Method for separating acid sophorolipids from lactone / acid sophorolipid-containing products

Abstract

The present invention relates to a method for separating acid-type sophorolipids or lactone-type sophorolipids, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids, from a lactone-type / acid-type sophorolipid-containing product, and to acid-type sophorolipids, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids, obtained by the method.

Description

[Technical Field]

[0001] The present invention relates to a method for separating acid-type sophorolipids or lactone-type sophorolipids, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids, from a lactone-type / acid-type sophorolipid-containing product, and to acid-type sophorolipids, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids, obtained by the method. [Background technology]

[0002] Sophorolipids are one of the important biosurfactants obtained by yeast fermentation. During the fermentation process, various by-products, such as fatty acids, salts, pigments, etc., are simultaneously produced. Therefore, the final product usually has a strong color and odor. To expand its application field to pharmaceuticals, foods, and cosmetics, specific downstream processes are required to purify or partially purify the target sophorolipid species.

[0003] Typically, the crude sophorolipid product of fermentation is a mixture of the acid and lactone forms of sophorolipids (i.e., acid sophorolipids and lactone sophorolipids, respectively). Both types of structures usually have some degree of acetylation of the OH groups on the sugar head groups. The acetyl groups on sophorolipids have been reported to be important for several biological functions, such as improved antiviral activity and cytokine stimulation. The acetyl groups also confer higher lipophilic properties, which may result in better skin delivery or active encapsulation properties.

[0004] However, the acetyl groups on sophorolipids are highly susceptible to hydrolysis in current downstream purification processes. In particular, the loss of acetyl groups in the acid form of sophorolipids is likely to be faster than in their lactone counterparts, as the molecule is more hydrophilic. Therefore, obtaining purified acetylated acid form sophorolipids from the crude sophorolipid product of fermentation remains a challenging task.

[0005] Chinese Patent Application Publication No. 109678914 discloses a method for purifying and separating different structures of sophorolipids. The method starts with a pretreated fermentation liquid, which is then subjected to filtration, treatment with cation exchange resins, anion exchange resins, and adsorption resins, desalting, and membrane ultrafiltration. The method uses various types of resins, including cation exchange resins. Such resins will provide a weak to strong basic environment during separation, which will cause hydrolysis of the acetyl groups on the sophorolipids and the lactone form of the sophorolipids.

[0006] JP 2014-140383 A discloses a method for obtaining a highly purified acid form of sophorolipid by starting from a partially purified acid form and purifying it by reverse-phase column chromatography. When used in the present invention, the starting material, "partially purified acid form of sophorolipid," is prepared by hydrolysis of an acid / lactone mixed sophorolipid, which cleaves the acetyl groups on the sophorolipid.

[0007] Traditional methods only yield fully hydrolyzed structures that lose some of their biological activity. Direct microbial synthesis is possible, but requires the use of engineered strains, limiting its acceptance in some applications. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there remains a need to provide a new method for obtaining acid sophorolipids, particularly acid sophorolipids enriched in acetylated acid sophorolipids, from crude sophorolipid products of fermentation. [Means for solving the problem]

[0009] An object of the present invention is to provide a method for separating acid-type sophorolipids or lactone-type sophorolipids, particularly acid-type sophorolipids enriched in acetylated acid-type sophorolipids, from a lactone-type / acid-type sophorolipid-containing product by chromatography, comprising: 1) optionally eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 10 to less than 40% by weight; 2) optionally eluting with an aqueous solution of a water-miscible organic solvent having a concentration of less than 40-60% by weight; 3) eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 50 to 70% by weight to collect an acid-type sophorolipid fraction enriched in acid-type sophorolipids, particularly acetylated acid-type sophorolipids; 4) optionally, eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 55 to 85% by weight to recover a lactone-type sophorolipid fraction; The method includes:

[0010] Furthermore, another object of the present invention is to provide acid-type sophorolipids obtained by the above-mentioned method, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids.

[0011] The present inventors have conducted extensive research to solve the above problems, and as a result, have surprisingly found that it is possible to obtain acid-type sophorolipids that are rich in acid-type sophorolipids, particularly acetylated acid-type sophorolipids, by subjecting crude sophorolipids or purified sophorolipids obtained from a fermentation process to chromatographic purification, particularly reverse-phase chromatographic purification.

[0012] Therefore, the present invention relates to a method for separating acid-type sophorolipids, particularly acid-type sophorolipids enriched in acetylated acid-type sophorolipids, from a lactone-type / acid-type sophorolipid-containing product by chromatographic purification, particularly reverse-phase chromatographic purification.

[0013] Furthermore, the present invention also relates to acid-type sophorolipids obtained by the above-mentioned method, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids.

[0014] The present invention provides an effective process for obtaining both acid-type sophorolipids and lactone-type sophorolipids with fully maintained acetyl groups. Meanwhile, it is possible to collect fractions of sophorolipid derivatives in one shot. The starting material can be crude sophorolipid from which only solid impurities have been removed. The final product is free of odor and color concerns, but yields white crystals upon drying. DETAILED DESCRIPTION OF THE INVENTION

[0015] It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0016] As used herein, the expression "stationary phase" refers to beads of porous polymeric material that readily absorb solvent and swell as a result.

[0017] As used herein, the term "mobile phase" refers to the solvent that passes through a liquid chromatography instrument.

[0018] As used herein, the term "fraction" refers to individual components collected based on differences in specific properties in a separation process in which a specific amount of a mixture (solid, liquid, solute, or suspension) is divided into several smaller amounts (fractions).

[0019] As used herein, the terms "loaded," "loading," and the like refer to the injection of a specific volume of a mixture into a column for the purpose of separating one component of the mixture from another component of the mixture.

[0020] As used herein, the term "column" refers to a column used in a chromatography process.

[0021] As used herein, the expression "bed volume" refers to the volume of chromatography media within a column.

[0022] As used herein, the expression "lactone-type / acid-type sophorolipid-containing product" refers to a product containing lactone-type sophorolipid and acid-type sophorolipid.

[0023] Furthermore, ranges defined throughout this specification are inclusive of the ranges defined therein, for example, a range of 1 to 10 means that both 1 and 10 are included within the range. For the avoidance of doubt, the applicants are entitled to all equivalents pursuant to applicable law.

[0024] In the following text, various aspects of the invention are defined in more detail. Each aspect so defined may be combined with any one or more other aspects, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0025] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "one embodiment" or "an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, although they may. Moreover, as will be apparent to one of ordinary skill in the art from this disclosure, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, as will be understood by one of ordinary skill in the art, when an embodiment described herein includes some features but not other features included in other embodiments, this means that combinations of features from different embodiments are within the scope of the present invention and form separate embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0026] The present invention provides a method for separating acid-type sophorolipids or lactone-type sophorolipids, particularly acid-type sophorolipids enriched in acetylated acid-type sophorolipids, from a lactone-type / acid-type sophorolipid-containing product by chromatography, comprising: 1) optionally eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 10 to less than 40% by weight; 2) optionally eluting with an aqueous solution of a water-miscible organic solvent having a concentration of less than 40-60% by weight; 3) eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 50 to 70% by weight to collect an acid-type sophorolipid fraction enriched in acid-type sophorolipids, particularly acetylated acid-type sophorolipids; 4) optionally, eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 55 to 85% by weight to recover a lactone-type sophorolipid fraction; The present invention relates to a method, including:

[0027] Sophorolipids Sophorolipids (SLs) are glycolipids consisting of hydroxyl fatty acids and sophorose or sophorose with its hydroxyl group partially acetylated. Note that sophorose is a sugar consisting of two glucose molecules linked via a β1→2 bond. Hydroxyl fatty acids are fatty acids with a hydroxyl group. Furthermore, SLs are mainly classified into acid type (general formula (1)) or lactone type (general formula (2)), in which the carboxyl group of the hydroxyl fatty acid is either free in the molecule or bound to the sophorose, respectively. SLs obtained by fermentation from specific yeast species (SL-producing yeasts) are usually a mixture of SLs shown in the following general formula (1) and SLs shown in the following general formula (2), and the 6'-position (R 2 ) and 6th place (R 1 ) with acetylation or protonation in the fatty acid chain length (R 3 ) is obtained as a collection of more than 30 structural homologs with different structures. [ka]

[0028] In general formula (1) or (2), R 0 is either a hydrogen atom or a methyl group. 1 and R 2 are each independently a hydrogen atom or an acetyl group. 3 is a saturated or unsaturated aliphatic hydrocarbon chain having at least one double bond, and may have one or more substituents. The substituents are not particularly limited as long as they do not interfere with the advantageous effects of the present invention, and examples thereof include halogen atoms, hydroxyl, lower (C 1~6 ) alkyl group, halo lower (C 1~6 ) alkyl group, hydroxy lower (C 1~6 ) alkyl group, halo lower (C 1~6 ) alkoxy groups. 3 typically has 11 to 20 carbon atoms, preferably 13 to 17 carbon atoms, and more preferably 14 to 16 carbon atoms.

[0029] SLs are obtained by fermentation by culturing microorganisms, such as yeasts, such as Starmerella (Candida) bombicola, C. apicola, C. petrophilum, Rhodotorula (Candia) bogoriensis, C. batistae, C. gropengiesseri, Wickerhamiella domercqiae, and Yarrowia lipolytica, using methods known in the art. The yeast may be a strain provided by a depository or obtained by serial subculture thereof. Commercial grade BioToLife yeast is preferred.

[0030] A preferred method for culturing yeast to produce SLs is to simultaneously provide high concentrations of sugar and a hydrophobic oily substrate. This method is not limited to this, and a wide variety of other methods known in the art can be applied as long as the beneficial effects of the present invention are not impaired. Examples of methods known in the art include those described in Japanese Patent Application Laid-Open No. 2002-045195. More specifically, this method may involve culturing Starmerella (Candida) bombicola as the producing yeast, using glucose as the sugar and a carbon source containing fatty acids and vegetable oil as the hydrophobic oily substrate.

[0031] Because the fatty acid moiety of SL is known to depend on the fatty acid chain length and the ratio of hydrophobic substrates, the fatty acid moiety can be controlled to some extent. For example, oleic acid or lipids containing a high proportion of oleic acid are suitable as hydrophobic substrates. Examples include vegetable oils such as palm oil, rice bran oil, rapeseed oil, olive oil, and safflower oil, as well as animal oils such as lard and beef tallow. Furthermore, when a mixed substrate of triglycerides and oleic acid is used as the hydrophobic substrate, sophorolipids containing a high proportion of oleic acid can be obtained in high yields in terms of quantity and efficiency. From the perspective of industrial applications, stable fermentation production of SL with high yields in terms of quantity and efficiency is required. In this case, a mixture of hydrophilic sugars and hydrophobic fats / oils is preferred as the carbon source. Glucose is frequently used as the hydrophilic substrate.

[0032] The liquid components are separated and removed from the resulting liquid culture by known solid-liquid separation methods such as centrifugation and decantation, and the solids are washed with water to obtain an SL-containing fraction (also called a crude sophorolipid product). The SL-containing fraction is a mixture of lactone-type SL and acid-type SL, and since the content of acid-type SL in the total amount of SL is less than 45 wt% (solid equivalent), it is classified as a lactone-type / acid-type SL-containing product.

[0033] According to the present invention, a lactone-type / acid-type sophorolipid-containing product useful as a starting material for separating acid-type sophorolipids, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids, may contain, for example, 20 to 40 wt % of acid-type SL, including non-acetylated and acetylated SL. Of the acid-type SL, acetylated acid-type SL may account for, for example, 15 to 35 wt %.

[0034] Furthermore, the lactone / acid sophorolipid-containing product as a starting material may contain, for example, 60 to 80% by weight of lactone SL, including non-acetylated and acetylated SL.

[0035] Chromatographic purification According to the present invention, chromatographic purification is used as a separation method that takes advantage of the amphiphilic structure of SLs.

[0036] The chromatography used in the present invention is preferably partition chromatography, in particular reversed phase chromatography.

[0037] Generally, the adsorbent as the stationary phase in chromatographic purification can be a hydrophobic macroporous resin. Macroporous resins are porous polymeric materials that have a non-foldable permanent pore structure in both the dry and solvated states, and have a pore size distribution that includes a significant proportion of macropores (i.e., pores with diameters greater than 1000 Å) or C8-C9. 18The adsorbent is a silica gel modified with an alkane. Macroporous resins can be prepared using a porogen or phase extender to create artificial porosity in a three-dimensional matrix. Once polymerization is complete, the porogen is removed from the matrix, leaving voids in the polymer structure. In one embodiment, the adsorbent comprises a cross-linked styrene, cross-linked poly(meth)acrylate, cross-linked polyacrylonitrile, or C18-modified silica gel (ODS) (e.g., COSMOSIL 40 C18-PREP, available from Nacalai Tesque, Inc.), preferably a macroporous resin based on styrene cross-linked with divinylbenzene (DVB), poly(meth)acrylate cross-linked with DVB, or polyacrylonitrile cross-linked with DVB, more preferably a macroporous resin based on polystyrene cross-linked with DVB, or poly(methyl methacrylate) (PMMA) cross-linked with DVB, most preferably a macroporous resin based on polystyrene cross-linked with DVB. The adsorbent used in the method of the present invention is preferably non-polar.

[0038] The shape of the adsorbent may be, for example, spherical or non-spherical beads, preferably roughly spherical beads, which typically have a diameter of 40 to 1000 microns, preferably 100 to 850 microns, more preferably 250 to 400 microns, even more preferably 250 to 350 microns, and most preferably 250 to 300 microns.

[0039] The adsorbent typically has a pore size of 100 to 1000 nm, preferably 200 to 800 nm, more preferably 300 to 700 nm, and most preferably 400 to 600 nm.

[0040] The eluent as the mobile phase in chromatographic purification can be a mixture of water and a water-miscible organic solvent.

[0041] In one embodiment, water-miscible organic solvents include C3-C4 ketones such as acetone and methyl ethyl ketone, cyclic ethers such as dioxane and tetrahydrofuran (THF), C1-C4-alkanols such as methanol, ethanol, n-propanol, isopropanol (iProH), n-butanol, tert-butanol, polyols and their mono- and dimethyl ethers such as ethylene glycol, propanediol, ethylene glycol monomethyl ether, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, glycerol, C2-C3-nitriles such as acetonitrile and propionitrile, acetic acid, dimethyl sulfoxide, dimethylformamide, formamide, acetamide, dimethylacetamide, butyrolactone, 2-pyrrolidone and N-methylpyrrolidone, preferably C1-C2-alkanols, more preferably ethanol.

[0042] In one embodiment, the ratio of inner diameter to height of the resin loaded in the chromatography column is 1:3 to 1:15, preferably 1:4 to 1:12, more preferably 1:5 to 1:10.

[0043] In one embodiment, the loading of sophorolipid on the resin, calculated by the weight of sophorolipid (g) to the volume of resin (ml), is 1:80 to 1:20, preferably 1:70 to 1:30, more preferably 1:60 to 1:35, and most preferably 1:50 to 1:40.

[0044] In one embodiment, 1 to 15 bed volumes (BV), preferably 3 to 12 BV, more preferably 4 to 10 BV, and most preferably 5 to 8 BV of mobile phase is applied for resin equilibration.

[0045] In one embodiment, 0.05 to 0.8 BV, preferably 0.08 to 0.7 BV, more preferably 0.1 to 0.6 BV, and most preferably 0.15 to 0.5 BV of mobile phase is used to dilute the lactone / acid sophorolipid-containing product to be separated (also referred to herein as "SL to be separated") and load it onto the resin.

[0046] In one embodiment, to achieve separation in step 3), 1-10 bed volumes (BV), preferably 2-8 BV, more preferably 2.5-6 BV, and most preferably 2.75-5 BV of mobile phase is applied to wash the resin.

[0047] For example, when an aqueous alcohol solution, particularly an aqueous ethanol solution, is used as the eluent, the SL to be separated is applied to an adsorbent (stationary phase), the acid-form SL is adsorbed onto the adsorbent, and impurities can be eluted and washed away by pouring in the eluent (aqueous ethanol solution) while intermittently or continuously increasing the ethanol concentration in the range of 10 to less than 60% by weight.Next, by eluting with an aqueous alcohol solution, particularly an aqueous ethanol solution, at a concentration of 50 to 70% by weight, it is possible to recover an acid-form SL fraction, particularly an acid-form SL fraction enriched in acetylated acid-form SL.

[0048] A particular embodiment of the method includes the following process. (1) The column is equilibrated by supplying an aqueous alcohol solution having a concentration of 10 to 30% by weight (for example, an aqueous ethanol solution having a concentration of 10 to 30% by weight) from the top of the column (hereinafter referred to as the top of the separation column). (2) The SL to be separated is diluted and loaded from the top of the separation column. (3) As an eluent, an aqueous alcohol solution (e.g., an aqueous ethanol solution) having a concentration of 10 to less than 40% by weight is poured from the top of the separation column. This process allows salts, including mainly acetic acid, and odorous components to be eluted. (4) The same eluent having a concentration of 40 to less than 60% by weight is poured from the top of the separation column. In this step, impurities such as pigments and odorous components, mainly containing acetic acid, can be eluted. (5) The same eluent having a concentration of 50 to 70 wt %, preferably 55 to 65 wt %, is poured into the top of the separation column to elute an acid-form SL fraction rich in acetylated acid-form SL. (6) Optionally, the same eluent having a concentration of 55 to 85% by weight, preferably 60 to 80% by weight, is poured into the top of the separation column to elute the lactone-type SL fraction.

[0049] It should be noted that in each of steps (1), (3), and (4), the alcohol (e.g., ethanol) concentration of the eluent can be increased over time within the above concentration range (i.e., gradient elution) or maintained at the same concentration (stepwise elution). The latter stepwise elution method is preferred, and an exemplary method is as follows: SLs are added to a column adsorbent equilibrated with an aqueous ethanol solution having an ethanol concentration of 10% by weight (step (1)) (step (2)), and then a certain amount of aqueous ethanol solution having an ethanol concentration of 10% by weight is poured into the column (step (3)). A certain amount of aqueous ethanol solution having an ethanol concentration of 50% by weight is poured into the column (step (4)). Subsequently, an aqueous ethanol solution having an ethanol concentration of 70% by weight is poured into the column to elute and collect the desired acid-form SL fraction (step (5)). Furthermore, step (3) above can be omitted, and step (4) can be initiated after step (2) by pouring in an aqueous ethanol solution having an ethanol concentration of about 40 to less than 60% by weight. In this case, since the salt and pigment components can be simultaneously eluted and removed in addition to the odorous acetic acid, the elution step can be shortened and the amount of ethanol used can be reduced.

[0050] A further particular embodiment of the method comprises the following process. (1) The column is equilibrated by supplying an aqueous alcohol solution having a concentration of 10 to 30% by weight (for example, an aqueous ethanol solution having a concentration of 10 to 30% by weight) from the top of the column (hereinafter referred to as the top of the separation column). (2) The SL to be separated is diluted and loaded from the top of the separation column. (3) As an eluent, an aqueous alcohol solution (e.g., an aqueous ethanol solution) having a concentration of 10 to less than 40% by weight is poured from the top of the separation column. This process allows salts, including mainly acetic acid, and odorous components to be eluted. (4) The same eluent having a concentration of 40 to less than 60% by weight is poured from the top of the separation column. In this step, impurities such as pigments and odorous components, mainly containing acetic acid, can be eluted. (5) The same eluent having a concentration of 55 to 65% by weight is poured into the top of the separation column to elute the acid-form SL fraction enriched in acetylated acid-form SL. (6) The same eluate having a concentration of 60 to 80% by weight is poured into the top of the separation column to elute the lactone-type SL fraction.

[0051] The obtained acid-form SL fraction enriched in acetylated acid-form SL contains 75 to 100 wt%, or 80 to 100 wt%, or 85 to 100 wt%, of acid-form SL, including acetylated and non-acetylated, based on the total weight of the acid-form SL fraction.

[0052] The obtained acid-form SL fraction enriched in acetylated acid-form SL mainly consists of, essentially consists of, or consists of acetylated acid-form sophorolipids. Preferably, the obtained acid-form SL fraction enriched in acetylated acid-form SL contains 75 to 100 wt%, 80 to 100 wt%, or 85 to 100 wt%, of acetylated acid-form SL, based on the total weight of the acid-form SL fraction.

[0053] If desired, the acid-form SL fraction enriched in acetylated acid-form sophorolipids may be subjected to further separation to provide acetylated acid-form sophorolipids.

[0054] Alternatively, the acid-form SL fraction enriched in acetylated acid-form sophorolipids may be optionally subjected to post-treatment, for example, the acid-form SL fraction enriched in acetylated acid-form sophorolipids may be subjected to conventional distillation, and the resulting distillation residue may be dried or precipitated.

[0055] The high-purity acid-form SL fraction obtained from the chromatography, which is rich in acetylated acid-form sophorolipids and contains water, is subjected to distillation to adjust the concentration of acid-form SL contained in the high-purity acid-form SL-containing solution. Furthermore, if the high-purity acid-form SL-containing solution obtained from the chromatography contains an organic solvent such as ethanol, this step also serves to remove the organic solvent.

[0056] The concentration of acid-form SL rich in acetylated acid-form sophorolipids contained in the liquid remaining after distillation (distillation residue liquid) is not particularly limited as long as the advantageous effects of the present invention are not hindered, and examples thereof include typically about 50% by weight or less, preferably 40% by weight or less, more preferably 30% by weight or less, and particularly preferably about 20% by weight. By adjusting the concentration of such acid-form SL rich in acetylated acid-form sophorolipids, advantageous effects can be obtained, such as the ability to easily form a solid or obtain a fine powder when used in the subsequent precipitation (pulverization) step.

[0057] The distillation method is not particularly limited as long as it does not interfere with the advantageous effects of the present invention, and any distillation method known in the art can be used. Examples of distillation methods include molecular distillation, vacuum distillation, steam distillation, etc. From an industrial viewpoint, vacuum distillation is preferred.

[0058] In the step of precipitating acid-form SL rich in acetylated acid-form sophorolipids from the obtained distillation residue liquid, the purified acid-form SL rich in acetylated acid-form sophorolipids of the present invention can be made into a powder form.

[0059] Examples of methods for precipitating acid-form SLs enriched in acetylated acid-form sophorolipids generally include freeze-drying (lyophilization), recrystallization, and spray-drying, but freeze-drying is preferred in the present invention. The acid-form SLs enriched in acetylated acid-form sophorolipids of the present invention are highly chemically stable, heat-resistant, and have a low risk of structural changes. Therefore, by utilizing spray-drying, which enables continuous production, it is possible to efficiently produce purified acid-form SLs enriched in acetylated acid-form sophorolipids of the present invention.

[0060] The solid (e.g., powder) purified acid-type SL thus obtained, which is rich in acetylated acid-type sophorolipids, has excellent storage stability, can be applied to various formulations, regardless of whether the formulations are water-based or oil-based, and is convenient in terms of handling.

[0061] Furthermore, the present invention also relates to acid-type sophorolipids, particularly acid-type sophorolipids rich in acetylated acid-type sophorolipids, obtainable by the method according to the present invention.

[0062] The obtained acid-type sophorolipid rich in acetylated acid-type sophorolipid contains 75 to 100% by weight, 80 to 100% by weight, or 85 to 100% by weight of acid-type sophorolipid, including acetylated and non-acetylated, based on the total weight of the acid-type sophorolipid.

[0063] The obtained acid-type sophorolipid rich in acetylated acid-type sophorolipid mainly consists of, essentially consists of, or consists of acetylated acid-type sophorolipid. Preferably, the obtained acid-type sophorolipid rich in acetylated acid-type sophorolipid contains 75 to 100% by weight, 80 to 100% by weight, or 85 to 100% by weight of acetylated acid-type sophorolipid based on the total weight of the acid-type sophorolipid.

[0064] Certain embodiments of the present invention relate to the preparation of purified glycerin-based sophorolipids, which may, by way of example, comprise the following steps: - Mix 20 g of BiotoLife (a partially hydrolyzed lactone / acid sophorolipid-containing product from BASF. Hydrolysis occurs at room temperature during storage. The lactone / acid ratio is listed in Table 2 below) with 10 g to 80 g of alcohol, such as ethanol, isopropanol, 1-2, pentanediol, propylene glycol, dipropylene glycol, or glycerin, and stir magnetically until homogeneous. - Add 1g to 10g of absorbent, such as silica gel, aluminum oxide, activated carbon, bleaching earth, polyacrylamide, polyvinylpyrrolidone, etc. to the BioToLife water / alcohol solution and stir for 30 minutes. The color and odor molecules are then partially absorbed by the absorbent. Using rotary evaporation of the solvent at -40-70 °C, the absorbent and the solid-like sophorolipid are filtered off to obtain a pale yellow, transparent solution. - The purified sophorolipid solid is dissolved in an equal mass ratio of glycerin and stirred under nitrogen purging until homogeneous. - Glycerin-based sophorolipids are obtained with reduced color and odor. Due to the absence of water, hydrolytic stability is greatly improved. [Example]

[0065] The following examples are set forth below to illustrate methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art.

[0066] Mobile phase: Thin layer chromatography (TLC) methods are used to determine the appropriate mobile phase to adequately separate the components in the sophorolipids.

[0067] TLC plates: TLC plates were prepared on sheets of aluminum foil coated with silica gel (purchased from Merck: TLC Silica Gel 60 RP-18 F 254 The experiment was carried out on 20 sheets of s package, sheet L x W 5 cm x 7.5 cm, aluminum support | Sigma-Aldrich (Sigmaaldrich.cn).

[0068] To quantify the results, the distance traveled by the substance is divided by the total distance traveled by the mobile phase. This ratio is called the retardation factor (RF). A large delta Rf for each component indicates good separation and can be used to estimate separation on a larger chromatographic column. Therefore, the delta Rf values ​​of lactone and acid sophorolipids in different solvent systems are compared.

[0069] Sample: 10 mg of lactone-type sophorolipid (purchased from Cyman) and 10 mg of acid-type sophorolipid (purchased from Carbosynth) were each dissolved in 10 mL of 95% by weight ethanol.

[0070] Mobile phase: 40 wt %, 60 wt %, and 80 wt % aqueous solutions were prepared using ethanol, isopropanol, acetone, and THF, respectively.

[0071] Development: Using a capillary tube, 10-20 μL of each sample solution was spotted onto a TLC plate. The mobile phase described above was used as the developing solvent in the separation chamber. The solvent moved upward on the plate, and the plate was removed from the chamber before the solvent front reached the top of the stationary phase.

[0072] Staining reagent and coloring: Prepare a staining reagent by mixing 71.7 mL of 95% ethanol, 2.7 mL of concentrated sulfuric acid, and 2 mL of anisaldehyde. The plate was immersed in the staining reagent for 1 second and then heated at 120°C for 1 minute.

[0073] Identification: The Rf values ​​of the spots were measured, and the delta values ​​of Rf between acid-type sophorolipids and lactone-type sophorolipids at different solvent concentrations are listed in Table 1.

[0074] [Table 1]

[0075] Example 1 - Resin pretreatment: 60 ml of resin (S-3 (chromatography No. 3), polystyrene cross-linked with divinylbenzene macroporous resin provided by Shanghai Huazhen Technology Co., Ltd.) was loaded into a column (inner diameter 2.4 cm, height 30 cm) and soaked in 100 wt% ethanol for 2 hours; then, it was washed with 8 BV (bed volume) of 100 wt% ethanol.

[0076] - Resin equilibration: The resin was washed with 2BV of 10 wt % ethanol in water.

[0077] - Loading: 1.5 g of BiotoLife (a partially hydrolyzed lactone / acid sophorolipid-containing product from BASF. Hydrolysis occurs at room temperature during storage. The lactone / acid ratios are listed in Table 2 below) was dissolved in 30 mL of 10 wt % ethanol in water and loaded onto the column. The loading of sophorolipid on the resin, calculated by the weight of sophorolipid (g) relative to the volume of resin (ml), is 1:40.

[0078] - Chromatography: The sophorolipid-loaded resin was washed with 5 BV of 60 wt% ethanol in water to collect the acid sophorolipid fraction, and then washed with 3.3 BV of 80 wt% ethanol in water to collect the lactone sophorolipid fraction.

[0079] - Characterization of fractions by HPLC-ELSD: Column: Aglient Eclipse C18 XDB (4.6 mm x 250 mm, 5 μm) Eluent: 0.01% by weight formic acid aqueous solution (A) - 100% by weight methanol (B) 0 to 5 minutes, 80 to 85 wt% methanol (B) (a mixed solution containing solution (A) and methanol (B), the weight percentage of (B) gradually increases from 80 wt% to 85 wt% over a period of 5 minutes) 5 to 7 minutes, 85 wt% methanol (B) (a mixed solution containing 15 wt% solution (A) and 85 wt% methanol (B)) 7 to 20 minutes, 85 to 100 wt% methanol (B) (a mixed solution containing solution (A) and methanol (B), the weight percentage of (B) gradually increases from 85 wt% to 100 wt% over a period of 13 minutes) 20–30 min, 100 wt% methanol (B) Flow rate: 1mL / min Injection volume: 5μL ELSD temperature: 100℃ Gas flow rate: 2L / min.

[0080] The results (purity) from HPLC-ELSD are shown in Table 2.

[0081] [Table 2]

[0082] Example 2 - Resin pretreatment: 100 mL of the same resin as in Example 1 was loaded into a column (inner diameter 2.4 cm, height 30 cm), immersed in 100 wt % ethanol for 2 hours, and then washed with 1 BV (bed volume) of 100 wt % ethanol.

[0083] - Resin equilibration: The resin was washed with 2BV of 10 wt % ethanol in water.

[0084] - Loading: 2.5 g of the same BioToLife as in Example 1 was dissolved in 10 mL of 10 wt % ethanol in water and loaded onto the column. The loading of sophorolipid on the resin, calculated by the weight of sophorolipid (g) relative to the volume of resin (ml), was 1:40.

[0085] - Chromatography: The sophorolipid-loaded resin was washed with 2.75 BV of 60 wt% ethanol in water to collect the acid sophorolipid fraction, washed with 0.5 BV of 60 wt% ethanol in water to collect the fraction of a mixture of lactone acid and acid sophorolipid (this fraction was not characterized by HPLC-ELSD and could be discarded later), washed with 2 BV of 60 wt% ethanol in water to collect the lactone acid sophorolipid fraction, and further washed with 1.25 BV of 80 wt% ethanol in water to collect the remaining lactone sophorolipid fraction.

[0086] - Characterization of fractions by HPLC-ELSD: Column: Aglient Poroshell 120 EC-C18 (2.1mm x 100mm, 2.7μm) Eluent: 0.1% by weight formic acid aqueous solution (A) - 100% by weight acetonitrile (B) 0-8 min, 80-85 wt% acetonitrile (B) (a mixed solution containing solution (A) and acetonitrile (B), the weight percentage of (B) gradually increases from 80 wt% to 85 wt% over a period of 8 min) 8 to 13 minutes, 85 to 100 wt% acetonitrile (B) (a mixed solution containing solution (A) and acetonitrile (B), the weight percentage of (B) gradually increases from 85 wt% to 100 wt% over a period of 5 minutes) Flow rate: 0.4mL / min Injection volume: 1μL ELSD temperature: 100℃ Gas flow rate: 2L / min.

[0087] The results (purity) from HPLC-ELSD are shown in Table 3.

[0088] [Table 3]

[0089] Example 3 (other than the present invention): - Resin pretreatment: The polystyrene cross-linked with D101 divinylbenzene macroporous resin provided by YILAISITE (Shanghai) New Material Technology Co., Ltd. is used. The column and pretreatment method are the same as in Example 1.

[0090] - Resin equilibration: The resin was washed with 4 to 5 BV of 10 wt % ethanol in water.

[0091] - Loading: 1.5 g of the same BioToLife as in Example 1 was dissolved in 30 mL of 10 wt % ethanol in water and loaded onto the column. The loading of the sophorolipid on the resin, calculated by the weight of the sophorolipid (g) relative to the volume of the resin (ml), was 1:40.

[0092] - Chromatography: The resin loaded with sophorolipids was washed with 6 to 8 BV of a 40 wt% ethanol solution, and the acid-type sophorolipid fraction was collected. It was then further washed with 3 to 4 BV of an 80 wt% ethanol solution, and the lactone-type sophorolipid fraction was collected.

[0093] - Characterization of fractions by HPLC-ELSD: Same as Example 1.

[0094] The results (purity) from HPLC-ELSD are shown in Table 4.

[0095] [Table 4]

[0096] The purity of the lactone-type sophorolipid fraction is low due to contamination with a large amount of acid-type sophorolipid, which results in a low yield of the acid-type sophorolipid fraction enriched in acetylated acid-type sophorolipid.

[0097] Example 4 (non-invention): - Resin pretreatment: An ion exchange resin (HZ202, macroporous anion resin, provided by Shanghai Huazhen Technology Co., Ltd.) was packed into a column (inner diameter 2.4 cm, height 30 cm), soaked in ethanol for 2-6 hours, then washed with water, rinsed with 2-5 BV of 1N NaOH solution, then washed with water until pH = 10, rinsed with 2-5 BV of 1N HCl solution, washed with water until pH = 4, and then rinsed with 2-5 BV of 1N NaOH solution to convert the counter ions to OH. - and washed with water until pH=8, then the resin is ready for use.

[0098] - Resin equilibration: The resin was washed with 4-5 BV of 100 wt% ethanol.

[0099] - Loading: 1 g of the same BioToLife as in Example 1 was dissolved in 50 mL of 100% by weight ethanol and loaded onto the column.

[0100] - Chromatography: The sophorolipid-loaded resin was washed with 6-7 BV of 100 wt% ethanol, and then with 3 BV of a KOH-ethanol / HO solution (2 mol / L, ethanol:HO = 9:1 by weight ratio).

[0101] - Characterization of fractions by HPLC-ELSD: Same as Example 1.

[0102] result: No target molecules were obtained in the eluent. It is assumed that lactone-type sophorolipids are hydrolyzed during the treatment and converted to acid-type sophorolipids. The acid-type may be absorbed by the resin.

Claims

1. A method for separating acid-type sophorolipids or lactone-type sophorolipids, particularly acid-type sophorolipids enriched in acetylated acid-type sophorolipids, from a lactone-type / acid-type sophorolipid-containing product by chromatography, comprising: 1) optionally eluting with an aqueous solution of a water-miscible organic solvent having a concentration of 10 to less than 40% by weight; 2) optionally eluting with an aqueous solution of said water-miscible organic solvent having a concentration of 40-60% by weight or less; 3) eluting with an aqueous solution of the water-miscible organic solvent having a concentration of 50 to 70% by weight, and collecting an acid-type sophorolipid fraction enriched in acid-type sophorolipid, particularly acetylated acid-type sophorolipid; 4) optionally, eluting with an aqueous solution of the water-miscible organic solvent having a concentration of 55 to 85% by weight to recover a lactone-type sophorolipid fraction; A method comprising:

2. 10. The method of claim 1, wherein the adsorbent for the chromatographic purification comprises a macroporous resin.

3. 3. The method of claim 2, wherein the macroporous resin is a silica gel modified with a C8 to C18 alkane.

4. The macroporous resin is a cross-linked styrene, a cross-linked poly(meth)acrylate, a cross-linked polyacrylonitrile, or C １８ 4. The method according to claim 2 or 3, comprising a modified silica gel, preferably a macroporous resin based on styrene cross-linked with divinylbenzene, poly(meth)acrylate cross-linked with divinylbenzene, polyacrylonitrile cross-linked with divinylbenzene, more preferably polystyrene cross-linked with divinylbenzene or poly(methyl methacrylate) (PMMA) cross-linked with divinylbenzene, most preferably a macroporous resin based on polystyrene cross-linked with divinylbenzene.

5. The method of any one of claims 2 to 4, wherein the macroporous resin is non-polar.

6. The method according to any one of claims 1 to 5, wherein the eluent used in the chromatographic purification is a mixture of water and a water-miscible organic solvent.

7. The water-miscible organic solvent is C ３ ~C ４ Ketones, such as acetone and methyl ethyl ketone, cyclic ethers, such as dioxane and tetrahydrofuran, C １ ~C ４ - alkanols, for example methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, polyols and their mono- and dimethyl ethers, for example ethylene glycol, propanediol, ethylene glycol monomethyl ether, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, glycerol, and also C ２ ~C ３ Nitriles, such as acetonitrile and propionitrile, acetic acid, dimethyl sulfoxide, dimethylformamide, formamide, acetamide, dimethylacetamide, butyrolactone, 2-pyrrolidone and N-methylpyrrolidone, preferably C １ ~C ２ The process according to claim 6, comprising an alkanol, more preferably ethanol.

8. The method according to any one of claims 1 to 7, wherein the lactone-type / acid-type sophorolipid-containing product contains 20 to 40 wt% of acid-type sophorolipids, including non-acetylated and acetylated sophorolipids.

9. The method according to claim 8, wherein the acetylated acid-type sophorolipid accounts for 15% by weight to 35% by weight of the acid-type sophorolipid.

10. 10. The method according to any one of claims 1 to 9, wherein for washing the resin to achieve separation in step 3) 1 to 10 bed volumes, preferably 2 to 8 bed volumes, more preferably 2.5 to 6 bed volumes, most preferably 2.75 to 5 bed volumes of eluent are applied.

11. An acid-type sophorolipid, particularly an acid-type sophorolipid rich in acetylated acid-type sophorolipid, obtained by the method according to any one of claims 1 to 10.

12. The acid-type sophorolipid-rich acid-type sophorolipid according to claim 11, comprising 75 to 100% by weight, 80 to 100% by weight, or 85 to 100% by weight of acetylated acid-type sophorolipid, based on the total weight of the acid-type sophorolipid.

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