Method for separating acid sophorolipid from lactone / acid sophorolipid-containing product

Abstract

The present invention relates to a method for separating acidic sophorolipids, or lactone sophorolipids, in particular acidic sophorolipids rich in acetylated acid sophorolipids, from products containing lactone / acid sophorolipids, and to the acidic sophorolipids, in particular acidic sophorolipids rich in acetylated acid sophorolipids, obtained by said method.

Description

Technical Field

[0001] The present invention relates to a method for separating an acid-type sophorolipid or a lactone-type sophorolipid, especially an acid-type sophorolipid enriched in acetylated acid-type sophorolipid, from a product containing a lactone-type / acid-type sophorolipid, and an acid-type sophorolipid, especially an acid-type sophorolipid enriched in acetylated acid-type sophorolipid, obtained by the method. Background Art

[0002] Sophorolipids are one of the important biosurfactants obtained from yeast fermentation. During the fermentation process, various by-products such as fatty acids, salts, pigments, etc. are produced at the same time. Therefore, the final product usually has a dark color and odor. In order to expand its application areas to pharmaceuticals, food and cosmetics, certain 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 acid-type and lactone-type sophorolipids (i.e., acid-type sophorolipids and lactone-type sophorolipids, respectively). Both types of structures typically have a certain degree of acetylation of the OH groups on the sugar head. It is reported that the acetyl groups on sophorolipids are critical for some biological functions (e.g., improved antiviral activity and stimulation of cytokines). The acetyl groups will also provide a more lipophilic property, which can lead to better skin delivery or active encapsulation properties.

[0004] However, the acetyl groups on sophorolipids are likely to be hydrolyzed during current downstream purification processes. Especially for acid-type sophorolipids, the loss of acetyl groups will be faster because the molecules are more hydrophilic than their lactone counterparts. Therefore, obtaining purified acetylated acid-type sophorolipids from fermented crude sophorolipid products remains a challenging task.

[0005] CN 109678914 A discloses a method for purifying and separating sophorolipids of different structures. The method starts with a pretreated fermentation broth, which is filtered, treated with cation exchange resins, anion exchange resins, and adsorption resins, desalted, and subjected to membrane ultrafiltration. The method uses different types of resins, including cation exchange resins. Such resins provide a weak to strong alkaline environment that causes hydrolysis of the acetyl groups on sophorolipids and lactone-type sophorolipids during separation.

[0006] JP 2014140383 A discloses a method for obtaining highly purified acid-type sophorolipids, which starts from partially purified acid-type sophorolipids and is purified by reverse phase column chromatography. The starting material used in the present invention, "partially purified acid-type sophorolipids", is prepared by hydrolysis of acid / lactone mixed sophorolipids. This will destroy the acetyl groups on the sophorolipids.

[0007] Conventional methods will only provide fully hydrolyzed structures that will have lost some biological activity. Direct microbial synthesis is possible, but requires the use of engineered strains, which will limit acceptance in some application areas.

[0008] Therefore, there is still a need to provide a novel method for obtaining acid-type sophorolipids, especially acid-type sophorolipids rich in acetylated acid-type sophorolipids, from a fermented crude sophorolipid product. Summary of the Invention

[0009] The object of the present invention is to provide a method for separating acid-type sophorolipids or lactone-type sophorolipids, especially acid-type sophorolipids rich in acetylated acid-type sophorolipids, from a product containing lactone-type / acid-type sophorolipids by chromatography, the method comprising the following steps:

[0010] 1) optionally eluting with an aqueous solution of a water-miscible organic solvent at a concentration of 10% to less than 40% by weight;

[0011] 2) optionally eluting with an aqueous solution of the water-miscible organic solvent at a concentration of 40% to less than 60% by weight;

[0012] 3) eluting with an aqueous solution of the water-miscible organic solvent having a concentration of 50% to 70% by weight to collect an acid-type sophorolipid, especially an acid-type sophorolipid fraction enriched in acetylated acid-type sophorolipid; and

[0013] 4) optionally eluting with an aqueous solution of the water-miscible organic solvent at a concentration of 55% to 85% by weight to collect a lactone-type sophorolipid fraction.

[0014] In addition, another object of the present invention is to provide an acid-type sophorolipid obtained by the method, especially an acid-type sophorolipid rich in acetylated acid-type sophorolipid.

[0015] To solve the above problems, the inventors have conducted intensive studies and have surprisingly found that by subjecting crude or purified sophorolipids from a fermentation process to chromatographic purification, in particular reverse-phase chromatographic purification, acid-type sophorolipids, in particular acid-type sophorolipids enriched in acetylated acid-type sophorolipids, can be obtained.

[0016] Therefore, the present invention relates to a method for separating acid-type sophorolipids, especially acid-type sophorolipids enriched in acetylated acid-type sophorolipids, from a lactone / acid-type sophorolipid-containing product by chromatographic purification, especially reverse phase chromatographic purification.

[0017] In addition, the present invention also relates to an acid-type sophorolipid obtained by the method, especially an acid-type sophorolipid rich in acetylated acid-type sophorolipid.

[0018] The present invention provides an effective method for producing both acid-type and lactone-type sophorolipids in which the acetyl groups are well maintained. At the same time, the fractions of the sophorolipid derivatives can be collected at one time. The starting material can be a crude sophorolipid from which only solid impurities have been removed. The final product obtained is white crystals (e.g., after drying) without worrying about odor and color. DETAILED DESCRIPTION

[0019] It is 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.

[0020] As used herein, the expression "stationary phase" means beads of porous polymeric material that readily absorb solvent and thereby swell.

[0021] As used herein, the expression "mobile phase" means the solvent that passes through the liquid chromatography instrument.

[0022] As used herein, the expression "fraction" refers to individual components collected based on differences in specific properties in a separation process where a quantity of a mixture (solid, liquid, solute, or suspension) is divided into multiple smaller quantities (fractions).

[0023] As used herein, the expression "loaded" or "load" refers to injecting a specific volume of a mixture into a column in order to separate one component from other components in the mixture.

[0024] As used herein, the expression "column" refers to a column used in a chromatography process.

[0025] As used herein, the expression "bed volume" means the volume of chromatography medium in a column.

[0026] As used herein, the expression "a product containing lactone-type / acid-type sophorolipids" refers to a product containing lactone-type sophorolipids and acid-type sophorolipids.

[0027] In addition, ranges defined throughout the specification also include the endpoints, for example, a range of 1 to 10 means that both 1 and 10 are included in the range. For the avoidance of doubt, applicants are entitled to obtain any equivalents under applicable law.

[0028] In the following paragraphs, different aspects of the present invention are defined in more detail. Unless explicitly indicated to the contrary, each aspect so defined can be combined with any one or more other aspects. In particular, any feature indicated as preferred or advantageous can be combined with any one or more other features indicated as preferred or advantageous.

[0029] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may.

[0030] Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art from this disclosure. Furthermore, although some embodiments described herein include some features but not others that are included in other embodiments, combinations of features from different embodiments are still intended to be within the scope of the invention claimed herein and to form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0031] The present invention relates to a method for separating acid-type sophorolipids or lactone-type sophorolipids, especially acid-type sophorolipids rich in acetylated acid-type sophorolipids, from a product containing lactone-type / acid-type sophorolipids by chromatography, the method comprising the following steps

[0032] 1) optionally eluting with an aqueous solution of a water-miscible organic solvent at a concentration of 10% to less than 40% by weight;

[0033] 2) optionally eluting with an aqueous solution of the water-miscible organic solvent at a concentration of 40% to less than 60% by weight;

[0034] 3) eluting with an aqueous solution of the water-miscible organic solvent having a concentration of 50% to 70% by weight to collect an acid-type sophorolipid, especially an acid-type sophorolipid fraction enriched in acetylated acid-type sophorolipid; and

[0035] 4) optionally eluting with an aqueous solution of the water-miscible organic solvent at a concentration of 55% to 85% by weight to collect a lactone-type sophorolipid fraction.

[0036] Sophorolipids

[0037] Sophorolipids (SL) are glycolipids composed of hydroxy fatty acids and sophorose or sophorose in which the hydroxyl groups are partially acetylated. It should be noted that sophorose is a sugar composed of two glucose molecules bonded by a β1→2 bond. Hydroxy fatty acids are fatty acids having a hydroxyl group. In addition, SL is mainly divided into an acid type (general formula (1)) or a lactone type (general formula (2)), in which the carboxyl group of the hydroxy fatty acid is free or bonded to the sophorose in the molecule, respectively. SL obtained by fermentation from a certain yeast (SL-producing yeast) is generally a mixture of SL represented by the following general formula (1) and SL represented by the following general formula (2), and is obtained as a mixture of SL with different fatty acid chain lengths (R 3 ) and at the 6′-position (R 2 ) and 6″-position (R 1 ) is obtained by a collection of 30 or more types of structural homologues with acetylation or protonation.

[0038]

[0039] In the general formula (1) or (2), R 0 is 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 aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond, and may have one or more substituents. The substituent is not particularly limited as long as it does not hinder the advantageous effects of the present invention, and examples thereof include halogen atoms, hydroxyl groups, lower (C 1-6 ) alkyl, halogenated lower (C 1-6 ) alkyl, hydroxy lower (C 1-6 ) alkyl, halogenated lower (C 1-6 ) alkoxy, etc. In addition, R 3 Typically it has from 11 to 20 carbon atoms, preferably from 13 to 17 carbon atoms, more preferably from 14 to 16 carbon atoms.

[0040] SL is obtained by fermentation, by culturing a microorganism, for example, by culturing yeast using methods known in the art, such as Starmerella (Candida) bombicola, C. apicola, C. petrofilum, Rhodotorula (Candia) bogoriensis, C. batistae, C. gropengiesseri, Wickerhamiella domercqiae, and Yarrowia lipolytica. The yeast may be a strain provided by a depository or a strain obtained by continuous subculture thereof. Commercial grade BioToLife is preferred.

[0041] As an example of a method for culturing yeast to produce SL, a preferred method is a method in which a high concentration of sugar and a hydrophobic oily substrate are simultaneously provided for culturing. This method is not limited thereto, and a wide range of other methods known in the art can be applied, as long as they do not hinder the advantageous effects of the present invention. Methods known in the art may include the method described in JP 2002-045195 A, etc. More specifically, this method may be a technique for culturing Bombus stamina (formerly Candida) as a production yeast using glucose as a sugar and a carbon source (including fatty acids and vegetable oils) as a hydrophobic oily substrate.

[0042] Since the fatty acid moiety of SL is known to depend on the ratio of fatty acid chain length and hydrophobic substrate, the fatty acid moiety can be controlled to a certain extent. For example, as a hydrophobic substrate, oleic acid or a lipid containing a high proportion of oleic acid is suitable. 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 tallow. In addition, when a mixed substrate of triglycerides and oleic acid is used as a hydrophobic substrate, a sophorolipid containing a high proportion of oleic acid can be obtained in high yield and high efficiency. From the perspective of industrial use, it is necessary to stably and fermentatively produce SL in high yield and high efficiency. In this case, a mixture of hydrophilic sugar and hydrophobic fat / oil is preferably used as a carbon source. Glucose is often used as a hydrophilic substrate.

[0043] For example, by separating and removing the liquid component from the obtained liquid culture by a hitherto known solid-liquid separation method (such as centrifugation and decantation), and washing the solid content with water, an SL-containing fraction (also referred to as a crude sophorolipid product) can be obtained. The SL-containing fraction is a mixture of lactone-type SL and acid-type SL, and is classified as a lactone-type / acid-type SL-containing product because the content ratio of its acid-type SL in the total amount of SL is less than 45% by weight (solid equivalent).

[0044] According to the present invention, a lactone-type / acid-type sophorolipid-containing product useful as a starting material for isolating acid-type sophorolipids, especially acid-type sophorolipids enriched in acetylated acid-type sophorolipids, may, for example, contain 20% to 40% by weight of acid-type SL, including unacetylated and acetylated acid-type SL. Among the acid-type SL, acetylated acid-type SL may, for example, account for 15% to 35% by weight.

[0045] In addition, the lactone-type / acid-type sophorolipid-containing product as a starting material may, for example, contain 60% to 80% by weight of lactone-type SL, including both unacetylated and acetylated lactone-type SL.

[0046] Chromatographic purification

[0047] According to the present invention, chromatographic purification was used as the separation method, taking advantage of the amphiphilic structure of SL.

[0048] The chromatography used in the present invention is preferably partition chromatography, in particular reverse phase chromatography.

[0049] The adsorbent used as the stationary phase in chromatographic purification is usually a hydrophobic macroporous resin. Macroporous resins are porous polymer materials with a permanent, non-foldable pore structure in both dry and solvated states, and have a large proportion of macropores (i.e., pores with a diameter greater than 100 nm). The pore size distribution of the pores) or the pore size distribution of C8 to C 18Alkane-modified silica gel. Macroporous resins can be prepared by using pore-forming agents or phase extenders to create artificial pores in a three-dimensional matrix. After polymerization is complete, the pore-forming agent is removed from the matrix, leaving gaps in the polymer structure. In one embodiment, the adsorbent includes a macroporous resin based on cross-linked styrene, cross-linked poly(meth)acrylate, cross-linked polyacrylonitrile, or C18-modified silica gel (ODS) (e.g., COSMOSIL 40C18-PREP provided by Nacalai Tesque, Inc.), preferably styrene cross-linked with divinylbenzene (DVB), poly(meth)acrylate cross-linked with DVB, 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 polystyrene cross-linked with DVB. The adsorbent used in the method of the present invention is preferably non-polar.

[0050] The adsorbent may be in the form of spherical or non-spherical beads, preferably substantially spherical beads. The diameter of such beads is typically 40 to 1000 microns, preferably 100 to 850 microns, more preferably 250 to 400 microns, even more preferably 250 to 350 microns, most preferably 250 to 300 microns.

[0051] The adsorbent typically has a pore size of 100 nm to 1000 nm, preferably 200 nm to 800 nm, more preferably 300 nm to 700 nm, most preferably 400 nm to 600 nm.

[0052] The eluent used as the mobile phase in chromatographic purification can be a mixture of water and a water-miscible organic solvent.

[0053] In one embodiment, the water-miscible organic solvent includes 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, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, glycerol, in addition to 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.

[0054] In one embodiment, the ratio of the inner diameter to the height of the chromatography column into which the resin is packed is 1:3 to 1:15, preferably 1:4 to 1:12, more preferably 1:5 to 1:10.

[0055] In one embodiment, the loading amount of sophorolipid on the resin is calculated as the weight of sophorolipid (g) to the volume of resin (ml), from 1:80 to 1:20, preferably from 1:70 to 1:30, more preferably from 1:60 to 1:35, and most preferably from 1:50 to 1:40.

[0056] In one embodiment, 1 to 15 bed volumes (BV), preferably 3 to 12 BV, more preferably 4 to 10 BV, most preferably 5 to 8 BV of mobile phase are applied for equilibration of the resin.

[0057] In one embodiment, the lactone-type / acid-type sophorolipid-containing product to be separated (also referred to herein as "SL to be separated") is diluted with 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 and loaded onto the resin.

[0058] In one embodiment, 1 to 10 bed volumes (BV), preferably 2 to 8 BV, more preferably 2.5 to 6 BV, most preferably 2.75 to 5 BV of mobile phase is applied for flushing the resin to perform the separation in step 3).

[0059] For example, when an aqueous alcohol solution, especially an aqueous ethanol solution, is used as the eluent, the SL to be separated can be applied to an adsorbent (stationary phase) by pouring the eluent (aqueous ethanol solution), eluting and washing away impurities, and simultaneously obtaining the acid-type SL adsorbed on the adsorbent, during which the ethanol concentration is intermittently or continuously increased in the range of 10% to less than 60% by weight, and then the acid-type SL fraction, especially the acid-type SL fraction enriched in acetylated acid-type SL, is collected by eluting with an aqueous alcohol solution, especially an aqueous ethanol solution, having a concentration of 50% to 70% by weight as the eluent.

[0060] A specific embodiment of the method includes the following process, wherein

[0061] (1) equilibrating the column by supplying an aqueous solution of alcohol having a concentration of 10% to 30% by weight (for example, an aqueous solution of ethanol having a concentration of 10% to 30% by weight) from the topmost portion of the column (hereinafter referred to as the separation column top);

[0062] (2) diluting the SL to be separated and loading it from the top of the separation column;

[0063] (3) Pour an aqueous solution of alcohol (e.g., an aqueous solution of ethanol) with a concentration of 10% to less than 40% by weight from the top of the separation column as an eluent. This process can elute salts and odorous components (mainly including acetic acid);

[0064] (4) Pour the same eluate having a concentration of 40% to less than 60% by weight from the top of the separation column. This step can elute impurities such as pigments and odorous components (mainly including acetic acid);

[0065] (5) pouring the same eluate having a concentration of 50% to 70% by weight, preferably 55% to 65% by weight, from the top of the separation column to elute an acid-type SL fraction enriched in acetylated acid-type SL; and

[0066] (6) Alternatively, the same eluent having a concentration of 55 to 85% by weight, preferably 60 to 80% by weight, is poured from the top of the separation column to elute the lactone-type SL fraction.

[0067] It should be noted that in each of steps (1), (3) and (4), the alcohol (e.g., ethanol) concentration of the eluent may be increased over time within the above-mentioned concentration range (i.e., gradient elution method), or may be maintained at the same concentration (stepwise elution method). The latter stepwise elution method is preferred, and an illustrative method is as follows. SL is added to a column adsorbent equilibrated with an aqueous solution of ethanol having an ethanol concentration of 10% by weight (step (1)) (step (2)), a certain amount of an aqueous solution of ethanol having an ethanol concentration of 10% by weight is poured (step (3)), a certain amount of an aqueous solution of ethanol having an ethanol concentration of 50% by weight is poured (step (4)), and then an aqueous solution of ethanol having an ethanol concentration of 70% by weight is poured to elute and collect the desired acidic SL fraction (step (5)). In addition, the above-mentioned step (3) may be omitted, and pouring of an aqueous solution of ethanol having an ethanol concentration of about 40% to less than 60% by weight as step (4) may be started after step (2). In this case, since the salt and pigment components can be eluted and removed at the same time in addition to the odorous acetic acid, the elution step can be shortened and the amount of ethanol used can be reduced.

[0068] A further embodiment of the method comprises the following process, wherein

[0069] (1) equilibrating the column by supplying an aqueous solution of alcohol having a concentration of 10% to 30% by weight (for example, an aqueous solution of ethanol having a concentration of 10% to 30% by weight) from the topmost portion of the column (hereinafter referred to as the separation column top);

[0070] (2) diluting the SL to be separated and loading it from the top of the separation column;

[0071] (3) Pour an aqueous solution of alcohol (e.g., an aqueous solution of ethanol) with a concentration of 10% to less than 40% by weight from the top of the separation column as an eluent. This process can elute salts and odorous components (mainly including acetic acid);

[0072] (4) Pour the same eluate having a concentration of 40% to less than 60% by weight from the top of the separation column. This step can elute impurities such as pigments and odorous components (mainly including acetic acid);

[0073] (5) pouring the same eluate having a concentration of 55% to 65% by weight from the top of the separation column to elute an acid-type SL fraction enriched in acetylated acid-type SL; and

[0074] (6) The same eluent having a concentration of 60 to 80% by weight was poured from the top of the separation column to elute the lactone-type SL fraction.

[0075] The obtained acid-type SL fraction enriched in acetylated acid-type SL contains 75 to 100% by weight, or 80 to 100% by weight, or 85 to 100% by weight of acid-type SL, including acetylated and unacetylated acid-type SL, based on the total weight of the acid-type SL fraction.

[0076] The obtained acid-type SL fraction enriched in acetylated acid-type SL mainly comprises, consists essentially of, or consists of acetylated acid-type sophorolipids. Preferably, the obtained acid-type SL fraction enriched in acetylated acid-type SL comprises 75% to 100% by weight, or 80% to 100% by weight, or 85% to 100% by weight of acetylated acid-type SL, based on the total weight of the acid-type SL fraction.

[0077] If necessary, the acid-type SL fraction enriched in acetylated acid-type sophorolipids may be subjected to further separation to provide acetylated acid-type sophorolipids.

[0078] Alternatively, the acid-type SL fraction rich in acetylated acid-type sophorolipids may be optionally subjected to post-treatment. For example, the acid-type SL fraction rich in acetylated acid-type sophorolipids may be subjected to conventional distillation, and the obtained distillation residue may be dried or precipitated.

[0079] The aqueous, acetylated acid-type sophorolipid-rich, liquid high-purity acid-type SL fraction obtained from the chromatography process is subjected to distillation to adjust the concentration of acid-type SL contained in the high-purity acid-type SL-containing solution. Furthermore, this is also a step for distilling off the organic solvent if an organic solvent such as ethanol is mixed in the high-purity acid-type SL-containing solution obtained from the chromatography process.

[0080] The concentration of the acid-type SL rich in acetylated acid-type sophorolipid contained in the liquid remaining after distillation (distillation residual liquid) is not particularly limited as long as the advantageous effects of the present invention are not hindered, and examples thereof generally include not more than about 50% by weight, preferably not more than 40% by weight, more preferably not more than 30% by weight, and particularly preferably about 20% by weight. By adjusting the concentration of the acid-type SL rich in acetylated acid-type sophorolipid in this way, advantageous effects can be obtained, such as being able to easily form a solid or obtain a fine powder when used in the next precipitation (powdering) step.

[0081] The distillation method is not particularly limited as long as it does not hinder the advantageous effects of the present invention, and distillation methods known in the art can be used. Examples of distillation methods include molecular distillation, vacuum distillation, steam distillation, and the like. From an industrial point of view, vacuum distillation is preferred.

[0082] In the step of precipitating the acid-type SL rich in acetylated acid-type sophorolipid from the obtained distillation residual liquid, the purified acid-type SL rich in acetylated acid-type sophorolipid of the present invention can be made into powder.

[0083] Although examples of methods for precipitating acid-type SL rich in acetylated acid-type sophorolipids generally include freeze-drying methods (lyophilization methods), recrystallization methods, spray-drying methods, etc., freeze-drying methods can be preferably performed in the present invention. Since the acid-type SL rich in acetylated acid-type sophorolipids of the present invention is chemically very stable and heat-resistant, and has a low risk of structural changes, the purified acid-type SL rich in acetylated acid-type sophorolipids of the present invention can be efficiently produced by using a spray-drying method, and this spray-drying method can achieve continuous production.

[0084] The solid (eg, powdered) purified acid-type SL rich in acetylated acid-type sophorolipids thus obtained has excellent storage stability, can be applied to various formulations, whether water-based or oil-based, and is convenient in handling.

[0085] In addition, the present invention also relates to an acid-type sophorolipid obtained by the method according to the present invention, in particular an acid-type sophorolipid enriched in acetylated acid-type sophorolipid.

[0086] The obtained acid-type sophorolipid enriched in acetylated acid-type sophorolipid comprises 75% to 100% by weight, or 80% to 100% by weight, or 85% to 100% by weight of acid-type sophorolipid, including acetylated and unacetylated acid-type sophorolipids, based on the total weight of the acid-type sophorolipids.

[0087] The obtained acid-type sophorolipid enriched in acetylated acid-type sophorolipid mainly comprises, consists essentially of or consists of acetylated acid-type sophorolipid. Preferably, the obtained acid-type sophorolipid enriched in acetylated acid-type sophorolipid comprises 75% to 100% by weight, or 80% to 100% by weight, or 85% to 100% by weight of acetylated acid-type sophorolipid based on the total weight of acid-type sophorolipid.

[0088] A specific embodiment of the present invention relates to the preparation of purified glycerol-based sophorolipids. As an example, the preparation may include the following steps:

[0089] 20 g of BiotoLife (a partially hydrolyzed lactone / acid-containing sophorolipid from BASF. Hydrolysis occurs at room temperature during storage. The ratios of lactone / acid-containing sophorolipids are listed in Table 2 below) were mixed with 10 to 80 g of an alcohol (e.g., ethanol, isopropanol, 1,2-pentanediol, propylene glycol, dipropylene glycol, glycerol) and stirred magnetically until homogeneous.

[0090] - Add 1 to 10 g of an absorbent (e.g. silica gel, alumina, 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.

[0091] A light yellow transparent solution was obtained by filtering the absorbent, and the solvent was evaporated by rotary evaporation at 40-70°C to obtain a solid sophorolipid.

[0092] -The purified sophorolipid solid was dissolved in glycerol in an equal mass ratio and stirred under nitrogen purge until homogeneous.

[0093] - Glycerol-based sophorolipids with reduced color and odor are obtained. Due to the absence of water, the hydrolytic stability is greatly improved.

[0094] Examples

[0095] The following examples are listed below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be comprehensive of all aspects of the subject matter disclosed herein, but are intended to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and modifications of the present invention, which will be apparent to those skilled in the art.

[0096] Mobile phase:

[0097] Thin layer chromatography (TLC) was used to determine the appropriate mobile phase to separate the components of the sophorolipids well.

[0098] TLC plates: TLC was performed on aluminum foil coated with silica gel (purchased from Merck: TLC silica gel 60RP-18F 254 s20 sheets per pack, sheet size L x W 5cm x 7.5cm, aluminum support | Sigma-Aldrich (sigmaaldrich.cn)

[0099] 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 ΔRf for each component indicates good separation and can be used to estimate separation on a larger column. Therefore, the ΔRf values of lactone and acid sophorolipids in different solvent systems were compared.

[0100] Sample: 10 mg of lactone-type sophorolipid (purchased from Cyman) and 10 mg of acid-type sophorolipid (purchased from Carbosynth) were dissolved in 10 mL of 95% by weight ethanol, respectively.

[0101] Mobile phase: 40% by weight, 60% by weight, and 80% by weight aqueous solutions were prepared using ethanol, isopropanol, acetone, and THF, respectively.

[0102] Spreading: 10-20 μL of each sample solution is spotted onto the TLC plate using a capillary tube. Use the mobile phase described above as the spreading solvent in the separation chamber. The solvent moves upward on the plate, and the plate is removed from the chamber before the solvent front reaches the top of the stationary phase.

[0103] Staining Reagent and Coloring: Prepare a mixture of 71.7 mL of 95% by weight ethanol, 2.7 mL of concentrated sulfuric acid, and 2 mL of anisaldehyde as a staining reagent. Soak the plate in the staining reagent for one second and then heat at 120°C for one minute.

[0104] Confirmation: The Rf values of the spots were measured, and the Δ values of Rf between the acid-type sophorolipid and the lactone-type sophorolipid at different solvent concentrations are listed in Table 1.

[0105] Table 1

[0106] ΔRf EtOH THF acetone iPrOH 40％ 0 0.05 0.06 - 60％ 0.4 0.14 0.21 0.22 80％ 0.17 0.03 0.19 0.19

[0107] Example 1

[0108] -Pretreatment of resin:

[0109] 60 mL of resin (S-3 (chromatographic No. 3), a polystyrene macroporous resin cross-linked with divinylbenzene (supplied by Shanghai Huazhen Technology Co., Ltd.) was packed into a column (inner diameter 2.4 cm, height 30 cm) and soaked in 100% by weight ethanol for 2 hours; then rinsed with 8 BV (bed volume) of 100% by weight ethanol.

[0110] -Resin balance:

[0111] The resin was rinsed with 2 BV of a 10% by weight ethanol solution in water.

[0112] -load:

[0113] 1.5 g of BioToLife (a partially hydrolyzed lactone / acid-containing sophorolipid from BASF. Hydrolysis occurs at room temperature during storage. The ratio of lactone / acid-containing sophorolipids is listed in Table 2 below) was dissolved in 30 mL of a 10% by weight aqueous solution of ethanol and loaded into the column. The loading amount of sophorolipid on the resin was 1:40, calculated based on the weight (g) of sophorolipid to the volume (ml) of the resin.

[0114] - Chromatography:

[0115] The sophorolipid-loaded resin was washed with 5 BV of a 60% by weight aqueous ethanol solution to collect the acid-type sophorolipid fraction; and then washed with 3.3 BV of an 80% by weight aqueous ethanol solution to collect the lactone-type sophorolipid fraction.

[0116] - Characterization of the fractions by HPLC-ELSD:

[0117] Column: Aglient Eclipse C18 XDB (4.6mm×250mm, 5μm)

[0118] Eluent: 0.01% by weight formic acid in water (A) – 100% by weight methanol (B)

[0119] 0-5 min, 80-85% by weight of methanol (B) (a mixed solution comprising solution (A) and methanol (B), with the weight percentage of (B) gradually increasing from 80% by weight to 85% by weight within 5 min)

[0120] 5-7 min, 85% by weight of methanol (B) (a mixed solution containing 15% by weight of solution (A) and 85% by weight of methanol (B))

[0121] 7-20 min, 85-100% by weight of methanol (B) (a mixed solution comprising solution (A) and methanol (B), with the weight percentage of (B) gradually increasing from 85% by weight to 100% by weight within 13 min)

[0122] 20-30 min, 100% by weight methanol (B)

[0123] Flow rate: 1 mL / min

[0124] Injection volume: 5 μL

[0125] ELSD temperature: 100°C

[0126] Gas flow rate: 2L / min.

[0127] The results from HPLC-ELSD (purity) are shown in Table 2:

[0128] Table 2

[0129]

[0130] a means that the acid-type sophorolipid in an amount of 29.74% by weight is composed of 26.10% by weight of acetylated acid-type sophorolipid and 3.64% by weight of other substances.

[0131] b means that the acid-type sophorolipid in an amount of 90.03% by weight is composed of 83.35% by weight of acetylated acid-type sophorolipid and 6.68% by weight of other substances.

[0132] Example 2

[0133] -Pretreatment of resin:

[0134] 100 mL of the same resin as in Example 1 was packed into a column (2.4 cm inner diameter, 30 cm height) and soaked in 100% by weight ethanol for 2 hours; then washed with 1 BV (bed volume) of 100% by weight ethanol.

[0135] -Resin balance:

[0136] The resin was rinsed with 2 BV of a 10% by weight ethanol solution in water.

[0137] -load:

[0138] 2.5 g of the same BioToLife as in Example 1 was dissolved in 10 mL of a 10% by weight ethanol aqueous solution and loaded into the column. The loading amount of the sophorolipid on the resin was 1:40, calculated based on the weight (g) of the sophorolipid to the volume (ml) of the resin.

[0139] - Chromatography:

[0140] The resin loaded with sophorolipids was washed with 2.75 BV of a 60% by weight aqueous ethanol solution to collect the acid-type sophorolipid fraction; washed with 0.5 BV of a 60% by weight aqueous ethanol solution to collect a fraction of a mixture of lactone-type and acid-type sophorolipids (this fraction was not characterized by HPLC-ELSD and can be processed later); washed with 2 BV of a 60% by weight aqueous ethanol solution to collect the lactone-type sophorolipid fraction; and further washed with 1.25 BV of an 80% by weight aqueous ethanol solution to collect the remaining lactone-type sophorolipid fraction.

[0141] - Characterization of the fractions by HPLC-ELSD:

[0142] Column: Aglient Poroshell 120EC-C18 (2.1mmx100 mm, 2.7um)

[0143] Eluent: 0.1% by weight formic acid in water (A) – 100% by weight acetonitrile (B)

[0144] 0-8 min, 80-85% by weight of acetonitrile (B) (a mixed solution comprising solution (A) and acetonitrile (B), with the weight percentage of (B) gradually increasing from 80% by weight to 85% by weight within 8 min)

[0145] 8-13 min, 85-100% by weight of acetonitrile (B) (a mixed solution comprising solution (A) and acetonitrile (B), with the weight percentage of (B) gradually increasing from 85% by weight to 100% by weight within 5 min)

[0146] Flow rate: 0.4 mL / min

[0147] Injection volume: 1 μL

[0148] ELSD temperature: 100°C

[0149] Gas flow rate: 2L / min.

[0150] The results from HPLC-ELSD (purity) are shown in Table 3:

[0151] Table 3

[0152]

[0153] a means that an amount of 29.74% by weight of the acid-type sophorolipid consists of 27.94% by weight of the acetylated acid-type sophorolipid, ie, the acetylated acid-type sophorolipid accounts for 100% by weight of the acid-type sophorolipid.

[0154] b means that an amount of 100.00% by weight of acid-type sophorolipid consists of 86.49% by weight of acetylated acid-type sophorolipid and 13.51% by weight of other substances.

[0155] Example 3 (not the present invention)

[0156] -Pretreatment of resin:

[0157] D101, a polystyrene macroporous resin cross-linked with divinylbenzene (supplied by YILAISITE (Shanghai) New Material Technology Co., Ltd.) was used. The column and pretreatment methods were the same as in Example 1.

[0158] -Resin balance:

[0159] The resin was rinsed with 4-5 BV of a 10% by weight ethanol solution in water.

[0160] -load:

[0161] 1.5 g of the same BioToLife as in Example 1 was dissolved in 30 mL of a 10% by weight ethanol aqueous solution and loaded into the column. The loading amount of the sophorolipid on the resin was 1:40, calculated based on the weight (g) of the sophorolipid to the volume (ml) of the resin.

[0162] - Chromatography:

[0163] The resin loaded with sophorolipid was washed with 6-8 BV of 40% by weight ethanol in water to collect the acid-type sophorolipid fraction; and further washed with 3-4 BV of 80% by weight ethanol in water to collect the lactone-type sophorolipid fraction;

[0164] - Characterization of the fractions by HPLC-ELSD:

[0165] Same as Example 1

[0166] The results from HPLC-ELSD (purity) are shown in Table 4:

[0167] Table 4

[0168]

[0169] a means that the acid-type sophorolipid in an amount of 29.74% by weight is composed of 26.10% by weight of acetylated acid-type sophorolipid and 3.64% by weight of other substances.

[0170] b means that the amount of 85.52% by weight of the acid-type sophorolipid consists of 85.52% by weight of the acetylated acid-type sophorolipid, ie the acetylated acid-type sophorolipid accounts for 100% by weight of the acid-type sophorolipid.

[0171] The purity of the lactone-type sophorolipid fraction was low due to the large amount of contamination with the acid-type sophorolipid, which resulted in a low yield of the acid-type sophorolipid fraction enriched in acetylated acid-type sophorolipid.

[0172] Example 4 (not the present invention)

[0173] -Pretreatment of resin:

[0174] Ion exchange resin (HZ202, macroporous anion resin, provided by Shanghai Huazhen Technology Co., Ltd.) was filled into a column (inner diameter 2.4 cm, height 30 cm) and soaked in ethanol for 2-6 hours and then washed with water; rinsed with 2-5 BV of 1N NaOH solution, and then washed with water until pH = 10; rinsed with 2-5 BV of 1N HCl solution, and washed with water until pH = 4; further rinsed with 2-5 BV of 1N NaOH solution to change the counter ion to OH - , washed with water until pH = 8, and then the resin is ready for use.

[0175] -Resin balance:

[0176] The resin was rinsed with 4-5 BV of 100% by weight ethanol.

[0177] -load:

[0178] 1 g of the same BioToLife as in Example 1 was dissolved in 50 mL of 100% by weight ethanol and loaded into the column.

[0179] - Chromatography:

[0180] The sophorolipid-loaded resin was washed with 6-7 BV of 100% by weight ethanol; and further washed with 3 BV of KOH-ethanol / H 2 O solution (2 mol / L, ethanol:H 2 O=9:1 by weight).

[0181] - Characterization of the fractions by HPLC-ELSD:

[0182] Same as Example 1

[0183] result:

[0184] No target molecule was obtained in the eluate. It is assumed that the lactone-type sophorolipid was hydrolyzed during the treatment and converted to the acid-type sophorolipid. The acid-type sophorolipid may be absorbed on the resin.

Claims

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

2. The method according to claim 1, wherein Adsorbents used for chromatographic purification include macroporous resins.

3. The method according to claim 2, wherein: The macroporous resin is a silica gel modified with C8 to C18 alkanes.

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

5. The method according to 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 chromatographic purification is a mixture of water and a water-miscible organic solvent.

7. The method according to claim 6, wherein: The water-miscible organic solvent includes C3-C4 ketones such as acetone and methyl ethyl ketone, cyclic ethers such as dioxane and tetrahydrofuran, C1-C4-alkanols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, polyols and their mono- and dimethyl ethers such as ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, glycerol, and further 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.

8. The method according to any one of claims 1 to 7, wherein The lactone / acid-type sophorolipid-containing product comprises 20% to 40% by weight of acid-type sophorolipids, including unacetylated and acetylated acid-type sophorolipids.

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

10. The method according to any one of claims 1 to 9, wherein 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 for flushing the resin for the separation in step 3).

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

12. The acid-type sophorolipid enriched in acetylated acid-type sophorolipid according to claim 11, comprising 75% to 100% by weight, or 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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