Method for isolating and / or purifying glycolipids

Abstract

To provide a method for temporally and spatially efficiently isolating glycolipids from various kinds of mixtures by efficiently using resources while achieving the purity and high yield of a preliminarily selected final product.SOLUTION: A method of isolating and / or purifying a glycolipid from a glycolipid-containing composition IM, the method comprising: providing a process apparatus 100 with an adsorbent R, wherein the adsorbent is a polymeric resin; and contacting the glycolipid-containing composition with the adsorbent to load the polymeric resin with a load material LM, wherein: A method comprising: a step in which a loading material comprises at least an amount of a glycolipid; a step in which an adsorbent is treated to recover a predetermined desired type of glycolipid, the treatment including contacting the adsorbent with a preselected recovery fluid RL to recover the predetermined desired type of glycolipid from the loading material on the adsorbent; and a step in which the recovered predetermined desired type of glycolipid is obtained from a process device.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for isolating and / or purifying glycolipids from a glycolipid-containing composition, which method can be used as an efficient purification method for biosurfactants, for example in the food, feed, or beverage industries, or in the cosmetic, detergent, industrial, and / or pharmaceutical industries. [Background technology]

[0002] Although biosurfactants such as glycolipids have great market potential, their success in large-scale applications is limited by economic limitations and purity requirements. High yield, scalability, and economic feasibility are essential for downstream processing, with purity undoubtedly a key aspect for certain applications. Thus, a significant portion of the manufacturing costs of biotechnological products is typically due to downstream processing costs and product losses.

[0003] A major challenge in downstream processing of biosurfactants is the complexity of the mixtures containing them. Depending on whether the biosurfactant is produced by fermentation, biocatalysis, plant biomass, or chemical synthesis, various types of impurities will be present in the complex mixture. These mixtures may contain microbial cells, proteins, enzymes, salts, ions, peptides, carbohydrates, fatty alcohols, diols, fatty acids, oils, unwanted biosurfactant by-products or precursors, etc. Proteins, peptides, and enzymes can cause allergic reactions if they remain in the final product, and neither genetically modified microorganisms nor toxic catalysts should be detected in the final product. Furthermore, while lipophilic compounds should not remain in the final product, it is well known that separating amphiphilic compounds, such as biosurfactants, from lipophilic compounds typically present in the final mixture is difficult. This is because lipophilic compounds are often used as substrates for biosurfactant production and are often added in excess. Different types of impurities, e.g., hydrophilic vs. hydrophobic contaminants, often require different process equipment in the purification method, making the overall purification of the biosurfactant slow and expensive and potentially resulting in product loss between process equipment.

[0004] The required purity varies depending on the intended use of the product. Pharmaceutical, food, and cosmetic applications require exceptional purity, while application areas such as (industrial) detergents, environmental remediation, and agriculture may have less stringent requirements. In most cases, it is essential to remove all cells, cellular debris such as DNA and RNA, and proteins / peptides that may potentially cause allergic reactions. Depending on the application, it may even be essential to separate glycolipid mixtures and purify specific glycolipid congeners. Existing applied purification methods for biosurfactants such as glycolipids often combine techniques and methods such as precipitation, lysis, washing, solvent extraction, membrane filtration, foam separation, crystallization, and ion exchange. However, such purification methods require various processing equipment and steps, resulting in product losses at each step. Summary of the Invention

[0005] The objective of the present invention is to provide a resource-efficient, time- and space-efficient technique for isolating glycolipids from a variety of mixtures while achieving a preselected purity and high recovery / yield of the final product.

[0006] Therefore, the present invention provides a method according to claim 1, more particularly a method for isolating and / or purifying glycolipids from a glycolipid-containing composition, the method comprising the steps of: supplying an adsorbent R to a process apparatus, the adsorbent being a polymer resin; contacting the glycolipid-containing composition with the adsorbent to load a load material LM onto the polymer resin, the load material comprising at least a certain amount of glycolipid LM1; treating the adsorbent to recover a predetermined desired type of glycolipid PLM1, the treatment comprising contacting the adsorbent with a preselected recovery liquid RL to recover the predetermined desired type of glycolipid from the load material on the adsorbent; and obtaining the recovered predetermined desired type of glycolipid PLM1 from the process apparatus.

[0007] The present invention is based on the insight that, by using appropriately designed process equipment, it is possible to select a predetermined type of glycolipid and isolate and / or purify said type of glycolipid from a complex mixture added to the process equipment. It has been discovered that a polymeric resin can adsorb certain types of components and at least a certain amount of glycolipid from a mixture fed to the process equipment. Due to the adsorption capacity of the polymeric resin, the components are loaded onto the adsorbent, forming a load material on the polymeric resin. The adsorbent to which the load material is adsorbed is then treated with a preselected recovery solution, allowing the recovery of a predetermined desired type of glycolipid from the material loaded on the adsorbent. In this manner, the desired type of glycolipid can be obtained from the mixture fed to the process equipment, thereby achieving high purity and recovery with little or no additional downstream processing steps. In this manner, product losses are significantly reduced and high purity can be obtained, which is suitable for most applications without the need for cumbersome, time-consuming, and resource-intensive additional processing and purification steps that result in suboptimal economics due to low final recovery / yield.

[0008] Preferably, the method further comprises the step of washing the adsorbent with a washing liquid WL to separate at least the non-loaded substances NLM from the adsorbent. It has been found that the use of a washing liquid makes it possible to remove the non-loaded substances from the adsorbent.

[0009] A cleaning liquid may be understood as a liquid used to remove non-loading materials NLM from the process equipment, i.e., the non-loading materials are not adsorbed on an adsorbent, and an exemplary cleaning liquid is RO water, but may also consist of a solution containing one or more cleaning liquids capable of removing non-loading materials from the process equipment.

[0010] The recovery liquid can be understood as a liquid used to desorb and / or remove substances from the adsorbent. The recovery liquid is typically used to desorb and recover the desired glycolipid. The recovery liquid can consist of a single liquid component or can consist of a solution containing one or more recovery liquids that can desorb and / or remove substances from the adsorbent.

[0011] A treatment liquid can be understood as a liquid used to treat the adsorbent so as to remove from the adsorbent a certain fraction of secondary components such as proteins, pigments, fatty acids, salts, sugars, cells, etc. adsorbed thereto, and / or as a treatment liquid used to modify the load substance, for example by partially hydrolyzing the load substance or by subjecting the load substance to a certain chemical modification(s), for example by glycosylation. The treatment liquid can consist of a single liquid component or can consist of a solution containing one or more treatment liquids capable of modifying the load substance and / or removing substances from the adsorbent.

[0012] The loading capacity described herein can be understood as the amount of a target component, such as a desired type of glycolipid, bound to the adsorbent divided by the total amount of adsorbent, and is typically expressed in terms of mass %, e.g., kg of desired glycolipid adsorbed to the polymer resin divided by the mass (kg) of polymer resin.

[0013] Product purity can be understood as the amount of a component of interest, such as a desired type of glycolipid, divided by the total amount of components present in the final product. Product purity is typically expressed as a % by weight.

[0014] Throughout this application, reference is made to loaded and unloaded substances. Within the context of the present invention, loaded substances are substances that adsorb onto the adsorbent. In contrast, unloaded substances are not adsorbed onto the adsorbent.

[0015] Preferably, the treatment step further includes contacting the adsorbent with one or more treatment solutions TL to modify at least a certain amount of glycolipids loaded on the adsorbent and / or remove one or more secondary components NPLM2 from the adsorbent. In this manner, the secondary components can be removed to purify desired types of glycolipids, and / or the loading substances can be converted into desired types of glycolipids by modifying at least a certain amount of glycolipids loaded on the adsorbent. In some cases, the cleaning solution and the treatment solution can be mixed, e.g., RO water and isopropanol, thereby simultaneously removing the secondary components and non-loaded substances. Preferably, the one or more treatment solutions TL are selected from polar solvents, nonpolar solvents, alkaline solvents, acidic solvents, neutral solvents, or combinations thereof. More specifically, the one or more treatment solutions TL are preferably selected from methanol, ethanol, propanol, isopropanol, butanol, hexane, heptane, ethyl acetate, KOH, NaOH, NH4OH, water, RO water, or combinations thereof. The treatment liquid can be selected to treat and handle the load material on the adsorbent in a predefined manner as further described herein.

[0016] The preselected recovery liquid RL is preferably selected from the group consisting of ionic liquids, liquid carbon dioxide, supercritical solvents, ethyl acetate, methanol, isopropanol, acetone, ethanol, heptane, tert-butyl methyl ether, diethyl ether, acetonitrile, phenoxyethanol, benzyl alcohol, phenethyl alcohol, hydrocinnamyl alcohol, tetrahydrofurfuryl alcohol, dimethyl isosorbide, methyl salicylate, eugenol, linalool, hexanol, glacial acetic acid, dimethyl carbonate, certain glycol ethers such as dipropylene glycol methyl ether and 1-propoxy 2-propanol, and lactate esters including ethyl lactate, butyl lactate, amyl lactate, ethylhexyl lactate, or combinations thereof, which liquids have been found or are expected to have the desired ability to remove a predetermined type of glycolipid from the adsorbent. Preferably, the glycolipid GL is selected from the group comprising glycosylated fatty acids, glycosylated fatty alcohols, glycosylated carotenoids, glycosylated hopanoids, glycosylated sterols, glycosylated paraconic acids, glyceroglycolipids, glycosphingolipids, lipopolysaccharides, phenolic glycolipids, glycopeptide lipids, nucleoside lipids.

[0017] In one embodiment, the glycolipid GL is selected from the group comprising sophorolipids, rhamnolipids, cellobiose lipids, xylolipids, trehalose lipids, mannosylerythritol lipids, glucolipids, fatty alcohol glucosides, alkyl polyglucosides, alkyl sophoroside, (anionic) alkyl glucosides, (anionic) alkyl pentosides, sucrose esters, sorbitan esters, methyl glucoside esters, fatty acid methyl glucamides, oligosaccharide fatty alcohols.

[0018] In one embodiment, the glycolipid GL is selected from acidic sophorolipid (ASL), lactone sophorolipid (LSL) in acetylated and / or non-acetylated form.

[0019] In a preferred embodiment, the glycolipid GL is selected from acetylated and / or non-acetylated bolasophorolipids (BSL), bolasophoroside (BSS), alkylsophoroside (ASS), alcohol glucoside (AGS), bolasophoroside (BSS), sucrose ester (SE), bolaglucoside (BGS), alkyl glucoside (ALGS), glucolipid, or combinations thereof.

[0020] In one embodiment of the present invention, the treatment step includes contacting the adsorbent R with a treatment solution TS2 containing a treatment solution at a first concentration c1, and contacting the adsorbent R with a recovery solution RS2 containing a recovery solution at a second concentration c2, where the first concentration c1 is such that essentially none of the glycolipid LM1 in the load material is removed from the adsorbent, and the second concentration c2 is such that essentially all of the glycolipid LM1 in the load material is removed from the adsorbent. In this manner, the adsorbent is first treated to remove components other than the desired glycolipid, and then the desired glycolipid can be recovered using the recovery solution. In some embodiments, the treatment solution and the recovery solution may be the same. In the above case, i.e., in the present invention, the treatment step includes contacting the adsorbent with a treatment solution containing a treatment solution having a first concentration c1 and contacting the adsorbent with a recovery solution containing a recovery solution having a second concentration c2, where the first concentration c1 of the treatment solution is low enough so that glycolipids in the load material are not efficiently removed from the adsorbent, and the recovery solution c2 is high enough so that glycolipids in the load material are efficiently removed from the adsorbent. By selecting the concentration of the recovery solution, certain secondary components can be selected and removed from the adsorbent using a recovery solution having a lower concentration c1, whereby a substantial amount of the desired glycolipid is not eluted from the adsorbent at the lower concentration. In this way, impurities such as specific proteins and pigments can be removed, and the desired glycolipid remains primarily in a purified state on the adsorbent. The purified desired glycolipid can then be obtained using a recovery solution having a higher concentration c2. In a preferred embodiment where the treatment solution and the recovery solution are the same, the impurities are removed using a recovery solution having a concentration of less than 45%, more preferably between 10% and 35%, and most preferably in the range of 15% to 30%. Preferably, the recovery solution comprises isopropanol diluted with RO water.

[0021] In one embodiment of the present invention, both the processing liquid and the recovery liquid are one of a polar solvent, a non-polar solvent, an alkaline solvent, an acidic solvent, a neutral solvent, or a combination thereof.

[0022] In one embodiment of the invention, the method is used to remove a first fraction of one or more loaded and / or unloaded secondary components from the adsorbent, wherein the first fraction is selected from impurities such as proteins, pigments, etc., or undesired types of glycolipids. In this way, the one or more secondary components, which are typically impurities or other types of glycolipids, are removed from the desired glycolipids.

[0023] In one embodiment of the present invention, the treatment step involves contacting the adsorbent with a first treatment solution, preferably an alkaline reagent, to at least partially hydrolyze / convert a certain amount of glycolipids loaded onto the adsorbent. In this manner, certain types of glycolipids, such as glycolipids containing ester functional groups, can be converted into desired types of glycolipids by partial hydrolysis of the ester functional groups. Thus, desired types of glycolipids can be produced or purified by conversion. In one aspect of the present invention, the method according to the present invention can be used to convert acetylated glycolipids into non-acetylated glycolipids. In another aspect, a method can be used to convert acetylated lactone-type sophorolipids and / or non-acetylated lactone-type sophorolipids into acetylated acidic sophorolipids and / or non-acetylated acidic sophorolipids. In another aspect, a method can be used to convert acetylated bola-type sophorolipids and / or non-acetylated bola-type sophorolipids into acetylated acidic sophorolipids and / or non-acetylated acidic sophorolipids and acetylated sophorose and / or non-acetylated sophorose.

[0024] In one embodiment of the present invention, the treatment step includes contacting the adsorbent with a second treatment liquid, preferably a non-polar solvent, more preferably a non-polar organic solvent, to remove a second fraction of one or more secondary components from the loaded material, wherein the second fraction is selected from non-polar components such as free fatty acids, fatty alcohols, alkanes, and / or oils. Preferably, the treatment liquid is selected from hexane, heptane, or methyl tert-butyl ether. Preferably, the treatment liquid is selected from hexane or heptane. The second treatment liquid may be further mixed with a polar solvent such as methanol, butanol, water, isopropanol, or a combination thereof. By using the second treatment liquid, the second fraction can be removed, thereby purifying the desired glycolipid loaded on the adsorbent.

[0025] In one embodiment of the present invention, the treatment step comprises contacting the adsorbent with a third treatment liquid, preferably a polar solvent, to remove a third fraction of one or more secondary components from the loaded material, wherein the third fraction is selected from hydrophilic impurities such as sugars, proteins, carbohydrates, salts, etc. By using the third treatment liquid, the third fraction can be removed, thereby purifying the desired glycolipid loaded onto the adsorbent.

[0026] Each of the above-mentioned processing steps can be performed alone or in combination with one or more of the above processing steps. In a preferred embodiment, all processing steps are performed. By selecting the first processing solution, the second processing solution, and the third processing solution, it is possible to selectively remove certain fractions from the mixture fed to the process equipment, as well as to remove certain fractions from the load material on the adsorbent. In this way, the process equipment can be used to isolate and / or purify the desired glycolipid to a high purity, preferably greater than 90%, and even more preferably greater than 93%, without having to rely on further purification steps.

[0027] In one embodiment of the present invention, the adsorbent is a neutral polymer resin. Thus, the desired glycolipid binds to the neutral polymer resin through hydrophobic interactions. The neutral polymer resin has a neutral overall charge, and ion exchange is not required to bind the desired glycolipid to the polymer resin. The hydrophobic interaction or affinity of the desired glycolipid can be altered by temperature changes. Preferably, the adsorbent is a polymer resin selected from the group consisting of polymethacrylate resins, acrylic resins, polystyrene resins, or combinations thereof, particularly styrene-divinylbenzene, polymethacrylate, chemically brominated polystyrene, acrylic esters, polystyrene, cross-linked polystyrene, methacrylic, porous polystyrene-divinylbenzene, styrene-divinylbenzene, styrene-divinylbenzene, acrylic esters, polymethacrylates, and chemically brominated polystyrene. The selected resins have been found to have a desired affinity for glycolipids such that at least a certain amount of glycolipid binds to the polymer resin upon contact. High affinity allows for high loading capacities to be achieved.

[0028] In one embodiment of the present invention, the process equipment has an input stream S1 and an output stream S2, and the cleaning step is preferably performed by introducing a cleaning solution WL into the process equipment via the input stream S1 until the output stream parameters are within a predetermined range of the input stream parameters. In one embodiment, the treatment step is performed by introducing a treatment solution TL into the process equipment via the input stream S1 until the output stream parameters are within a predetermined range of the input stream parameters. For example, RO water is introduced to remove hydrophilic impurities such as sugars, proteins, peptides, polyols, organic acids, inorganic acids, carbohydrates, and salts. In one embodiment, the parameters are selected from conductivity, refractive index, pH, protein content, sugar content, test swab values, or a combination thereof. Of course, when considering conductivity, for example, the cleaning step or treatment step is performed until the output stream conductivity reaches a value lower than 150% of the input stream conductivity.

[0029] In one embodiment of the present invention, the method further comprises evaporating the recovery liquid and, if present, the treatment liquid. By evaporating the recovery liquid and / or treatment liquid used to recover the desired type of glycolipid from the adsorbent, the recovery liquid and / or treatment liquid can be removed and preferably reused. In this way, the desired glycolipid can be obtained in a highly purified form. It has been found that a purity of more than 98% by mass can be achieved. In one embodiment of the present invention, the method comprises recycling at least one of the recovery liquid RL, treatment liquid TL, and optionally the wash liquid WL. Practical testing has shown that recycling the recovery liquid and / or treatment liquid, and optionally the wash liquid, allows for more efficient use of resources.

[0030] It is preferred to recirculate at least one of the recovery liquid RL, the processing liquid TL and optionally the cleaning liquid WL, in this way the efficient use of resources can be improved.

[0031] In one embodiment, the glycolipid-containing composition is a plant extract or the end product of a fermentative, enzymatic, plant biomass, or chemical production process, hi one embodiment, the process is carried out in combination with a fermentation process, preferably in an in situ product recovery facility.

[0032] According to one embodiment, the process is carried out in a batch, continuous or semi-continuous manner, with continuous or semi-continuous being preferred.

[0033] According to one embodiment, the glycolipid-containing composition is passed through a reactor containing an adsorbent.

[0034] According to one embodiment, the glycolipid-containing composition is passed through a column packed with a polymeric adsorbent.

[0035] Referring now specifically to the figures, it is emphasized that the details shown are by way of example and for the purpose of illustrative discussion only of different embodiments of the present invention. They are presented to provide what is believed to be the most useful and facile explanation of the principles and conceptual aspects of the present invention. In this regard, no attempt has been made to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the invention. The description, together with the drawings, will make apparent to those skilled in the art how several forms of the present invention may be embodied in practice. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows a flow chart including the steps of the method. [Figure 2] FIG. 2 illustrates an exemplary embodiment of the method. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present application relates to a method for isolating glycolipids from a glycolipid-containing composition, typically said composition being of biological origin, said method being based on the steps set forth in claim 1, which method is further described below.

[0038] Isolated glycolipids can be used as biosurfactants, exhibiting unique properties compared to their classical counterparts, which are typically fossil-derived, such as milder production conditions, lower toxicity, higher biodegradability and skin and environmental compatibility, and (completely) biologically derived nature. Microbial biosurfactants in particular offer several advantages, as they can be produced from local (waste) biomass streams, avoiding dependence on tropical (e.g., palm oil) and / or fossil resources, with their obvious drawbacks. These numerous advantages have encouraged applications in the food, agrochemical, cosmetic, and pharmaceutical industries, as well as in environmental protection and energy-saving technologies. While biosurfactants have great market potential, their success in large-scale applications is limited by economic and technical limitations and stringent purity requirements in some markets. In upstream processing, selective and efficient (high yield relative to substrate) conversion of inexpensive raw materials and high productivity are required. In downstream processing, high yield (recovery rate) is essential. In many applications, the purity of the desired glycolipid is important and secondary components such as allergens should be removed. In many cases, a large portion of the production costs of biotechnological products can be attributed to downstream processing costs and corresponding product losses.

[0039] Some major challenges in downstream processing of biosurfactants are related to the complexity of the mixture, which often contains a relatively low concentration of the desired product. Depending on whether the biosurfactant is produced through fermentation, biocatalysis, plant biomass, or chemical synthesis, various impurities will be present in the mixture. Various production methods generally require not only hydrophilic substrates (e.g., glycerol, glycose, sucrose, etc.) but also hydrophobic substrates (e.g., alkanes, fatty acids, triacylglycerides, fatty alcohols, etc.) to produce glycolipid structures. While some biochemical production methods, such as fermentation production methods, do not strictly require the provision of both hydrophilic and hydrophobic substrates, combining these substrates generally results in higher productivity, making it a preferred option.

[0040] Fermentation production processes typically produce complex mixtures because the fermentation broth may contain substrates, cells, salts, carbohydrates, polyols, proteins, peptides, intracellular and extracellular metabolites, etc. Hydrophobic substrates, such as oils and fatty acids, are often solubilized in the broth by manufactured biosurfactants.

[0041] Therefore, a major challenge in processing biosurfactants is the complexity of the mixtures, often containing relatively low product concentrations, and the presence of numerous impurities in the mixtures. It is necessary to remove any impurities, such as cells, debris, DNA, RNA, and proteins. This is due to the potential for allergic reactions, particularly caused by proteins, peptides, or enzymes, and the DNA / RNA that may be derived from GM organisms. Until now, certain isolation techniques, such as chromatography and / or adsorption, have often been limited to the isolation and purification of low-concentration biosurfactant-containing mixtures, mostly in research and development laboratory facilities, due to highly inefficient methods, low adsorption capacity of resins, equipment size, and high investment and operating costs.

[0042] It is frequently stated in the art that the low adsorption capacity of prior art adsorbents and their lack of separation ability between biosurfactants and other components in a mixture, the high cost of the resins, and the large size of the required equipment make adsorption unsuitable for most applications. Furthermore, the use of adsorption as a purification method for purifying glycolipids (produced by fermentation), such as borasophorolipids (BSL), borasophoroside (BSS), alkylsophoroside (ASS), alcohol glucoside (AGS), sucrose ester (SE), boraglycoside (BGS), alkylglucosides (ALGS), glucolipids, etc., in either acetylated or nonacetylated forms, has not been reported.

[0043] In accordance with the present invention, the inventors have described an improved method for isolating glycolipids from a mixture containing glycolipids, resulting in a high recovery rate and a highly pure product, allowing the isolated glycolipid to be used in several applications while minimizing the risk of allergic reactions. Furthermore, the desired purity of the isolated glycolipid can be modified by flexible washing and / or treatment steps. Furthermore, glycolipids isolated by the present method can be further modified during the purification process, thereby eliminating the need for additional treatment and / or purification steps in downstream process equipment that may be required when glycolipids are purified after conventional derivatization. Thus, the present method results in a more efficient method for converting and / or isolating biosurfactants.

[0044] An exemplary embodiment of the method according to the invention will be explained in more detail using the process flow chart shown in Figure 1 and the method flow chart shown in Figure 2. The process equipment comprises a polymeric resin as adsorbent R.

[0045] As used herein, the terms complex mixture, glycolipid-containing composition, and input mixture may be used interchangeably unless the context states or suggests otherwise.

[0046] As shown in the process flow chart of FIG. 1, the method can begin with an equilibration step 10. Typically, a wash solution WL is used to equilibrate the polymer resin. A preferred wash solution is reverse osmosis water, also known as RO water. Typically, the wash solution WL is added in an amount of 3 to 5 bed volumes. Additionally or alternatively, a resin equilibration step 11 is performed in which the polymer resin is exposed to a resin equilibration solution REL. Typically, the equilibration solution is similar to or the same as the recovery solution used later in the method to recover the desired type of glycolipid. The wash solution is used to remove non-loaded material NLM from the process equipment. That is, non-loaded material is not adsorbed to the adsorbent. A complex mixture IM, including glycolipids GL, is fed to the process equipment. This mixture may be pretreated in a pretreatment step 15, such as a microfiltration step, a gravimetric separation step, or a centrifugation step. The glycolipids are loaded onto the adsorbent (20), and the remaining non-loaded material can be removed via a waste stream 60. An optional recycle stream can be added in between for efficient resource use.

[0047] After the loading step 20, the adsorbent can be subjected to a series of treatment steps (31-34) using a treatment liquid TL to remove the secondary components NPLM2 from the adsorbent. Typical secondary components to be removed are selected from the group comprising ions, salts, sugars, (oligo)saccharides, cells, oils, fatty acids, proteins, peptides, amino acids, residual hydrophobic substrates and / or hydrophobic substances, pigments, or combinations thereof.

[0048] To efficiently use resources, the treated liquid TL can be added back to the process equipment via a recycle stream 62. The treated liquid TL is preferably selected from isopropanol, methanol, ethanol, propanol, butanol, hexane, heptane, ethyl acetate, NaOH, NH4OH, water, RO water, or a combination thereof. By selecting a predetermined treated liquid TL, a predetermined fraction of the load material NPLM2 can be removed from the adsorbent. In this manner, the desired glycolipid can be purified. The desired glycolipid can then be recovered via a recovered liquid RL. Removal of the recovered liquid RL during the purification step 42 allows the desired glycolipid to be obtained in a highly purified form 61.

[0049] A feature of the method of the present invention is that there may be several processing steps, and all processing steps may be carried out in the same process equipment. In the above cases, the adsorbent to which glycolipids are adsorbed may be treated with water, a buffer solution, a solvent, or a mixture thereof to remove certain impurities. Aqueous washing is typically sufficient to remove hydrophilic impurities present in the fraction not adsorbed to the adsorbent. Such impurities include various organic or inorganic salts and acids, sugars, carbohydrates, polyols, proteins, peptides, and various pigments / color components. Washing with a buffer solution may improve the washout of poorly water-soluble compounds or may help ensure product stability. Washing with a water-solvent mixture (such as, but not limited to, methanol, ethanol, isopropanol, and acetone) may facilitate the washout of other impurities weakly adsorbed to the adsorbent.

[0050] In process step 31, the adsorbent is treated with treatment solution TS2 to remove the first fraction F1 from the adsorbent. It has been found that, typically, if the treatment solution capable of eluting the desired glycolipid from the adsorbent has a lower concentration than that used to elute the desired glycolipid from the adsorbent, impurities such as proteins and coloring pigments can be removed from the adsorbent, while the desired glycolipid remains primarily on the adsorbent. In this manner, the desired glycolipid can be purified. That is, the first concentration is not high enough to efficiently desorb the desired glycolipid, so the desired glycolipid remains on the adsorbent. A preferred treatment solution is an alcohol-solvent solution, where the alcohol is a C1-C7 alcohol diluted in an aqueous solution such as water, preferably at a concentration of 20% to 40% by volume, more preferably 24% to 34% by volume, and most preferably 25% to 30% by volume. A preferred treatment solution is propanol, and even more preferred isopropanol. The desired glycolipid can then be removed in a recovery step 40 by recovering the desired type of glycolipid PLM1 using recovery solution RS2. The recovery solution can be similar to the treatment solution, but with a higher concentration high enough to remove the desired glycolipid. Methodologically, this is advantageous when using similar or the same type of solution.

[0051] The inventors have also found that the adsorbent can be subjected to a treatment step 32 in which the adsorbent is treated with a second treatment liquid TL2, preferably a nonpolar organic solvent. In this manner, a second fraction F2 containing nonpolar components can be removed from the adsorbent using a nonpolar organic solvent. In this manner, components such as free fatty acids, fatty alcohols, diols, dicarboxylic acids, triacylglycerides, oils, etc. can be removed from the adsorbent, while the desired glycolipids remain on the adsorbent. By removing the second fraction, the desired glycolipids can be obtained in a purer form by subsequently recovering them by exposing the adsorbent to a recovery liquid. Hexane and heptane are preferred organic solvents. Impurities adsorbed to the polymeric adsorbent can be washed out using treatment with pure nonpolar solvents or mixtures of nonpolar solvents, such as, but not limited to, hexane, heptane, cyclohexane, and diethyl ether, which have poor solubilization properties for glycolipids. This is particularly useful for the separation of hydrophobic substrates such as free fatty acids, oils, fatty alcohols, fats, diols, dicarboxylic acids, triacylglycerides, etc., some of which are used as substrates in the production of glycolipids and / or are co-produced by the microbial production strain, when these impurities co-elute with the glycolipids.

[0052] A further treatment step 33 can be used to purify the desired glycolipids. The inventors have found that further purification of the desired glycolipids can be achieved by treating the adsorbent with a third treatment solution TL3, preferably a polar solvent such as RO water, during treatment step 33. In this case, it has been found that hydrophilic impurities such as sugars, proteins, peptides, organic acids, polyols, carbohydrates, salts, etc. can also be removed from the adsorbent, while the desired type of glycolipid remains loaded on the adsorbent.

[0053] Even more surprisingly, it has been found that treatment step 34 can be used to modify and / or convert glycolipids while they are adsorbed to the adsorbent. Examples of such modifications include chemical hydrolysis of ester and glycosidic bonds, or other chemical modifications such as esterification, glycosylation, and (bio)chemical derivatization routes described in the art, for example, but not limited to, (Delbeke, 2016; Delbeke et al., 2018; Delbeke, Lozach, et al., 2016; Delbeke, Movsisyan, et al., 2015; Delbeke, Roelants, et al., 2016; Delbeke, Roman, et al., 2015; D. Develter & Fleurackers, 2008; Gross et al., 2013; Van Bogaert et al., The modifications may be carried out by, but are not limited to, glycosylation, acylation, alkylation, amidation, amination, arylation, biotinylation, 15-carbamoylation, carbonylation, cycloaddition, coupling reactions, etherification, esterification, glycosylation, halogenation, metallation, metathesis, nitrile formation, olefination, oxidation, phosphinylation, phosphonylation, phosphorylation, quaternization, rearrangement reactions, reduction, silylation, thiolation, thionation, or any combination thereof, towards ω-quaternary ammonium SLs (QASLs), ω-SS amine oxides, ω-SS amines, ω20 bolaamphiphile SS, etc., as described in the art for wild-type SLs described by [End Page 111] (Wang et al., 2011). Such modifications can be carried out by passing an aqueous solution or solvent containing a (bio)catalyst, optionally in combination with a donor / acceptor required in a (bio)chemical reaction, over the adsorbent, during which the (bio)catalyst reacts with the adsorbed glycolipid. In a preferred embodiment, the adsorbent and the load material thereon are contacted with an alkaline agent to partially modify a certain amount of glycolipids present in the load material. In this manner, modification and / or conversion can be used to further purify a desired type of glycolipid by converting other types of glycolipids on the adsorbent to the desired type of glycolipid.By conversion, desired types of glycolipids can be produced or purified. This method can be used to convert acetylated glycolipids to non-acetylated glycolipids and / or lactone-type glycolipids to acidic glycolipids. Non-acetylated lactone-type sophorolipids can be converted to acetylated acidic sophorolipids and / or non-acetylated acidic sophorolipids. In another embodiment, this method can be used to convert acetylated bola-type sophorolipids and / or non-acetylated bola-type sophorolipids to acetylated acidic sophorolipids and / or non-acetylated acidic sophorolipids, as well as acetylated sophorose and non-acetylated sophorose.

[0054] FIG. 2 shows an exemplary embodiment of a method flowchart. A process apparatus 100 includes an adsorbent R. In the exemplary embodiment shown, the adsorbent is provided in a column 100a with an input stream SC1 and an output stream SC2. A wash liquid WL, a treatment liquid TL, and a recovery liquid RL can be supplied to the reactor in a controlled flow, which can be continuous or semi-continuous. The conduits can be pressurized with a pressurized gas, such as N2 gas. Additionally or alternatively, a pump, such as a membrane pump, can be used to control the liquid supplied to the adsorbent. In one embodiment, multiple columns 100a can be used, and these columns can be arranged in series or in parallel with each other. During start-up, a glycolipid-containing mixture IM is typically supplied to column 100a after screening the adsorbent R with the wash liquid WL. The glycolipid-containing mixture is supplied to column 100a to load the adsorbent with glycolipids. By selecting the type of recovery liquid RL, the desired type of glycolipid can be recovered and isolated by desorbing the glycolipids with the recovery liquid RL. The recovery liquid and / or the treatment liquid can be recycled via a recycle stream 200a.

[0055] The present invention typically features a method for isolating glycolipids from a glycolipid-containing composition IM. The composition can be fed as an input mixture IM to a process unit of the method. In one embodiment, the glycolipid-containing composition is the end product of a fermentation process, an enzymatic production process, a plant biomass production process, or a chemical production process.

[0056] In a further embodiment, the glycolipid-containing composition is the end product of a plant extraction. In another embodiment, the glycolipid-containing composition in the methods according to various embodiments of the present invention is the end product of an enzymatic derivatization method. In another embodiment, the glycolipid-containing composition in the methods according to various embodiments of the present invention is the end product of a chemical derivatization method. Derivatization by chemical (derivatization) routes described in the art include, but are not limited to, Fischer synthesis, for example (Delbeke, 2016; Delbeke et al., 2018; Delbeke, Lozach, et al., 2016; Delbeke, Movsisyan, et al., 2015; Delbeke, Roelants, et al., 2016; Delbeke, Roman, et al., 2015; D. Develter & Fleurackers, 2008; Gross et al., 2013; Van Bogaert et al., The present invention also encompasses, but is not limited to, glycosylation, acylation, alkylation, amidation, amination, arylation, biotinylation, 15-carbamoylation, carbonylation, cycloaddition, coupling reactions, etherification, esterification, glycosylation, halogenation, metallation, metathesis, nitrile formation, olefination, oxidation, phosphinylation, phosphonylation, phosphorylation, quaternization, rearrangement reactions, reduction, silylation, thiolation, thionation, or any combination thereof, directed to ω-quaternary ammonium SLs (QASLs), ω-SS amine oxides, ω-SS amines, ω20 bolaamphiphile SSs, and the like, as described in the art for wild-type SLs described by [End Page 111] (Wilson, 2011).

[0057] In a specific embodiment, the method of the present invention involves isolating polymeric glycolipids from a glycolipid-containing composition IM. In yet another embodiment, the glycolipid is selected from the group consisting of glycosylated fatty acids, glycosylated fatty alcohols, glycosylated carotenoids, glycosylated hopanoids, glycosylated sterols, glycosylated paraconates, glyceroglycolipids, glycosphingolipids, lipopolysaccharides, phenolic glycolipids, glycopeptide lipids, and nucleoside lipids. In yet a further embodiment, the method of the present invention involves isolating sophorolipids, rhamnolipids, xylolipids, trehalose lipids, mannosylerythritol lipids, glucolipids, fatty alcohol glucosides, alkyl polyglucosides, alkyl sophorosides, (anionic) alkyl glucosides, (anionic) alkyl pentosides, sugar esters, fatty acid methyl glucamides, and oligosaccharide fatty alcohols from a glycolipid-containing composition IM, wherein the specific glycolipids are adsorbed onto a polymeric adsorbent and subsequently desorbed from the polymeric adsorbent. In yet further specific embodiments, the present invention discloses a method for isolating sophorolipids from a sophorolipid-containing composition, the method comprising adsorbing the sophorolipids onto a polymeric adsorbent and desorbing the sophorolipids from the polymeric adsorbent.

[0058] In a preferred embodiment, the glycolipid composition comprises a glycolipid such as a bora-glucoside, an acetylated bora-glucoside, an alkyl sophoroside, an acetylated alkyl sophoroside, an alkyl glucoside, an acetylated alkyl glucoside, an alcohol glucoside, an acetylated alcohol glucoside, a bora-sophoroside, an acetylated bora-sophoroside, and a bora-sophorolipid, as described in WO 2020 / 104582, which is incorporated herein by reference.

[0059] In one embodiment, a chemically brominated polystyrene-based resin is used to adsorb glycolipids from the input mixture IM. An alcohol solvent is used as the recovery solution to selectively desorb the bolaform SL. In this way, bolaform SLs can be isolated from other types of glycolipids, such as acidic bolaform SLs.

[0060] In one embodiment, the acidic SLs are isolated by removing other components, such as secondary components and / or other types of glycolipids, such as bolaform SLs, from the adsorbent. After the removal of other types of glycolipids, the SLs are recovered with a recovery solution RL, preferably a non-acidic recovery solution, such as isopropanol. The acidic SLs are then recovered with an acidic recovery solution RL, which may be a mixture containing hydrogen halides, such as acetic acid, citric acid, and / or hydrochloric acid, or an aqueous solution containing a mixture thereof.

[0061] In one embodiment, non-polar, poorly water-soluble SLs, such as lactone-type SLs, are isolated from complex mixtures by absorbing the non-polar SLs onto an adsorbent, preferably a polymeric resin, more preferably a polymethacrylate resin and / or an acrylic ester-based resin.

[0062] In one embodiment, the input mixture IM comprises a fermentation broth dissolved in an alcohol solution, such as an ethanol solution. Preferably, the alcohol solution comprises C1, C2, C3, and C4 alcohols at a concentration of 80% or greater.

[0063] In one embodiment of the present invention, alkylsophorosides (ASS) are recovered from absorbent R using a recovery liquid containing a solvent mixture of a low-polarity solvent and a non-polar solvent. Preferably, the low-polarity solvent has a water solubility measured at 20°C of between 5 g / 100 mL and 80 g / 100 mL, preferably between 7 g / 100 mL and 80 g / 100 mL, and most preferably between 7 g / 100 mL and 80 g / 100 mL. Preferably, the non-polar solvent has a water solubility measured at 20°C of between 0 g / 100 mL and 5 g / 100 mL, preferably between 0 g / 100 mL and 1 g / 100 mL. Those skilled in the art can refer to water solubility parameters according to the ILO International Chemical Safety Card. Preferred non-polar solvents are alkanes such as hexane, octane, and heptane. Preferred low-polarity solvents are esters such as ethyl acetate. Preferably, the mixture further comprises an alcohol, preferably any one of a C1 alcohol, a C2 alcohol, a C3 alcohol, a C4 alcohol, such as methanol and / or butanol.

[0064] The Glycolipid-Containing Composition IM may further comprise any additional compounds or substances. In a further embodiment, the Glycolipid-Containing Composition IM comprises a glycolipid and one or more additional secondary compounds selected from the group comprising ions, salts, (oligo)saccharides, cells, organic and / or inorganic acids, oils, fatty acids, proteins, enzymes, peptides, amino acids, (residual) hydrophobic substrates, residual hydrophobic substances, pigments, and / or allergens, or combinations thereof.

[0065] The method according to an embodiment of the present invention is characterized in that secondary components such as proteins, peptides, (residual) hydrophobic substrates, residual hydrophobic substances, salts, and / or organic acids are removed from the glycolipid-containing composition, followed by desorption of the glycolipid from the polymeric adsorbent. The (residual) hydrophobic substances can be selected from fatty acids, fatty alcohols, fatty diols, dicarboxylic acids, triacylglycerides, alkanes, oils, fats, etc. It has been found that the components can be removed by the treatment solution TL.

[0066] Typically, glycolipids are first adsorbed onto a polymeric adsorbent R, followed by desorption from the polymeric adsorbent. In particular, glycolipids are desorbed using a recovery solution RL, which may contain one or more solvents, preferably one or more organic or inorganic solvents. The inventors have discovered that adsorption of glycolipids onto the polymeric adsorbent is based on the affinity between the glycolipid and the adsorbent. The adsorbent is a polymeric resin. It has been found that certain polymeric resins can achieve higher loading capacities, particularly loading capacities greater than 5%, and even up to 10% or more. Preferred polymeric resins are selected from the group consisting of polymethacrylate resins, acrylic resins, polystyrene resins, or combinations thereof, particularly polymeric resins selected from styrene-divinylbenzene, polymethacrylate, chemically brominated polystyrene, acrylic acid esters, polystyrene, crosslinked polystyrene, methacrylic, porous polystyrene-divinylbenzene, styrene-divinylbenzene, styrene-divinylbenzene, acrylic acid esters, polymethacrylate, and chemically brominated polystyrene.

[0067] The method according to various embodiments is further characterized in that desorption of glycolipids is carried out using a recovery liquid RL, preferably comprising one or more solvents, preferably one or more organic or inorganic solvents, selected from the group consisting of ionic liquids, liquid carbon dioxide, supercritical solvents, ethyl acetate, methanol, isopropanol, acetone, ethanol, heptane, tert-butyl methyl ether, diethyl ether, acetonitrile, phenoxyethanol, benzyl alcohol, phenethyl alcohol, hydrocinnamyl alcohol, tetrahydrofurfuryl alcohol, dimethyl isosorbide, methyl salicylate, eugenol, linalool, hexanol, glacial acetic acid, dimethyl carbonate, certain glycol ethers such as dipropylene glycol methyl ether and 1-propoxy 2-propanol, and lactate esters including ethyl lactate, butyl lactate, amyl lactate, and ethylhexyl lactate. In another embodiment of the present invention, the total concentration of the one or more solvents described above is between 20% and 100%, preferably between 50% and 100%, and even more preferably between 70% and 100%, where the concentrations are expressed as volumetric concentrations. Furthermore, glycolipids can be recovered from the polymeric adsorbent by desorption or elution by passing a recovery solution, particularly a water-solvent mixture or a pure solvent, over the adsorbent. In this case, it is essential to select a solvent in which the glycolipid exhibits sufficiently high solubility. The efficiency of desorption, and thus elution, depends on the contact time, temperature, solubility of the glycolipid in the solvent / eluent, volume of the solvent / eluent, and the specific matrix of the polymeric adsorbent.

[0068] The methods according to various embodiments of the present invention are typically characterized by adsorbing glycolipids to a polymeric adsorbent during isolation of the glycolipids from the composition. In certain embodiments, the polymeric adsorbent is a polymeric adsorption resin. In yet other embodiments, the polymeric adsorbent is selected from the group including polymethacrylate resin, acrylic resin, polystyrene resin, or mixtures thereof. In yet further particular embodiments, the polymeric adsorbent is a polystyrene resin.

[0069] In another embodiment of the present invention, the method further comprises one or more treatment steps. Preferably, at least one treatment step is carried out after adsorption of glycolipids onto the polymeric adsorbent and before desorption of glycolipids from the polymeric adsorbent. The washing step in the method of the present invention can be carried out using water, a buffer solution, a solvent, or a combination thereof.

[0070] Furthermore, different treatment solutions with different hydrophobicities can be used to achieve stepwise desorption of the loading substances, for example, acidic sophorolipids and / or lactone sophorolipids and / or mixtures thereof can be separated from fatty acids, or different congeners of sophorolipids can be separated from each other, for example, bola-SLs from acidic SLs.

[0071] The method according to the present invention involves isolating glycolipids from a glycolipid-containing composition, in which the glycolipids are adsorbed onto a polymeric adsorbent followed by desorption of the glycolipid from the polymeric adsorbent. In certain embodiments, during the method, the adsorbed glycolipids can be (bio)chemically and / or enzymatically modified, followed by desorption of the glycolipids from the polymeric adsorbent using a treatment solution TL. The chemical and / or enzymatic modification of the adsorbed glycolipids can be selected from chemical and / or enzymatic hydrolysis of ester bonds, chemical and / or enzymatic hydrolysis of glycosidic bonds, esterification, etherification, glycosylation, polymerization, amidation, (reductive) amination, quaternization, oxidation, and epoxidation. In this way, the glycolipids loaded onto the absorbent can be modified and at least a certain amount of the loaded glycolipid can be converted into the desired type of glycolipid within the same process equipment, without the need to rely on additional and / or downstream modification steps. In this way, both the modification and purification of the desired glycolipid can be performed within the same process equipment. Furthermore, by modifying the loading substance to a desired glycolipid, the purity of the desired glycolipid can be improved.

[0072] At the end of the isolation process, the desired glycolipid is collected from the eluted fraction, for example, by evaporation, preferably vacuum evaporation, or by a combination of ultrafiltration or nanofiltration and diafiltration. As shown in FIG. 2, the process and / or recovery liquid can be separated from the desired glycolipid PLM1 in an evaporation vessel 102. By reducing the pressure in the liquid-filled vessel 102 to below the vapor pressure of the process and / or recovery liquid, the liquid is evaporated at a lower temperature than usual. To optimize the use of resources, the evaporated liquid is preferably recycled back to the beginning of the process. Tests have shown that the process and / or recovery liquid can be reused in this way, achieving efficient use of resources.

[0073] In another embodiment, the polymeric adsorbent R can be regenerated and prepared for the next isolation. The resin can be regenerated semi-infinitely. Tests have shown that the ability to regenerate the resin allows it to be reused for multiple process runs, which is beneficial from the perspective of efficient resource use. Regeneration can be carried out using a combination of water, buffer, and / or solvent. While this washing step is not strictly required, it can be advantageous when impurities accumulate on the adsorbent and may reduce its loading capacity.

[0074] In another aspect of the invention, the method for isolating glycolipids from a glycolipid-containing composition is carried out in a batch mode. In another embodiment, the method is carried out in a continuous mode. In yet another embodiment, the method is carried out in a semi-continuous mode.

[0075] In further embodiments, the method according to the invention is carried out in combination with fermentation, biocatalytic, and / or chemical methods. In the above embodiments, production of the glycolipid-containing composition by fermentation is carried out in combination with glycolipid isolation, e.g., in combination with fermentation in an in situ product recovery configuration. In certain configurations, the polymeric adsorbent and the glycolipid-containing composition are mixed in a reactor. In further embodiments, in the above configurations, the method according to the invention is carried out in a reactor.

[0076] In one embodiment, glycolipids loaded onto the absorbent are chemically modified by partial hydrolysis. In a specific embodiment, bolasophorolipids (bolaSLs) were used to produce acidic sophorolipid SLs and sophorose, as described, for example, in WO 2015 / 028278, incorporated herein by reference. The loaded material containing the bolaSLs is exposed to an alkaline agent, such as NaOH. The pH of the alkaline agent is preferably greater than 8, more preferably greater than 12. A higher pH has been found to improve treatment times by increasing the reaction rate. Typically, the absorbent is washed with water to remove sophorose and base, followed by desorption of the acidic sophorolipids.

[0077] In another configuration, during the process of the present invention, the glycolipid-containing composition is passed through a reactor containing a polymeric adsorbent. In one embodiment, a polymeric resin as described herein can be mixed with the glycolipid-containing mixture IM in the reactor in which the process of the present invention is carried out. In a preferred embodiment, during the process according to various embodiments of the present invention, the glycolipid-containing composition is passed through a column packed with a polymeric adsorbent. In another embodiment, the lactone-form SL is absorbed onto an absorbent in the process equipment and modified with an alkaline agent to form the acid-form SL. Typically, the adsorbent is washed with water to remove acetate and base.

[0078] The method according to the present application can be used for the isolation of glycolipids in the food industry, the cosmetic industry, the pharmaceutical industry, the environmental protection industry, or the energy saving industry. [Example]

[0079] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these or any of the examples, and various modifications can be made by those skilled in the art within the scope of the present invention. The present invention will be illustrated by the following examples.

[0080] Liquid concentrations in the examples are typically expressed as volume percent, which means the volume of a liquid that can be mixed with and / or diluted by another liquid to form a mixture, divided by the total volume of that mixture.

[0081] Example 1 - Purification and isolation of acidic sophorolipids (ASL) and borasophorolipids (BSL) from complex mixtures using adsorption resins Initial resin screening was performed by mixing resins (polystyrene, styrene-divinylbenzene, polymethacrylate, acrylic ester, dextran-based coal, and bituminous coal) with a solution containing SL. The resins were first equilibrated sequentially with RO water, ethanol, and then RO water again. At each stage, the mixture was placed in an incubator at 200 rpm for 15 min. The loading capacity was estimated by comparing the concentration of SL in the liquid before and after the addition of the resin. Subsequently, the resin was removed from the mixture by filtration through a 100 μm cutoff sieve and washed with RO water until the conductivity of the wash water reached less than 100 μS / cm. This ensured the removal of all hydrophilic impurities such as sugars and salts.

[0082] All resins were able to retain some amount of sophorolipids, also abbreviated as SL(s) herein. Mixing the adsorbed resin with acetone for 30 minutes at 200 rpm in an incubator yielded recovery rates up to 77%. Other solvents, such as ethanol, isopropanol, and ethyl acetate, were also able to elute SLs.

[0083] Using a chemically brominated polystyrene-based resin, we were able to elute all bolaform SLs without any acidic SLs using 40%–50% isopropanol. This is also possible with cross-linked polystyrene resin, but the affinity difference is smaller, making separation more difficult.

[0084] Although bituminous coal adsorbed only acidic SL and not bora-SL, its loading capacity was significantly lower, making it more suitable as a final purification step at the end of the process to remove small amounts of acidic SL impurities remaining in the bora-SL product.

[0085] Although bolaform SLs are uncharged, they are also adsorbed by the hydrophobic backbone of the ionic resin. This property can be used to separate the acidic and bolaform SLs by first eluting the bolaform SLs with a solvent, followed by regeneration of the ion exchange resin to elute the acidic SLs.

[0086] Ion exchange resins were screened by mixing the SL solution with each resin. All ionic resins were able to retain both SLs, and the weakly anionic cross-linked polystyrene resin was observed to have acceptable loading capacity. While bolaform SLs could be eluted with 70% isopropanol, similar to the cross-linked polystyrene adsorption resin, acidic SLs remained associated with the resin by ionic bonds even when 96% isopropanol was applied. The same principle can be applied to remove fatty acids from bolaform SLs or acidic SLs from lactone SLs. Acetic acid (CH3COOH) and citric acid (C6H8O7) were able to partially remove acidic SLs. The best results were obtained using 5% aqueous hydrogen chloride (HCl) solution.

[0087] Additional elution tests were performed using a vacuum manifold holding small 10-15 mL columns packed with resin. Selected sorption resins were sequentially equilibrated with RO water, isopropanol, and RO water. Cell-free supernatant containing SLs produced by fermentation was then added to each column. The columns were placed in an incubator at 30°C and 200 rpm for 15 minutes. This incubation step was repeated for each loading, elution, and washing step. Fractions were collected in 15 mL test tubes using a vacuum chamber. Washing steps were performed with RO water, and elution was performed with a 50% to 96% isopropanol solution. Even with a short contact time of just 15 minutes, loading capacities of up to 10% were recorded for the chemically brominated polystyrene resin.

[0088] The optimal polystyrene resin with relevant techno-economic properties was tested in a glass laboratory-scale column with a volume of 300 mL. Samples were taken periodically to monitor SL concentration, conductivity, pH, and Brix. Protein concentration was determined in all fractions using a standard BCA protein assay. The experiments were always performed in the following order: Wash with RO water. Condition the resin (new resin only). The product is loaded. Rinse with RO water until conductivity is less than 100 μS / cm. The product is eluted with 50% to 100% solvent (e.g., ethanol). (Regenerate the resin with 90%-100% solvent, e.g., ethanol).

[0089] A cell-free fermentation broth containing primarily bolaform SL and some non-acetylated acidic SL was processed using the above method. Using a polystyrene resin and eluting with 96% ethanol, a loading capacity of over 7.5% and a recovery rate of over 80% were obtained. This recovery rate could be further increased by extending the elution step. Purity could also be increased, as a significant peak in the protein was detected during elution. This example is further illustrated in Example 2.

[0090] Example 2 - Purification of acidic and bolaform SL from proteins and colored pigments using adsorption resins After rinsing the product-loaded polystyrene resin with RO water, SL elution using a high ethanol concentration desorbs all SLs, but also some proteins, as described in Example 1. While most of the proteins and color pigments were already removed during the loading and RO water wash steps, more hydrophobic proteins and color pigments remained due to their affinity for the resin. The 300 mL experiment in Example 1 was repeated, but this time the resin was washed with a lower ethanol concentration. More proteins and color pigments could be removed by first washing the resin with 25% to 30% ethanol, which was sufficient to eliminate most of the color pigments and proteins but low enough not to desorb any SLs. SLs were then eluted using 50% to 96% ethanol.

[0091] Increased purity of SL was achieved without affecting high recovery by applying the following sequence: Wash with RO water. Condition the resin (new resin only). The product is loaded. Wash with 25%–30% ethanol / isopropanol. Rinse with RO water until conductivity is less than 100 μS / cm. Elute the product with 50%–100% ethanol / isopropanol. (Regenerate the resin with 90%-100% solvent, e.g., ethanol).

[0092] If increased purity is desired, the adsorption step described above can be substituted or added to the glycolipid purification method.

[0093] This method was also evaluated on a 30 L pilot-scale column containing a polystyrene resin absorbent. After subjecting the fermentation broth to microfiltration to remove cells, the filtrate was treated with the adsorption resin. 96% ethanol was used to elute the SLs to maximize recovery, and the column was completely regenerated, all in one step. Loading capacities of greater than 8% were systematically recorded, resulting in recoveries of greater than 95% of the bola-type SLs. Comparing this purification strategy with combined 50 kDa and 10 kDa PES ultrafiltration purification methods on the same cell-free filtrate produced by fermentation, both recovery and purity can be significantly enhanced, as shown in Table 1.

[0094] [Table 1]

[0095] Finally, the adsorbent was used to 3 SL was purified from the fermentation of 1.1 ml of SL. Cells were removed by microfiltration, and the filtrate was used to 3 A column was loaded with polystyrene resin. SLs were eluted using a solution of 70% isopropanol. Several batches were performed, resulting in an overall recovery of 95% for bolaform SLs.

[0096] Example 3 - Purification of lactone-form SL (water-insoluble) from a complex mixture using adsorption resin Samples containing mixtures of acidic and lactone SLs with various degrees of acetylation were prepared. Diacetylated lactone SLs were the most prominent. Nine different adsorption resins (polystyrene, styrene-divinylbenzene, acrylic ester, and polymethacrylate-based) were screened using both the fermentation broth and the supernatant, the latter obtained by centrifugation of the same broth.

[0097] Laboratory-scale tests were performed using a vacuum manifold holding small 10-15 mL columns packed with resin. Prior to loading, the resin was rinsed with RO water and shaken at 200 rpm for 15 minutes. After removing the water by applying a vacuum, the resin was loaded with broth or supernatant and shaken at 200 rpm for 30 minutes. The resin was washed with RO water to remove hydrophilic impurities before elution. 70% aqueous isopropanol was used as the eluent. While styrene-divinylbenzene-based resins showed some adsorption, the best results were obtained with polymethacrylate- and acrylate-based resins, reaching a maximum loading capacity of 8%. Resins with larger surface areas were able to adsorb more SLs, but elution of the SLs proved much more difficult.

[0098] Several other solvents were evaluated in mixtures of acidic and lactone-form SLs dissolved in water. The reference elution solvent was 70% isopropanol. Both SLs could be eluted with 70% ethanol, 70% methanol, 100% butanol, 100% acetonitrile, 70% acetone, and 100% tetrahydrofuran, with butanol performing slightly better than isopropanol. Nonpolar solvents such as hexane and heptane did not elute any SLs and may be suitable for separating free fatty acids, fatty alcohols, and oils during adsorption.

[0099] After screening the resins for adsorption capacity and elution efficiency, they were scaled up to a 300 mL column. Experiments were performed using the same fermentation broth and supernatant containing SL. The column was packed with a polymethacrylate-based adsorption resin. The column was rinsed with RO water to a conductivity below 20 μS / cm before loading with the broth or supernatant. Prior to elution, the resin was washed with RO water to a conductivity of 50 μS / cm. Elution was performed with a 70% isopropanol solution.

[0100] Due to promising results, purification of lactone-form SL was tested in a 5 L glass column. A 5 L column was packed with a polymethacrylate-based adsorption resin. The resin was rinsed with RO water until the conductivity of the effluent dropped below 20 μS / cm.

[0101] The fermentation broth was mixed with an equal volume of 96% ethanol to dissolve as much lactone-form SL as possible. The broth was then mixed with diatomaceous earth filter aid and poured onto a filter bed to remove all cells and debris. The ethanol in the filtrate was evaporated before loading onto the resin to improve adsorption efficiency. After loading the column, the resin was washed with RO water until the conductivity dropped below 50 μS / cm. Elution was performed using a 70% isopropanol solution. Samples were taken every 5 minutes to monitor SL concentration, conductivity, pH, and Brix. The overall SL recovery reached 89%.

[0102] Example 4 - Purification and isolation of borasophorosides (BSS), alkylsophorosides, and alcohol glucosides from complex mixtures using adsorption resins A 10-15 mL laboratory-scale column was packed with polystyrene-based resin and rinsed with RO water. Subsequently, the fermentation broth was microfiltrated at high temperature to obtain a cell-free filtrate. This filtrate contained alkyl sophorosides (alkyl SS), alcohol glucosides (alcohol GS), and bora-type sophorosides (bora-SS). After the filtrate was loaded onto the column, the resin was washed with RO water until the conductivity was less than 100 μS / cm. Various solvent mixtures, including methyl tert-butyl ether (MTBE) and isopropanol, or a mixture of heptane, ethyl acetate, methanol, butanol, and water, were added to the resin and shaken at 200 rpm for 30 minutes, followed by elution.

[0103] A mixture of 20% heptane (water solubility of 0.0022 g / 100 ml at 25°C), 27.5% ethyl acetate (EA) (water solubility of 8.7 g / 100 ml at 20°C), 2.5% butanol, 2.5% methanol, and 37.5% water appeared to be most selective for alkyl SS, eluting 93%, while only 10% of alcohol GS and no bola-SS were eluted. Isopropanol was not at all selective, eluting all three glycolipids.

[0104] Methyl tert-butyl ether (MTBE) (water solubility 4.2 g / 100 ml) and diethyl ether (water solubility 6.9 g / 100 ml at 20 °C) showed some selective properties but were less efficient, eluting 40% of alkyl SS along with 13% of alcohol GS and 3% of bora SS.

[0105] The genetically engineered Starmerella bombicola strain that produces this mixture of sophorosides and glucolipids also produces de novo fatty acids, which also need to be removed because they co-elute with the SS when using the solvent mixtures mentioned above.

[0106] After evaporating the solvent, the precipitated SS was dispersed in RO water and reloaded onto a column packed with polystyrene-based resin. After washing the column with RO water, each column was eluted with a different solvent (mixture). The following heptane to ethyl acetate ratios were applied: 100:0, 90:10, 80:20, 60:40, and 40:60. Using 100% heptane did not remove de novo fatty acid derivatives. The hexane solution appeared to be too nonpolar. The 90:10 solution eluted some of the de novo fatty acid derivatives and 39.6% of the alkyl SS, while the 80:20 solution seemed most promising for removing most of the de novo fatty acid derivatives. Unfortunately, coelution of 59% of the alkyl SS was also observed. Both the 60:40 and 40:60 solutions were able to elute not only all the alkyl SS but also the de novo fatty acid derivatives. However, the second ratio also coeluted a fraction of the alcohol GS. Although further optimization is required, this experiment demonstrates that selective elution from the adsorption resin allows for the separation of de novo fatty acid derivatives, alkyl SS, and alcohol GS.

[0107] Example 5 - Purification of acetylated borasophorosides (ABSS) from complex mixtures using adsorption resins The adsorption capacity of acetylated borasophoroside on polystyrene resin was estimated by filling a 5 mL Eppendorf tube with resin and loading various known product concentrations (5 m / m% to 12 m / m%) onto the resin. Solutions were prepared by dissolving the required amount of lyophilized pure product in RO water. After shaking at 200 rpm for 45 min, a sample of the solution was taken and analyzed by TLC. When the resin's loading capacity was exceeded, a product spot was observed on the TLC plate, indicating that not all of the product was able to bind to the resin.

[0108] The conclusion of this experiment was that the adsorption capacity of acetylated bora-sophorosides on this resin was between 5 m / m% and 6 m / m%, which is significantly lower than the adsorption capacity of the same resin for bora-SL (8 m / m%). The hypothesis was that the extra space was occupied by the acetyl groups, allowing less product to be adsorbed. However, when the experiment was repeated, even the highest bora-SS ratio tested, 12 m / m%, was fully adsorbed onto the resin after overnight shaking. It is believed that multilayer adsorption occurs when sufficient contact time is applied.

[0109] Purification of acetylated bolaform SS from fermentation broth was first demonstrated in a 300 mL column and then in a 30 L column using the method described in Example 1 for bolaform SL. Prior to use, the resin-packed column was rinsed with RO water, 96% isopropanol, and RO water. After loading the cell-free filtrate onto the column, the resin was again washed with RO water to a conductivity of 20 μS / cm. The bolaform SS was eluted with 70% isopropanol. A recovery of 88% ABSS was achieved with a purity of over 95%, consistent with the results obtained for bolaform SL in Example 1.

[0110] [Table 2]

[0111] Example 6 - Purification of sucrose esters (SE) using adsorption resin Adsorption was evaluated for E473 sucrose esters, which are soluble in warm water. Laboratory-scale tests were performed using a vacuum manifold holding small 10-15 mL columns packed with polymethacrylate-based resin. Prior to loading, the resin was rinsed with RO water and shaken at 200 rpm for 15 min. After removing the water by applying a vacuum, the resin was loaded with the sucrose ester solution and shaken at 200 rpm for 30 min, which was sufficient to adsorb all SEs. The resin was again washed with RO water to remove hydrophilic impurities before elution. Elution with 70% isopropanol was successful, but with lower recovery rates than for the acidic and lactone forms of SLs; this can be compensated for by increasing the isopropanol concentration or increasing the elution time.

[0112] Example 7 - Chemical (Partial) Hydrolysis of Glycolipids 1) Preparation of acidic SL and sophorose by (partial) hydrolysis of bola-SL on a column Bola-SLs have ester bonds that are susceptible to (alkaline) hydrolysis. Because glycosidic bonds can withstand alkaline hydrolysis, the resulting products are non-acetylated acid-SLs and sophorose, both of which are interesting molecules. By performing hydrolysis on a column, acid-SLs and sophorose can be produced and purified in the same equipment run, even when starting from a complex mixture such as a cell-free fermentation broth containing bola-SLs. Because purification occurs during the same equipment run, partial hydrolysis can also be performed to obtain a desired ratio between bola-SLs and acid-SLs. Sophorose will elute during hydrolysis. Sophorose can be further purified by ion exchange, nanofiltration, or dialysis polishing steps to remove ions and salts. A preferred conversion of bola-SLs to acid-SLs and sophorose is illustrated below. chemical formula 1 [ka]

[0113] To evaluate sodium hydroxide and ammonia hydroxide as bases for chemical hydrolysis, purified bolaform SL was first mixed with several concentrations of each base (0.01 M to 0.5 M and 0.1 M to 1 M, respectively) and shaken at 200 rpm for 1 h. When using NaOH, a minimum concentration of 0.05 M or maintenance of pH 12 was required to achieve complete conversion. With NH4OH, a significantly higher concentration of 1 M was required. The advantage of using NH4OH is that it is volatile and can be removed from sophorose, eliminating the need for complex purification. Volatility is also a disadvantage, as irritating ammonia vapors are generated during the process. Hydrolysis begins as early as pH 8 but becomes significantly faster at pH 12. Due to the formation of acidic SL, the pH decreases during hydrolysis, and the pH must be increased again to maintain a high reaction rate.

[0114] [Table 3]

[0115] Next, hydrolysis was tested in a 300 mL adsorption column packed with polystyrene resin. The resin was equilibrated as described in Example 1 and washed with RO water to a conductivity of less than 50 μS / cm. The resin was then loaded with a 10 m / m% SL solution. Two bed volumes were tested with NaOH at concentrations of 0.1 M and 0.25 M, and with NH4OH at 0.5 M, all at a flow rate of approximately 3 bed volumes per hour. Hydrolysis could be followed due to the increase in conductivity and Brix value, the latter value being related to the amount of sophorose. The resin was washed with RO water to a conductivity of 50 μS / cm, followed by elution of the SLs using 96% ethanol. No bolaform SLs were detected at either NaOH concentration, whereas 0.5 M NH4OH was not sufficient to complete hydrolysis at the current contact time.

[0116] The method was scaled up to a 30 L column using the same SL load and two bed volumes of 0.1 M NaOH solution at a flow rate of approximately two bed volumes per hour. During hydrolysis, the pH in the effluent rose to 12.5. After washing with RO water, the SLs were eluted using 50% ethanol. As expected, no bolaform SLs were detected, indicating complete hydrolysis.

[0117] Alternatively, hydrolysis can be performed first in a reactor followed by adsorption, which allows for easier tracking and control. However, when using complex compositions such as cell-free fermentation broths, reactions with other products also occur, resulting in unexpected by-products and higher base consumption. Therefore, adsorption after hydrolysis tends to be used for already isolated products with higher purity.

[0118] 2) On-column chemical (partial) hydrolysis of lactone-type SL Lactone-form SLs are also susceptible to (alkaline) hydrolysis to form acidic SLs. However, lactone-form SLs (wild-type) produced by fermentation are usually doubly acetylated. It is preferable to remove acetate and base ions after hydrolysis. By combining hydrolysis and adsorption, non-acetylated acidic SLs can be produced and purified directly from pure product or cell-free fermentation broth containing a mixture of acetylated acidic SLs and lactone-form SLs.

[0119] Hydrolysis tests were performed on a laboratory scale using the same vacuum manifold and methodology as described in Example 1. The difference was that one bed volume of 0.1 M NaOH solution was added to the column between the loading and rinsing steps. The hydrolysis reaction was carried out overnight at 50°C in a thermomixer. After 24 hours, the acetate and salt-containing solution was removed by applying a vacuum. After rinsing with RO water, the non-acetylated acidic form of SL was eluted with 70% isopropanol. Complete conversion was observed under these conditions.

[0120] 3) Removal of acetylation from glycolipids by chemical (partial) hydrolysis Acetylation can dramatically affect the properties of glycolipids. They can be (partially) removed by (alkaline) hydrolysis. Preferably, acetate and base ions are removed after hydrolysis. By combining hydrolysis and adsorption, glycolipids can be (partially) deacetylated on a column and purified directly from the pure product or cell-free fermentation broth containing acetylated glycolipids.

[0121] Acetylated bora-type SS The genetically engineered Starmerella bombicola strain used to produce acetylated bora-type SS also produces small amounts of bora-type SL. As described above in Example 7, alkaline hydrolysis can be applied on a column to convert the bora-type SL to acid-type SL and sophorose. However, this also removes acetylation, resulting in different products. The bora-type SS can then be separated from the acid-type SL using the method in Example 1.

[0122] Cell-free broth from a 150 L fermentation containing acetylated bolaform SL was hydrolyzed to remove acetylation by adding 30% NaOH solution to a pH of 12. The solution was incubated for 1 h, continuously monitored, and the pH was adjusted to 12, after which the solution was stabilized at pH 4–5 by adding 4.5 M H2SO4.

[0123] The hydrolyzed solution was loaded onto a 30 L column containing polystyrene resin. After rinsing with RO water to a conductivity of less than 50 μS / cm, the SL was eluted with 70% isopropanol to give pure acetylated bolaform SS.

[0124] [Table 4]

[0125] Drawing translation Figure 1 Process unit

Claims

1. A method for isolating and / or purifying a glycolipid (GL) from a glycolipid-containing composition (IM), comprising: A step of supplying an adsorbent (R) to a process device (100), the adsorbent is a polymeric resin; a step of contacting the glycolipid-containing composition with the adsorbent to load a loading substance (LM) onto the polymer resin, The loading substance comprises at least a certain amount of glycolipid (LM1); treating the adsorbent to recover a predetermined desired type of glycolipid (PLM1), The treatment includes contacting the adsorbent with a preselected recovery liquid (RL) to recover the predetermined desired type of glycolipids from the load material on the adsorbent; Obtaining the recovered predetermined desired type of glycolipid (PLM1) from the process equipment; A method comprising:

2. The method comprises: washing the adsorbent with a washing liquid (WL) to separate at least non-loaded materials (NLM) from the adsorbent; The method of claim 1 further comprising:

3. The processing step comprises: contacting the adsorbent with one or more treatment liquids (TL) to modify at least a certain amount of glycolipids (LM1) and / or remove one or more secondary components (NPLM2) from the adsorbent (R); 3. The method of claim 1 or 2, further comprising:

4. The method according to any one of claims 1 to 3, wherein the one or more treatment liquids (TL) are selected from polar solvents, non-polar solvents, alkaline solvents, acidic solvents, neutral solvents, or combinations thereof.

5. The one or more treatment liquids (TL) may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, hexane, heptane, ethyl acetate, KOH, NaOH, NH 4 5. The method of claim 3 or 4, wherein the oxidizing agent is selected from OH, water, RO water, or a combination thereof.

6. 6. The method of any one of claims 1 to 5, wherein the preselected recovery liquid (RL) is selected from the group comprising ionic liquids, liquid carbon dioxide, supercritical solvents, ethyl acetate, methanol, isopropanol, acetone, ethanol, heptane, tert-butyl methyl ether, diethyl ether, acetonitrile, phenoxyethanol, benzyl alcohol, phenethyl alcohol, hydrocinnamyl alcohol, tetrahydrofurfuryl alcohol, dimethyl isosorbide, methyl salicylate, eugenol, linalool, hexanol, glacial acetic acid, dimethyl carbonate, certain glycol ethers such as dipropylene glycol methyl ether and 1-propoxy 2-propanol, and lactate esters including ethyl lactate, butyl lactate, amyl lactate, ethylhexyl lactate, or combinations thereof.

7. 7. The method of any one of claims 1 to 6, wherein the glycolipid (GL) is selected from the group comprising glycosylated fatty acids, glycosylated fatty alcohols, glycosylated carotenoids, glycosylated hopanoids, glycosylated sterols, glycosylated paraconates, glyceroglycolipids, glycosphingolipids, lipopolysaccharides, phenolic glycolipids, glycopeptide lipids, and nucleoside lipids.

8. 8. The method according to any one of claims 1 to 7, wherein the glycolipid (GL) is selected from the group comprising sophorolipids, rhamnolipids, cellobiose lipids, xylolipids, trehalose lipids, mannosylerythritol lipids, glucolipids, fatty alcohol glucosides, alkyl polyglucosides, alkyl sophoroside, (anionic) alkyl glucosides, (anionic) alkyl pentosides, sucrose esters, sorbitan esters, methyl glucoside esters, fatty acid methyl glucamides, oligosaccharide fatty alcohols.

9. The method according to any one of claims 1 to 8, wherein the glycolipid (GL) is selected from acidic sophorolipid (ASL) and lactone sophorolipid (LSL) in acetylated or non-acetylated form.

10. 10. The method of any one of claims 1 to 9, wherein the glycolipid (GL) is selected from acetylated or non-acetylated bolasophorolipids (BSL), bolasophoroside (BSS), alkylsophoroside (ASS), alcohol glucoside (AGS), sucrose ester (SE), bolaglycoside (BGS), alkyl glucoside (ALGS), glucolipid, or a combination thereof.

11. The processing step comprises: contacting the adsorbent (R) with a treatment solution (TS2) containing a treatment solution having a first concentration c1; Contacting the adsorbent (R) with a recovery solution (RS2) containing a recovery solution having a second concentration c2; where: The first concentration c1 is a concentration such that essentially all of the glycolipid (LM1) in the load material is not removed from the adsorbent; and The method of any one of claims 3 to 10, wherein the second concentration c2 is a concentration such that essentially all of the glycolipid (LM1) in the load material is removed from the adsorbent.

12. 12. The method according to any one of claims 1 to 11, wherein both the treatment liquid (TL2) and the recovery liquid (RL2) are one of a polar solvent, a non-polar solvent, an alkaline solvent, an acidic solvent, a neutral solvent, or a combination thereof.

13. 13. Use of the method according to claim 10 or 12 for removing a first fraction (F1) of one or more loaded and / or unloaded secondary components (NPLM2) from the adsorbent (R), wherein the first fraction is selected from impurities such as proteins, pigments, etc.

14. The processing step comprises: contacting the adsorbent (R) with a first treatment liquid (TL1), preferably an alkaline reagent, to at least partially modify the glycolipids (GL) loaded on the adsorbent; The method according to any one of claims 3 to 12, comprising:

15. 15. Use of the method according to claim 14 for converting acetylated glycolipids into non-acetylated glycolipids.

16. Use of the method according to claim 14 for converting acetylated lactone-type sophorolipid and / or non-acetylated lactone-type sophorolipid (LSL) into acetylated acidic sophorolipid and / or non-acetylated acidic sophorolipid (ASL).

17. Use of the method according to claim 14 for converting acetylated bolasophorolipids and / or non-acetylated bolasophorolipids (BSL) into acetylated acidic sophorolipids and / or non-acetylated acidic sophorolipids (ASL) and acetylated sophorose and / or non-acetylated sophorose.

18. The processing step comprises: contacting the adsorbent (R) with a second treatment liquid (TL2), preferably a non-polar solvent, more preferably a non-polar organic solvent, to remove a second fraction (F2) of the one or more secondary components from the load material; where:

15. The method according to any one of claims 1 to 12 and 14, wherein the second fraction is selected from non-polar components such as free fatty acids, fatty alcohols, triacylglycerides, dicarboxylic acids, diols, alkanes, and / or oils.

19. 19. The method according to any one of claims 1 to 12, 14 and 18, wherein the second treatment liquid (TL2) is selected from hexane, heptane or methyl tert-butyl ether.

20. The processing step comprises: contacting the adsorbent (R) with a third treatment liquid (TL3), preferably a polar solvent, to remove a third fraction (F3) of the one or more secondary components from the load material; where:

20. The method according to any one of claims 1 to 12, 14, 18 and 19, wherein the third fraction is selected from hydrophilic impurities such as sugars, proteins, peptides, polyols, organic acids, inorganic acids, carbohydrates, salts, etc.

21. 21. The method according to any one of claims 1 to 12, 14 and 18 to 20, wherein the polymer resin is selected from the group comprising polymethacrylate resin, acrylic resin, polystyrene resin or a combination thereof, in particular selected from styrene-divinylbenzene, polymethacrylate, chemically brominated polystyrene, acrylic ester, polystyrene, cross-linked polystyrene, methacrylic, porous polystyrene-divinylbenzene, styrene-divinylbenzene, styrene-divinylbenzene, acrylic ester, polymethacrylate, chemically brominated polystyrene.

22. The method of any one of claims 1 to 12, 14 and 18 to 21, wherein the polymer resin is a neutral resin.

23. 23. The method according to claim 2 and any one of claims 3 to 12, 14 and 18 to 22, wherein the process equipment comprises an input stream (S1) and an output stream (S2), and the cleaning step is performed by introducing the cleaning liquid (WL) into the process equipment via the input stream until parameters of the output stream are within a predetermined range of parameters of the input stream.

24. 24. The method according to claim 3 and any one of claims 4 to 12, 14 and 18 to 23, wherein the process equipment comprises an input stream (S1) and an output stream (S2), and the treating step is carried out by introducing the treatment liquid (TL) into the process equipment via the input stream until parameters of the output stream are within a predetermined range of parameters of the input stream.

25. 25. The method of claim 23 or 24, wherein the parameter is selected from conductivity, refractive index, pH, protein content, sugar content, test swab value, or a combination thereof.

26. evaporating the recovery liquid (RL); evaporating said treatment liquid (TL), if present; The method of any one of claims 1 to 12, 14 and 18 to 25, further comprising:

27. recirculating at least one of the recovery liquid (RL), the processing liquid (TL), and optionally the cleaning liquid (WL); The method of any one of claims 1 to 12, 14 and 18 to 26, further comprising:

28. 28. The method according to any one of claims 1 to 12, 14 and 18 to 27, wherein the glycolipid-containing composition is a plant extract or an end product of a fermentative, enzymatic or chemical production process.

29. 29. The method according to any one of claims 1 to 12, 14 and 18 to 28, wherein the method is carried out in combination with a fermentation process, preferably in combination with a fermentation process in an in-situ product recovery facility.

30. 30. The method of any one of claims 1 to 12, 14 and 18 to 29, wherein the method is carried out in a batch, continuous or semi-continuous manner.

31. The method according to any one of claims 1 to 12, 14 and 18 to 30, wherein the glycolipid-containing composition is passed through a reactor containing the adsorbent.

32. The method according to any one of claims 1 to 12, 14 and 18 to 31, wherein the glycolipid-containing composition is passed through a column packed with the polymer adsorbent.

Images