LCPs. Therefore, LCPs can be manufactured to contain additives, such as bioactive molecules.

[0288] Table 10

[0289] Example 11 - Immobilized Enzyme - Two-Step Method

[0290] LCP was produced on a larger scale using a method that hydrolyzes immobilized lipases and a rotating bed reactor (RBR, SpinChem RBR S2).

[0291] Approximately 8 grams of immobilized TL IM (Novozymes) were added to the RBR.

[0292] In the first step, sunflower seed oil was hydrolyzed (see Example 9). 120 mL of oil was poured into the RBR, and 15 mL of water was added. The temperature was set to 60°C, and the bed was allowed to rotate at 600 RPM for 2 hours. After hydrolysis, TL IM was removed from the reactor.

[0293] The second step consists of adding approximately 120g of sunflower seed lecithin (see Example 9) and maintaining the reactor at a low RPM (approximately 50) until the lecithin is fully mixed with the oil. Visual inspection revealed no residual turbidity and no visible particles in the mixture, indicating good mixing.

[0294] Finally, about 200 ml of tap water was added to the reactor and the temperature was lowered to 40°C. The mixing rate was reduced to 50 RPM and the reaction was allowed to proceed for 24 hours.

[0295] Approximately 300-400 g of LCP was generated. SAXS analysis confirmed that the LCP possessed an Fd3m structure.

[0296] The experiment was repeated using rapeseed oil (see Example 9), with the same results.

[0297] Example 12 - Immobilized Enzyme

[0298] LCP was prepared using a general method: 300 mg of oat oil containing 40% polar lipids (PL-40) was mixed with 1.2 ml of water. The enzyme was then added in immobilized form (commercial formulation TL IM, Novozymes). After incubation at 700 RPM for 16 hours at 40°C, the enzyme was removed, and the product was examined and found to contain the Fd3m phase. In this case, the product was completely enzyme-free, which may be desirable in some applications. Furthermore, the removal of the enzyme ensured that enzymatic hydrolysis did not continue thereafter.

[0299] Example 13 – Formation of LCP using a mixture of hydrolysates

[0300] A mixture of hydrolysates was prepared according to this disclosure. Water was then added to form an LCP.

[0301] Optical microscopy is used to image hydrolysate mixtures before the addition of water and 5–20 seconds after the addition of water. See [link to documentation]. Figure 3 .

[0302] It is evident that LCP formation is observed within the first 20 seconds after water is added to the hydrolysate mixture.

[0303] Example 14 – Formation of LCP using coconut oil

[0304] Coconut oil (purchased from ICA, food grade) was hydrolyzed in a rotating bed reactor using TL IM immobilized lipase until approximately 85% of the TAG was hydrolyzed, as analyzed by GC-MS and GC-FID methods. The hydrolyzed coconut oil was mixed with varying amounts (wt%) of sunflower seed lecithin (ICA) to form a hydrolysate mixture.

[0305] Then 2 mL of water was added to the 300 mg hydrolysate mixture. Additionally, an anhydrous reference sample was prepared.

[0306] Then, SAXS measurements were performed after a 12-hour reaction time to determine the structure of the formed LCP (see Table 11).

[0307] Table 11

[0308] Based on this, it is evident that various polar lipids can be added when aiming to form an LCP structure. Lower amounts of polar lipids lead to the formation of the Fd3m phase, while higher amounts lead to the formation of the HII phase.

[0309] Example 15 – LCP with additives

[0310] The hydrolysate mixture containing 52 wt% of polar lipids from Example 14 was further processed.

[0311] Different levels of hemoglobin were dispersed in the hydrolysate mixture. The hydrolysate mixture containing the dispersed hemoglobin was then added to water, followed by SAXS analysis to determine the LCP structure. 3 wt% hemoglobin in the hydrolysate mixture: HII 11 wt% hemoglobin in the hydrolysate mixture: HII 24 wt% hemoglobin in the hydrolysate mixture: HII It is evident that directly incorporating additives from 3 wt% to 24 wt% into the hydrolysate mixture does not prevent the subsequent formation of LCP by adding water.

[0312] Example 16 – Formation of LCP using MCT oil

[0313] MCT (medium chain triglyceride) oil (AAK, pharmaceutical grade) was hydrolyzed in a rotating bed reactor using TL IM immobilized lipase until approximately 50-75% of the TAG was hydrolyzed, with approximately 50-25% remaining – as analyzed by GC-MS and GC-FID methods.

[0314] Hydrolyzed MCT oil was mixed with 50 wt% soybean lecithin. NatraPhosTM Soya PC100-C Pharma (CRODA, pharmaceutical grade) and egg yolk lecithin NatraPhos™ PL95EN PHARMA (CRODA, pharmaceutical grade) One of the mixtures is used to obtain a mixture of two hydrolysates.

[0315] Upon addition of water to each hydrolysate mixture, an LCP with a HII structure was formed. Therefore, hydrolysate mixtures from different polar lipids can be successfully prepared and subsequently converted into LCPs by adding water.

Claims

1. A method for generating a stable liquid crystal phase, said stable liquid crystal phase comprising lipids organized in the Fd3m and / or HII space groups, said method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and; ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add an aqueous solvent to the starting mixture and mix; as well as c. Add at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase, and then mix; This results in a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

2. The method of claim 1, wherein step (a) precedes steps (b) and (c), and wherein steps (b) and (c) may be performed in any order or simultaneously.

3. The method according to claim 1 or 2, wherein step (b) precedes step (c).

4. The method according to claim 1 or 2, wherein step (c) precedes step (b) so that a hydrolysate mixture is formed after step (c), which is converted into a stable liquid crystal phase after water is added in step (b).

5. The method according to claim 1 or 2, wherein the at least one lipase is contained in an aqueous solvent.

6. The method according to any one of the preceding claims, wherein the oil and polar lipids of the starting mixture have been premixed.

7. A method for generating a stable liquid crystal phase, said stable liquid crystal phase comprising lipids organized in space groups Fd3m and / or HII, said method comprising the following steps: a1. Contacting an oil with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by the at least one lipase to obtain an oil hydrolysate; b1. Optionally, remove at least one lipase from the oil hydrolysate. c1. The oil hydrolysate from step a1 or b1 is mixed with a polar lipid to form a hydrolysate mixture consisting of: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and The oil hydrolysate and polar lipids are well dispersed in each other; d1. Add an aqueous solvent to the hydrolysate mixture and mix further; This results in a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

8. The method according to any of the preceding claims, wherein the volume ratio of the aqueous solvent to the starting mixture is in the range of 0.01:1 to 10:1, for example from 0.2:1 to 10:1, for example from 0.3:1 to 6:

1.

9. The method according to any one of the preceding claims, wherein the aqueous solvent comprises at least 70 vol% water, such as at least 80 vol% water, such as at least 90 vol% water, such as at least 95 vol% water, such as at least 99 vol% water.

10. The method according to any one of the preceding claims, wherein the aqueous solvent is composed of water.

11. The method according to any one of the preceding claims, wherein the starting mixture contains less than 15 wt% of other components.

12. The method according to any of the preceding claims, wherein the liquid crystal phase formation is allowed to continue for at least 12 hours, for example at least 14 hours, preferably at least 16 hours.

13. The method according to any one of the preceding claims, wherein at least 50 wt% of the lipids from the starting mixture are incorporated into the liquid crystal phase.

14. The method according to any of the preceding claims, wherein at least 50 wt% of the lipid structure in the liquid crystal phase is in the Fd3m or HII space group.

15. The method according to any one of the preceding claims, wherein the stable liquid crystal phase maintains its crystal structure at 25°C for at least 4 days.

16. The method according to any one of the preceding claims, wherein the oil is selected from vegetable oils, microbial oils, marine oils, animal oils, and any combination thereof.

17. The method according to any one of the preceding claims, wherein the polar lipid comprises one or more phospholipids.

18. The method according to any of the preceding claims, wherein the polar lipid comprises one or more glycolipids.

19. The method according to any of the preceding claims, wherein the polar lipid comprises or is composed of one or more lecithin, such as one or more plant lecithin, or is composed of the same.

20. The method according to any of the preceding claims, wherein, under the conditions of step (c) or step (a1), the primary activity of the at least one lipase is the hydrolysis of triglycerides.

21. The method according to any one of the preceding claims, wherein at least one lipase comprises, or is composed of, a triglyceride lipase.

22. The method of claim 21, wherein the triglyceride lipase has sn1,3-position specificity.

23. The method according to any one of the preceding claims, wherein the at least one lipase is selected from the group consisting of: from R. Miehei Lipase, from R. Arrhizus lipase and from Thermomyces languniosus Lipase.

24. The method according to any one of the preceding claims, wherein the at least one lipase comprises, or is composed of, an immobilized lipase.

25. The method according to any one of the preceding claims, wherein at least one additive is added, resulting in a liquid crystal phase comprising said additive.

26. The method of claim 25, wherein if the additive is lipophilic, the at least one additive is added to the starting mixture or hydrolysate mixture; or if the additive is water-soluble, the at least one additive is added together with the aqueous solvent.

27. The method according to any one of claims 25 or 26, wherein the at least one additive is selected from bioactive molecules, pharmaceutically active ingredients, colorants, flavorings, and seasonings.

28. A liquid crystal phase, which is obtained or can be obtained by any of the preceding claims.

29. A liquid crystal phase, obtained or obtainable by any of the preceding claims, the liquid crystal phase comprising 30 to 65 wt% of polar lipids and having a lipid structure characterized by small-angle X-ray scattering (SAXS) spectra defined by peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44 for Fd3m space group, or 1, √3, 2, √7, 3, √12 for HII space group.

30. A liquid crystal phase comprising lipids organized in space groups Fd3m and / or HII, said liquid crystal phase comprising a phase mixture comprising the following components: i. Polar lipids in the range of 30 to 65 wt%, ii. Oils in the range of 35 to 70 wt%; and iii. Aqueous solvents; The liquid crystal phase described herein has a lipid structure characterized by small-angle X-ray scattering (SAXS) spectra, which are defined by peak positions at q values ​​at the following ratios: √3, √8, √11, √12, √16, √19, √24, √27, √32, √44 for Fd3m space group, and / or 1, √3, 2, √7, 3, √12 for HII space group.

31. The liquid crystal phase according to any one of claims 28-30, wherein the volume ratio of the aqueous solvent to the phase mixture is at least 0.01:1, for example at least 0.2:

1.

32. The liquid crystal phase according to any one of claims 28-31, wherein the lipid structure is in the Fd3m space group.

33. The liquid crystal phase according to any one of claims 28-32, wherein the ratio between triglycerides (TAG) and free fatty acids (FFA) is equal to or less than 1:

5.

34. The liquid crystal phase according to claim 33, wherein the ratio between TAG and FFA is equal to or less than 1:10, for example equal to or less than 1:20, for example equal to or less than 1:30, for example equal to or less than 1:40, for example equal to or less than 1:50, for example equal to or less than 1:60, for example equal to or less than 1:70, for example equal to or less than 1:80, for example equal to or less than 1:90, for example equal to or less than 1:95, for example equal to or less than 1:

99.

35. The liquid crystal phase according to any one of claims 28-34, wherein the liquid crystal phase is substantially free of TAG.

36. The liquid crystal phase according to any one of claims 28-35, wherein the liquid crystal phase comprises less than 15 wt% of other components.

37. The liquid crystal phase according to any one of claims 28-36, further comprising one or more additives.

38. The liquid crystal phase according to claim 37, wherein the one or more additives are selected from bioactive molecules, pharmaceutically active ingredients, colorants, flavorings, and tasters.

39. A method for forming a mixture of hydrolysates, comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase, and then mix.

40. A method for forming a mixture of hydrolysates, comprising the following steps: a1. Contacting an oil with at least one lipase under conditions that allow lipase activity, such that at least 1 wt% of the triglycerides in the oil are hydrolyzed by the at least one lipase to obtain an oil hydrolysate; b1. Optionally, remove at least one lipase from the oil hydrolysate. c1. The oil hydrolysate from step a1 or b1 is mixed with a polar lipid to form a hydrolysate mixture, the hydrolysate mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and The oil hydrolysate and polar lipids are well dispersed in each other.

41. A mixture of hydrolysates formed as claimed in any one of claims 4 and 39-40.

42. A mixture of hydrolysates, comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oil hydrolysates in the range of 35 to 70 wt%; and iii. Other components less than 15 wt%.

43. A method for forming a liquid crystal phase, comprising the following steps: i. Provide a mixture of hydrolysates; ii. Add an aqueous solvent to the hydrolysate mixture and mix; This results in the formation of a stable lipid-based liquid crystal phase.

44. The method according to claim 43, wherein, The aqueous solvent is added at a ratio of at least 0.01:1 relative to the volume of the hydrolysate mixture, for example at least 0.2:

1.

45. A liquid crystal phase, which may be obtained by the method according to claims 43-44.

46. ​​Use of the liquid crystal phase according to any one of claims 28-38 in the preparation of food, feed, cosmetics or nutritional supplements.

47. The liquid crystal phase according to claims 28-38, used as a pharmaceutical.

48. A product selected from food, feed, cosmetics and nutritional supplements, wherein the product comprises a liquid crystal phase according to any one of claims 28-38.

49. Use of a triglyceride lipase with sn1,3-position specificity in the production of a liquid crystal phase according to any one of claims 28-36.

50. The use according to claim 49, wherein the use is in the method according to any one of claims 1-27.

51. A method for generating a stable liquid crystal phase, said stable liquid crystal phase comprising lipids organized in space groups Fd3m and / or HII, said method comprising the following steps: a. Provide a starting mixture comprising: i. Polar lipids in the range of 30 to 65 wt%, and ii. Oils in the range of 35 to 70 wt%; The oil and polar lipids are well dispersed in each other; b. Add water to achieve a ratio of at least 0.2:1 relative to the volume of the initial mixture, and mix; c. Contact the mixture from step (b) with at least one triglyceride lipase having sn1,3-position specificity under conditions allowing lipase activity, such that at least 50 wt% of the triglycerides in the oil are hydrolyzed by said at least one lipase; and d. Further mix the mixture from step (c); This results in a stable liquid crystal phase containing lipids from tissues in the Fd3m and / or HII space groups.

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