# US 11,306,339 B2 · Method for the production of a rhamnolipid > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,339 B2 - Document ID: US11306339B2 - Status: Granted - Kind: B2 - Application number: 16/310,152 - Filed: 2017-06-16 - Published: 2019-08-08 - Granted: 2022-04-19 - Earliest claimed: 2016-06-16 - Assignees: Superbrewed Food Inc. - Applicants: Superbrewed Food, Inc.; Aharon M. Eyal; Bryan P. Tracy; Christopher Joseph McWilliams - Inventors: Aharon M. Eyal; Bryan P. Tracy; Christopher Joseph McWilliams - Web page: https://patentgrep.com/patent/US11306339B2 ## CPC - C12P 19/44: Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} - A61K 8/046: {Aerosols; Foams} - A61K 8/602: {Glycosides, e.g. rutin} - A61K 9/0014: {Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00)} - A61K 9/122: {Foams; Dry foams (edible foams A61K9/0056)} - A61Q 19/00: Preparations for care of the skin - C07H 1/08: from natural products - C07H 15/04: attached to an oxygen atom of the saccharide radical - C12P 7/6409: Fatty acids - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) ## Abstract A method for the production of a rhamnolipid. The method includes culturing in a fermentation medium an organism capable of producing a rhamnolipid to form a fermentation broth comprising at least one rhamnolipid and having a pH>5; extracting at least one lipophilic impurity from said fermentation broth; acidulating said fermentation broth to form an aqueous medium of pH<5 comprising the at least one rhamnolipid; extracting the at least one rhamnolipid from said acidulated fermentation broth; and separating the at least one rhamnolipid to obtain a purified rhamnolipid. ## Claims ### Claim 1 A method for production of a rhamnolipid, comprising (i) providing an aqueous fermentation medium comprising a carbon source and a nitrogen source; (ii) culturing in said fermentation medium an organism capable of producing a rhamnolipid, wherein said carbon source is metabolized to form a fermentation broth having a pH greater than 5 and comprising at least one rhamnolipid, at least one lipophilic impurity, and at least one hydrophilic impurity; (iii) extracting at least one lipophilic impurity from said fermentation broth with a first extractant to form (a) a lipophilic-impurity-depleted fermentation broth comprising the at least one rhamnolipid and (b) a first extract comprising said first extractant and said at least one lipophilic impurity; (iv) separating the first extractant and the at least one lipophilic impurity from the first extract; (v) acidulating said lipophilic-impurity-depleted fermentation broth to form an acidulated lipophilic-impurity-depleted aqueous medium of pH less than 5 comprising the at least one rhamnolipid; (vi) extracting the at least one rhamnolipid from said acidulated lipophilic-impurity-depleted aqueous medium with a second extractant to obtain a second extract comprising said second extractant and the at least one rhamnolipid; and (vii) separating the at least one rhamnolipid from said second extract to obtain a purified rhamnolipid; wherein said first extractant, said second extractant, or both comprise at least one of (a) a hydrocarbon having a boiling point under 20° C. at atmospheric pressure, and (b) an oxygenated organic compound having a boiling point under 20° C. at atmospheric pressure, wherein said oxygenated organic compound has a Hansen solubility parameter polarity component in the range between 2 Mpa0.5 and 8 Mpa0.5 and a Hansen solubility parameter H-bond component in the range between 2 Mpa0.5 and 8 Mpa0.5. ### Claim 2 (depends on 1) A method according to claim 1, wherein said at least one rhamnolipid comprises one rhamnose moiety in combination with two fatty acid chains and/or two rhamnose moieties in combination with two fatty acid chains. ### Claim 3 (depends on 1) A method according to claim 1, wherein rhamnolipid concentration in said fermentation broth having a pH greater than 5 is in the range between 1 g/l and 70 g/l. ### Claim 4 (depends on 1) A method according to claim 1, wherein said first extractant, said second extractant or both comprise said oxygenated organic compound and an olefin with a boiling point under 20° C. while at atmospheric pressure. ### Claim 5 (depends on 1) A method according to claim 1, wherein said first extractant comprises an olefin and said second extractant comprises said oxygenated organic compound. ### Claim 6 (depends on 1) A method according to claim 1, wherein said separating said first extractant, said separating said second extractant, or both comprises evaporation. ### Claim 7 (depends on 6) A method according to claim 6, further comprising liquefying at least a fraction of the separated first and/or separated second extractant, wherein said liquefying comprises subjecting said at least a fraction of the separated first and/or separated second extractant to a refrigerant circuit. ### Claim 8 (depends on 7) A method according to claim 7, wherein a refrigerant in the refrigerant circuit is selected from the group consisting of R-11, R-12, R-13, R-14, R-21, R-22, R-23, R-41, R-113, R-114, R-115, R-116, R-123, R-124, R-125, R-134a, R-141b, R-142b, R-143a, R-152a, R-218, R-227ea, R-236ea, R-245ca, R-365mfc, RC318, R-406a, R-410a, R-414a, R-500, R-502, R-503, R-1301, and ammonia. ### Claim 9 A method for production of a rhamnolipid, comprising (i) providing an aqueous fermentation medium comprising a carbon source and a nitrogen source; (ii) culturing in said fermentation medium an organism capable of producing a rhamnolipid, wherein said carbon source is metabolized to form a fermentation broth having a pH greater than 5 and comprising at least one rhamnolipid, at least one lipophilic impurity and at least one hydrophilic impurity; (iii) acidulating said fermentation broth to form an acidulated aqueous medium of pH less than 5; (iv) extracting the at least one rhamnolipid and the at least one lipophilic impurity from said acidulated aqueous medium with a first extractant, thereby obtaining a hydrophilic-impurity-depleted first extract comprising the at least one rhamnolipid, the at least one lipophilic impurity and the first extractant; (v) separating said first extractant from said at least one rhamnolipid and said at least one lipophilic impurity in said first extract to obtain a separated medium comprising said at least one rhamnolipid and said at least one lipophilic impurity; (vi) adjusting the pH of the separated medium to a pH greater than 5; and (vii) extracting the at least one lipophilic impurity from said pH-adjusted separated medium with a second extractant to obtain purified rhamnolipid wherein said first extractant, said second extractant, or both comprise at least one of (a) a hydrocarbon having a boiling point under 20° C. while at atmospheric pressure and (b) an oxygenated organic compound having a boiling point under 20° C. at atmospheric pressure, wherein said oxygenated organic compound has a Hansen solubility parameter polarity component in the range between 2 MPa0.5 and 8 MPa0.5 and a Hansen solubility parameter H-bond component in the range between 2 MPa0.5 and 8 MPa0.5. ### Claim 10 (depends on 9) A method according to claim 9, wherein said extracting the at least one lipophilic impurity comprises forming a second extract comprising the second extractant and the at least one lipophilic impurity. ### Claim 11 (depends on 10) A method according to claim 10, further comprising separating the second extractant and the at least one lipophilic impurity from the second extract. ### Claim 12 (depends on 9) A method according to claim 9, wherein said at least one rhamnolipid comprises one rhamnose moieties in combination with two fatty acid chains, and/or two rhamnose moieties in combination with two fatty acid chains. ### Claim 13 (depends on 9) A method according to claim 9, wherein rhamnolipid concentration in said fermentation broth is in the range between 1 g/l and 70 g/l. ### Claim 14 (depends on 9) A method according to claim 9, wherein said separating said first extractant comprises evaporation. ### Claim 15 (depends on 14) A method according to claim 14, further comprising liquefying at least a fraction of the separated first extractant, wherein said liquefying comprises subjecting said at least a fraction of the separated first extractant to a refrigerant circuit. ### Claim 16 (depends on 15) A method according to claim 15, wherein a refrigerant in the refrigerant circuit is selected from the group consisting of R-11, R-12, R-13, R-14, R-21, R-22, R-23, R-41, R-113, R-114, R-115, R-116, R-123, R-124, R-125, R-134a, R-141b, R-142b, R-143a, R-152a, R-218, R-227ea, R-236ea, R-245ca, R-365mfc, RC318, R-406a, R-410a, R-414a, R-500, R-502, R-503, R-1301, and ammonia. ### Claim 17 A method for the production of a rhamnolipid, comprising (i) providing a fermentation medium comprising a carbon source and a nitrogen source; (ii) culturing in said fermentation medium an organism capable of producing a rhamnolipid, wherein said carbon source is metabolized to form a fermentation broth having a pH greater than 5 and comprising at least one rhamnolipid, at least one lipophilic impurity, and at least one hydrophilic impurity; (iii) extracting at least one lipophilic impurity from said fermentation broth with a first extractant, whereby (a) a lipophilic-impurity-depleted fermentation broth comprising the at least one rhamnolipid and (b) a first extract comprising said first extractant and said at least one lipophilic impurity are formed; (iv) separating said first extractant and said at least one lipophilic impurity from said first extract; (v) acidulating said lipophilic-impurity-depleted fermentation broth to a pH less than 5, whereby a rhamnolipid-enriched phase is formed; and (vi) separating said rhamnolipid-enriched phase from the acidulated fermentation broth wherein said first extractant, said second extractant, or both comprise at least one of (a) a hydrocarbon having a boiling point under 20° C. while at atmospheric pressure and (b) an oxygenated organic compound having a boiling point under 20° C. at atmospheric pressure, wherein said oxygenated organic compound has a Hansen solubility parameter polarity component in the range between 2 MPa0.5 and 8 MPa0.5 and a Hansen solubility parameter H-bond component in the range between 2 MPa0.5 and 8 MPa0.5.