EP1028969A2

EP1028969A2 - New sophoroselipids, method for their production and use

Info

Publication number: EP1028969A2
Application number: EP98955474A
Authority: EP
Inventors: Siegmund Lang; Andreas Brakemeier; Dieter Wullbrandt; Andreas SEIFFERT-STÖRIKO
Original assignee: Aventis Research and Technologies GmbH and Co KG
Current assignee: Aventis Research and Technologies GmbH and Co KG
Priority date: 1997-11-07
Filing date: 1998-10-17
Publication date: 2000-08-23
Also published as: JP2001522597A; DE19749413A1; WO1999024448A3; WO1999024448A2; US6433152B1

Abstract

The invention relates to sophoroselipids of formula (I) wherein R<4> represents H, -CH2CH3, -CH2CH2CH3, n is an integer number from 2 to 27, R<1> and R<2> represent, independently from each other, H or a group of formula (II), and R<3> represents H or -OH. The invention also relates to a method for producing sophoroselipids of formula (I) wherein R<1>, R<2>, R<3> and n have the aforementioned meanings and R<4> represents -CH3, -CH2CH3 or -CH2CH2CH3. According to this method, a yeast capable of secreting a sophoroselipid in the form of a lactone in the culture excess, is fermented in a culture medium containing a glycerin, a succinate, a mono-, di- and/or tri-saccharide and a lipid precursor, said sophoroselipid being then isolated from the culture solution. The inventive method is characterised in that the lipid precursor contains one or more 3-alkanols, 4-alkanols or an alkanone with a chain length from 6 to 30 carbon atoms or mixtures of said alkanols/alkanone. Furthermore, according to a variant of the aforementioned method for producing sophoroselipids of formula (I), wherein R<1>, R<2>, R<3> and n have the meanings aforementioned and R<4> represents -CH3, -CH2CH3 or -CH2CH2CH3, the culture medium is maintained during fermentation under a reduced oxygen concentration. The new sophoroselipids may be used, e.g., as surfactants, cosmetics, disinfecting agents or pharmaceutical products.

Description

description

INNOVATIVE SOPHOROSELIPIDS, METHOD FOR THE PRODUCTION AND USE THEREOF

The present invention relates to novel sophorose lipids, processes for their biotechnological production and their use as surfactants, cosmetics, disinfectants or pharmaceuticals

Microbial glycolipids have been known for a number of years as biosurfactants with a wide range of uses in cosmetics, the detergent and cleaning agent sector, the food sector, medicine and environmental protection Better degradability is caused by bacteria, yeasts or fungi when growing on long-chain petroleum products, on vegetable oils and fats or their derivatives or on

Mono- and Ohgosacchaπden formed A targeted modification of the molecular structures of these products by varying the carbon source has so far been achieved only to a small extent Since its discovery in 1961, the sophorose lipid has been one of the intensively researched microbial glycolipids [PA Gönn, JFT Spencer and AP Tuiloch, Can J Chem, 39 (1961), 846-855]

According to various reports, the sophorose lipid can be prepared from various yeasts of the genus Candida (Torulopsis) as a secondary metabolite using a substrate from the carbon sources mentioned above. Suitable yeast strains are Candida bombicola, Candida bogonensis, Candida magnoliae,

Candida gropengiessen and Candida apicola are described [R Hommel, Biodegradation, 1, (1991), 107]

The sophorose lipids formed by the genus Candida have the structure shown below (1) R

R ¹ = -C (O) CH _3l HR ² = -CH ₃ , HR ³ = - (CH ₂ ) _n -, n = 13-16, - (CH ₂ ) _n -CH = CH- (CH ₂ ) _n n = 5-7

Structure 1

In addition to this main lactonic product with both glycosidic and ester-like hydroxy fatty acid, non-cyclized intermediates are also found in small proportions. Depending on the substrate used, the hydroxy fatty acid can be saturated, but can also be polyunsaturated. In addition, the 6'-O and 6 "-O positions of the glucose units are acetylated to different degrees. With these sophorose lipids, only slight differences in the fatty acids of the side chain are observed.

To produce a glycolipid with an amphiphilic structure, ie high interfacial activity, the sophorose lipid lactones have to be converted into the sophorose lipid esters or amides via complex synthesis and purification steps become [p. Inoue, et al. U.S. patent. 4,215,213, 1990; Y. Ishigami, JP application: Toku Kai Hei 6-100581].

It is known that in the fermentation of yeasts of the Candida genus using 2-alkanols instead of vegetable oils and fats, fatty acids or their alkyl esters, non-cyclized glucose and sophorose lipids with surface-active properties are accessible (see DE 195 18 982.5) . However, the corresponding 2-alkanols are expensive or complex to produce. It has not previously been possible to use less expensive, more accessible 1-alcohols, since these are known to be largely converted to the corresponding acids before they are converted to glycolipids (see, for example, DF Jones, R.Howe, J. Chem. Soc. -C-, (1968), 2801-2808). There were only indications of access to these substances that were never isolated or characterized in more detail (Davila, A.-M., Marchai, R., Vandecasteele, J.-P., J. Industr. Microbioi., (1994) 249-257).

Surprisingly, it has now been found that non-cyclized glucose and sophorose lipids with surface-active properties are also accessible when using 3-alkanols, 4-alkanols, 2-alkanones, 3-alkanones and 4-alkanones.

In addition, the successful use of 1-alcohols and alkanals to obtain the novel products is possible if the culture medium has a reduced oxygen concentration during the fermentation of the microorganisms.

The invention thus relates to compounds of the formula I wherein

RH, -CH ₂ CH ₃ , -CH ₂ CH CH ₃ , one an integer from 2 to 27,

R and R independently of one another H or CH and

R ^{3 is} H or -OH.

Preferred embodiments of the present invention are shown below.

1. A compound of the formula I in which R ^{4 is} H, where R ¹ and R ² together are -C (O) CH ₃ , R ¹ H and R ² -C (O) CH ₃ ,

R ¹ -C (O) CH ₃ and R ² H or

R ¹ and R ^{2 are} H,

R ^{3 represents} H and where n is an integer from 6 to 14, preferably 8 to

12 means.

2. A compound of the formula I in which R ^{4 is} -CH ₂ CH ₃ or -CH ₂ CH ₂ CH ₃ , where R ¹ and R ² together are -C (O) CH ₃ ,

R ^{1 represents} H and R ² -C (O) CH ₃ , R ¹ -C (O) CH ₃ and R ² H or R ¹ and R ² H, R ^{3 represents} H or -OH and where n is an integer from 6 to 14, * preferably 8 to 12.

The present invention further relates to a process for the preparation of sophorose lipids according to formula I, the radicals R ¹ , R ² and R ³ and n having the abovementioned meaning and R ⁴ -CH ₃ , -CH ₂ CH ₃ or -CH ₂ CH ₂ CH ₃ means in which a yeast with the ability to secrete sophorose lipids in the form of a lactone in the culture supernatant is fermented in a culture medium which contains glycerol, succinate, a mono-, a di- and / or a trisaccharide and a lipid - Contains precursor, and then the sophorose lipid is isolated from the culture solution, characterized in that the lipid precursor has one or more 3-alkanols, 4-alkanols or alkanones with a chain length of 6 to 30 carbon atoms or mixtures of these alkanols / alkanones.

The chain length of the alkanol / alkanone is preferably 10 to 18

Carbon atoms, with which a compound of the formula I can be prepared, in which n is an integer from 4 to 14, 2-dodecanone or 3-dodecanone is particularly preferably used as the alkanone, whereby a compound of the formula I with n = 8 and R ⁴ = -CH ₃ or with n = 7 and R ⁴ = -CH ₂ CH _{3 can be} produced, where R ¹ , R ² and R ^{3 have} the meaning given above.

The present invention further relates to a process for the preparation of sophorose lipids according to formula I, where the radicals R ¹ , R ² and R ³ and n have the meaning given above and R ^{4 is} H, -CH ₃ , -CH ₂ CH ₃ or - CH ₂ CH ₂ CH ₃ means in which a yeast with the ability to sophorose lipids in the form of a lactone in the

To secrete culture supernatant is fermented in a culture medium which contains glycerol, succinate, a mono-, a di- and / or a trisaccharide and a lipid precursor, and then the sophoroselipid is isolated from the culture solution, characterized in that the lipid Precursor one or more alkanols, alkanals or alkanones with a chain length of 6 to 30 carbon atoms or

Mixtures of these alkanols / alkanals / alkanones and the culture medium is kept under a reduced oxygen concentration during the fermentation. The chain length of the alkanol / alkanone / alkanal is preferably from 10 to 18 carbon atoms, which can be used to prepare a compound of the formula I in which n is an integer from 4 to 15; preference is given to a 1-alkanol or an alkanal as lipid Precursor used, particularly preferred as alkanol 1-

Dodecanol used, whereby a compound of formula I with n = 9 and R ⁴ = H can be prepared, where R ¹ , R ² and R ^{3 have} the meaning given above.

Lipid precursor is to be understood here as meaning compounds which have an alkyl chain of 6 to 30 carbon atoms and at least one as a functional group

Contain hydroxy and / or a carbonyl group.

Examples of lipid precursors are 1-alkanols, 2-alkanols, 3-alkanols, 4-alkanols, alkanals, 2-alkanones, 3-alkanones, 4-alkanones with a chain length of 6 to 30 carbon atoms.

Examples of 1-alkanols are, without being restricted thereby, 1-hexanol, 1-octanol, 1-decanol, 1-undecanol, 1-dodecanol, 1-tetradecanol, 1-pentadecanol, 1-octadecanol, 1-eicosanol or 1-triacontanol, preference is given to 1-dodecanol and 1-tetradecanol.

Examples of 2-alkanols are, without being restricted thereby, 2-hexanol, 2-octanol, 2-decanol, 2-undecanol, 2-dodecanol, 2-tetradecanol, 2-pentadecanol, 2-octadecanol, 2-eicosanol or 2-triacontanol, preference is given to 2-dodecanol and 2-tetradecanol.

Examples of 3-alkanols are, without being restricted thereby, 3-hexanol, 3-octanol, 3-decanol, 3-undecanol, 3-dodecanol, 3-tetradecanol, 3-pentadecanol, 3-octadecanol, 3-eicosanol or 3-triacontanol, preference is given to 3-dodecanol and 3-tetradecanol.

Examples of 4-alkanols are, without being restricted thereby, 4-octanol, 4-decanol, 4-undecanol, 4-dodecanol. 4-tetradecanoi, 4-pentadecanol, 4-octadecanol, 4-eicosanol or 4-triacontanol, 4-dodecanol and 4-tetradecanol are preferred.

Examples of alkanals are, without being restricted thereby, hexanal, octanal, decanal, undecanal, dodecanal, tetradecanal, pentadecanal,

Octadecanal, Eicosanal or Triacontanal, dodecanal and tetradecanal are preferred.

Examples of 2-alkanones are, without being restricted thereby, 2-hexanone, 2-octanone, 2-decanone, 2-undecanone, 2-dodecanone, 2-tetradecanone,

2-pentadecanone, 2-octadecanone, 2-eicosanone or 2-thacontanone, preference is given to 2-dodecanone and 2-tetradecanone.

Examples of 3-alkanones are, without being restricted thereby, 3-hexanone, 3-octanone, 3-decanone, 3-undecanone, 3-dodecanone, 3-tetradecanone,

3-pentadecanone, 3-octadecanone, 3-eicosanone or 3-triacontanone, preference is given to 3-dodecanone and 3-tetradecanone.

Examples of 4-alkanones are, without being restricted thereby, 4-octanone, 4-decanone, 4-undecanone, 4-dodecanone, 4-tetradecanone, 4-

Pentadecanone, 4-octadecanone, 4-eicosanone or 4-triacontanone, preference is given to 4-dodecanone and 4-tetradecanone.

The extent of the formation of the new type of structure, which arises especially when using 1-alkanols / alkanals, is limited to the

Oxygen supply bound during the production phase. By limiting the oxygen concentration of the culture medium with the aid of suitable methods, the oxidation-minimizing oxidation of the fatty alcohol to the fatty acid can be almost prevented and the formation of the desired products is thus increased.

Suitable methods for limiting the oxygen concentration are, for example, the lowering, without this being intended to be a limitation the fumigation rate and the reduction of the stirrer speed, whereby oxygen depletion can also be brought about by the metabolism of the yeasts. The oxygen concentration is accessible by measuring the oxygen partial pressure. This is 5 to 40%, preferably 5 to 15% of the saturation value during the product formation phase of the fermentation. The saturation value relates to air (oxygen content approx. 21%, based on the volume of the gas mixture), which is passed through the culture solution under normal pressure at the respective fermentation temperature. It can be determined with a suitable probe at the beginning of the fermentation.

In the method according to the present invention, all yeast strains can be fermented which secrete the sophorose lipids described in the literature in lactone form (structure 1) into the culture supernatant.

A yeast of the genus Candida is particularly suitable for the process according to the invention, preferably Candida bombicola, Candida bogoriensis, Candida magnoliae, Candida gropengiesseri or Candida apicola, which are commercially available.

Glucose or sucrose are particularly suitable as carbon sources.

The invention is described in detail below. Furthermore, it is determined by the content of the claims.

By using alkanols, alkanals or alkanones with chain lengths of

C6 to C30 as a hydrophobic carbon source in addition to a further carbon source such as glycerol, succinate or mono-, di- and trisaccharides such as sucrose, mannose, fructose, glucose and D-mannitol or other sugar alcohols, preferably glucose or sucrose, the sophorose lipids can be obtained with a variation in the structure of the hydrophobic part of the molecule. The purified products mostly contain lipid components of the chain length of the substrate used, the latter either being incorporated directly into the glycolipid (compounds of structures 2 with R ³ = H) or after a (ω-1) hydroxylation as alkanediol is bound to the sugar component via a glycosidic bond (compounds of structure 3 with R ³ = OH). In this way, only non-ionic surfactants with a typical surfactant structure are produced.

The products are not mono- or diacetylated in the sugar portion in the 6'-O- and 6 "-O-position of the glucose unit. The compounds according to structures 2 and 3 with n = 2 to 27 are obtained by varying the chain length of the alkanols or alkanals / alkanones used (C6 to C30) The acetylated sophorose lipids can be converted into the non-acetylated compounds by an alkaline hydrolysis.

Accordingly, for example, when using

1-hexanol a compound of the formula I with n = 3, 1-octanol a compound of the formula I with n = 5,

1-decanol a compound of formula I with n = 7,

1-undecanol a compound of formula I with n = 8, 1-dodecanol a compound of formula I with n = 9,

1-tetradecanol a compound of formula I with n = 1 1, 1-pentadecanol a compound of formula I with n = 12

1-octadecanol a compound of formula I with n = 15,

- 1-Eicosanol produce a compound of formula I with n = 17 or when using 1-triacontanol a compound of formula I with n = 27, where R ¹ , R ² , R ³ and R ^{4 have} the meaning given above.

Most of the 1-alkanols, the 3-alcohols and the 4-alcohols as well as the alkanals, 2-alkanones, 3-alkanones and 4-alkanones of chain length C6 to C30 are commercially available, some of them can be obtained from the corresponding, inexpensive alkenes represent. The alcohols are also available from the alkanones and the aldehydes (Organikum, 16th edition, VEB-Deutscher Verlag der Wissenschaften 1986 or other corresponding textbook on applied organic chemistry).

Structure 2 Structure 3

2, 3: n = 2-27; R ¹ , R ² = H or -C (O) CH ₃ ; R ⁼ H, CH ₃ , CH CH ₃ , CH ₂ CH CH ₃

2a: n = 9; R ¹ , R ² = -C (O) CH ₃ ; R ⁴ = H (compound 1, see example 1)

2b: n = 9; R ¹ , R ² = H; R ⁴ = H (compound 2, see example 3)

To isolate the formed sophorose lipids of structures 2 and 3, the culture solution is neutralized after separating the biomass by centrifugation or filtration with an alkali and with an organic solvent such as. B.

Carboxylic acid esters, such as ethyl acetate, butyl acetate or ethers, such as tert-butyl methyl ether and diethyl ether or other known to those skilled in the art

Extracted solvent exhaustively. The organic phases are separated, combined and over a drying agent such as e.g. Dried sodium sulfate. To

Removal of the extractant in vacuo after azeotropic removal of the

Water obtained a yellow-brown raw product.

The products acetylated in the 6'-O- and 6 "-O-positions can be converted into the corresponding hydroxy compounds by alkaline saponification with bases (for example aqueous NaOH) or alkanolates (for example sodium methoxide). The producer strains used are fermented in a medium with alkanols, alkanals or alkanones with a chain length of C6 to C30, preferably C10 to C18. The concentration of alkanols, alkanals or alkanones can be set at the start of the fermentation or selected by continuous replenishment according to the conversion rate, the

Postdosing is preferred. The use of sugars, preferably glucose or sucrose, has proven useful for providing an additional carbon source. In addition to the carbon source, the medium should also contain one or more nitrogen sources, sulfate and magnesium as well as potassium, sodium, calcium and chloride ions, one or more phosphate sources and a growth-promoting complex substrate such as e.g. Yeast extract included.

The sugar is used in concentrations of 30 to 200 g / l nutrient solution, with concentrations between 80 and 150 g / l being preferred.

Known to the person skilled in the art can be used as a nitrogen source, such as e.g. B. urea, ammonium chloride or ammonium sulfate can be used in concentrations of 0.1 to 5 g / l nutrient solution, preferably a concentration of 0.5 - 2.5 g / l is selected.

A 0.001 to 0.1 molar sodium phosphate or potassium phosphate buffer or a mixture of the two metal ions is used as the phosphorus source and for buffering the medium.

The optimal temperature for fermentation is in the range between 20 and 40 ° C, preferably 25 to 30 ° C.

The pH is unregulated and drops during fermentation. At the beginning of the fermentation, it is adjusted to a value of approximately 5 to 7, preferably 5.5 to 6.5, by the buffer. The pH during the product formation phase is preferably in the range from 2.5 to 4.

The sophorose lipids obtained as explained above have a significantly increased effect Solubility (chain length of the alcohol <C22) relative to the classic products a greater reduction in the surface tension of water.

The use of primary alcohols, aldehydes and ketones can significantly reduce the costs of the products, so that they can be used in fields of application for which products which can be obtained from the 2-alcohols are too expensive.

The novel sophorose lipids have excellent surface and interfacial activity. They are very biodegradable and have a bactericidal effect.

The products can be used as surfactants, emuigators, co-surfactants and as a moisture-retaining agent. They therefore have application prospects in the detergent and cleaning agent sector. Since they have a low toxicity, they can be used for the production of cosmetics and used in the food sector. As biodegradable biosurfactants, they can be used in environmental protection. Because of their microbicidal activity, they can be used in medicine, for example, as pharmaceuticals or as disinfectants.

Furthermore, enantiomerically pure alcohols can be obtained from the biosurfactant. The microorganisms convert the ketones or racemic secondary alcohols used into optically active alcohols. The enantiomeric excess achieved is considerably greater than 95%.

The release of the optically active alcohols can be achieved, for example, by acid methanolysis, without this being intended to restrict it.

The invention is explained in more detail below using exemplary embodiments. example 1

To produce the dodecyl sophorosides in a 500 ml Erlenmeyer flask with baffles, 100 ml of a culture medium of the following composition are placed:

Glucose H ₂ O 150.00 g / l

Sodium citrate-3 H ₂ O 5.00 g / l

Yeast extract (granulated, Merck, Darmstadt) 4.00 g / l

Ammonium chloride 1.54 g / l

Potassium dihydrogen phosphate 1.00 g / l

Magnesium sulfate-7 H ₂ O 0.70 g / l

Sodium chloride 0.50 g / l

Calcium chloride-2 H ₂ O 0.27 g / l

Dipotassium hydrogen phosphate - 3 H ₂ O 0.16 g / l

The medium is inoculated with the yeast Candida bombicola ATCC 22214 and incubated on a rotary shaker at 100 rpm and a temperature of 30 ° C. After a cultivation period of 48, 72 and 96 h, 5 g / l 1 -dodecanol are added to the culture solution under aseptic conditions.

The culture management between and after the addition of alcohol takes place under unchanged conditions. The measured dry biomass concentration at the end of the cultivation is 17 g / l. The pH of the culture suspension drops over the entire range of cultivation. After a cultivation period of 10 d, the amount of alcohol offered has been converted; the cultivation is then stopped.

To isolate the products, the culture suspension is neutralized with 1 N sodium hydroxide solution and then extracted twice with twice the volume of ethyl acetate. The organic phases are separated, combined and dried over anhydrous sodium sulfate. After the desiccant has been separated off through a paper filter, the solvent is reduced at a reduced rate Distilled off pressure on the rotary evaporator. The solidified, almost water-free, yellow-brown crude product is obtained in a yield of 8 g / l, corresponding to 0.53 g per g of 1-dodecanol.

In addition to the dodecyl sophorosides (compound 1: formula I, wherein n = 9; R ¹ , R ² = -C (O) CH ₃ ; R ³ = H and R ⁴ = H (molecular structure 2a) and compound 2 : Formula I, in which n = 9; R ¹ , R ² = H; R ³ = H and R ⁴ = H (molecular structure 2b)) also small amounts of the sophoroselipid lactone and can be on silylated silica gel (RP-18) in the solvent mixture Separate methanol water 90: 0 (v / v) by thin layer chromatography into its individual substances with characteristic Rf values.

Nuclear magnetic resonance spectroscopy and FAB mass spectrometry as well as a combined gas chromatographic-mass spectrometric analysis of the hydrophobic part of the molecule (after acidic methanolysis) of the compounds can be used to demonstrate that the individual compounds are based on the molecular structures shown in the figures. The main product of cultivation is

Compound 1 (molecular structure 2a).

R _f (RP-18; methanol / water 90:10 v / v) 0.46 [compound 1]

Spectroscopic data for compound 1:

MS (FAB, matrix glycerol, neg.): M / z = 593 (100, [MH] ^" ), 551 (30, [M-COCH ₃ ] ^" ), 509 (6, [M-2 COCH ₂ -HD

Characterization of the product mixture based on the relative proportions of its lipid components using GC-MS (FS = fatty acid):

Glycolipid mixture based on glucose / 1 -dodecanol:

1 -dodecanol 69.8%

FS C 12: 0 10.0%

FS 15-OH-C 16: 0 1.1%

FS 16-OH-C 16: 0 1.4% FS 17-OH-C 18: 1 8.3%

FS 17-OH-C 18: 0 ..5.5%

FS 18-OH-C 18: 1 1.6%

FS 18-OH-C 18: 0 0.4%

FS C 16: 0 0.2%

FS C 16: 1 0.2%

FS C 18: 0 0.1%

FS C 18: 1 1, 1%

Other 0.3%

Example 2

To produce the biosurfactants in a bioreactor (V _tot = 2.5 I), 2 I of a culture medium of the following composition are placed:

Glucose H ₂ O 140.00 g / l

Sodium citrate-3 H ₂ O 5.00 g / l

Yeast extract (granulated, Merck Darmstadt) 4.00 g / l ammonium chloride 1.54 g / l

Potassium dihydrogen phosphate 1.00 g / l

Magnesium sulfate-7 H ₂ O 0.70 g / l

Sodium chloride 0.50 g / l

Calcium chloride-2 H ₂ O 0.27 g / l dipotassium hydrogen phosphate-3 H ₂ O 0.16 g / l

The medium is inoculated with the yeast Candida bombicola ATCC 22214 and incubated for 40 h under the specified conditions until the biomass (18 g / l) has completely developed. After growth is complete, 1.5 g / l 1 -dodecanol is supplied sterile via a metering pump. The yeast culture reacts to the metering in by spontaneously lowering the dissolved oxygen concentration from previously 75 to 10% pO ₂ . At the same time, the carbon dioxide formation rate and the Oxygen consumption rate significantly. After further addition of 2 g / l of 1-dodecanol, it takes about 30 hours until the added fatty alcohol is completely metabolized. The depletion of the substance leads to a renewed increase in the dissolved oxygen concentration and a decrease in the rate of oxygen consumption and the formation of carbon dioxide, before, due to the new addition of 1-dodecanol, the conditions are restored before the metabolism is complete. With repeated repetition of such dodecanol dosages, the cultivation is continued until it is stopped. After a cultivation period of 160 h, 50 g / l glucose in solid, non-sterile form is replenished in order to prevent a limitation of this energy source. Already 25 hours after the first

When the alcohol is added, the novel sophorose lipid can be detected in the culture broth using HPLC technology. Its concentration increases to 14.3 g / l (corresponding to 0.64 per g 1 -dodecanol) by the end of the cultivation. Furthermore, the pH of the culture suspension drops over the entire product formation period. After a cultivation period of 235 h, a total amount of 22.5 g / l of the

Alcohol implemented, the cultivation is then stopped. Figure 1 summarizes the course of the bioreactor cultivation based on all analysis parameters.

In Figure 1, BTM mean dry biomass, SL sophorose lipid, Q CO ₂

Carbon dioxide formation rate, QO ₂ oxygen consumption rate, RQ respiration quotient and pO ₂ oxygen partial pressure, given as a percentage of the saturation value.

To isolate the products, the culture suspension is neutralized with 1 N sodium hydroxide solution and then twice with twice the volume

Extracted ethyl acetate. The organic phases are separated, combined and dried over anhydrous sodium sulfate. After the desiccant has been separated off using a paper filter, the solvent is distilled off under reduced pressure on a rotary evaporator. The crude glycolipid product is obtained in a total yield of 14.3 g / l.

As determined by thin-layer chromatographic investigations on silylated silica gel (RP-18) in the mobile solvent mixture methanol / water 90:10 (v / v), the product mixture consists of the compounds already described in Example 1.

Example 3

For basic hydrolysis of the sophorose lipid obtained according to Example 2, 20 g of the glycolipid mixture are dissolved in 400 ml of 1 N sodium hydroxide solution and refluxed with stirring for 4 h.

The reaction solution is then adjusted to pH 4 with concentrated hydrochloric acid and cooled to 4 ° C. for 12 h. The precipitate which separates out is separated off using a paper filter, washed with 500 ml of ice-cold water and taken up again in 300 ml of water for recrystallization (60 ° C. → 4 ° C.). The precipitated product is filtered off again and then freeze-dried.

The solid can be separated into two substances by means of chromatographic separation on silylated silica gel (RP-18) in the solvent system methanol / water 80:20 (v / v). The spectroscopic studies of the separated substances show that the non-polar main component has the structure of compound 2 (molecular structure 2b). The more polar

The component is the deacetylated sophorose lipid predominantly with free 17-hydroxyoctadecenoic acid as the hydrophobic molecular component.

R _f (RP-18: methanol / water 90:10 v / v): 0.54 [compound 2]

Spectroscopic data for compound 2: MS (FAB, matrix glycerol, neg.): M / z = 509 (100, [MH] ^" ), 347 (10, [M-glucose + H ₂ OH] ^" ) Example 4

For the production of the alkyl sophorosides from 2-, 3- and 4-dodecanone as well as 4-tetradecanol in a 500 ml Erlenmeyer flask with baffles, 100 ml of a culture medium of the following composition are placed in each case:

Glucose H ₂ O 150.00 g / l

Sodium citrate-3 H ₂ O 5.00 g /

Yeast extract 4.00 g / ammonium chloride 1.54 g / l

Potassium dihydrogen phosphate 1.00 g /

Magnesium sulfate-7 H ₂ O 0.70 g /

Sodium chloride 0.50 g / l

Calcium chloride-2 H ₂ O 0.27 g / l dipotassium hydrogen phosphate-3 H ₂ O 0.16 g /

The medium is inoculated with the yeast Candida bombicola ATCC 22214 and incubated on a rotary shaker at 100 rpm and a temperature of 30 ° C. After a cultivation period of 48, 72 and 96 h, 3.3 g / l of the various hydrophobic C substrates are added to the culture solution under aseptic conditions (total amount 10 g / l). The culture management between and after the addition of the substrates takes place under unchanged conditions. The pH of the culture suspension drops over the entire range of cultivation. After a cultivation period of 12 d, the offered amounts of 2- and 3-dodecanone and 4-tetradecanol were implemented without residue; substrate residues remain in the culture broth of the 4-dodecanone cultivation. All cultivations will then be stopped.

To isolate the products, the culture suspensions are neutralized with 1 N sodium hydroxide solution and then twice with twice the volume

Extracted ethyl acetate. The organic phases are separated, combined and dried over anhydrous sodium sulfate. After removing the Desiccant through a paper filter, the solvent is distilled off under reduced pressure on a rotary evaporator. To remove the substrate residues, the product of the 4-dodecanone cultivation is washed repeatedly with hexane. The remaining, educt-free and highly viscous raw products are mixed with n-butanol to remove the bound water azeotropically. It is then completely distilled off again under reduced pressure. The product mixtures are obtained in the following yields:

2-dodecanone: 14 g / l corresponding to 1.4 g per g of 2-dodecanone

3-dodecanone: 17 g / l corresponding to 1.7 g per g of 3-dodecanone

4-dodecanone: 3 g / l corresponding to 0.3 g per g of 4-dodecanone

4-tetradecanol: 17 g / l corresponding to 1.7 g per g of 4-tetradecanol

Determined by GC-MS, they contain to a large extent the 2-, 3- or 4-alkanols homologous to the substrates as lipid components in addition to more or less small amounts of hydroxy fatty acids.

Glycolipid mixture based on glucose / 2-dodecanone:

2-dodecanol 84.5%

FS 15-OH-C 16: 0 0.3%

FS 16-OH-C 16: 0 0.3%

FS 17-OH-C 18: 1 4.1%

FS 17-OH-C 18: 0 2.2%

FS 18-OH-C 18: 1 0.6%

FS C 16: 0 0.9%

FS C 16: 1 0.5%

FS C 18: 1 5.0%

FS C 18: 0 0.7%

Other 0.9% Glycolipid mixture based on glucose / 3-dodecanone:

3-dodecanol 80.0%

2-undecanol 3.4%

FS 15-OH-C 16: 0 0.6%

FS 16-OH-C 16: 0 0.7%

FS 17-OH-C 18: 1 7.5%

FS 17-OH-C18: 0 2.5%

FS 18-OH-C 18: 1 1, 2%

FS18-OH-C 18: 0 0.2%

FSC 16: 0 0.3%

FSC 16: 1 0.3%

FSC 18: 1 2.6%

FSC 18: 0 0.3%

Other 0.4%

Glycolipid mixture based on glucose / 4-dodecanone:

4-dodecanol 41.5%

Other C12 22.0%

FS 15-OH-C 16: 0 1.4%

FS 16-OH-C 16: 0 1.1%

FS 17-OH-C 18: 1 12.6%

FS 17-OH-C 18: 0 1.6%

FS 18-OH-C 18: 1 0.3%

FSC 16: 0 0.9%

FSC 16: 1 1.3%

FSC 18: 0 0.3%

FSC 18: 1 3.7%

Others 8.4% Glycolipid mixture based on glucose / 4-tetradecanol:

4-tetradecanol 23.8%

FS 15-OH-C 16: 0 4.7%

FS 16-OH-C 16: 0 4.8%

FS 17-OH-C 18: 1 39.9%

FS 17-OH-C 18: 0 17.4%

FS 18-OH-C 18: 1 6.5%

FS 18-OH-C 18: 0 1.1%

Others 1, 8%

Example 5

The sophorose lipid obtained from 2-dodecanone according to Example 4 was used to determine the enantiomeric excess of the fatty alcohol. The 2-dodecanol was obtained from the sophorose lipid by release using acidic methanolysis. To determine the enantiomeric excess, the optical rotation value of the isolated 2-dodecanol was measured at 25 ° C. and a wavelength of 589 nm. The rotation value [a] ²⁵ ₅₈₉ is +7.4 (5 g / 100 ml measured in

Ethanol) [<- c = 5].

In the literature, a rotation value of +6.9 (5 g / 100 ml measured in ethanol at 25 ° C.) is given for the (S) -2-dodecanol (Kirchner et al., J. Am. Chem. Soc, 107 , (1985), 7072-7076). According to the authors, this corresponds to one

95% enantiomeric excess. It follows from this that the enantiomeric excess of the 2-dodecanol according to the invention should be substantially greater than 95%.

Claims

claims

1. sophorose lipids of the formula I,

wherein

RH, -CH ₂ CH ₃ , -CH CH CH ₃ , one an integer from 2 to 27,

R 1 and R 9 independently of one another are H or ^C CH 3 and R ^{3 are} H or -OH.

2. Sophorose lipids according to claim 1, characterized in that n is an integer from 6 to 14, in particular from 8 to 12.

3. A process for the preparation of sophorose lipids according to formula I, wherein the

R ¹ , R ² and R ³ and n are as defined in claim 1 and R ^{4 is} -CH ₃ , -CH ₂ CH ₃ or -CH ₂ CH ₂ CH ₃ , in which a yeast with the

Ability to secrete sophorose lipids in the form of a lactone in the culture supernatant, fermented in a culture medium containing glycerol, succinate, a mono-, a di- and / or a trisaccharide and a lipid precursor, and then the sophorose lipid from the culture solution is isolated, characterized in that the lipid precursor is one or has several 3-alkanols, 4-alkanols or alkanones with a chain length of 6 to 30 carbon atoms or mixtures of these alkanols / alkanones.

4. The method according to claim 3, characterized in that the chain length of the alkanol / alkanone is 10 to 18 carbon atoms.

5. The method according to claim 3, characterized in that a 2-alkanone, a 3-alkanone, a 4-alkanone or mixtures thereof are used as alkanone.

6. The method according to claim 3, characterized in that 2-dodecanone or 3-dodecanone is used as alkanone.

7. A process for the preparation of sophorose lipids according to formula I, wherein the

R ¹ , R ² and R ³ and n have the meaning given in Claim 1 and R ^{4 is} H, -CH ₃ , -CH ₂ CH ₃ or -CH ₂ CH ₂ CH ₃ , in which a yeast with the ability to To secrete sophorose lipids in the form of a lactone into the culture supernatant, fermenting in a culture medium which contains glycerol, succinate, a mono-, a di- and / or a trisaccharide and a lipid

Contains precursor, and then the sophorose lipid is isolated from the culture solution, characterized in that the lipid precursor has one or more alkanols, alkanals or alkanones with a chain length of 6 to 30 carbon atoms or mixtures of these alkanols / alkanals / alkanones and the culture medium during the fermentation is kept under a reduced oxygen concentration.

8. The method according to claim 7, characterized in that the oxygen partial pressure of the culture solution is less than 40% of the saturation value, preferably less than 15% of the saturation value.

9. The method according to claim 7, characterized in that as a lipid precursor a 1-alkanol or an alkanal is used.

10. The method according to claim 7, characterized in that 1-dodecanol is used as alkanol.

11. The method according to any one of claims 3 to 10, characterized in that a yeast of the genus Candida, preferably Candida bombicola, Candida bogoriensis, Candida magnoliae, Candida gropengiesseri or Candida apicola, is fermented.

12. The method according to any one of claims 3 to 11, characterized in that glucose or sucrose is used as saccharide.

13. Use of the sophorose lipids according to one of claims 1 and 2 or produced by the method according to one of claims 3 to 12 as

Surfactant, cosmetics, disinfectant or pharmaceutical.