Field of the invention

The invention relates to a mixture composition comprising glucolipids, to its use for producing formulations and to formulations comprising this mixture composition.

Prior art

EP2787065 discloses formulations comprising rhamnolipids, where the content of di-rhamnolipids is bigger than the content of mono-rhamnolipids, and that the excess of di-rhamnolipids increases the rate of foam formation and/or for foam stabilization.

Matsuyama T., Tanikawa T., Nakagawa Y. (2011 ) Serrawettins and Other Surfactants Produced by Serratia. In: Soberon-Chavez G. (eds) Biosurfactants. Microbiology Monographs, vol 20. Springer, Berlin, Heidelberg disclose a structure of rubiwettin RG1 to be beta-D-glucopyranosyl 3-(3'- hyd roxytetrad ecanoy loxy )d eca noate .

DE19648439 discloses mixtures used for the preparation of washing-up liquids comprising glycolipids and surfactants.

It was an object of the invention to provide bio-based compositions which have the same adventages as di-rhamnolipids, while having a simpler structure and/or lower molecular weight.

Description of the invention

Surprisingly, it has been found that the mixture composition described below is able to achieve the object addressed by the invention.

The present invention therefore provides mixture compositions comprising certain glucolipids in defined weight ratios.

The present invention further provides formulations comprising the mixture compositions according to the invention.

One advantage of the mixture compositions according to the invention is their excellent foam stability under aqueous conditions.

A further advantage of the mixture compositions according to the invention is their outstanding foam volume under aqueous conditions. A further advantage of the mixture compositions according to the invention is their exceptional foaming behaviour.

A further advantage of the mixture compositions according to the invention is their simple formulatability in any desired aqueous surface-active systems.

A further advantage of the mixture compositions according to the invention is their good thickenability with conventional thickeners in formulations.

A further advantage is their good ability to wash off skin and hair.

A further advantage of the mixture compositions according to the invention is their mildness and good physiological compatibility, in particular characterized by a high value in the red blood cell (RBC) test.

A further advantage is their good skin feel during and after washing.

A further advantage of the mixture compositions according to the invention is that they leave behind a soft skin feel after washing.

A further advantage of the mixture compositions according to the invention is that they leave behind a smooth skin feel after washing.

A further advantage of the mixture compositions according to the invention is that they have a refatting effect on the skin.

A further advantage of the mixture compositions according to the invention is that they can be synthesized essentially free from oil.

A further advantage is that the mixture compositions according to the invention can be produced with higher space-time yield, higher carbon yields, and higher product concentration than di- rhamnolipids.

In connection with the present invention, the term“glucolipid” is understood as meaning compounds of the general formula (I) or salts thereof,

formula (I)

where

m = 1 or 0,

R ¹ and R ² = independently of one another identical or different organic radical having 2 to 24 carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxy- substituted, optionally unsaturated, in particular optionally mono-, di- or triunsaturated, alkyl radical, preferably one selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH ₂ )o-CH3 where o = 1 to 23, preferably 4 to 12.

Distinct glucolipids are abbreviated according to the following nomenclature:

“GL-CXCY” is understood as meaning glucolipids of the general formula (I) in which one of the radicals R ¹ and R ² = (CH ₂ )o-CH3 where o = X-4 and the remaining radical R ¹ or R ² = (CH ₂ )o-CH3 where o = Y-4.

The nomenclature used thus does not differentiate between“CXCY” and“CYCX”.

If one of the aforementioned indices X and/or Y is provided with“:Z”, then this means that the respective radical R ¹ and/or R ² = an unbranched, unsubstituted hydrocarbon radical with X-3 or Y-3 carbon atoms having Z double bonds.

In connection with the present invention, the“pH” is defined as the value which is measured for a corresponding substance at 25°C after stirring for five minutes using a pH electrode calibrated in accordance with ISO 4319 (1977).

In connection with the present invention, the term“aqueous medium” is understood as meaning a composition which comprises at least 5% by weight of water, based on the total composition under consideration.

Unless stated otherwise, all the stated percentages (%) are percentages by mass.

The present invention provides a mixture composition comprising glucolipids of the general formula (I) or salts thereof

formula (I),

where

R ¹ and R ² = independently of one another identical or different organic radical having 2 to 24 carbon atoms,

characterized in that the mixture composition comprises

at least 51 % by weight to preferably 98% by weight, preferably 60% by weight to 95% by weight, more preferably 70% by weight to 90% by weight, particularly preferably 75% by weight to 85% by weight, glucolipids GL-C10C10 of the general formula (I) with R ¹ and R ² = (CH ₂ )6-CH3, where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present. A preferred mixture composition according to the invention is characterized in that the pH of the mixture composition at 25°C is from 3.5 to 9, preferably from 5 to 7 and particularly preferably from 5.6 to 6.6.

The glucolipids present in the mixture composition according to the invention are present at least partially as salts on account of the given pH.

In preferred mixture compositions according to the invention the cations of the glucolipid salts present are selected from the group comprising, preferably consisting of, Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ , Al ³⁺ , NH ₄ ⁺ , primary ammonium ions, secondary ammonium ions, tertiary ammonium ions and quaternary ammonium ions.

Exemplary representatives of suitable ammonium ions are tetramethylammonium,

tetraethylammonium , tetrapropylammonium, tetrabutylammonium and [(2- hydroxyethyl)trimethylammonium] (choline) and also the cations of 2-aminoethanol (ethanolamine, MEA), diethanolamine (DEA), 2,2',2"-nitrilotriethanol (triethanolamine, TEA), 1-aminopropan-2-ol (monoisopropanolamine), ethylenediamine, diethylenetriamine, triethylenetetramine,

tetraethylenepentamine, 1 ,4-diethylenediamine (piperazine), aminoethylpiperazine and aminoethylethanolamine.

The mixtures of the abovementioned cations may also be present as cations of the glucolipid salts present according to the invention.

Particularly preferred cations are selected from the group comprising, preferably consisting of, Na ⁺ , K ⁺ , NH ₄ ⁺ and the triethanolammonium cation.

It may be advantageous and is therefore preferred if the mixture composition according to the invention comprises

1 % by weight to 30% by weight, preferably 5% by weight to 25% by weight, particularly preferably 10% by weight to 20% by weight, of GL-C8C10,

where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present.

A preferred mixture composition according to the invention is characterized in that the mixture composition comprises

0.5% by weight to 20% by weight, preferably 3% by weight to 17% by weight, particularly preferably 5% by weight to 15% by weight, of GL-C10C12: 1 ,

where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present.

A further preferred mixture composition according to the invention is characterized in that the mixture composition comprises

0.5% by weight to 20% by weight, preferably 2% by weight to 15% by weight, particularly preferably 3% by weight to 12% by weight, of GL-C10C12 where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present.

A particularly preferred mixture composition according to the invention is characterized in that the mixture composition comprises

1 % by weight to 30% by weight, preferably 5% by weight to 25% by weight, particularly preferably 10% by weight to 20% by weight, of GL-C8C10,

0.5% by weight to 20% by weight, preferably 3% by weight to 17% by weight, particularly preferably 5% by weight to 15% by weight, of GL-C10C12: 1 ,

0.5% by weight to 20% by weight, preferably 2% by weight to 15% by weight, particularly preferably 3% by weight to 12% by weight, of GL-C10C12

where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present.

A very particularly preferred mixture composition according to the invention is characterized in that the mixture composition comprises

10% by weight to 20% by weight, of GL-C8C10,

5% by weight to 15% by weight, of GL-C10C12: 1 ,

3% by weight to 12% by weight, of GL-C10C12

where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present.

Over and above this, it is preferred if the mixture composition according to the invention comprises glucolipids of the formula GL-CX in only small amounts. In particular, the mixture composition according to the invention comprises preferably

0% by weight to 5% by weight, preferably 0,01 % by weight to 4% by weight, particularly preferably 0, 1 % by weight to 3% by weight, of GL-C10,

where the percentages by weight refer to the sum of all of the glucolipids of the general formula (I) present.

The mixture composition according to the invention preferably contains at least 60% by weight, preferably at least 80% by weight, particularly preferably at least 90% by weight, of glucolipids of the general formula (I), where the percentages by weight refer to the total dry mass of the overall mixture composition.

The term“total dry mass” in the context of the present invention is understood to mean the portion of the mixture composition according to the invention which remains - naturally in addition to water - after the mixture composition according to the invention has been freed of the components which are liquid at 25°C and 1 bar. The mixture compositions according to the invention can advantageously be incorporated into cosmetic formulations in particular.

Consequently, a further subject matter of the present invention is the use of the mixture compositions according to the invention for producing formulations, in particular cosmetic formulations, and also the formulations, in particular cosmetic formulations, which comprise the mixture composition according to the invention.

The formulation according to the invention preferably contains 0,5% by weight to 20% by weight, preferably 2% by weight to 15% by weight, particularly preferably 3% by weight to 12% by weight, of glucolipids of the general formula (I), where the percentages by weight refer to the overall formulation.

Besides the mixture compositions according to the invention, preferred formulations according to the invention comprise at least - next to the glucolpid - one further surfactant, it being possible to use, for example, anionic, nonionic, cationic and/or amphoteric surfactants. Preferably, from an applications-related point of view, preference is given to mixtures of anionic and nonionic surfactants. The total surfactant content of the formulation is preferably 5 to 60% by weight and particularly preferably 15 to 40% by weight, based on the total formulation.

The nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, in particular primary alcohols having preferably 8 to 18 carbon atoms and on average 1 to 12 mol of ethylene oxide (EO) per mol of alcohol, in which the alcohol radical can be linear or preferably 2-position methyl-branched or can contain linear and methyl-branched radicals in a mixture, as are customarily present in oxo alcohol radicals. In particular, however, alcohol ethoxylates with linear radicals from alcohols of native origin having 12 to 18 carbon atoms, for example from coconut, palm, tallow fat or oleyl alcohol, and on average 2 to 8 EO per mol of alcohol are preferred. The preferred ethoxylated alcohols include, for example, C12-C14-alcohols with 3 EO, 4 EO or 7 EO, C9-C11 -alcohol with 7 EO, C13-C15-alcohols with 3 EO, 5 EO, 7 EO or 8 EO, C12-C 18-alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as mixtures of C12-C14-alcohol with 3 EO and C12-C18-alcohol with 7 EO. The stated degrees of ethoxylation are statistical average values which can be an integer or a fraction for a specific product. Preferred alcohol ethoxylates have a narrowed homolog distribution. In addition to these nonionic surfactants, it is also possible to use fatty alcohols with more than 12 EO. Examples thereof are tallow fatty alcohol with 14 EO, 25 EO, 30 EO or 40 EO. Nonionic surfactants which contain EO and PO (propylene oxide) groups together in the molecule can also be used. In this connection, it is possible to use block copolymers with EO-PO block units or PO-EO block units, but also EO-PO-EO copolymers or PO-EO-PO copolymers.

It is of course also possible to use mixed alkoxylated nonionic surfactants in which EO and PO units are not distributed blockwise, but randomly. Such products are obtainable as a result of the simultaneous action of ethylene oxide and propylene oxide on fatty alcohols.

Furthermore, alkyl glycosides can also be used as further nonionic surfactants.

A further class of preferably used nonionic surfactants, which are used either as the sole nonionic surfactant or in combination with other nonionic surfactants, are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, preferably having 1 to 4 carbon atoms in the alkyl chain, in particular fatty acid methyl esters, as are described for example in the Japanese patent application JP 58/217598 or which are preferably prepared by the process described in the international patent application WO-A-90/13533.

Nonionic surfactants of the amine oxide type, for example N-cocoalkyl-N,N-dimethylamine oxide and N-tallowalkyl-N,N-dihydroxyethylamine oxide, and of the fatty acid alkanolamide type may also be suitable. The amount of these nonionic surfactants is preferably not more than that of the ethoxylated fatty alcohols, in particular not more than half thereof.

Further suitable surfactants are polyhydroxy fatty acid amides; the polyhydroxy fatty acid amides are substances which can usually be obtained by reductive amination of a reducing sugar with ammonia, an alkylamine or an alkanolamine and subsequent acylation with a fatty acid, a fatty acid alkyl ester or a fatty acid chloride.

The anionic surfactants used are, for example, those of the sulphonate and sulphate type. Suitable surfactants of the sulphonate type here are preferably C9-C13-alkylbenzenesulphonates, olefinsulphonates, i.e. mixtures of alkene- and hydroxyalkanesulphonates, and also disulphonates, as are obtained, for example, from C12-C18-monoolefins with a terminal or internal double bond by sulphonation with gaseous sulphur trioxide and subsequent alkaline or acidic hydrolysis of the sulphonation products. Also of suitability are alkanesulphonates which are obtained from C12-C18- alkanes, for example by sulphochlorination or sulphoxidation with subsequent hydrolysis or neutralization. Similarly, the esters of a-sulpho fatty acids (ester sulphonates), for example the o sulphonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids, are also suitable.

Further suitable anionic surfactants are sulphated fatty acid glycerol esters. Fatty acid glycerol esters are to be understood as meaning the mono-, di- and triesters, and also mixtures thereof, as are obtained in the preparation by esterification of a monoglycerol with 1 to 3 mol of fatty acid or in the transesterification of triglycerides with 0.3 to 2 mol of glycerol. Preferred sulphated fatty acid glycerol esters here are the sulphation products of saturated fatty acids having 6 to 22 carbon atoms, for example of caproic acid, caprylic acid, capric acid, myristic acid, lauric acid, palmitic acid, stearic acid or behenic acid.

Preferred alk(en)yl sulphates are the alkali metal and in particular the sodium salts of the sulphuric acid half-esters of the C12-C18-fatty alcohols, for example from coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or the C10-C20-oxo alcohols and those half-esters of secondary alcohols of these chain lengths. Furthermore, preference is given to alk(en)yl sulphates of the specified chain length which contain a synthetic straight-chain alkyl radical prepared on a petrochemical basis, and which have an analogous degradation behaviour to the suitable compounds based on fatty chemical raw materials. From the point of view of washing, the C12-C16-alkyl sulphates and C12-C18-alkyl sulphates and also C14-C18-alkyl sulphates are preferred. 2,3-Alkyl sulphates, which are prepared for example in accordance with the US patent specifications 3,234,258 or 5,075,041 and can be obtained as commercial products of the Shell Oil Company under the name DAN®, are also suitable anionic surfactants. The sulphuric acid monoesters of the straight-chain or branched C7-C20-alcohols ethoxylated with 1 to 6 mol of ethylene oxide, such as 2-methyl-branched C9-C11 -alcohols having on average 3.5 mol of ethylene oxide (EO) or C12-C18-fatty alcohols with 1 to 4 EO, are also suitable. On account of their high foaming behaviour, they are used in cleaning compositions only in relatively small amounts, for example in amounts of from 1 to 5% by weight.

Further suitable anionic surfactants are also the salts of alkylsulphosuccinic acid, which are also referred to as sulphosuccinates or as sulphosuccinic acid esters and constitute the monoesters and/or diesters of sulphosuccinic acid with alcohols, preferably fatty alcohols and in particular ethoxylated fatty alcohols. Preferred sulphosuccinates contain C8-C18-fatty alcohol radicals or mixtures of these. Particularly preferred sulphosuccinates contain a fatty alcohol radical which is derived from ethoxylated fatty alcohols. In this connection, sulphosuccinates whose fatty alcohol radicals are derived from ethoxylated fatty alcohols with a narrow homolog distribution are particularly preferred in turn. It is likewise also possible to use alk(en)ylsuccinic acid having preferably 8 to 18 carbon atoms in the alk(en)yl chain or salts thereof.

Particularly preferred anionic surfactants are soaps. Also of suitability are saturated and unsaturated fatty acid soaps, such as the salts of lauric acid, myristic acid, palmitic acid, stearic acid, (hydrogenated) erucic acid and behenic acid, and also soap mixtures derived in particular from natural fatty acids, for example coconut, palm kernel, olive oil or tallow fatty acid.

The anionic surfactants including the soaps can be in the form of their sodium, potassium or ammonium salts, as well as soluble salts of organic bases, such as mono-, di- or triethanolamine. Preferably, the anionic surfactants are in the form of their sodium or potassium salts, in particular in the form of the sodium salts.

Amphoteric surfactants which can be used according to the invention are those surface-active compounds which carry at least one quaternary ammonium group and at least one -COO - or -SO3 group in the molecule. Particularly preferred amphoteric surfactants in this connection are betaine surfactants such as alkyl- or alkylamidopropylbetaines. In particular, betaines such as the N-alkyl- N,N-dimethylammonium glycinates, e.g. the cocoalkyldimethylammonium glycinate,

N-acylaminopropyl-N,N-dimethylammonium glycinates, e.g. the

cocoacylaminopropyldimethylammonium glycinate, the C12-C18-alkyldimethylacetobetaine, the cocoamidopropyldimethylacetobetaine, 2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines and sulphobetaines having in each case 8 to 18 carbon atoms in the alkyl or acyl group, and also the cocoacylaminoethylhydroxyethylcarboxymethyl glycinate are preferred here. A particularly preferred zwitterionic surfactant is the N,N-dimethyl-N-(lauroylamidopropyl)ammoniumacetobetaine known under the INCI name Cocamidopropyl Betaine.

Further suitable amphoteric surfactants are formed by the group of amphoacetates and amphodiacetates, in particular, for example, coco- or laurylamphoacetates or -diacetates, the group of amphopropionates and amphodipropionates, and the group of amino acid-based surfactants such as acyl glutamates, in particular disodium cocoyl glutamate and sodium cocoyl glutamate, acyl glycinates, in particular cocoyl glycinates, and acyl sarcosinates, in particular ammonium lauroyl sarcosinate and sodium cocoyl sarcosinate.

Furthermore, the formulations according to the invention can comprise at least one additional component selected from the group of

emollients,

emulsifiers,

thickeners/viscosity regulators/stabilizers,

UV photoprotective filters,

antioxidants,

hydrotropes (or polyols),

solids and fillers,

film formers,

pearlescent additives,

deodorant and antiperspirant active ingredients,

insect repellents,

self-tanning agents,

preservatives,

conditioners,

perfumes,

dyes,

odour absorbers,

cosmetic active ingredients,

care additives,

superfatting agents,

solvents.

Substances which can be used as exemplary representatives of the individual groups are known to the person skilled in the art and can be found for example in the German application

DE 102008001788.4. This patent application is hereby incorporated by reference and thus forms part of the disclosure.

As regards further optional components and the amounts of these components used, reference is made expressly to the relevant handbooks known to the person skilled in the art, for example K. Schrader,“Grundlagen und Rezepturen der Kosmetika [Fundamentals and Formulations of Cosmetics]”, 2nd edition, page 329 to 341 , Hijthig Buch Verlag Heidelberg.

The amounts of the respective additives are dependent on the intended use.

Typical guide formulations for the respective applications are known prior art and are contained for example in the brochures of the manufacturers of the respective base materials and active ingredients. These existing formulations can generally be adopted unchanged. If required, however, the desired modifications can be undertaken without complication by means of simple experiments for the purposes of adaptation and optimization. The mixture compositions according to the invention and the formulations according to the invention comprising the mixture composition according to the invention can advantageously be used for the cleaning of surfaces. In this form of the use according to the invention, the surface is preferably the surface of a living being, in particular of a person, with such surfaces particularly preferably being selected from skin and hair. In the context of the inventive use on the surface of a living being, the inventive use is a non-therpeutic use, preferably a cosmetic use.

The examples listed below describe the present invention by way of example without any intention of limiting the invention, the scope of application of which arises from the entire description and the claims, to the embodiments specified in the examples.

The following figures form part of the description:

Figure 1 : Foam volume of different lipids vs. time

Examples:

Example 1: Construction of an expression vector for the P. aeruginosa gene rhIA and S. rubidaea gene rbwB

For the heterologous expression of the gene rhIA (SEQ ID NO: 3) from P. aeruginosa and rbwB (SEQ ID NO: 1 ) from S. rubidaea the plasmid pACYC_rhlA_Pa rbwB_Srub was constructed. The synthetic operon consisting of rhlA_Pa (SEQ ID NO: 7) which encodes a 3-(3- hydroxyalkanoyloxy)alkanoic acid (HAAs) synthase (RhIA, SEQ ID NO: 4) and a

glucosyltransferase (RbwB, SEQ ID NO: 2), respectively, was cloned under the control of the rhamnose inducible promoter Pr _ha into the vector pACYCATh-5. Downstream of the synthetic operon a terminator sequence is located. The genes were amplified from genomic DNA of P. aeruginosa and S. rubidaea respectively via PCR. The P _Rha promoter cassette (SEQ ID NO: 5) and the terminator sequence (SEQ ID NO: 6) were amplified from E. coli K12 genomic DNA. The vector is based on pACYC184 (New England Biolabs, Frankfurt /Main, Germany) and carries a p15A origin of replication for E. coli and a pVS1 origin of replication for the replication in P. putida KT2440. The pVS1 origin comes from the Pseudomonas plasmid pVS1 (Itoh Y, Watson JM, Haas D, Leisinger T, Plasmid 1984, 11 (3), 206-20). rhIA and rbwB were fused via cross-over PCR to generate an optimized operon. For amplification the Phusion™ High-Fidelity Master Mix from New England Biolabs (Frankfurt/Main, Germany) was used according to manufacturer’s manual. In the next step the fusion construct was cloned into the vector pACYCATh-5 using the restriction sites Apa\IPspX\. The ligated product was transformed into chemically competent E. coli DH5a cells (New England Biolabs, Frankfurt/Main, Germany). Procedure of PCR purification, cloning and transformation were carried out according to manufacturer’s manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting plasmid was named pACYC_rhlA_Pa rbwB_Srub (SEQ ID NO: 8).

The P. putida strain KT2440 was transformed with the plasmid pACYC_rhlA_Pa rbwB_Srub by means of electroporation (Iwasaki K, et al., Biosci. Biotech. Biochem. 1994. 58(5):851-854)) and plated onto LB-agar plates supplemented with kanamycin (50 pg/nnL). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named BS-PP-368 (P. putida KT2440 pACYC_rhlA_Pa rbwB_Srub).

Example 2: Production of glucolipid with strain BS-PP-368 (P. putida KT2440 pACYC_rhlA_Pa rbwB_Srub)

For the production of glucolipid the DASGIP® parallel bioreactor system from Eppendorf

(Hamburg, Germany) was used. The fermentation was performed using 1 L reactors. pH and p02 were measured online for process monitoring. OTR/CTR measurements served for estimating the metabolic activity and cell fitness, inter alia.

The pH electrodes were calibrated by means of a two-point calibration using standard solutions of pH 4.0 and pH7.0, as specified in DASGIP’s technical instructions. The reactors were equipped with the necessary sensors and connections as specified in the technical instructions, and the agitator shaft was fitted. The reactors were then filled with 300 ml water and autoclaved for 20 min at 121 °C to ensure sterility. The pO _å electrodes were connected to the measuring amplifiers and polarized overnight (for at least 6 h). Thereafter, the water was removed under a clean bench and replaced by fermentation medium (2.2 g/L (NH4)2S04, 0.02 g/L NaCI, 0.4 g/L MgS04 X 7Hå0, 0.04 g/L CaCl2 x 2Hå0, sterilized separately: 2 g/L KH2PO4, 8.51 g/L KH2PO4, 20 g/L glucose, 10 mL/L trace elements solution M12 (sterile-filtered: 0.2 g/L ZnS04 x 7 H2O, 0.1 g/L MnCL x 4Hå0, 1.5 g/L Nas-Citrat x 2 H2O, 0.1 g/L CuS0 ₄ x 5 H2O, 0.002 g/L N1CI2 x 6 H2O, 0.003 g/L Na ₂ Mo0 ₄ x 2 H2O, 0.03 g/L H3BO3, 1 g/L FeS04 x 7 H2O). Thereafter, the p02 electrodes were calibrated to 100 % with a one-point calibration (stirrer: 600 rpm/aeration 10 sl/h air), and the feed, correction agent and induction agent lines were cleaned by“cleaning in place” as specified in the technical instructions. To this end, the tubes were rinsed first with 70 % ethanol, then with 1 M NaOH, then with sterile fully-demineralized water and, finally, filled with the respective media.

Using the P. putida strain BS-PP-368, 25 ml LB1 medium (10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCI, pH 7.0) supplemented with kanamycin (50 pg/nnL) in a baffeled shake flask were inoculated with 100 pi of a glycerol stock solution and incubated for ~18 h over night at 30 °C and 200 rpm. The first preculture was used to inoculate 50 ml seed medium (autoclaved: 4.4 g/L Na2HP04 ^* 2 H2O, 1.5 g/L KH2PO4, 1 g/L NH4CI, 10 g/L yeast extract, sterilized separately: 20 g/L glucose, 0.2 g/L MgS04 ^* 7 H2O, 0.006 g/L FeCL, 0.015 g/L CaCL, 1 ml/L trace elements solution SL6 (sterile-filtered: 0.3 g/L H3BO3, 0.2 g/L C0CI2 x 6 H2O, 0.1 g/L ZnS0 x 7 H2O, 0.03 g/L MnCI ₂ x 4H2O, 0.01 g/L CuCI ₂ x 2 H2O, 0.03 g/L Na ₂ Mo0 x 2 H2O, 0.02 g/L NiCI ₂ x 6 H ₂ 0) in a 500 ml baffeled shake flask (starting Oϋboo 0.2). The culture were incubated for ~7 h at 200 rpm and 30°C. In order to inoculate the reactors with an optical density of 0.7, the Oϋboo of the second preculture stage was measured and the amount of culture required for the inoculation was calculated.

The required amount of culture was added with the help of a 30 ml syringe through a septum into the heat-treated and aerated reactor.

The standard program shown in Table 1 is used: a)

b)

c)

Table! Standard program used for heated and aerated reactor The pH was adjusted unilaterally to pH 7.0 with 12.5 % strength ammonia solution. During the growth phase and the biotransformation, the dissolved oxygen (pO _å or DO) in the culture was adjusted to at least 30 % via the stirrer speed and the aeration rate. After the inoculation, the DO dropped from 100 % to these 30 %, where it was maintained stably for the remainder of the fermentation.

The fermentation was carried out as a fed batch. The feed starts with a 2.5 g/L*h glucose feed, composed of 500 g/L glucose, and was triggered via the DO peak which indicates the end of the batch phase. 3 h after the feed start, the expression of glucolipid production was induced with 0.2 % (w/v) rhamnose. The inducer concentration referred to the volume at the beginning of fermentation. For both sugars stock solution of 220 g/L was used. The production of glucolipid started with the induction. At specified time points samples were taken from the fermenter to determine the concentration of glucolipids produced. After 65 h fermentation 1 1.1 g/L glucolipid was produced.

Example 3: HPLC-based quantification of glucolipids

Quantification of glucolipids was carried out by means of HPLC. Using a displacement pipette (Combitip), 900 pi of 70 % (v/v) n-propanol was introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed for minimization of evaporation. The addition of 100 pi fermentation broth followed. After shaking for 1 min in a Retsch mill at a frequency of 30 Hz, the resulting crude extract mixture was centrifuged for 5 min at 13,000 rpm, and 800 mI of the clear supernatant was transferred into an HPLC vial. Further dilutions of cell broth were carried out in 55 % (v/v) propanol. Samples were stored at -20°C before measurement.

For the detection and quantification of lipids an evaporation light scattering detector (Sedex LT- ELSD Model 85LT) was used. The measurement was carried out by means of Agilent

Technologies 1200 Series (Santa Clara, Calif.) and a Zorbax SB-C8 Rapid Resolution column (4,6 x 150 mm, 3,5 pm, Agilent). The injection volume was 5.0 pi and the run time was 20 min. Mobile phase A: aqueous 0.1 % TFA (trifluoracetic acid, solution); mobile phase B: methanol. The column temperature was 40 °C. The ELSD (detector temperature 60 °C) and the DAD (diode array, 210 nm) were used as detectors.

Gradient:

t [min] Flow [I ml/min]

0.00 70 % 1.00

15.00 100 % 1.00

15.01 70 % 1.00

20.00 70 % 1.00

Table 2. Gradient of mobile phases of A and B over time The gradient used starts with 70 % B in A to 100 % B within 15 minutes at a flow rate of 1 mL/min followed by 5 minutes of re-equilibration with 70 % B in A (see Table 2). Reference materials were used whose identity and purity were checked by HPLC-MS/MS and NMR.

The product of example 2 has a composition as described below and is called“production example 1” from now on:

GL-C10C10: 63.7%,

GL-C8C10: 15.5%,

GL-C10C12:1 : 7.9%,

GL-C10C12: 6.6%,

GL-C8C8: 1.8%,

GL-C10C10:1 : 1.3%,

GL-C12C12:1 : 1.1 %,

GL-C12C12: 0.8%

GL-C10: 1.3%,

Example 4: Evaluation of foaming properties using the SITA Foam Tester

Foamability of surfactants and surfactant based products is an important consumer-perceived attribute. Fast flash foaming and high foam volumes are indications to the consumers that the product is an efficacious quality product. Both parameters can be determined using the“SITA foam tester R-2000” measuring device from SITA Messtechnik GmbH. In this device, foam is generated by introducing air into a defined volume of a surfactant solution through a special rotor. The total volume of liquid and resulting foam is measured over time by means of a computer controlled sensing technique.

Using this method, production example 1 (A) was evaluated for its foamability in comparison to a composition of di-Rhamnolipids (B) and a composition of mono-Rhamnolipids (C), both bearing a comparable fatty acid substitution.

(B) and (C) are prepared by means of preparative column chromatography.

For this10 g of freeze-dried RL mixture (JBR 505, Jeneil Biosurfactants, initially ~ 5% by weight total rhamnolipid concentration) are dissolved in 5% by weight concentration in water-saturated ethyl acetate which comprises 1 % by weight of acetic acid. 750 g of a silica 60 gel (200-500 pm; 35-70 mesh; Sigma-Aldrich, Germany) are suspended in water-saturated ethyl acetate (acidified with 1 % by weight of acetic acid) and poured into a column (diameter = 65 mm, maximum fill level = 600 mm, 1 I solvent reservoir). 2-3 cm of acid-treated sea sand (Riedel de Haen, Seelze, Germany) are coated as protective layer over the stationary phase. The eluent used is likewise water-saturated ethyl acetate which comprises 1 % by weight of acetic acid. The rhamnolipid solution is placed onto the prepared column. The eluent flow rate is adjusted to 15 ml/min. The eluate is collected in 100 ml fractions and analysed by means of thin-film chromatography and HPLC. The mono- and di-rhamnolipid forms can be separated this way. Fractions of identical composition are combined and the solvent is stripped off on a rotary evaporator. Then, the residue is dissolved in water and freeze-dried. In order to obtain adequate amounts, this procedure is carried out several times. The purity of the resulting fractions is determined as > 99% by weight by means of ¹ H-NMR and HPLC.

The composition produced in example 2 (A) and the rhamnolipids (B) and (C) were each diluted to a concentration of 0.5% active surfactant matter with water of a total hardness of 10° dH (German hardness). The pH of each test solution was adjusted to 6.0. 300 ml of each test solution were then tested for their foamability at 30°C using a constant stirring speed of 1500 rpm for 10 sec. A total of 10 such measurement intervals were carried out for each test solution. Figure 1 illustrated the foam volume over time for each test solution:

Measurement parameters: temperature: 30° C ± 0,5° C; sample volume/measurement: 300 ml; concentration of test sample: 0.5% in water; water: 10 °dH (= german hardness), pH: 6, stirring speed: 1500 rpm; stirring time: 10 sec; number of intervals: 10; number

Surprisingly, as seen in figure 1 , the composition according to the invention (A) shows the best performance in the SITA foam test, as it shows the fastest flash foaming and generates the highest foam volumes in the SITA foam test.

Production example 1 (A) performs very much better than the composition of mono- rhamnolipids (C), and also to lesser extentd but still significantly better than the composition of di- Rhamnolipids (B). This finding is very surprising as there is a much greater structural similarity between (A) and (C) than between (A) and (B).

Example 5: Example formulations

The following examples are for the purpose of illustration and are not to be construed as limitations of the present invention since many variations thereof are possible without departing from its scope.

The example formulations are made using conventional methods. If necessary, the pH value is adjusted by addition of either aqueous sodium hydroxide or citric acid at the end of the

manufacturing process.

The term“Glucolipid” as used in the example formulations always refers to production example 1. The term“Glycolipids” as used in the example formulations always refers to Rhamnolipids, which are commercially available as Rheance One from Evonik Nutrition & Care GmbH.

Shampoo Example formulation 1 : Volume and Body Shampoo

Example formulation 2: Repair Shampoo

Example formulation 3: Anti-Dandruff Shampoo Parfum, Dyes q. s.

Example formulation 4: Strengthening Shampoo

Example formulation 5: Shampoo

Example formulation 6: Moisturising Shampoo

Example formulation 7: Shampoo

Example formulation 8: Cleansing Oil Shampoo Parfum, Dyes q. s.

Example formulation 9: Shine Shampoo

Example formulation 10: Shampoo

Example formulation 11 : Reinforcing Shampoo

Example formulation 12: Shampoo

Example formulation 13: Fortifying Shampoo

Example formulation 14: Shampoo

Example formulation 15: Brunette Shampoo

Example formulation 16: 2 in 1 Shampoo

Example formulation 17: Shampoo

Example formulation 18: Foam Shampoo

Example formulation 19: Anti-Dandruff Shampoo

Example formulation 20: Shampoo Example formulation 21 : Intensive Care Oil-Shampoo

Example formulation 22: Shampoo

Example formulation 23: Shampoo Example formulation 24: Shampoo

Example formulation 25: Shampoo for Children

Example formulation 26: Intensive Cream Shampoo

Ingredient /

Example formulation 27: Shampoo

Example formulation 28: Caffeine Shampoo

Example formulation 29: Power Grey Shampoo

Example formulation 30: Shampoo

Example formulation 31 : Caffeine Shampoo

Example formulation 32: Shampoo

Example formulation 33: Shampoo

Example formulation 34: Volume Shampoo

Example formulation 35: Shampoo

Example formulation 36: Shampoo

Example formulation 37: Shampoo Example formulation 38: Shampoo

Example formulation 39: Conditioning Shampoo Example formulation 40: Conditioning Shampoo

Example formulation 41 : Conditioning Shampoo

Example formulation 42: Conditioning Shampoo

Example formulation 43: Natural Shampoo

Example formulation 44: Natural Shampoo

Example formulation 45: Natural Shampoo

Example formulation 46: Natural Shampoo

Ingredient /

Example formulation 47: Natural Shampoo

Example formulation 48: Natural Shampoo

Example formulation 49: Anti-Hair Loss Tonic

Example formulation 50: Shower Oil

Example formulation 51: Shower Mousse Example formulation 52: Shower Jelly

Example formulation 53: Shower Jelly

Example formulation 54: Shower Scrub

Example formulation 55: Cellulose Scrub

Ingredient /

Example formulation 56: Exfoliating Body Scrub

Example formulation 57: Dry Shampoo

Example formulation 58: Dry Shampoo

Shower Example formulation 59: Shower Gel

Example formulation 60: Body Wash Example formulation 61 : Cool Down Shower Gel

Example formulation 62: Shower Gel

Example formulation 63: Shower Gel

Example formulation 64: Body Wash

Example formulation 65: Anti-Aging Body Wash

Example formulation 66: Shower Gel

Example formulation 67: Shower Gel

Example formulation 68: Shower Gel for Men

Example formulation 69: Moisturising Shower Cream

Example formulation 70: Shower Cream

Example formulation 71: Shower Cream Example formulation 72: Shower Cream

Example formulation 73: Shower Gel Example formulation 74: Shower Gel

Example formulation 75: Oil Shower

Example formulation 76: Shower Gel

Example formulation 77: Showerl

Example formulation 78: Shower Scrub

Example formulation 79: Cellulose Scrub

Example formulation 80: Exfoliating Body Scrub

Example formulation 81 : Washing Powder

Example formulation 82: Washing Powder

Example formulation 83: Powder Cleanser

Example formulation 84: Powder Cleanser Soap

Example formulation 85: Gentle Liquid Soap

Example formulation 86: Soap

Example formulation 87: Caring Liquid Soap

Example formulation 88: Cream Soap

Ingredient /

Example formulation 89: Liquid Soap

Example formulation 90: Liquid Soap

Example formulation 91 : Liquid Soap

Example formulation 92: Cream Soap

Example formulation 93: Jelly Soap

Example formulation 94: Soap

Example formulation 95: Soap

Example formulation 96: Painting Soap

Example formulation 97: Painting Soap

Bath

Example formulation 98: Bath

Example formulation 99: Creme Oil Bath

Example formulation 100: Relaxing Good Sleep Bath

Example formulation 51: Pampering Oil Bath

Example formulation 101: Aroma Bath

Example formulation 102: Bath Bomb

Example formulation 103: Bath Bomb

Example formulation 104: Bath Salt

Example formulation 105: Bath Salt

x-in-1

Example formulation 106: Shower Gel and Shampoo Example formulation 107: Shower, Shampoo and Shave

Example formulation 108: 2-in-1 Body Wash and Shampoo

Example formulation 109: 2-in-1 Bubble Bath and Wash

Example formulation 110: Body and Face Wash

Example formulation 111 : Hair and Body Shampoo

Example formulation 112: After-Sun Hair and Body Shampoo

Ingredient /

Example formulation 1 13: Hair and Body Shampoo & Shower Gel

Facial Cleansing-Example formulations

Example formulation 114: Eye Make-Up Remover

Example formulation 115: Eye Make-Up Remover

Example formulation 116: Micellar Lotion

Example formulation 117: Waterproof Eye Make-Up Removal Example formulation 118: Waterproof Eye Make-Up Removal

Example formulation 119: Micellar Gel

Example formulation 120: Micellar Gel

Example formulation 121 : Micellar Make-Up Remover

Example formulation 122: Facial Cleansing Foam Example formulation 123: Facial cleansing Foam

Example formulation 124: Cleansing Foam Example formulation 125: Cleansing Foam

Example formulation 126: Cleansing Foam

Example formulation 127: Pump Foam for Facial Cleansing

Example formulation 128: Pump Foam for Facial Cleansing

Example formulation 129: Micellar Water

Example formulation 130: Refreshing Micellar Water

Example formulation 131 : Oil-infused Micellar Water

Example formulation 132: Facial Tonic

Example formulation 133: Facial Tonic

Example formulation 134: Facial Tonic

Ingredient /

Example formulation 135: Micellar Water

Example formulation 136: Micellar Water Example formulation 137: Micellar Water

Example formulation 138: Micellar Water Example formulation 139: Micellar Water

Example formulation 140: Micellar Water

Example formulation 141 : Face Cleanser and Scrub

Microemulsionen

Example formulation 142: Microemulsion for Make-Up Removal Example formulation 143: Microemulsion for Make-Up Removal

Toothpaste

Example formulation 144: Toothpaste

Example formulation 145: Toothpaste

Example formulation 146: Toothpaste

Example formulation 147: Toothpaste

Example formulation 148: Toothpaste

Example formulation 149: Toothpaste

Example formulation 150: Toothpaste

Example formulation 151 : Toothpaste

Example formulation 152: Toothpaste

Example formulation 153: Toothpaste

Example formulation 154: Toothpaste

Example formulation 155: Toothpaste

Example formulation 156: Toothpaste

Example formulation 157: Toothpaste

Example formulation 158: Toothpaste

Example formulation 159: Toothpaste

Example formulation 160: Toothpaste

Example formulation 161: Toothpaste Example formulation 162: Toothpaste

Example formulation 163: Toothpaste Dyes, pH adjuster, preservative q. s.

Example formulation 164: Toothpaste

Example formulation 165: Toothpaste

Example formulation 166: Toothpaste

Example formulation 167: Toothpaste

Example formulation 168: Toothpaste

Example formulation 169: Toothpaste

Example formulation 170: Toothpaste

Example formulation 171 : Toothpaste

Example formulation 172: Toothpaste

Example formulation 173: Toothpaste

Example formulation 174: Toothpaste for Kids

Example formulation 175: Toothpaste for Children

Example formulation 176: Baby Toothpaste

Example formulation 177: Toothpaste for Children

Example formulation 178: Kids Toothpaste

Example formulation 179: 2 in 1 Toothpaste + Mouthwash

Example formulation 180: 2 in 1 Toothpaste + Mouthwash Mouthwash

Example formulation 181 : Mouthrinse without Alcohol

Example formulation 182: Mouthrinse without Alcohol Example formulation 183: Mouthrinse without Alcohol

Example formulation 184: Mouthrinse without Alcohol

Example formulation 185: Mouthrinse

Example Example formulation 43: Mouthrinse without Alcohol

Example formulation 186: Mouthrinse without Alcohol

Example formulation 187: Mouthrinse without Alcohol

Example formulation 188: Mouthrinse without Alcohol

Example formulation 189: Mouthrinse without Alcohol

Example formulation 190: Mouthrinse without Alcohol

Example formulation 191 : Mouthrinse without Alcohol Dyes, pH adjuster, Preservative q. s.

Example formulation 192: Mouthrinse without Alcohol

Example formulation 193: Mouthrinse

Example formulation 194: Mouthrinse

Example formulation 195: Mouthrinse

Example formulation 196: Mouthrinse

Example formulation 55: Mouthrinse

Example formulation 197: Mouthrinse

Example formulation 198: Mouthrinse

Example formulation 199: Mouthrinse

Example formulation 200: Mouthrinse

Ingredient /

Example formulation 201: Mouthrinse Example formulation 202: Mouthrinse

Example formulation 203: Mouthrinse

Example formulation 204: Mouthrinse

Example formulation 205: Mouthrinse

Example formulation 206: Mouthrinse

Example formulation 207: Mouthrinse Concentrate

Example formulation 208: Mouthrinse Concentrate

Example formulation 209: Mouthrinse Concentrate

Example formulation 210: Peroxide Mouthrinse

Example formulation 211 : Fluoride Gel

Example formulation 212: Fluoride Gel

Example formulation 213: Dental Floss

Example formulation 214: Teeth Wipe

Example formulation 215: Chewing Gum

Example formulation 216: Mouthwash Tabs

Example formulation 217: Tooth Tab Dyes, pH adjuster, Preservative q. s.

Example formulation 218: Powder Toothpaste

Further example formulations:

Parfums, Dyes, Preservatives

Example formulations for use in household care: F1 to F5 Hand dishwashing formulations

SLES = Texapon® N 70 (trade name of BASF SE, sodium lauryl ether sulphate with 2 EO)

SL 18: Sophorolipid from Ecover was used, which has an acid to lactone ratio of 70:30 and a lactone form to oleic acid ratio of 60:1.

CAPB = Tego® Betaine C 60 (trade name of Evonik Industries AG, cocoamidopropylbetaine)

LAO= cocamine oxide. (OXIDET DM-246, trade name of Kao Chemicals)

Thickener: Keltro®! T (Xanthan Gum, trade name of CP Kelco)

F6 to F9 solid dishwashing formulations

F10 Powder detergent

F1 1 Liquid detergent

F12 Liquid detergent concentrate

F13 Laundry formulation