Etheric sophorolipids with pH and temperature stability and their use

Abstract

The present invention provides modified sophorolipids of formula (I) that are more pH and temperature stable than their lactonic counterparts. Further, the compounds have decreased HLB parameters, as well as improved wetting and solubilization parameters, water-in-oil emulsifying ability, surface tension lowering activity and / or antibacterial activity compared to their acidic or even lactonic counterparts. TIFF2025518694000013.tif86128

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 350,093, filed on June 8, 2022, which is incorporated herein by reference in its entirety.

Background Art

[0002] Background of the Invention Sophorolipids are classified as glycolipid biosurfactants because they are produced by the fermentation of microorganisms such as Starmerella bombicola using carbohydrates and lipids as carbon sources.

[0003] 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 via a β - 1,2 bond. The sophorose in sophorolipids can be acetylated, particularly at the 6' and / or 6'' positions. The fatty acid in sophorolipids is glycosidically linked to the sophorose molecule through a hydroxyl group, and its terminal carboxylic acid group is either free (as shown in Figure 1) or, generally at the 4'' position, internally esterified (i.e., in the lactone form as shown in Figure 2). The fatty acids of sophorolipids generally have 16 or 18 carbon atoms and one unsaturated bond. However, it can consist of C12 - C22 chain lengths with various degrees and positions of unsaturation.

[0004] Sophorolipids are often produced as a mixture of related molecules. The differences between related molecules mainly arise from their fatty acid structures (degree of unsaturation, chain length, position of unsaturation, and position of hydroxylation), whether they are produced in the free or lactone form, acetylation patterns, the presence of stereoisomers, and / or whether the glycosidic bond on the fatty acid is at the ω - position (terminal) or ω - 1 - position (sub - terminal).

[0005] Sophorolipids have many advantageous features that make them superior to synthetic surfactants, such as biodegradability, low toxicity, high surface and interfacial activity, and stability over a wide range of temperatures, pressures, and ionic strengths. Therefore, sophorolipids have been reported to have various applications in, for example, food preservation, biomedicine, cosmetics, environmental purification by microorganisms, purification of heavy metal contamination, cement slurries, and agriculture and livestock.

[0006] On the other hand, the functional properties of sophorolipids can differ in their lactonic and free (also called "acidic" or "linear") forms. For example, acidic sophorolipids generally have a higher hydrophilic-lipophilic balance (HLB) than lactonic sophorolipids, while lactonic sophorolipids generally have a lower HLB and greater surface tension reduction properties than acidic sophorolipids. Furthermore, acidic sophorolipids typically have very high water solubility due to their free carboxylic acid groups. Combining lactonic and acidic sophorolipids in different ratios can affect, for example, the size of emulsion droplets, viscosity reduction properties, and surface / interfacial tension reduction properties. In other cases, only lactonic sophorolipids may be required for certain applications due to their properties.

[0007] One problem associated with lactonic sophorolipids is their sensitivity to the surrounding environment. Specifically, lactonic sophorolipids are prone to ring opening, which converts them to their acidic form depending on the surrounding environment, such as temperature and pH. For example, when the environmental pH changes outside the range of about 4 - 7, the ester bond of the lactone ring may be hydrolyzed, increasing the relative proportion of acidic sophorolipids in the mixture.

[0008] Therefore, there is a need for lactonic sophorolipids or their equivalents with improved stability. Such improvements would allow for better control and prediction of the properties of a given sample of sophorolipids, making quality control easier in the various fields in which sophorolipids are utilized. Furthermore, the sophorolipids with improved stability would expand the possibilities of their use in a variety of other applications that were previously considered impossible due to environments in which lactonic sophorolipids are unstable or can become unstable.

SUMMARY OF THE INVENTION

[0009] One aspect of the present invention is a modified sophorolipid of formula (I): TIFF2025518694000002.tif67128 is provided, wherein R1 and R2 are each independently hydrogen, ethyl (-CH2CH3), or acetyl (-COCH3); R3 is hydrogen or methyl; A is an optionally substituted saturated or unsaturated aliphatic chain.

[0010] In some embodiments, the aliphatic chain A has 10 to 21 carbons such that the total number of carbons in the aliphatic chain A, R3, and the carbon to which R3 is attached is 12 to 22 carbons.

[0011] The present invention encompasses all of the compounds represented by general formula (I), including their hydrates, their geometric and optical isomers, and their polymorphic forms.

[0012] The compounds according to the present invention are stable to pH and temperature compared to their lactonic counterparts. Furthermore, the compounds of formula (I) have a reduced HLB parameter, as well as improved wetting and solubilization parameters, water-in-oil emulsifying ability, surface tension lowering activity, and / or antibacterial activity compared to their acidic or even lactonic counterparts. Thus, the compounds have a wide range of uses in various industries, including but not limited to food, environmental purification by microorganisms, petroleum, agriculture, livestock, and aquaculture.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0014] Detailed Description of the Invention Modified Sophorolipid In one aspect, the present invention provides a modified sophorolipid of general formula (I) which is a cyclic ether analog of lactonic sophorolipid.

[0015] The present invention provides a compound of formula (I): TIFF2025518694000003.tif64128, where R1 and R2 are each independently hydrogen, ethyl (-CH2CH3), or acetyl (-COCH3); R3 is hydrogen or methyl (-CH3); A is an optionally substituted saturated or unsaturated aliphatic chain.

[0016] In a preferred embodiment, the aliphatic chain A has 10 - 21 carbons such that the total number of carbons in the aliphatic chain A, at the carbon to which R3 is attached, and the carbon to which R3 is attached (the "ring chain") is 12 - 22 carbons.

[0017] In some embodiments, the total number of carbons in the ring chain is 18. The ring chain can be either saturated or unsaturated. In a specific embodiment, the unsaturated ring chain has a C=C bond at the 8th or 9th position and can be a cis or trans isomer.

[0018] The aliphatic chain may be substituted. For example, substitution at the 8th or 9th carbon in the ring chain is possible through olefin chemistry. The substituents include a halogen atom, hydroxyl, lower (C1-C6) alkyl group, halo lower (C1-C6) alkyl group, hydroxy lower (C1-C6) alkyl group, halo lower (C1-C6) alkoxy group, etc. Preferred halogen atoms, or halogen atoms bonded to an alkyl group or alkoxy group, include fluorine, chlorine, bromine, and iodine. When determining the total number of carbons in the ring chain, the number of carbons in the substituents is not considered.

[0019] According to some embodiments, both R1 and R2 are ethyl, R3 is methyl, and A is an unsaturated aliphatic chain having 1, 2, or 3 unsaturations and 16 carbons.

[0020] According to some embodiments, both R1 and R2 are ethyl, R3 is hydrogen, and A is an unsaturated aliphatic chain having 1, 2, or 3 unsaturations and 17 carbons.

[0021] According to some embodiments, R1 is ethyl, R2 is hydrogen, R3 is methyl, and A is an unsaturated aliphatic chain having 1, 2, or 3 unsaturations and 16 carbons.

[0022] According to some embodiments, R1 is ethyl, R2 is hydrogen, R3 is hydrogen, and A is an unsaturated aliphatic chain having 1, 2, or 3 unsaturations and 17 carbons.

[0023] According to some embodiments, R1 is hydrogen, R2 is ethyl, R3 is methyl, and A is an unsaturated aliphatic chain having 1, 2, or 3 unsaturations and 16 carbons.

[0024] According to some embodiments, R1 is hydrogen, R2 is ethyl, R3 is hydrogen, and A is an unsaturated aliphatic chain having 17 carbons with 1, 2, or 3 unsaturations.

[0025] According to some embodiments, both R1 and R2 are hydrogen, R3 is methyl, and A is an unsaturated aliphatic chain having 16 carbons with 1, 2, or 3 unsaturations.

[0026] According to some embodiments, both R1 and R2 are hydrogen, R3 is hydrogen, and A is an unsaturated aliphatic chain having 17 carbons with 1, 2, or 3 unsaturations.

[0027] In another embodiment, R1 and R2 are each independently hydrogen, ethyl, or acetyl, R3 is methyl, and A is an unsaturated aliphatic chain having 16 carbons with 1 unsaturation.

[0028] In another embodiment, R1 and R2 are each independently hydrogen, ethyl, or acetyl, R3 is hydrogen, and A is an unsaturated aliphatic chain having 17 carbons with 1 unsaturation.

[0029] In a further embodiment, R1 and R2 are each independently hydrogen, ethyl, or acetyl, R3 is methyl, and A is an unsaturated aliphatic chain having 16 carbons with 2 unsaturations.

[0030] In a further embodiment, R1 and R2 are each independently hydrogen, ethyl, or acetyl, R3 is hydrogen, and A is an unsaturated aliphatic chain having 17 carbons with 2 unsaturations.

[0031] In a further embodiment, R1 and R2 are each independently hydrogen, ethyl, or acetyl, R3 is methyl, and A is an unsaturated aliphatic chain having 16 carbons with 3 unsaturations.

[0032] According to a further aspect, each of R1 and R2 is independently hydrogen, ethyl, or acetyl, R3 is hydrogen, and A is an unsaturated aliphatic chain having 3 unsaturations and 17 carbons.

[0033] According to another aspect, each of R1 and R2 is independently hydrogen, ethyl, or acetyl, R3 is methyl, and A is a fully saturated aliphatic chain having 16 carbons.

[0034] According to another aspect, each of R1 and R2 is independently hydrogen, ethyl, or acetyl, R3 is hydrogen, and A is a fully saturated aliphatic chain having 17 carbons.

[0035] The present invention encompasses all of the compounds represented by general formula (I), including their hydrates, their geometric and optical isomers, and their polymorphic forms. Preferably, the compounds of general formula (I) are derived from lactonic sophorolipids. In some aspects, the lactonic sophorolipids are produced as a result of the fermentation of sophorolipid-producing organisms.

[0036] The cyclic ether structure reduces the pH instability and temperature sensitivity associated with the corresponding lactonic sophorolipids. The compounds of the present invention are stable at a pH of about 3 to about 14 and / or a temperature up to about 100 °C. Further, the compounds of formula (I) have a reduced HLB value compared to their lactonic counterparts. This results in improved wetting and solubilization parameters, water-in-oil emulsifying ability, surface tension lowering activity, and / or antibacterial activity compared to the acidic or lactonic counterparts.

[0037] Sophorolipid In some embodiments, the modified sophorolipids according to the present invention are prepared by chemically modifying sophorolipids. Sophorolipids are generally obtained from microbial fermentation, and pure fatty acids, fatty acid mixtures, pure fatty acid esters, mixtures of fatty acid esters, triglycerides are used as carbon sources together with carbohydrate sources such as corn syrup, dextrin and glucose. The microorganism can be a wild-type or genetically engineered yeast strain. As shown in Figure 1, sophorolipids generally include sophorose, which may or may not be acetylated at the 6' and 6'' positions, and hydroxylated fatty acid tails with various degrees of unsaturation. Most commonly, the fatty acid consists of 16 or 18 carbon atoms and at least one unsaturated bond, but the fatty acid structure (e.g., degree of unsaturation, chain length) varies depending on the carbon source provided during production and the microorganism used for fermentation.

[0038] The use of renewable substrates and different microbial species, as well as the production of sophorolipids using various culture parameters (culture time, stirring speed, pH of the medium and added nutrients), enables the acquisition of compounds with clearly different structural and physical properties. This allows the production of a wide range of compounds with different physical, chemical, biochemical, and / or biophysical properties.

[0039] The hydrophobic fatty acid terminus of sophorolipids is usually glycosidically linked to the sophorose molecule at the terminus or sub-terminus. The fatty acid carboxylic acid group in sophorolipids is either free (also called "acidic" or "linear", see Figure 1) or generally intramolecularly esterified at the 4'' position (e.g., see Figure 2). The degree of lactonization of sophorolipids and acetylation of the sophorose moiety can be affected, among other things, by the carbon source supplied during the production of sophorolipids. For example, it has been found that sophorolipids derived from rapeseed, sunflower, and palm oil rich in C18:0 and C18:1 fatty acids are formed with a higher concentration of diacetyl lactone than sophorolipids produced from the corresponding fatty acid ester raw materials.

[0040] Sophorolipids have environmental compatibility, high biodegradability, low toxicity, high selectivity, and specific activity under a wide range of temperature, pH, and salinity conditions.

[0041] Fermentation In some embodiments, the modified sophorolipids according to the present invention are prepared by chemically modifying sophorolipids produced by a fermentation process known in the art in the presence of selected fatty acid and carbohydrate raw materials. As used herein, "fermentation" refers to the growth of cells under controlled conditions. The growth can be aerobic or anaerobic. Fermentation processes known in the art include, but are not limited to, solid-state fermentation, liquid-state fermentation, or modifications, hybrids, and / or combinations thereof.

[0042] The sophorolipids according to the present invention can be derived via a fermentation process from recombinant organisms or by strains that inherently produce sophorolipids. Non-limiting examples of sophorolipid-producing organisms include Candida bombicola, Candida apicola, Candida bogoriensis, Yarrowia lipolytica, Starmerella bombicola, Starmerella clade, Rhodotorula bogoriensis, Wickerhamiella domericqiae, and Wickerhamomyces anomalus. Some recombinant sophorolipid-producing microorganisms have been reported to control the sophorolipid structure. As a non-limiting example, certain recombinant S. bombicola strains may be utilized to produce sophorolipids that are either completely lactonic or completely acidic (see, for example, Roelants et. al., Towards the Industrialization of New Biosurfactants: Biotechnological Opportunities for the Lactone Esterase Gene from Starmerella Bombicola, 113 Biotechnology and Bioengineering 3, 550-559 (2015)). As an additional non-limiting example, recombinant Candida bombicola strains with the acetyltransferase gene knocked out can be used to produce sophorolipids without acetylation (see, for example, WO2012 / 080116).

[0043] The target microorganisms can be cultured on-site, either on a small or large scale, and can be utilized even while mixed with their culture media. The modified sophorolipids of the present invention can be produced in large quantities even at the required locations. The growth vessels used to grow sophorolipid-producing organisms can be any fermenter or culture reactor for industrial use.

[0044] In one embodiment, a single type of microorganism is grown within a reactor system. In an alternative embodiment, multiple microorganisms that can be grown together without adversely affecting the growth or the resulting product can be grown within a single reactor system. For example, two or more different types of microorganisms may be grown simultaneously within a single reactor. In some embodiments, more than one type of microorganism grows symbiotically within the reactor.

[0045] In one embodiment, the vessel may have or be connected to a functional control device / sensor for measuring important elements in the culture process, such as pH, oxygen, pressure, temperature, output of the stirring shaft, humidity, viscosity, and / or microorganism density and / or metabolite concentration.

[0046] The culture may be supplemented with a carbon source. The carbon source is typically a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and / or maltose, preferably glucose; an organic acid such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and / or pyruvic acid; an alcohol such as ethanol, isopropyl, propanol, butanol, pentanol, hexanol, isobutanol, and / or glycerol; an oil or fat such as soybean oil, rice bran oil, canola oil, olive oil, corn oil, sesame oil, and / or linseed oil, etc. These carbon sources may be used alone or in combination of two or more.

[0047] The culture can be supplemented with a nitrogen source. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and / or ammonium chloride. These nitrogen sources may be used independently or in combination of two or more.

[0048] Oxygen can also be supplied to the growth medium in the culture. In one embodiment, air is slowly moved to remove air with low oxygen content and introduce air containing oxygen. In the case of liquid-phase fermentation, the air containing oxygen may be ambient air that is replenished daily through a mechanism including an impeller for mechanically agitating the liquid and an air sparger for supplying gas bubbles to the liquid to dissolve oxygen in the liquid.

[0049] In one embodiment, the microorganism can be grown on a solid or semi-solid substrate such as, for example, corn, wheat, soybeans, chickpeas, beans, oatmeal, pasta, rice, and / or powders or meals of these or other similar substances.

[0050] In one embodiment, the medium contains growth factors and micronutrients for the microorganism. This is particularly preferred when culturing microorganisms that cannot produce all the necessary vitamins. Inorganic nutrients containing trace elements such as iron, zinc, copper, manganese, molybdenum and / or cobalt may also be included in the medium. Furthermore, sources of vitamins, essential amino acids, and microelements may be included in the form of powders or meals such as, for example, corn powder, or in the form of extracts such as yeast extract, potato extract, beef extract, soy extract, banana peel extract, or in purified form. For example, amino acids such as those useful for protein biosynthesis may also be included.

[0051] In one embodiment, inorganic salts may also be included. Usable inorganic salts can 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 combination of two or more.

[0052] In some embodiments, for example, when the microorganism used for seeding the substrate is in the form of spores (e.g., bacterial spores), a germination promoter can be added to the substrate. Examples of the germination promoter according to the present invention include, but are not limited to, L-alanine, manganese, L-valine and L-asparagine, or any other known germination promoter.

[0053] In some embodiments, additional acids and / or antibacterial agents in the liquid medium may be added before and / or during the culture process. Antibacterial agents or antibiotics are used to protect the culture from contamination. Further, when gas is generated during the culture, an antifoaming agent may also be added to prevent the formation and / or accumulation of foam.

[0054] The pH of the mixture should be suitable for the target microorganism. Buffers and pH adjusters such as carbonates and phosphates may be used to stabilize the pH near a preferred value. When metal ions are present at high concentrations, it may be necessary to use a chelating agent in the liquid medium.

[0055] The methods and apparatuses for culturing microorganisms and generating microbial by-products can be carried out in batch, semi-continuous, or continuous processes.

[0056] In one embodiment, the culture of the microorganism is carried out at about 5°C to about 100°C, preferably about 15°C to about 60°C, more preferably about 25°C to about 50°C. In a further embodiment, the culture may be carried out continuously at a constant temperature. In another embodiment, the culture may be subjected to temperature changes.

[0057] In one aspect, the method and the apparatus used in the culture process are sterilized. Culture apparatuses such as reactors / containers are separated from, but may be connected to, a sterilization unit, for example, an autoclave. The culture apparatus may also have a sterilization unit that sterilizes in situ before starting seeding. 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 container. In another aspect, the medium may be pasteurized or, optionally, no heat may be applied at all, where the use of low water activity and low pH may control unwanted bacterial growth.

[0058] In one aspect, at the end of the culture (e.g., when a desired cell density or a density of a specified metabolite is achieved), all of the microbial culture composition is removed. In this batch procedure, an entirely new batch is started upon the recovery of the first batch.

[0059] In another aspect, only a portion of the fermentation product is removed at any given time. In this aspect, the biomass containing live cells remains in the container as an inoculum for a new culture batch. The composition removed may be a cell-free medium or may contain cells, spores, hyphae, conidia, or other microbial propagules. In this way, a semi-continuous system is created.

[0060] In some aspects, the sophorolipids produced by the target microorganism may be retained within the microorganism or secreted into their growth medium. The sophorolipid content can be, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0061] The growth medium may contain compounds that stabilize the activity of the sophorolipids. The sophorolipids can be purified or used in a crude form, meaning that they are not separated from the fermentation broth in which they are produced.

[0062] In certain embodiments, the sophorolipids are isolated and / or purified from the growth medium resulting from the fermentation of a biosurfactant-producing microorganism. Isolation and purification can be readily achieved using standard methods or techniques described in the literature. If desired, the sophorolipids can be further concentrated.

[0063] As used herein, the terms "isolated," "purified," or variations thereof, when used in reference to a biological or natural substance such as a glycolipid, mean that the substance is substantially free of other compounds such as cellular material with which it is associated in nature. That is, the substance does not occur naturally without these other compounds and / or has different or characteristic properties compared to the properties found in the natural substance.

[0064] In certain embodiments, the purified compound is present at least 60% by weight of the compound of interest. Preferably, the preparation has at least 75%, more preferably at least 90%, and most preferably at least 99% (w / w) of the desired compound of interest by weight. For example, a purified compound is a compound in which the compound of interest is present at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) by weight. Purity is measured by any suitable standard method, for example, by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis.

[0065] The ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 - 20 covers any number, combination of numbers, or sub - range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, as well as all decimal values intervening between said integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub - ranges, “nested sub - ranges” extending from either endpoint of the range are specifically contemplated. For example, nested sub - ranges within the exemplary range of 1 - 50 may include, in one direction, 1 - 10, 1 - 20, 1 - 30, and 1 - 40, or in the other direction, 50 - 40, 50 - 30, 50 - 20, and 50 - 10.

[0066] Modification method In some embodiments, the modified sophorolipids according to the present invention are obtained by applying synthetic methods known in the art for modifying sophorolipids obtained from the products of fermentation by sophorolipid - producing microorganisms. The sophorolipids to be chemically modified may be obtained by the fermentation methods and processes described above and / or other methods known in the art. As discussed above, purification methods known for purifying sophorolipids from the growth medium may be utilized. The sophorolipids to be modified may be obtained in a substantially lactonic form, a substantially acidic form, or a mixture of lactonic and acidic forms. As used herein, lactonic sophorolipids encompass any isomers of sophorolipids containing a cyclic ester.

[0067] In some embodiments, the starting material for the modified sophorolipids according to the present invention may be a pure lactonic sophorolipid substantially free of acidic sophorolipids. Advantageously, using a pure lactonic sophorolipid as the starting material for chemical conversion reduces possible side reactions and enables the reaction product to be obtained as a pure cyclic ether of general formula (I) substantially free of acyclic counterparts. The pure lactonic sophorolipid may be obtained directly as a purified product of fermentation, or may be obtained after subjecting a fermentation product containing a mixture of lactonic and acidic sophorolipids to a chemical process known in the art for lactonizing the acidic sophorolipids. In another embodiment, the lactonic sophorolipid can be isolated from a mixture of lactonic and acidic sophorolipids obtained from fermentation. This can be achieved, for example, by utilizing standard liquid-liquid extraction techniques that exploit the differences in the chemical characteristics of lactonic and acidic sophorolipids. That is, while acidic sophorolipids contain a carboxylic acid functional group, lactonic sophorolipids do not contain readily ionizable groups.

[0068] In another embodiment, the lactonic sophorolipid to be modified is not isolated from a mixture of lactonic and acidic forms prior to modification. Advantageously, in this way, it is not necessary to separate the lactonic sophorolipid from the acidic sophorolipid before modification. Modification of the lactonic sophorolipid can be carried out by extracting a mixture of lactonic and acidic sophorolipids into a selected organic solvent and then adding the required reducing reagent thereto.

[0069] The modification of the lactonic sophorolipids according to the present invention involves selectively reducing at least the carbonyl (C=O) group of the fatty acid in the sophorolipid to an alkyl (-CH2-) group without reducing another type of functional group present in the molecule, to obtain a cyclic ether compound of formula (I). A non-limiting and exemplary schematic of the reduction of lactonic sophorolipids is as follows: Shown in TIFF2025518694000004.tif49152, where Ra and Rb are each independently hydrogen or acetyl. Further, A' is a saturated or unsaturated aliphatic chain having one less carbon than A in chain length. A' may be optionally substituted. When an acetyl group is present in Ra and / or Rb, as a result of the reduction process, this may also be reduced to form an ether bond with the adjacent oxygen. Thus, R1 and R2 can be hydrogen, acetyl, or ethyl.

[0070] According to some embodiments of the invention, the reduction of the lactonic sophorolipid to obtain the compound of formula (I) is achieved by the use of a reducing agent and a Lewis acid catalyst in an organic solvent. The reaction may be carried out at any suitable temperature. In some embodiments, the reaction is carried out at approximately 60 °C.

[0071] The reducing agent according to the invention is any suitable agent capable of reducing the carbonyl group in the lactonic sophorolipid. Suitable reducing agents include, without limitation, hydrosilane sources such as triethylsilane (Et3SiH), tris(trimethylsilyl)silane (TTMS), tetraphenyldisilane (TPDS), and / or polymethylhydrosilane (PHMS).

[0072] The Lewis acid catalyst according to the invention may be any acid compatible with the reducing agent that catalyzes the reduction of the carbonyl group in the lactonic sophorolipid. Suitable Lewis acid catalysts include, without limitation, indium bromide (InBr3), bismuth tribromide (BiBr3), titanium alkoxide (e.g., titanium isopropoxide), boron tribromide (BBr3), aluminum bromide (AlBr3), and zinc(II) bromide (Zn(II)Br2). In some embodiments, the Lewis acid catalyst is indium bromide. The Lewis acid catalyst may be present in an amount of about 1 mol% to about 25 mol%.

[0073] The organic solvent according to the invention may be any non-polar organic solvent that can be used as a reaction medium and is suitable for the reduction of lactonic sopholipids. 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.

[0074] Figure 3 shows a non-limiting and exemplary reaction scheme according to the present invention. The starting material is a lactonic sopholipid provided either in pure form or as a mixture containing lactonic and acidic sopholipids. The reduction is carried out at 60 °C in CHCl3 using a reagent combination of Et3SiH and InBr3. The compound labeled "4" is a modified sopholipid according to the present invention.

[0075] Aliphatic chain A The aliphatic chain A according to the present invention can be of any length as long as it is suitable for intramolecular ring formation with the sophose residue to form a compound of formula (I). Preferably, the total number of carbons in the aliphatic chain A, R3, and the carbon to which R3 is attached (the "ring chain") is from 12 to 22 carbons. In certain embodiments, the aliphatic chain A has from 10 to 21 carbons. More preferably, the aliphatic chain A has 14 and 17 carbons such that the total number of carbon atoms in the ring chain is 16 - 18. In some embodiments, the total number of carbon atoms in the ring chain is 18.

[0076] Furthermore, when the aliphatic chain A is unsaturated, it can have one or more double and / or triple bonds in the chain as long as the unsaturation does not render intramolecular ring formation sterically impossible. When the aliphatic chain A has at least one double bond, the geometric isomers of the alkene are fully consistent with the scope and purpose of the present invention. That is, when the aliphatic chain A has at least one degree of unsaturation, the compound of formula (I) encompasses both cis and trans isomers.

[0077] Also, the degree and / or position of unsaturation and the desired isomers in the aliphatic chain A can be artificially introduced or can be inherent to the fatty acid from which the aliphatic chain A is derived. In some embodiments, the unsaturation in the ring chain is removed via known chemical processes. Further, substituents may be introduced into the unsaturation (e.g., double bonds) in the ring chain through synthetic methods known in the art. In some embodiments, the degree and position of unsaturation in the aliphatic chain A are inherent to the corresponding fatty acid. For example, when the aliphatic chain A is derived from oleic acid (18:1 cis-9) or elaidic acid (18:1 trans-9), it has the corresponding isomers at the same position (i.e., cis-9 or trans-9, respectively).

[0078] Non-limiting examples of fatty acids from which the aliphatic chain A can be derived include caprylic acid, capric acid, lauric acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, and arachidonic acid. A preferred fatty acid is oleic acid.

[0079] Industrial Applicability Another aspect of the invention is directed to the use of the compounds represented by general formula (I) in a wide range of applications. The compounds may be used not only in fields where conventional sophorolipids are employed or can be employed, but also in various other fields where their application has been considered impossible due to environments in which lactonic sophorolipids become unstable. Since the cyclic ether structure of the compounds of formula (I) is more stable to pH and temperature than the lactonic counterparts, some embodiments of the invention provide that the compounds of formula (I) replace or complement conventional sophorolipids, particularly when the general properties (other than stability issues) of lactonic sophorolipids are desired or preferred. In some embodiments, the compounds of formula (I) have a lower HLB value than their corresponding starting compounds of formula (I').

[0080] In another aspect, the compounds of formula (I) are utilized in fields where a decreased HLB value resulting in improved wetting and solubilization parameters and / or improved water-in-oil emulsifying ability is desirable or required. In a further aspect, the compounds of formula (I) can be used when an enhancement of surface tension lowering activity and / or antibacterial activity is desirable or required.

[0081] Exemplary uses of the compounds of formula (I) include, but are not limited to, their use as food additives or supplements, pharmaceutically active substances or adjuvants, cosmetic ingredients, and ingredients in cleaning products. The compounds of formula (I) can also be used in environmental purification by microorganisms and purification of heavy metal pollution, as well as in the petroleum industry, for example, in drilling, cement slurries, crushing, improving oil recovery, preventing scale formation, acidification, demulsification of crude oil, corrosion inhibition, reducing oil viscosity, equipment cleaning, water injection methods, and / or foam and steam injection methods. Further, in agriculture, aquaculture, and livestock, the compounds of formula (I) can be used, for example, as soil conditioners; broad-spectrum biopesticides, antiviral, antifungal, and antibacterial agents; feed additives such as to improve nutrient absorption or feed preservation; nutrient sources; and / or therapeutic agents for diseases or infections in plants, fish, and livestock. The compounds of formula (I) can also be used in environmental fields such as for reducing greenhouse gas emissions.

[0082] 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 material not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified substance or step and those that do not materially affect the "basic and novel characteristics of the claimed invention, e.g., the ability to improve the bioavailability of a substance". The use of the term "comprising" contemplates other aspects "consisting of" or "consisting essentially of" the recited components.

[0083] Unless otherwise specifically recited or apparent from the context, as used herein, the term "or" is understood to be inclusive. Unless otherwise specifically recited or apparent from the context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0084] Unless otherwise specifically recited or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the mean value. 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 recited value.

[0085] The recitation of a list of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of the listed groups. The recitation of a variable or an aspect of an aspect herein includes that aspect as any single aspect or in combination with any other aspect or a part thereof.

[0086] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

Examples

[0087] The following are examples of the present invention, which are presented for illustrative purposes and are not intended to limit the invention. Various modifications or changes within the spirit and scope of the present application will be suggested to and understood by those skilled in the art.

[0088] Example 1: Exemplary Compounds The numbers in the leftmost column represent compound numbers. The present invention encompasses all compounds represented by general formula (I), including their hydrates, their geometric and optical isomers, and their polymorphic forms. TIFF2025518694000005.tif67161TIFF2025518694000006.tif234163TIFF2025518694000007.tif180162

[0089] Example 2: Production of Lactonic Sophorolipids Preparation A stainless-steel fermentation reactor is used for the production of sophorolipids. The reactor contains approximately 150 gallons of water, to which a medium containing dextrose (25 - 150 g / L), yeast extract (1 - 10 g / L), canola oil (25 ml / L - 110 ml / L), and urea (0.5 - 5 g / L) is added.

[0090] The reactor includes a mixing device for continuous stirring and mixing of the culture broth. The reactor containing the medium is steam-treated at 100 °C for approximately 60 minutes to sterilize the reactor and the growth medium.

[0091] The reactor is then cooled. When the temperature of the reactor reaches approximately 35 °C, an antibiotic is added to the medium to prevent bacterial contamination. The other pipes and openings of the reactor are sterilized by spraying with isopropyl alcohol (IPA).

[0092] A small reactor is used to grow the recombinant Starmerella bombicola inoculum culture. The culture is grown in the small reactor at 26 - 28 °C for at least 42 - 48 hours. The S. bombicola strain produces S. bombicola lactone esterase, which catalyzes the esterification of linear sophorolipids to produce lactonic sophorolipids.

[0093] When the stainless-steel fermentation reactor reaches 30 °C, approximately 25 L of the inoculum culture is inoculated.

[0094] Fermentation The fermentation temperature is maintained at 23 - 28 °C. After about 22 - 26 hours, the pH of the culture broth is set to about 3.0 - 4.0, or about 3.5, using 20% NaOH. A computer that controls the pump used to monitor the pH and add base to maintain the pH at 3.5 is included in the fermentation reactor.

[0095] After culturing for about 6 - 7 days (120 hours + / - 1 hour), if a 7.5 ml layer of sophorolipid can be seen, the batch is in a recoverable state. In some cases, a small amount of oil may also be visible and a small amount of glucose may also be detected (e.g., about 0% - 0.5%).

[0096] Recovery and purification The culture broth is collected in a first collection container and left to stand for 24 - 48 hours. A sophorolipid layer rich in lactone precipitates at the bottom of the first collection container, which can be collected and purified, for example, by liquid - liquid extraction to isolate lactonic sophorolipid. After purification, the sophorolipid is in a state where further modification can be carried out.

Claims

1. A compound of formula (I): wherein R 1 and R 2 are each independently hydrogen, ethyl, or acetyl; R 3 is hydrogen or methyl; A is an optionally substituted saturated or unsaturated aliphatic chain, a compound of formula (I).

2. The aliphatic chain A, R 3 and R 3 wherein the total number of carbons at the carbon to which they are attached is from 12 to 22 carbons, and the aliphatic chain A has from 10 to 21 carbons excluding substituents, the compound according to claim 1.

3. The aliphatic chain is saturated, the compound according to claim 2.

4. The aliphatic chain is unsaturated, the compound according to claim 2.

5. A is an unsaturated aliphatic chain having 16 or 17 carbon atoms and 1 double bond, the compound according to claim 1.

6. A is an unsaturated aliphatic chain having 16 or 17 carbon atoms and 2 double bonds, the compound according to claim 1.

7. A is an unsaturated aliphatic chain having 16 or 17 carbon atoms and 3 double bonds, the compound according to claim 1.

8. A is further substituted with halogen, hydroxyl, lower (C1 - 6) alkyl, halo lower (C1 - 6) alkyl, hydroxy lower (C1 - 6) alkyl, and / or halo lower (C1 - 6) alkoxy, the compound according to claim 1.

9. The compound according to claim 1, synthesized from sophorolipids produced by fermentation of sophorolipid-producing organisms using capric acid, capric acid, lauric acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, and / or arachidonic acid.

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

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

12. The aliphatic chain A, R 3 and R 3 The method according to claim 11, wherein the aliphatic chain A has 10 to 21 carbons excluding substituents such that the total number of carbons at the carbon to which and the carbon to which R binds is 12 to 22 carbons.

13. The method according to claim 12, wherein the aliphatic chain is saturated.

14. The method according to claim 12, wherein the aliphatic chain is unsaturated.

15. The method according to claim 11, wherein A is an unsaturated aliphatic chain having 16 or 17 carbon atoms and 1 double bond.

16. The method according to claim 11, wherein A is an unsaturated aliphatic chain having 16 or 17 carbon atoms and 2 double bonds.

17. The method according to claim 11, wherein A is an unsaturated aliphatic chain having 16 or 17 carbon atoms and 3 double bonds.

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

19. The method according to claim 11, wherein the compound of formula (I) has a decreased HLB value compared to the compound of formula (I').

20. The following: compound.

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