# US 2014/0194336 A1 · Sophorolactone production

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- Number: US 2014/0194336 A1
- Document ID: US20140194336A1
- Status: Published application
- Kind: A1
- Application number: 14/123,858
- Filed: 2012-06-06
- Published: 2014-07-10
- Earliest claimed: 2011-06-06
- Assignees: Ecover Co-Ordination Center N.V.
- Inventors: Dirk Develter; Steve Fleurackers
- Web page: https://patentgrep.com/patent/US20140194336A1

## CPC

- C12P 19/445: {The saccharide radical is condensed with a heterocyclic radical, e.g. everninomycin, papulacandin}
- C07C 59/01: Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
- C07H 15/04: attached to an oxygen atom of the saccharide radical
- C11D 1/662: {Carbohydrates or derivatives}
- C11D 1/825: Mixtures of compounds all of which are non-ionic
- C11D 3/2096: {Heterocyclic compounds}
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/64: Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P 17/181: {Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin}
- C12P 19/12: Disaccharides
- C12P 19/44: Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)}
- 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)}
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- 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)
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a process for selectively producing sophorolactone without use of organic solvent, comprising the steps of: -pre-cultivating cells of a Candida species capable of producing sophorolactone, in absence of an oily substrate until a stationary growth phase is obtained, -cultivating said pre-cultivated cells in an aqueous medium in the presence of at least one fermentable sugar and substrate; the reaction mixture of sugar, substrate and pre-cultivated cells being present in an amount and conditions such that the cells metabolize the sugar and substrate thereby forming sophorolactone and fatty acid, -continuously feeding said substrate to said cells thereby suppressing the formation of fatty acid and keeping fatty acid levels in the reaction mixture below 10 g/l, resulting in the crystallization of at least part of the sophorolactone present in the reaction mixture, -warming the reaction mixture to a temperature between 60° C. and 90° C., thereby melting the sophorolactone crystals, -allowing the molten sophorolactone to settle and to provide a crude sophorolactone composition, and -removing the crude sophorolactone composition from the remainder of the reaction mixture without use of an organic solvent.

## Claims

### Claim 1

Process for the selective production of sophorolactone in absence of an organic solvent, comprising the steps of:
pre-cultivating cells of a Candida species capable of producing sophorolactone, in absence of an oily substrate until a stationary growth phase is obtained,
cultivating said pre-cultivated cells in an aqueous medium in the presence of at least one fermentable sugar and substrate; the reaction mixture of sugar, substrate and pre-cultivated cells being present in an amount and conditions such that the cells metabolize the sugar and substrate thereby forming sophorolactone and fatty acid,
continuously feeding said substrate to said cells thereby suppressing the formation of fatty acid and keeping fatty acid levels in the reaction mixture below 10 g/l, resulting in the crystallization of at least part of the sophorolactone present in the reaction mixture,
warming the reaction mixture to a temperature between 60° C. and 90° C., thereby melting the sophorolactone crystals,
allowing the molten sophorolactone to settle and to provide a crude sophorolactone composition, and
removing the crude sophorolactone composition from the remainder of the reaction mixture without use of an organic solvent.

### Claim 2 (depends on 1)

Process according to claim 1, comprising the steps of:
cooling the crude sophorolactone composition thereby producing a solid, such as a paste,
dispersing the solid in water, preferably decalcified water, at a temperature of 5-25° C., using a mass ratio of water to solid between 0.5 and 15, thereby obtaining an aqueous dispersion of sophorolactone crystals,
separating the crystals from the water,
repeating the dispersion and separation steps from 0 to 10 times keeping the temperature of the water below 40° C.,
drying the resulting sophorolactone composition thereby providing dried sophorolactone.

### Claim 3 (depends on 2)

Process according to claim 2, wherein prior to drying water is removed from the crystals by melting the crystals to a temperature between 35° C. and 55° C., thereby providing a lower layer comprising sophorolactone and an upper layer of supernatant water; separating the lower layer from the upper layer to obtain a sophorolactone composition with increased active matter content.

### Claim 4 (depends on 3)

Process according to claim 3, wherein drying is by spray drying, drum drying, convection drying, thin film evaporation, vacuum drying, flaking, extruding or casting.

### Claim 5 (depends on 2)

Process according to claim 2, wherein the water separated from the crystals, is re-used in said dispersing step.

### Claim 6 (depends on 1)

Process according to claim 1, wherein the sophorolactone composition separated off the reaction mixture is in the form of an aqueous sophorolipid mixture comprising at least 30 wt %, expressed on the dry matter content of the aqueous sophorolipid mixture.

### Claim 7 (depends on 6)

Process according to claim 6, wherein the sophorolactone is comprised in a composition comprising at most 60 weight percent water.

### Claim 8 (depends on 1)

Process according to claim 1, wherein the sugar and substrate are fed to the pre-cultivated cells in a fed batch mode.

### Claim 9 (depends on 1)

Process according to claim 1, wherein the substrate comprises corn steep liquor.

### Claim 10 (depends on 1)

Process according to claim 1, wherein the substrate is a triglyceride.

### Claim 11 (depends on 1)

Process according to claim 1, wherein the substrate is isostearic acid or isostearic alcohol.

### Claim 12 (depends on 1)

Process according to claim 1, wherein the substrate is a shielded ester of formula H—(CH2)n—O—CO—(CH2)m—COOH with an carbon chain length of 16 to 18.

### Claim 13 (depends on 1)

Process according to claim 1, further comprising the step of filtering the sophorolactone composition separated off the reaction mixture by depth filtration.

### Claim 14 (depends on 1)

Process according to claim 1, whereby the fermentation is executed in a vessel at an overpressure of between 0-1.5 bar.

### Claim 15 (depends on 1)

Process according to claim 1, wherein the substrate comprises a carbon chain length lower than C14 and the fermentation medium comprises cells of a Candida species modified to improve the conversion of said substrate into sophorolactone.

### Claim 16 (depends on 15)

Process according to claim 15, wherein the Candida is Candida bombicola mutant strain M18, M30 or M33; preferably Candida bombicola M30.

### Claim 17 (depends on 1)

Sophorolactone composition in the form of a solid or paste, obtainable by a method according to claim 1, consisting of at least 60 weight percent sophorolactone, 0-30 wt % sophorolipid acid, 0-1 wt % residual substrate, including less than 0.2 wt % fatty acid, and remainder water.

### Claim 18 (depends on 17)

Sophorolactone composition according to claim 17, consisting of 70 wt %-100 wt % sophorolipid lactone, 0-1 wt % sophorolipid acid, less than 0.1% residual substrate including fatty acid, and remainder water.

### Claim 19 (depends on 18)

Formulation obtained by dissolving the sophorolactone composition of claim 18 in a solvent with a polarity parameter delta P between 5 and 9.5 and a hydrogen bonding parameter delta H between 8 and 15.

### Claim 20 (depends on 19)

Formulation according to claim 19, wherein the solvent is selected from the group consisting of phenoxyethanol, benzylalcohol, phenetyl alcohol, hydrocinnamylalcohol, dimethylisosorbide, methyl salicylate and eugenol.

### Claim 21 (depends on 18)

Dry solid product for laundry or dishwashing, comprising the composition of claim 18, wherein the pH of the product is 8.0-14.0, as measured on a 1% solution of the product in water.

### Claim 22 (depends on 1)

Process for the preparation of partially hydrolysed sophorolactone comprising the steps of: providing a sophorolactone or sophorolactone composition obtainable by a process according to claim 1; and hydrolysing at least part of the sophorolactone, either by adding to the sophorolactone 0.5 to 1 equivalents of a base or by heating the sophorolactone for more than 4 hours, thereby hydrolysing at least part of the sophorolactone to sophorolipid acid and/or its salt.

### Claim 23 (depends on 1)

Process for the preparation of a ω hydroxy fatty acid of formula CH2OH—CH2-(CH2)n-COOH or a ω-1 hydroxy fatty acid of formula CH3-CHOH—(CH2)n-COOH wherein the acid is derived from a sophorolipid, with n corresponding to an integer between 2 and 16, comprising the step of:
providing a sophorolactone or sophorolactone composition obtainable by a method according to claim 1,
hydrolysing the sophorolactone to sophorolipid acid using a base,
separating the sugar moiety and the ω and ω-1 hydroxy fatty acid moiety of the sophorolipid acid; preferably by hydrolysis with a weak acid or by enzymatic reaction, and
recovering the sugar moieties and/or the w and w-1 hydroxy fatty acid obtained.

### Claim 24 (depends on 23)

Process according to claim 23, whereby the enzymatic reaction is carried out in the presence of endoglycoceramidase II.

### Claim 25 (depends on 23)

Hydroxy fatty acid obtained by a method according to claim 23, characterized by a concentration of non hydroxylated fatty acids below 0.8% expressed on the basis of the total hydroxy fatty acid content.