Technical field The present invention relates to a fermentative process for producing mannosylerythritol lipids, and recovering mannosylerythritol lipids by heat treatment and extraction with alcohol-like solvent.

Background of the invention Microbial surfactants have unique characteristics (i. e. mild production conditions, biodegradability, wide range of biological activities) in comparison with their chemical counterparts. They may be applied at extreme temperatures, acidity, and salt concentrations. Due to these advantages they can be used not only as ingredients in food, cosmetics and pharmaceutics, but also in environmental and energy-saving technology (Banat et al. , Appl. Microbiol. Biotech. , 2000,53, page 495-508).

Mannosylerythritol lipids (= MEL) are examples of such microbial surfactants.

Like a typical natural product, mannosylerythritol lipids do not possess a single defined structure but consist of similar compounds depending on the producing microorganism and the substrate used.

DE 198 02 450 describes a process and an application for ustilipids. These are mannosylerythritol lipids produced by micro-organisms of the genus Ustilago. In particular, a fermentation process in presence of a deposited strain Ustilago maydis DSM 11494 is described. The product is isolated by applying solvent extraction or chromatography. The crude extract contained 5.3 g/L ustilipids. After purification the yield reduced to 0.8 g/L.

In Agric. Biol. Chem (1990,54 (1), page 31-36) D. Kitamoto describes the accumulation of mannosylerythritol lipids by a Candida antartica strain.

In Agric. Biol. Chem. (1990,54 (1), page 37-40) D. Kitamoto describes mannosylerythritol lipids production by different strains of Candida antarctica. Under the optimal conditions in a shake culture, the concentration of the total lipids amounted to about 40 g/L after 8 days, corresponding to a productivity of 5 g/Ld. The same yield and productivity was also reported by Kim et al. (Biotechnol. Letters, 2002,24, pages 225- 229) but they used Candida antarctica (SY16) as production strain with oleic acid and glycerol as substrates. Kitamoto et al. focused the subsequent work on a specific strain, Pseudozyma (Candida) antarctica T 34, and succeeded in the production of 140 g/L of mannosylerythritol lipids (MEL) by additional feeding of n-octadecane (Biotechnol.

Letters, 2001,23, page 1709-1714). However, the fermentation time was 30 days, which is equivalent to a productivity of 4.6 g/Ld. All these fermentations were performed in shake flasks.

Kim et al. (Appl. Microbiol. Biotechnol. , 1999,52, pages 713-722) reported the production of 100 g/L crude MEL using Candida sp. SY16 (Candida antarctica KCTC 7804) with soybean oil as substrate performed in a 5 L bioreactor. However, the crude product contained only 4% (w/w) pure MEL. Hitherto, Adamczak and Bednarski in Biotechnol. Letters, 2000,22, 313-316) described the highest yield of MEL obtained in a bioreactor (=46 g/L) with a productivity of 7.6 g/Ld, by using Candida antartica ATCC 20509.

Accordingly, a need exists for a process for producing mannosylerythritol lipids in high yields, high productivity and followed by an easy downstream processing allowing high recovery yields of mannosylerythritol lipids.

The current invention provides such a process.

Summary of the invention The present invention discloses a fermentative process for producing and recovering mannosylerythritol lipids, said process comprising the following steps: a) Preparing a pre-culture containing a carbon source and nitrogen source for a micro-organism of the species Pseudozyma aphidis, b) preparing a fermentation culture medium comprising a nitrogen source and a fatty oil, c) adding the micro-organisms containing pre-culture to the fermentation culture medium, d) allowing the micro-organisms to grow until at least 45 g/L mannosylerythritol lipids are obtained in the fermentation medium, e) recovering of mannosylerythritol lipids.

The current invention relates to a process wherein the fermentation culture medium is prepared from a nitrogen source based on nitrate and the fatty oil is selected from the group consisting of soybean oil, sunflower oil, rapeseed oil, coconut oil, palm or palm kernel oil, canola oil, corn oil, cottonseed oil, olive oil, peanut oil, rice bran oil, safflower oil, sesame oil, almond kernel oil or any other oil suitable for use in cosmetic or food products, and mixtures of two or more thereof.

The current invention relates to a process wherein productivity of step d) of said process is higher than 8 g/Ld.

The current invention further relates to a process wherein recovering mannosylerythritol lipids from fermentation medium comprises the following steps: a) heating the fermentation medium to disintegrate the emulsified fermentation medium, b) cooling to obtain phase separation into liquid and solid phase, c) treating the solid phase with alcohol-like solvent to obtain dissolution of mannosylerythritol lipids, d) collecting mannosylerythritol lipids from the alcohol-like solution, e) optionally treating the liquid phase of step b) with alcohol-like solvent to obtain mannosylerythritol lipids, and f) optionally combining collected mannosylerythritol lipids of step d) and e).

The current invention discloses a process wherein the fermentation medium is heated to a temperature above 100°C, preferably 105°C-120°C and the alcohol-like solvent is ethanol.

The current invention relates to a process wherein the recovery yield of mannosylerythritol lipids is higher than 85%, preferably higher than 90%.

Furthermore, the current invention relates to a fermentative process comprising the following steps: a) Preparing a pre-culture containing a nitrate as nitrogen source and glucose as carbon source for the micro-organism Pseudozyma aphidis deposited on 16/04/2002 under number DSM 14930, b) preparing a fermentation culture medium comprising a glucose, nitrate and a fatty oil, c) adding the micro-organisms containing pre-culture to the fermentation culture medium, d) allowing the micro-organisms to grow until at least 70 g/L mannosylerythritol lipids are obtained in the fermentation medium, e) heating the fermentation medium to a temperature above 100°C to disintegrate the emulsified fermentation medium, f) cooling to obtain phase separation into liquid and solid phase, g) treating the solid phase with ethanol to obtain dissolution of mannosylerythritol lipids, h) collecting mannosylerythritol lipids from step g), i) treating the liquid phase of step f) with ethanol to obtain mannosylerythritol lipids, and j) combining mannosylerythritol lipids of step h) and i) to obtain a recovery yield of at least 85%, preferably higher than 90%.

The current invention relates to a process wherein in step d) the productivity is at least 10 g/Ld.

The current invention discloses a Pseudozyma aphidis strain characterised in that it produces more than 70 g/L mannosylerythritol lipids.

The current invention relates to a process wherein in step d) at least 120 g/L mannosylerythritol lipids with a productivity of more than 12 g/Ld are obtained.

The current invention further discloses a Pseudozyma aphidis strain deposited under the Budapest Treaty at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen by Technische Universitat Braunschweig on 16/04/2002 under number DSM 14930.

Furthermore, the current invention relates to the use of mannosylerythritol lipids in cosmetics for skin care and in food products.

Brief description of the drawings Figure 1 is a microscope (Nikon Optiphot; amplification: 1500) picture of Pseudozyma aphidis DSM 14930 grown on glucose and said picture is taken with a microscope. It is shown that the cells contain prominent highly refractive globules, which were even much more abundant if soybean oil was used as carbon source.

Detailed description of the invention The present invention discloses a fermentative process for producing and recovering mannosylerythritol lipids, said process comprising the following steps: . 5 a) Preparing a pre-culture containing a carbon source and nitrogen source for a micro-organism of the species Pseudozyma aphids, b) preparing a fermentation culture medium comprising a nitrogen source and a fatty oil, c) adding the micro-organisms containing pre-culture to the fermentation culture medium, d) allowing the micro-organisms to grow until at least 45 g/L mannosylerythritol lipids are obtained in the fermentation medium, e) recovering of mannosylerythritol lipids.

The pre-culture can be prepared from glucose as carbon source and ammonium nitrate as nitrogen source. Preferably, the pre-culture is prepared from 20 to 30 g/L glucose and 0.2-1. 8 g/L ammonium nitrate. The preculture can be enriched with 0.1 to 0.9 g/L potassium dihydrogen phosphate, 0.1 to 0.9 g/L magnesium sulphate and 0.5 to 3.0 g/L yeast extract. The pre-cultivation can be performed in 2 L shake flasks filled with 500 ml medium at 30°C and at 100 rpm on a rotary shaker.

The current invention relates to a process wherein the fermentation culture medium is prepared from a nitrogen source based on nitrate. The fatty oil can be any fatty oil and preferably is selected from the group consisting of soybean oil, sunflower oil, rapeseed oil, coconut oil, palm or palm kernel oil, canola oil, corn oil, cottonseed oil, olive oil, peanut oil, rice bran oil, safflower oil, sesame oil, almond kernel oil or any other oil suitable for use in cosmetic or food preparations, and mixtures of two or more thereof.

At least 45 g/L mannosylerythritol lipids with a productivity higher than 8 g/Ld is obtainable by applying a fermentation culture medium which is containing sodium nitrate. The fermentation can be performed in a bioreactor. Conductivity sensors can be used for the controlled addition of fatty oils as foam inhibitors. The controlled addition of these compounds, which also act as substrates, enables the microorganism to enhance the yield and productivity.

6 High yields of mannosylerythritol lipids are obtainable by applying soybean oil as fatty oil and sodium nitrate as nitrogen source.

The current invention further relates to a process wherein recovering mannosylerythritol lipids from fermentation medium comprises the following steps: a) heating the fermentation medium to disintegrate the emulsified fermentation medium, b) cooling to obtain phase separation into liquid and solid phase, c) treating the solid phase with alcohol-like solvent to obtain dissolution of mannosylerythritol lipids, d) collecting mannosylerythritol lipids from the alcohol-like solution, e) optionally treating the liquid phase of step b) with alcohol-like solvent to obtain mannosylerythritol lipids, and f) optionally combining collected mannosylerythritol lipids of step d) and e).

Mannosylerythritol lipids are surfactant-like or emulsifying products. By virtue of the nature of these products it is very difficult to separate and recover these products in high yields and high purity. The current invention discloses a recovery process wherein the emulsified fermentation medium is disintegrated by a heating step.

The current invention discloses a process wherein the fermentation medium is heated to a temperature above 100°C, preferably 105°C-120°C.

The fermentation medium is heated to a temperature above 100°C, preferably 110°C during 10 minutes. After cooling, phase separation occurs into a liquid phase containing the cell debris and a solid sticky phase comprising mannosylerythritol lipids.

An alcohol-like solvent, such as methanol, ethanol, propanol and/or butanol can be applied to collect the mannosylerythritol lipids. Preferably, ethanol is added to the solid sticky phase and mannosylerythritol lipids can be collected from the alcohol-like solution by filtration. In addition, the liquid phase containing the cell debris can likewise be 7 treated with alcohol-like solvent, preferably ethanol. To recover residual mannosylerythritol lipids from this fraction, centrifugation can be applied.

Both fractions containing mannosylerythritol lipids are combined and residual ethanol can be removed by evaporation. After drying, mannosylerythritol lipids are recovered.

The current invention relates to a process wherein the recovery yield of mannosylerythritol lipids is higher than 85%, preferably higher than 90%.

The mannosylerythritol lipids, thus recovered, have a purity of at least 90%.

Furthermore, the current invention relates to a fermentative process comprising the following steps: a) Preparing a pre-culture containing a nitrate as nitrogen source and glucose as carbon source for the micro-organism Pseudozyma aphidis deposited on 16/04/2002 under number DSM 14930, b) preparing a fermentation culture medium comprising glucose, a nitrate and a fatty oil, c) adding the micro-organisms containing pre-culture to the fermentation culture medium, d) allowing the micro-organisms to grow until at least 70 g/L mannosylerythritol lipids are obtained in the fermentation medium, e) heating the fermentation medium to a temperature above 100°C to disintegrate the emulsified fermentation medium, f) cooling to obtain phase separation into liquid and solid phase, g) treating the solid phase with ethanol to obtain dissolution of mannosylerythritol lipids, h) collecting mannosylerythritol lipids from step g), i) treating the liquid phase of step f) with ethanol to obtain mannosylerythritol lipids, and j) combining mannosylerythritol lipids of step h) and i) to obtain a recovery yield of at least 85%, preferably higher than 90%.

The current invention relates to a process wherein in step d) the productivity is at least 10 g/Ld. In fact, it relates to a process wherein in step d) at least 120 g/L mannosylerythritol lipids with a productivity of more than 12 g/Ld are obtained.

By applying Pseudozyma aphidis deposited on 16/04/2002 under number DSM 14930, at least 70 g/L mannosylerythritol lipids are obtained in the fermentation medium and recovery yield is at least 85% with a purity of 90%. The feed solution can contain high concentrations of substrates such as glucose at 200-400 g/L, a nitrate, preferably sodium nitrate, at 10-30 g/L, and yeast extract at a concentration of 10-30 g/L.

Furthermore by applying Pseudozyma aphidis deposited on 16/04/2002 under number DSM 14930, it is possible to obtain at least 165 g/L mannosylerythritol lipids with a productivity of 14g/Ld in the bioreactor medium and recovery yield is at least 85%, preferably higher than 90% with a purity of at least 90%.

The strain Pseudozyma aphidis is synonym to Sterigmatos aphidis which was <BR> <BR> described in detail by Henninger and Windisch (Arch. Microbiol. , 1975,105, page 49- 50). The strain was isolated from the secretions of Aphididae on leaves of Solanum pseudocapsicum and is able to assimilate both inositol and potassium nitrate. A broad range of carbon sources can be used by this fungi such as pentoses, hexoses, sugar alcohols, soluble starch, ethanol and organic acids. Urease reaction is positive. Staining with Dazionium Blue B salt is also positive. Initial growth on malt-extract agar at 28°C shows elongated cells (1.4-3. 6) x (4.3-11. 5 um) often pointed at one or both ends. Very long cells up to 40 Am are also observed. After growth is completed the agar is covered with a thin aerial mycelium made up of ramifying, acropetal chains of fusiform blastconidia originating from short denticles, sterigma-like structures or attenuating hyphae. Microscopically it shows septated hyphae with the cytoplasm retracted in some cells and with retraction septa. The streak culture is variable, sometimes powdery, mostly 9 rough and flat with rough margin. The colour is cream to yellow. The fungi is anamorph to Ustilaginales because the comparison of the 26S ribosomal DNA placed it in the same group with Ustilago maydis (Boekhout, J. Gen. Appl. Microbiol., 1995,41, page 359- 366).

The current invention further discloses a Pseudozyma aphidis strain deposited under the Budapest Treaty at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen by Technische Universität Braunschweig on 16/04/2002 under number DSM 14930. Pseudozyma aphidis DSM 14930 was isolated from soil.

Furthermore, the current invention relates to the use of mannosylerythritol lipids in cosmetics for skin care. Essential criteria for identifying products that are applicable as cosmetics for skin care are skin compatibility and protection, efficacy, sensory properties, no critical impurities and environmental compatibility. Mannosylerythritol lipids fulfil these criteria.

The current invention further relates to the use of mannosylerythritol lipids (as an ingredient or additive) in food products and preparations. For example, the mannosylerythritol lipids of the present invention can be used as non-toxic emulsifiers in salad dressings, mayonnaise, margarine and other fat-based products, and in dairy (e. g. ice-cream), confectionery (e. g. chocolate) and bakery products.

The current invention has the following advantages: - fermentation process results in high fermentation yields of mannosylerythritol lipids, especially when performed in a bioreactor, - the process results in high productivity of mannosylerythritol lipids, - foam formation is prevented by the controlled addition of fatty oils which simultaneously act as substrates, - downstream processing is comprising a simple heating step followed by cooling with simultaneous formation of a sticky solid phase enriched with mannosylerythritol lipids, - alcohol-like solvent can be added for further purification, - high recovery yields of mannosylerythritol lipids are obtainable by the current disclosed process, - mannosylerythritol lipids are obtainable in high purity because nearly all of the triglycerides added and enzymatically released fatty acids are consumed at the end of the cultivation run.

The current invention is illustrated by way of the following examples.

Example 1 1. Preculture preparation Medium: Glucose H20 66 g/L NH4NO3 1 g/L KH2PO4 0.5 g/L MgSO4 7H20 0.2 g/L Yeast extract 1 g/L 132 g Glucose H2O was dissolved in 1 L tap water. The sugar solution was prepared in double concentration and had to be sterilized separately from the likewise double concentrated salt and yeast extract solution. After cooling down the two 1 L solutions were combined at a ratio of 1: 1 under aseptic conditions in order to yield 500 ml medium filled in two shake flasks equipped with two baffles. The starting pH was 6.

The medium was inoculated with a few colonies of Pseudozyma aphidis DSM 70725, provided by the DSMZ Deutschen Stammsammlung für Mikroorganismen und Stammkulturen, Braunschweig, and incubated for 3 days at 27°C and 100 rpm on a rotary shaker.

2. Bioreactor cultivation Medium: Soybean oil 80 ml/L NaN03 2 g/L Yeast extract 1 g/L KH2PO4 0.2 g/L MgS04-7 H20 0.2 g/L The 72 L bioreactor was equipped with two Rushton turbines of 14.5 cm diameter. The upper impeller was installed 5 cm below the medium surface. The bioreactor was filled with 30 L tap water and the salts and yeast extract were added in appropriate concentrations. The soybean oil was sterilized separately and added aseptically to the medium. The resulting pH of 6.2 was not adjusted throughout the cultivation. The pre-culture, separately prepared, was added and fermentation was continued for 9 days. During the cultivation the stirrer speed and aeration rate were gradually reduced from 300 rpm to 250 rpm and 720 L/h to 108 L/h, respectively, in order to reduce foam formation and to manually control the p02 at approximately 60%.

After 0.8 days N-liinitation occurred with simultaneous cessation of growth indicated by the beginning stationary phase. Cell protein was chosen as equivalent for bio dry mass because the gravimetric determination of bio dry mass was erroneous due to the incorporation of storage compounds inside the cell which leads to an increase of biomass after N-limitation. The mass ratio between cell protein and bio dry mass before N- limitation was approximately 1: 5 so that a cell protein concentration of 2.5 g/L is roughly equivalent to a bio dry mass of 12.5 g/L. Cell protein was determined using the well known method of Lowry after solvent extraction of the biomass and subsequent alkaline cell disruption (Spoeckner et al., Appl. Microbiol. Biotechnol, 1999,51, pages 33-39).

After 2.6 days the addition of soybean oil was started at a rate of 0.35 ml/Lh and stopped after 7.7 days. After one day addition of soybean oil, the formation of MEL- beads indicated the enhanced product formation. Overall 123 mIJL soybean oil (80 ml/L initial, 43 ml/L added) was converted into maximum 74 g/L of mannosylerythritol lipids resulting in a yield coefficient of 0.65 (g/g) and a maximum productivity of 9.6 g/Ld.

3. Recovery of mannosylervthritol lipids The fermentation medium was heated to 110°C for 10 minutes followed by cooling to room temperature. The heating procedure divided the culture suspension into four separate phases: the upper phase contained triglycerides and fatty acids, the second phase was aqueous, the third was composed of cells and cell debris, the solid fourth phase pasted at the bottom and was enriched with mannosylerythritol lipids (=MEL). The former heavier than water MEL-beads containing mannosylerythritol lipids and unconsumed fatty acids and triglycerides turned to a sticky solid phase enriched by MEL and depleted by fatty acids and triglycerides. Lower temperatures or a shorter processing time did not lead to a clear formation of the new sticky solid phase. Small amounts of the MEL were additionally found in the cell containing phase. After discarding the upper oil and aqueous phase, as well as the upper part of the third cell containing phase, the residue was treated as follows: the aqueous phase containing cell debris was decanted and extracted by ethanol. Cell debris were separated by centrifugation. The solid sticky phase was dissolved in ethanol and cell debris were separated by filtration. Both mannosylerythritol lipids containing fractions were combined and ethanol was removed by vacuum evaporation. The residual highly viscous product was lyophilized and mannosylerythritol lipids were recovered at 88% recovery yield with a purity of 91%.

Purity of mannosylerythritol lipids was determined by HPLC analysis on a silica column with evaporative light scattering detector and applying a gradient solvent program of chloroform and methanol at a flow rate of 1 ml/min.

Example 2 1. Preculture preparation The preculture was prepared as described in Example 1, but the medium was inoculated with a few colonies of Pseudozyma aphidis deposited on 16/4/2002 under number DSM 14930.

2. Bioreactor cultivation Medium: Glucose 30 g/L Soybean oil 20 ml/L 14 NaN03 3 g/L Yeast extract 1 g/L KH2PO4 0. 2 g/L MgS04-7 H20 0. 2 g/L Cultivations conditions: temperature 27°C, stirrer speed 300 rpm, aeration rate 1080 1/h.

The 72 L bioreactor was equipped with two Rushton turbines of 14.5 cm diameter. The upper impeller was installed 5 cm below the medium surface. The bioreactor was filled with 30 L tap water and the salts, glucose and yeast extract were added in appropriate concentration. The soybean oil was sterilized separately and added aseptically to the medium. Glucose was used as additional carbon source beside soybean oil. The resulting pH of 6.2 was not adjusted throughout the cultivation. The pre-culture, separately prepared, was added and fermentation was continued for 12 days.

At 1.2 days nitrate was completely consumed and growth turned to the stationary phase. In order to initiate regrowing of the microorganism the addition of a concentrated solution (3.1 L) of glucose (342 g/L), sodium nitrate (20 g/L) and yeast extract (14 g/L) was started at 1.75 days and at a rate of 2. 2 ml/min. The complete solution was fed after 2.8 days. The addition of soybean oil for foam reduction was necessary between 0.75 and 3.75 days and in total 6.12 L soybean oil was added under controlled conditions. After 11.75 days, 165 g/L mannosylerythritol lipids were formed with a yield coefficient of 0.86 (w/w) and a productivity of 14.04 g/Ld.

3. Recovery of mannosvlerythritol lipids Mannosylerythritol lipids were recovered as described in example 1.