Ether sophorolipid with stable pH value and temperature and application thereof

Abstract

The present invention provides modified sophorolipids of formula I, which have a higher pH and temperature stability than a lactone counterpart thereof. Furthermore, the compounds have a lower HLB parameter, as well as higher wetting and solubilizing parameters, water-in-oil emulsifying ability, surface tension reducing activity, and / or antibacterial activity compared to its acidic or even lactone counterpart. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 350,093, filed on June 8, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Sophorolipids are classified as glycolipid biosurfactants because they are fermented by microorganisms (e.g., Starmerella bombicola) using carbohydrates and lipids as carbon sources.

[0004] Sophorolipids are amphiphilic molecules containing a hydrophilic carbohydrate group (sophorose) and a hydrophobic fatty acid. Sophorose is a disaccharide consisting of two glucose molecules linked by a β-1,2 bond. The sophorose in sophorolipids can be acetylated, especially at the 6' and / or 6" positions. The fatty acids in sophorolipids are glycosidically linked to the sophorose molecule via the hydroxyl groups and their terminal carboxylic acid groups are either free (e.g. Figure 1 ), or are usually internally esterified at the 4" position (i.e. Figure 2 The lactone form shown). The fatty acid of the sophorolipid usually contains 16 or 18 carbon atoms and one degree of unsaturation. However, it can be composed of chain lengths of C12 to C22 with different degrees of unsaturation and positions of unsaturation.

[0005] Sophorolipids are often produced as mixtures of related molecules. The differences between related molecules mainly stem from the following: fatty acid structure (unsaturation, chain length, position of unsaturation and position of hydroxylation); whether it is produced in free form or lactone form; acetylation pattern; the presence of stereoisomers; and / or whether the glycosidic bond on the fatty acid is at the ω position (terminal) or the ω1 position (subterminal).

[0006] Sophorolipids have many favorable properties that make them superior to synthetic surfactants, such as biodegradability, low toxicity, high surface activity and interfacial activity, and stability over a wide range of temperatures, pressures, and ionic strengths. Therefore, sophorolipids are reported to have a wide range of uses in food preservation, biomedicine, cosmetics, bioremediation, heavy metal remediation, cement slurries, and agricultural and livestock production.

[0007] On the other hand, the functional properties of sophorolipids may differ between the lactone form and the free form (also called "acidic" or "linear"). For example, acidic sophorolipids generally have a higher hydrophilic-lipophilic balance (HLB) than lactone sophorolipids, while lactone sophorolipids generally have a lower HLB and greater surface tension reducing properties than acidic sophorolipids. In addition, acidic sophorolipids are generally highly water-soluble due to their free carboxylic acid groups. Combining lactone sophorolipids and acidic sophorolipids in different ratios can affect properties such as emulsion droplet size, viscosity reducing properties, and surface / interfacial tension reducing properties. In other cases, certain uses may only require lactone sophorolipids due to their properties.

[0008] One problem associated with lactone sophorolipids is their sensitivity to the surrounding environment. Specifically, lactone sophorolipids are prone to ring opening, thereby converting to an acidic form depending on the surrounding environment (e.g., temperature and pH). For example, changes in the pH value of the environment outside the range of about 4 to 7 may cause the ester bond of the lactone ring to hydrolyze, thereby increasing the relative proportion of acidic sophorolipids in the mixture.

[0009] Therefore, there is a need for lactone sophorolipids or their equivalents with higher stability. Such improvements will provide better control and predictability of the properties of a given sophorolipid sample, making quality control easier in different fields where sophorolipids are used. In addition, sophorolipids with higher stability will expand their potential use in various other applications that were previously considered impossible because the environment of the lactone sophorolipid is or may be unstable. Summary of the invention

[0010] One aspect of the present invention provides a modified sophorolipid of formula I:

[0011]

[0012] in:

[0013] R1 and R2 are each independently hydrogen, ethyl (—CH2CH3) or acetyl (—COCH3);

[0014] R3 is hydrogen or methyl; and

[0015] A is an optionally substituted saturated fatty chain or an unsaturated fatty chain.

[0016] In some embodiments, the fatty chain A has 10 to 21 carbons, such that the total number of carbons to which the fatty chain A, R3, and R3 are attached is 12 to 22 carbons.

[0017] The present invention includes all compounds represented by the general formula I, including their hydrates, geometric isomers, optical isomers and polymorphs.

[0018] Compared with its lactone counterpart, compound according to the present invention is more stable in pH value and temperature. In addition, compared with its acidity or even lactone counterpart, formula I compound has reduced HLB parameter, and improved wetting and solubilizing parameter, water-in-oil emulsifying ability, surface tension reducing activity and / or antimicrobial activity. Therefore, this compound has a wide range of uses in multiple industries, including but not limited to food, bioremediation, oil, agriculture, animal husbandry and aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 An exemplary general structure of a sophorolipid in free acid form is shown, wherein: Y is hydrogen or methyl; R and R' are each independently hydrogen or acetyl; R c It refers to a saturated or unsaturated aliphatic hydrocarbon chain usually about C10 to C20 in length, with varying degrees of unsaturation and positions of unsaturation.

[0020] Figure 2 An exemplary general structure of a lactone sophorolipid is shown, wherein Y, R, R' and R c like Figure 1 shown.

[0021] Figure 3 An exemplary reaction scheme for the reduction of a lactone sophorolipid (SLP) to obtain a modified sophorolipid according to the present invention is shown. DETAILED DESCRIPTION

[0022] Modified Sophorolipids

[0023] In one aspect, the present invention provides a modified sophorolipid of formula I, which is a cyclic ether analog of a lactone sophorolipid.

[0024] The present invention provides a compound of formula I:

[0025]

[0026] in:

[0027] R1 and R2 are each independently hydrogen, ethyl (—CH2CH3) or acetyl (—COCH3);

[0028] R3 is hydrogen or methyl (—CH3); and

[0029] A is an optionally substituted saturated fatty chain or an unsaturated fatty chain.

[0030] In a preferred embodiment, the fatty chain A has 10 to 21 carbons, so that the total of the fatty chain A, R3, and the carbon to which R3 is attached (ring chain) is 12 to 22 carbons.

[0031] In some embodiments, the total number of carbons in the ring chain is 18 carbons. The ring chain can be a saturated ring chain or an unsaturated ring chain. In a specific embodiment, the unsaturated ring chain has a C=C bond at position 8 or 9 and can be a cis isomer or a trans isomer.

[0032] Alternatively, the aliphatic chain is substituted. For example, the substitution at carbon position 8 or 9 in the ring chain can be achieved by olefin chemistry. Substituents include halogen atoms, hydroxyl, low (C1-C6) alkyl, halo low (C1-C6) alkyl, hydroxyl low (C1-C6) alkyl, halo low (C1-C6) alkoxyl and the like. Preferred halogen atoms or halogen atoms combined with alkyl or alkoxyl groups include fluorine, chlorine, bromine and iodine. When determining the total number of carbons in the ring chain, the number of carbons in the substituent is not considered.

[0033] According to some embodiments, R1 and R2 are both ethyl, R3 is methyl, and A is an unsaturated fatty chain having 16 carbon atoms and one, two or three degrees of unsaturation.

[0034] According to some embodiments, R1 and R2 are both ethyl, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbons and one, two, or three degrees of unsaturation.

[0035] According to some embodiments, R1 is ethyl, R2 is hydrogen, R3 is methyl, and A is an unsaturated fatty chain having 16 carbon atoms and one, two or three degrees of unsaturation.

[0036] According to some embodiments, R1 is ethyl, R2 is hydrogen, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbons and one, two or three degrees of unsaturation.

[0037] According to some embodiments, R1 is hydrogen, R2 is ethyl, R3 is methyl, and A is an unsaturated fatty chain having 16 carbon atoms and one, two or three degrees of unsaturation.

[0038] According to some embodiments, R1 is hydrogen, R2 is ethyl, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbon atoms and one, two or three degrees of unsaturation.

[0039] According to some embodiments, R1 and R2 are both hydrogen, R3 is methyl, and A is an unsaturated fatty chain having 16 carbons and one, two, or three degrees of unsaturation.

[0040] According to some embodiments, R1 and R2 are both hydrogen, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbons and one, two, or three degrees of unsaturation.

[0041] According to other embodiments, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is methyl, and A is an unsaturated fatty chain having 16 carbon atoms and one degree of unsaturation.

[0042] According to other embodiments, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbon atoms and one degree of unsaturation.

[0043] According to a further embodiment, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is methyl, and A is an unsaturated fatty chain having 16 carbon atoms and two degrees of unsaturation.

[0044] According to a further embodiment, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbon atoms and two degrees of unsaturation.

[0045] According to a further embodiment, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is methyl, and A is an unsaturated fatty chain having 16 carbon atoms and three degrees of unsaturation.

[0046] According to a further embodiment, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is hydrogen, and A is an unsaturated fatty chain having 17 carbon atoms and three degrees of unsaturation.

[0047] According to other embodiments, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is methyl, and A is a fully saturated fatty chain having 16 carbons.

[0048] According to other embodiments, R1 and R2 are each independently hydrogen, ethyl or acetyl, R3 is hydrogen, and A is a fully saturated fatty chain having 17 carbons.

[0049] The present invention covers all compounds represented by the general formula I, including hydrates, geometric isomers and optical isomers thereof and polymorphs thereof. Preferably, the compound of the general formula I is derived from a lactone sophorolipid. In some embodiments, the lactone sophorolipid is produced by fermentation of a sophorolipid-producing organism.

[0050] The cyclic ether structure reduces the pH instability and temperature sensitivity associated with the corresponding lactone sophorolipids. The compounds of the present invention are stable at pH values ​​of about 3 to about 14 and / or temperatures up to about 100° C. In addition, the HLB values ​​of the compounds of formula I are lower than those of the lactone counterparts. This can improve wetting and solubilization parameters, oil-in-water emulsification ability, surface tension reduction activity and / or antibacterial activity compared to the acidic or lactone counterparts.

[0051] Sophorolipids

[0052] In some embodiments, the modified sophorolipids according to the present invention are prepared by chemically modifying sophorolipids. Sophorolipids are usually obtained by fermentation of microorganisms, which use pure fatty acids, fatty acid mixtures, pure fatty acid esters, mixtures of fatty acid esters, triglycerides, and carbohydrate sources such as corn syrup, dextrin and glucose as carbon sources. The microorganism can be a wild-type yeast strain or an engineered yeast strain. Figure 1 As shown, sophorolipids generally include sophorose that may or may not be acetylated at the 6' and 6" positions and a hydroxylated fatty acid tail with varying degrees of unsaturation. Most commonly, the fatty acid consists of 16 or 18 carbon atoms and has at least one degree of unsaturation, but the fatty acid structure varies depending on the carbon source provided during the production process (e.g., degree of unsaturation, chain length) and the microorganism undergoing the fermentation.

[0053] The production of sophorolipids using renewable substrates and different microbial species, as well as the variation of culture parameters (culture time, stirring speed, medium pH and added nutrients), can obtain compounds with different structural characteristics and physical properties. This makes it possible to produce a variety of compounds with different physical, chemical, biochemical and / or biophysical properties.

[0054] The hydrophobic fatty acid tail of the sophorolipid is usually glycosidically linked to the sophorose molecule at the terminal or subterminal end. The fatty acid carboxylic acid groups in the sophorolipid are either free (see Figure 1 , also called the "acidic" or "straight-chain" form), or are usually intramolecularly esterified at the 4'-position (see Figure 2 ). The degree of lactonization of sophorolipids and the degree of acetylation of the sophorose moiety can be influenced, inter alia, by the carbon source added during the production of the sophorolipids. For example, it has been found that sophorolipids derived from rapeseed oil, sunflower oil and palm oil rich in C18:0 and C18:1 fatty acids contain higher levels of diacetylated lactones than sophorolipids produced from the corresponding fatty acid ester feedstocks.

[0055] Sophorolipids have environmental compatibility, high biodegradability, low toxicity, high selectivity and specific activity under a wide range of temperature, pH and salinity conditions.

[0056] Fermentation

[0057] In some embodiments, the modified sophorolipid according to the present invention is prepared by chemically modifying a sophorolipid, which is produced by a fermentation process known in the art in the presence of selected fatty acids and carbohydrate raw materials. As used herein, "fermentation" refers to the growth of cells under controlled conditions. Growth can be aerobic growth or anaerobic growth. Fermentation methods known in the art include, but are not limited to, solid-state fermentation, submerged fermentation, or modifications, hybridizations, and / or combinations thereof.

[0058] The sophorolipids of the present invention can be derived from a strain of a naturally occurring sophorolipid by a fermentation process of a recombinant organism. Non-limiting examples of organisms producing sophorolipids include Candida bombicola, Candida apicola, Candida bogoriensis, Yarrowia lipolytica, Starmerella bombicola, Starmerella clade, Rhodotorula bogoriensis, Wickerhamiella domercqiae, and Wickerhamomyces anomalus. It is reported that some recombinant sophorolipid-producing microorganisms are able to control the structure of sophorolipids. As a non-limiting example, certain recombinant Bombus spp. strains can be used to produce only lactone sophorolipids or only acidic sophorolipids (see, e.g., Roelants et al., Towards the Industrialization of New Biosurfactants: Biotechnological Opportunities for the Lactone Esterase Gene from Starmerella Bombicola, Biotechnology and Bioengineering, Vol. 113, pp. 550 to 559 (2015)). As another non-limiting example, recombinant Bombus spp. strains with acetyltransferase gene knockout can be used to produce sophorolipids that are not acetylated (see WO 2012 / 080116).

[0059] The microorganisms of interest can be cultured and utilized on a small or large scale on site, even while still mixed with their culture medium. The sophorolipids of the present invention can also be mass-produced at the desired site. The growth vessel used to culture the organisms that produce sophorolipids can be any fermenter or culture reactor for industrial use.

[0060] In one embodiment, a single type of microorganism is grown in a reactor system. In other embodiments, multiple microorganisms can be grown in a single reactor system, and these microorganisms can be grown together without adversely affecting growth or the resulting products. For example, 2 to 3 or more different microorganisms can be grown simultaneously in a single reactor. In some embodiments, more than one microorganism is grown symbiotically in a reactor.

[0061] In one embodiment, the vessel may have functional controls / sensors, or may be connected to functional controls / sensors to measure important factors in the culture process, such as pH, oxygen, pressure, temperature, stir shaft power, humidity, viscosity and / or microbial density and / or metabolite concentration, etc.

[0062] Carbon sources may be supplemented during cultivation. Carbon sources are usually carbohydrates, such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol and / or maltose (preferably glucose); organic acids, such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid and / or pyruvic acid; alcohols, such as ethanol, isopropanol, propanol, butanol, pentanol, hexanol, isobutanol and / or glycerol; fats and oils, such as soybean oil, rice bran oil, rapeseed oil, olive oil, corn oil, sesame oil and / or linseed oil. These carbon sources may be used alone or in combination of two or more.

[0063] A nitrogen source may also be supplemented during cultivation. The nitrogen source may be potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and / or ammonium chloride, etc. These nitrogen sources may be used alone or in combination of two or more.

[0064] The growing culture in culture may also be oxygenated. One embodiment utilizes slow movement of air to remove air low in oxygen content and introduce oxygenated air. In the case of submerged fermentation, the oxygenated air may be ambient air that is replenished daily by a variety of mechanisms including impellers for mechanically agitating the liquid and air distributors for supplying gas bubbles to the liquid to dissolve oxygen into the liquid.

[0065] In one embodiment, the microorganisms may be grown on a solid or semisolid substrate, such as corn, wheat, soybeans, chickpeas, soy, oatmeal, pasta, rice, and / or flours or meals of these or other similar materials.

[0066] In one embodiment, the growth factors and micronutrients of the microorganisms are included in the culture medium. This is particularly preferred when the growing microorganisms cannot generate all the vitamins they need. Inorganic nutrients may also be included in the culture medium, and the inorganic nutrients include trace elements such as iron, zinc, copper, manganese, molybdenum and / or cobalt. In addition, the source of vitamins, essential amino acids and trace elements can be included in the culture medium in the form of fine powder or coarse powder (for example, corn flour) or in the form of extracts (for example, yeast extract, potato extract, beef extract, soybean extract, banana peel extract, etc.) or in purified form. Amino acids (for example, amino acids that can be used for protein biosynthesis) may also be included.

[0067] In one embodiment, the culture medium may also include an inorganic salt. Available inorganic salts may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate, sodium chloride and / or sodium carbonate. These inorganic salts may be used alone or in a combination of two or more.

[0068] In some embodiments, for example, when the microorganism used to inoculate the substrate is in spore form (e.g., bacterial endospores), a germination promoter may be added to the substrate. Examples of germination promoters according to the present invention include, but are not limited to, L-alanine, manganese, L-valine, and L-asparagine or any other known germination promoters.

[0069] In some embodiments, additional acids and / or antimicrobials may be added to the liquid culture medium before and / or during the culture process. Antimicrobials or antibiotics are used to protect the culture from contamination. In addition, defoamers may be added to prevent the formation and / or accumulation of foam when gas is generated during the culture process.

[0070] The pH of the mixture should be appropriate for the microorganism of interest. Buffers and pH adjusters (e.g., carbonates and phosphates) can be used to stabilize the pH near the preferred value. When metal ions are present in high concentrations, it may be necessary to use chelating agents in the liquid culture medium.

[0071] The methods and apparatus for the cultivation of microorganisms and the production of microbial byproducts may be performed as a batch, quasi-continuous or continuous process.

[0072] In one embodiment, the cultivation of the microorganisms is carried out at about 5° to about 100° C., preferably 15° C. to 60° C., more preferably 25° C. to 50° C. In another embodiment, the cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subjected to temperature changes.

[0073] In one embodiment, the equipment used in the method and the culture process is a sterile device. The culture equipment (e.g., reactor / vessel) can be separated from a sterilizing device (e.g., autoclave), but connected thereto. The culture equipment can also have a sterilizing device that sterilizes on the spot before starting inoculation. Air can be sterilized by methods known in the art. For example, ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the culture medium can be pasteurized, or alternatively not heated at all, but low water activity and low pH value can be used to control the growth of undesirable bacteria.

[0074] In one embodiment, all of the microbial culture composition is removed when the culture is complete (eg, when a desired cell density or density of a particular metabolite is reached). In this batch process, a completely new batch is started when the first batch is harvested.

[0075] In another embodiment, only a portion of the fermentation product is removed at any one time. In this embodiment, the biomass with living cells remains in the vessel as an inoculant for a new culture batch. The removed composition can be a culture medium without microorganisms or a composition containing cells, spores, mycelium, conidia or other microbial propagules. A quasi-continuous system is created in this way.

[0076] In some embodiments, the sophorolipids produced by the microorganisms of interest can be retained in the microorganisms or secreted into their growth medium. The content of the sophorolipids can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, etc.

[0077] The growth medium may contain compounds that stabilize the activity of the sophorolipids.The sophorolipids may be purified or the sophorolipids may be used in crude form, meaning that they are not separated from the fermentation broth in which they are produced.

[0078] In certain embodiments, the sophorolipid is isolated and / or purified from a growth medium produced by a microbial fermentation that produces a biosurfactant. Isolation and purification can be easily achieved using standard methods or techniques described in the literature. If necessary, the sophorolipid can be further concentrated.

[0079] As used herein, the terms "isolated," "purified," or variations thereof, when used in connection with biological or natural materials such as glycolipids, mean that the material is substantially free of other compounds, such as cellular material, with which it is associated in nature. That is, the material would not naturally exist without these other compounds and / or has different or unique characteristics compared to those found in the natural material.

[0080] In some embodiments, the purified compound accounts for at least 60% of the compound of interest by weight. Preferably, the preparation is at least 75%, more preferably at least 90%, most preferably at least 99% (w / w) of the desired compound. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100% (w / w) of the desired compound by weight. Purity is measured by any appropriate standard method such as column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC) analytical method.

[0081] The ranges provided herein are understood to be shorthand expressions of all numerical values ​​within the range. For example, a range of 1 to 20 may be understood to include any number, combination of numbers, or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, as well as all decimal values ​​between the above integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With regard to subranges, "nested subranges" extending from either end of the range are particularly contemplated. For example, a nested subrange of an exemplary range of 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in another direction.

[0082] Modification method

[0083] In some embodiments, the modified sophorolipids of the present invention are obtained by modifying a sophorolipid obtained from a microbial fermentation product that produces sophorolipids by applying a synthetic method known in the art. The sophorolipid to be chemically modified can be obtained by the fermentation method and process described above and / or any other method known in the art. As described above, the sophorolipids can be purified from the growth medium using known purification methods. The sophorolipid to be modified can be obtained in a substantially lactone form, a substantially acidic form, or a mixture of a lactone form and an acidic form. As used herein, a lactone sophorolipid includes any isomer of a sophorolipid containing a cyclic ester.

[0084] In some embodiments, the starting material of the modified sophorolipid according to the present invention may be a pure lactone sophorolipid substantially free of acidic sophorolipids. Advantageously, the use of pure lactone sophorolipids as the starting material for chemical transformation can reduce potential side reactions and allow the reaction product to be obtained as a pure cyclic ether of the general formula I substantially free of acyclic counterparts. Pure lactone sophorolipids can be obtained directly as a purified product of fermentation, or by lactonization of a fermentation product containing lactone sophorolipids and acidic sophorolipids using chemical methods known in the art, thereby obtaining pure lactone sophorolipids. In other embodiments, lactone sophorolipids can be separated from a mixture of lactone sophorolipids and acidic sophorolipids obtained by fermentation. This can be achieved, for example, by using standard liquid-liquid extraction techniques that take advantage of the differences between the chemical properties of lactone sophorolipids and acidic sophorolipids. That is, acidic sophorolipids contain carboxylic acid functional groups, while lactone sophorolipids do not contain easily ionized groups.

[0085] In other embodiments, the lactone sophorolipid to be modified is not separated from the mixture of lactic acid form and acidic form prior to modification. Advantageously, this method eliminates the need to separate the lactone sophorolipid from the acidic sophorolipid prior to modification. The modification of the lactone sophorolipid can be carried out by adding the necessary reducing agent to the mixture of the lactone sophorolipid and the acidic sophorolipid after extraction into the selected organic solvent.

[0086] The modification of the lactone sophorolipid according to the present invention involves the selective reduction of at least the carbonyl group (C=O) of the fatty acid in the sophorolipid to an alkyl group (—CH2—), rather than reducing other types of functional groups present in the molecule to obtain a cyclic ether compound of formula I. A non-limiting exemplary scheme of the reduction of the lactone sophorolipid is shown below:

[0087]

[0088] Wherein Ra and Rb are each independently hydrogen or acetyl. In addition, A' is a saturated fatty chain or unsaturated fatty chain whose chain length is one carbon shorter than A. A' may be optionally substituted. If acetyl is present at Ra and / or Rb, it may also be reduced to form an ether bond with the adjacent oxygen due to the reduction process. Therefore, R1 and R2 may be hydrogen, acetyl or ethyl.

[0089] According to some embodiments of the present invention, the lactone sophorolipid is reduced by using a reducing agent and a Lewis acid catalyst in an organic solvent to obtain a compound of formula I. The reaction can be carried out at any suitable temperature. In some embodiments, the reaction is carried out at about 60°C.

[0090] The reducing agent according to the present invention is any suitable reagent capable of reducing the carbonyl group in the lactone sophorolipid. Suitable reducing agents include, but are not limited to, hydrosilane sources such as triethylsilane (Et3SiH), tris(trimethylsilyl)silane (TTMS), tetraphenyldisilane (TPDS) and / or polymethylhydrosilane (PHMS).

[0091] The Lewis acid catalyst according to the present invention can be any acid that is compatible with a reducing agent to catalyze the reduction of a carbonyl group in a lactone sophorolipid. Suitable Lewis acid catalysts include, but are not limited to, indium bromide (InBr3), bismuth bromide (BiBr3), titanium alkoxides (e.g., titanium isopropoxide), boron tribromide (BBr3), aluminum bromide (AlBr3), and zinc bromide (Zn(II)Br2). In some embodiments, the Lewis acid catalyst is indium bromide. The Lewis acid catalyst can be present in a concentration of about 1 mol% to about 25 mol%.

[0092] The organic solvent according to the present invention can be any non-polar organic solvent that can be used as a reaction medium and is suitable for reducing the lactone sophorolipid. Suitable organic solvents include, but are not limited to, chloroform (CHCl3), dichloromethane, toluene, 1,4-dioxane, dibromomethane and bromoform. Preferably, the organic solvent is chloroform.

[0093] Figure 3 A non-limiting exemplary reaction scheme according to the present invention is shown. The starting material is a lactone sophorolipid provided in pure form or in the form of a mixture comprising a lactone sophorolipid and an acidic sophorolipid. Reduction is carried out at 60° C. using a reagent combination of Et3SiH and InBr3 in CHCl3. The compound marked as "4" is a modified sophorolipid of the present invention.

[0094] Fatty Acid Chain

[0095] The fatty chain A according to the present invention can be of any length as long as the length is suitable for forming an intramolecular ring with the sophorose residue to form a compound of formula I. Preferably, the total number of carbon atoms in the carbon to which the fatty chain A, R3 and R3 are attached (the "ring chain") is 12 to 22 carbons. In certain embodiments, the fatty chain A has 10 to 21 carbons. More preferably, the fatty chain A has 14 and 17 carbons, so that the total number of carbon atoms in the ring chain is 16 to 18. In some embodiments, the total number of carbon atoms in the ring chain is 18.

[0096] In addition, if the fatty chain A is an unsaturated fatty chain A, one or more double bonds and / or triple bonds may be present in the chain, as long as the degree of unsaturation does not make the stereo formation of the intramolecular ring impossible. In the case where the fatty chain A has at least one double bond, the geometric isomers of the olefin are fully within the scope of the present invention. That is, if the fatty chain A has at least one degree of unsaturation, the compound of formula I encompasses cis-isomers and trans-isomers.

[0097] In addition, the degree and / or position of unsaturation in fatty chain A and the desired isomerism can be artificially introduced or inherent in the fatty acid derived from fatty chain A. In some embodiments, the unsaturation in the ring chain is removed by known chemical processes. In addition, substituents can be introduced at the unsaturation (e.g., double bond) in the ring chain by synthetic methods known in the art. In some embodiments, the degree and position of unsaturation in fatty chain A are inherent to the corresponding fatty acid. For example, if fatty chain A is derived from oleic acid (18:1 cis-9) or elaidic acid (18:1-trans-9), it retains the corresponding isomerism (i.e., cis-9 or trans-9, respectively) at the same position.

[0098] Non-limiting examples of fatty acids from which fatty chain A can be derived include caprylic acid, capric acid, lauric acid, stearic acid, arachidic acid, eicosanoic acid, behenic acid, tetracosanoic acid, hexacosanoic acid, myristoleic acid, palmitic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, and arachidonic acid. A preferred fatty acid is oleic acid.

[0099] Industrial Applicability

[0100] Other aspects of the invention relate to the use of compounds represented by formula I in various applications. The compounds can be used not only in areas where conventional sophorolipids are used or can be used, but also in various other areas where their use is considered infeasible due to the unstable environment of lactone sophorolipids. Since the cyclic ether structure of the compound of formula I is more stable than its lactone counterpart in pH and temperature, some embodiments of the invention replace or supplement conventional sophorolipids with compounds of formula I, especially when the general properties of lactone sophorolipids (except for their stability issues) are desired or preferred. In some embodiments, the HLB value of the compound of formula I is lower than that of its counterpart, the starting compound of formula I'.

[0101] In other embodiments, the compounds of formula I are used in areas where it is desirable or necessary to reduce the HLB value to improve wetting and solubilization parameters and / or improve water-in-oil emulsification. In further embodiments, the compounds of formula I can be used where it is desirable or necessary to enhance surface tension reduction activity and / or antimicrobial activity.

[0102] Exemplary applications of compounds of formula I include, but are not limited to, use as food additives or supplements, active pharmaceutical ingredients or adjuvants, cosmetic ingredients, and ingredients in cleaning products. Compounds of formula I can also be used for bioremediation and remediation of heavy metals, as well as for drilling, cement slurry, fracturing, enhanced oil recovery, scale prevention, acidification, crude oil demulsification, corrosion inhibition, oil viscosity reduction, equipment cleaning, water injection and / or foam and steam flooding and other oil industries. In addition, in agriculture, aquaculture and animal husbandry production, compounds of formula I can be used as soil conditioners; broad-spectrum biopesticides, antiviral, antifungal and antibacterial drugs; feed additives, such as additives for enhancing nutrient absorption or feed preservation; nutritional sources; and / or therapeutic agents for plant, fish and livestock diseases or infections. Compounds of formula I can also be used in the field of the environment, for example, for reducing greenhouse gases.

[0103] The transitional term "comprising," which is synonymous with "including" or "containing," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic, novel property," such as the ability to increase the bioavailability of a substance. Use of the term "comprising" refers to other embodiments where the recited components are "consisting of" or "consisting essentially of."

[0104] Unless otherwise specified or obvious from the context, the term "or" as used herein should be understood to be inclusive. Unless otherwise specified or obvious from the context, the terms "a", "an", and "the" as used herein should be understood to be singular or plural.

[0105] Unless otherwise specified or apparent from the context, as used herein, the term "about" should be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. As a further example, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the specified value.

[0106] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of the variable as any single group or combination of the listed groups. The recitation of an embodiment for a variable or aspect herein includes the embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0107] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0108] Example

[0109] The following are examples of the present invention, which are provided by way of illustration and are not intended to limit the present invention. Those skilled in the art will suggest and understand various modifications or changes within the spirit and scope of the present application.

[0110] Example 1: Exemplary compounds

[0111] The numbers in the leftmost column represent the compound numbers. The present invention covers all compounds represented by the general formula I, including their hydrates, geometric isomers and optical isomers and polymorphs thereof.

[0112]

[0113]

[0114]

[0115] Example 2: Production of lactone sophorolipids

[0116] preparation

[0117] A stainless steel fermentation reactor was used to produce sophorolipids. The reactor contained about 150 gallons of water, to which a culture medium containing dextrose (25 to 150 g / L), yeast extract (1 to 10 g / L), rapeseed oil (25 ml / L to 110 ml / L), and urea (0.5 to 5 g / L) was added.

[0118] The reactor included a mixing device for continuous stirring and mixing of the culture. The reactor with the culture medium was steamed at 100°C for about 60 minutes to sterilize the reactor and the growth medium.

[0119] The reactor was then allowed to cool. Once the reactor reached approximately 35°C, antibiotics were added to the culture medium to prevent bacterial contamination. Other reactor pipes and openings were sterilized using isopropyl alcohol (IPA).

[0120] The mini-reactor is used to grow the recombinant Candida bombus seed culture. The culture is grown at 26° C. to 28° C. in the mini-reactor for at least 42 hours to 48 hours. The Candida bombus strain produces Candida bombus lactone esterase, which catalyzes the esterification of linear sophorolipids to produce lactone sophorolipids.

[0121] Once the stainless steel fermentation reactor reached 30°C, it was then inoculated with approximately 25 L of inoculum culture.

[0122] Fermentation

[0123] The fermentation temperature was maintained at 23° C. to 28° C. After about 22 to 26 hours, the pH of the culture was set to about 3.0 to 4.0, or about 3.5 using 20% ​​NaOH. The fermentation reactor included a computer that monitored the pH and controlled the pumps used to apply alkali to maintain the pH at 3.5.

[0124] After about 6 to 7 days of cultivation (120 hours + / - 1 hour), if 7.5 ml of the sophorolipid layer is visible, the batch is ready for harvest. In some cases, visible oil and little glucose is detected (e.g., about 0% to 0.5%).

[0125] Harvesting and Purification

[0126] The culture is harvested into a first collection vessel and allowed to stand for 24 to 48 hours. A layer of lactone-rich sophorolipids is deposited at the bottom of the first collection vessel, which is collected and can be purified by liquid-liquid extraction or the like to separate the lactone sophorolipids. After purification, the sophorolipids are ready for further modification.

Claims

1. A compound of formula I: in: R1 and R2 are each independently hydrogen, ethyl or acetyl; R3 is hydrogen or methyl; and A is an optionally substituted saturated fatty chain or an unsaturated fatty chain.

2. The compound of claim 1, wherein the aliphatic chain A has 10 to 21 carbons, excluding substituents, such that the total number of the aliphatic chain A, R3, and the carbon to which R3 is attached is 12 to 22 carbons.

3. The compound according to claim 2, wherein the fatty chain is a saturated fatty chain. The compound according to claim 2 , wherein the fatty chain is an unsaturated fatty chain.

5. The compound according to claim 1, wherein A is an unsaturated fatty chain having 16 or 17 carbon atoms and containing one double bond. The compound according to claim 1 , wherein A is an unsaturated fatty chain having 16 or 17 carbon atoms and containing two double bonds.

7. The compound according to claim 1, wherein A is an unsaturated fatty chain having 16 or 17 carbon atoms and containing three double bonds.

8. The compound according to claim 1, wherein A is further substituted by halogen, hydroxy, lower (C1-C6) alkyl, halo-lower (C1-C6) alkyl, hydroxy-lower (C1-C6) alkyl and / or halo-lower (C1-C6) alkoxy.

9. The compound of claim 1, wherein the compound is synthesized by fermenting sophorolipids produced by an organism that produces sophorolipids using caprylic acid, capric acid, lauric acid, stearic acid, eicosanoic acid, behenic acid, tetracosanoic acid, hexacosanoic acid, myristoleic acid, palmitic acid, hexadecenoic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, trans-linoleic acid, and / or arachidonic acid.

10. The compound of claim 1, which is stable at a pH of about 3 to 14 and / or a temperature up to about 100°C.

11. A method for preparing the compound according to claim 1, the method comprising: In the presence of a reducing agent and a Lewis acid catalyst, reducing at least one carbonyl group adjacent to the fatty chain A' in the compound of formula I': wherein Ra and Rb are each independently hydrogen or acetyl, and A' is a saturated fatty chain or an unsaturated fatty chain having one less carbon than the fatty chain A.

12. The method of claim 11, wherein the fatty chain A has 10 to 21 carbons, excluding substituents, such that the total of the fatty chain A, R3, and the carbon to which R3 is attached is 12 to 22 carbons.

13. The method of claim 12, wherein the fatty chain is a saturated fatty chain.

14. The method of claim 12, wherein the fatty chain is an unsaturated fatty chain.

15. The method of claim 11, wherein A is an unsaturated fatty chain having 16 or 17 carbon atoms and containing one double bond.

16. The method of claim 11, wherein A is an unsaturated fatty chain having 16 or 17 carbon atoms and containing two double bonds.

17. The method of claim 11, wherein A is an unsaturated fatty chain having 16 or 17 carbon atoms and containing three double bonds.

18. The method of claim 11, wherein A is further substituted by halogen, hydroxy, lower (C1-C6) alkyl, halo-lower (C1-C6) alkyl, hydroxy-lower (C1-C6) alkyl and / or halo-lower (C1-C6) alkoxy.

19. The method of claim 11, wherein the compound of formula I has a lower HLB value than the compound of formula I'.

20. A compound:

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