Compositions containing high concentrations of iturin-based lipopeptides

Abstract

The present invention relates to a composition having a high concentration of iturin-based lipopeptides. In particular, the composition according to the present invention has 20-150 g / L of iturin-based lipopeptides, and the composition is stable and homogeneous due to the presence of a surfactant.

Description

[Technical field]

[0001] The present invention relates to compositions containing high concentrations of iturinic lipopeptides. In particular, the composition according to the invention has a concentration of iturin-based lipopeptide between 5 and 200 g / L and is stable and homogeneous due to the presence of surfactants and / or hydrotropic molecules. [Background technology]

[0002] Iturin-based lipopeptides are molecules produced by various strains of Bacillus species, more specifically, iturin-based lipopeptides are produced by Bacillus subtilis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus thermoamylovorans ), Bacillus thermocloacae, Bacillus firmus, Bacillus mojavensis, Bacillus bere Illustrative examples include molecules produced by strains of Bacillus velenzensis and Bacillus valissmortis. 633, W23, ATCC19659, DSM23117, QST713 or AQ713, FMBJ, 3-10, RB14, BH072 and their derivatives, Bacillus amyloliquefaciens FZB42, KB3, SYBC H47, GA1 and their derivatives.

[0003] The series of these molecules is Iturin A, A L and C, mojavensin, mycosa mycosubtillin, and bacillomycins A, B, C, D, F, L and L C These ingredients are primarily known for their antifungal properties, but may also have antibacterial properties. These properties result from their amphipathic nature, which allows them to interact with different membrane components.

[0004] Iturin-based molecules can be produced in solution in the fermentation broth by the various strains mentioned above, and can then be recovered by extraction, concentration and purification from the culture supernatant according to methods known to those skilled in the art. Nevertheless, their behavior in solution depends on the concentration of the iturin-based molecules. In fact, above a critical micelle concentration (10-20 mg / L), iturin-based compounds are known to form more or less complex micelles, like other lipopeptide molecules. Moreover, the size of these micelles evolves to form increasingly complex structures as the concentration of the iturin-based molecules increases from an average size of 10 nm to concentrations below 500 mg / L (Jauregi et al. 2013), and even above 1 g / L. At a concentration of 10 g / L, they develop into vesicular structures with an average diameter of 150 nm (Grau et al. 2001), which then develop into bilayer structures at a concentration of 10 g / L (Hamley et al. 2013). (Hamley et al. 2013) and on iturin A (Grau et al. 2001). As reported in the literature, these superstructures have not been found in other lipopeptide molecules, for example in the surfactin family. Furthermore, increasing the concentration of iturin-based compounds tends to increase their interactions with proteins present in the medium, thus increasing the size of the structures and their insolubility (Jauregi et al. 2013).

[0005] These physicochemical properties of iturin-based compounds therefore pose solubility problems and result in unstable compositions prone to precipitation or gelation when the concentration of the iturin-based lipopeptide in the compound is increased.

[0006] Selected prior art to the present invention.

[0007] Few prior art documents refer to compositions with iturin-based lipopeptides with a concentration above 5 g / L, and even less to compositions with a concentration above 20 g / L, mainly because those skilled in the art know that iturin-based lipopeptides tend to form high concentration micelles, which prevent good solubility and make the composition unstable.

[0008] However, according to the prior art, Japanese Patent Publication No. 2003-128512 (Showa Denko), cosmetic compositions containing surfactants to which iturin and surfactin are added for antibacterial properties are known. However, there is no mention of the stability and homogeneity of iturin in those compositions. This is because iturin is not the main compound in various compositions.

[0009] Also known is US Patent Publication No. 2016 / 183537, which describes a composition derived from Bacillus amyloliquefaciens containing iturin, surfactin, and fengycin to improve plant growth or plant protection. The document does not mention the concentrations of the molecules contained in the composition. There is no mention of high iturin concentrations.

[0010] Also known is a publication by Choukri HBID et al., which describes the influence of lipopeptide extracts on oxygen transfer during fermentation. These extracts correspond to a mixture of iturin / surfactin in concentrations up to 4 g / L. The possibility of higher iturin concentrations is not mentioned in this document.

[0011] Thus, none of the prior art documents encourages the skilled artisan to make compositions with high concentrations of iturin-based lipopeptides, because they know that they are of poor use due to their instability and heterogeneity. Summary of the Invention [Problem to be solved by the invention]

[0012] The inventors have shown that it is possible to prepare stable and homogeneous compositions with concentrations of iturin-based lipopeptides above 5 g / L by adding molecules with surfactant properties. In fact, the inventors have shown that surfactants improve the solubility of iturin-based lipopeptides above 5 g / L, in particular at 10 g / L and 20 g / L, and up to 50, 100 and even 200 g / L, without the appearance of precipitation or gels.

[0013] The invention has application in the production of concentrated solutions of antifungal, antibacterial or biosurfactant biopesticide molecules for the plant protection industry, but also in the food, cosmetic, pharmaceutical and petroleum sectors. [Means for solving the problem]

[0014] Detailed Description of the Invention The general concept of the present invention relates to a stable, homogenous liquid composition comprising a mixture of lipopeptides, the concentration of which is greater than 5 g / L of iturin-based lipopeptide.

[0015] In such compositions, the lipopeptide is combined with a molecule that has surfactant properties so that the concentrated iturin-based lipopeptide composition remains stable and homogeneous.

[0016] The first subject of the present invention is a method for the preparation of lipopeptides of the iturin family at a concentration of more than 20 g / L and one or more surfactants selected from the group consisting of anionic surfactants, nonionic surfactants or oils. The present invention relates to a stable and homogenous composition comprising an active agent.

[0017] By "stable and homogeneous" composition is meant a composition that does not contain precipitate and remains homogeneous between 4° C. and 40° C. Lack of solidification, i.e., the solution is liquid and not gel-like, is also an important criterion.

[0018] The term "liquid" must be understood within the context of the present invention as a fluid composition, ie a composition that has a low viscosity (ratio of shear rate to shear stress).

[0019] The term "homogeneous" should be understood to mean a mixture in which the different components are indistinguishable with the naked eye after stirring. Homogeneity can be assessed by the uniformity of concentration at any point in the mixture and the absence of structure or microstructure (caking or precipitation) observable with the naked eye.

[0020] Such homogeneity of the mixture is preferred as it allows for a uniform concentration of the iturin-based lipopeptide which can then be diluted in an aqueous solution.

[0021] The term "stable" must be understood as the fact that the composition remains homogeneous over time. Thus, a stable homogeneous mixture is one that has a uniform concentration that remains constant over time.

[0022] According to a preferred embodiment, the composition according to the invention comprises 20-200 g / L of iturin-based lipopeptide. It may for example comprise 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 200 g / L of iturin-based lipopeptide. Preferably, the concentration of iturin-based lipopeptide in the composition is 20-150 g / L or 20-100 g / L. More preferably, the concentration of iturin-based lipopeptide is more than 50 g / L, i.e. 50-150 g / L, or 50-100 g / L, or 50-80 g / L.

[0023] "Iturin-based molecules" include iturin A, A L and C, Mohabensin, Mycosubtilis. and bacillomycin A, B, C, D, F, L and L C means.

[0024] The compositions of the present invention include molecules that have surfactant properties.

[0025] "Molecules having surfactant properties" are also referred to as "surface-active agents." Also known as surfactants or simply "surfactants," amphiphilic molecules Surfactants, surfactin, fengycin (or plipastatin) These molecules include lipopeptides, chemical surfactants, and biological surfactants such as rhamnolipids and polysaccharides. These can be used alone or in combination.

[0026] Surfactants include heparin, hyaluronic acid, dextran, amylose, chitosan, anionic surfactants derived from amino acids, nonionic surfactants derived from polyglycosides, hydrotrope surfactants, surfactant isomers and / or fengycin (or primathone). These include lipopeptides such as tatin isomers, rhamnolipids and vegetable oils.

[0027] More particularly, the present invention relates to anionic, nonionic and oil surfactants.

[0028] Surfactants in the anionic surfactant group include surfactin, fengycin or amino The compound is selected from the group consisting of acid derivatives.

[0029] By "surfactin family molecules" is meant surfactin A, B, C, ichenysin and pumilacidin.

[0030] By "fengycin family of molecules" is meant fengycin A and B, plipastatin A and B and agrastatin.

[0031] The surfactants of the non-ionic surfactant group are selected from the following: Fatty alcohol oxyalkylates, pentylene glycol and its derivatives, hydrotropic molecules of alkylpolyglycoside type (alkylpolyglycosides and alkylethoxypolyglycosides), molecules of polyglycoside texturizing agent type (xanthan gum, gum arabic, gum tragacanth, guar gum, locust bean gum, talimandu gum, pectin, gel gum, carrageenates, agar, algins, etc.) salts).

[0032] The surfactants of the oil group are selected from: The oils and modified oil extracts (acidified oils, methylated oils, esterified oils) are selected from the following oils and modified oil extracts: almond, peanut, argan, avocado, rapeseed, Lorenzo, neem, hazelnut, cashew nut, macadamia nut, olive, pistachio, rice, oleic sunflower, Camelina, flax, borage, safflower, hemp, cotton, wheat germ, corn, walnut, poppy, evening primrose, barley, pumpkin seed, grape seed, pea, sesame, soybean, sunflower.

[0033] Iturin-based lipopeptides are microemulsified in these oily compositions or fats to obtain the surfactant effect from the modified and extracted oils.

[0034] The compositions of the present invention may also contain additives such as lipid sources, salts and solvents. These additives can participate in solubilization without being surfactants. Examples of solvents include ethanol, methanol, acetonitrile, dimethylsulfoxide, butanol, pentanol, and acetone.

[0035] According to a first aspect, the present invention relates to a composition comprising 5-50 g / L of an iturin-type lipopeptide and 1-40 g / L of a surfactant.

[0036] In a particular composition, the iturin-based lipopeptide is mycosubtilin. In another particular composition, the amount of iturin-based lipopeptide is preferably 10 g / L to 50 g / L, preferably 20 to 50 g / L, more preferably 30 to 50 g / L.

[0037] According to a second aspect, the present invention relates to a composition comprising 5-100 g / L of an iturin-based lipopeptide, 1-40 g / L of a surfactant and 1-100 g / L of fengycin (or plipastatin).

[0038] In certain compositions, the amount of iturin-based lipopeptide is preferably 10-80 g / L, preferably 20-80 g / L, most preferably 30-80 g / L.

[0039] According to a third aspect, the present invention relates to a composition comprising 5-100 g / L of an iturin-based lipopeptide, 1-40 g / L of surfactin and 1-30 g / L of a chemical surfactant.

[0040] In certain compositions, the amount of iturin-based lipopeptide is preferably 10-80 g / L, preferably 20-80 g / L, preferably 30-80 g / L.

[0041] In certain embodiments, the chemical surfactant may be a polyglycoside-based nonionic surfactant or an anionic surfactant derived from an amino acid. Preferably, the chemical surfactant is composed of a mixture of an anionic surfactant and a nonionic surfactant.

[0042] According to a fourth aspect, the present invention relates to a composition comprising 5-100 g / L of an iturin-based lipopeptide, 1-40 g / L of a surfactant, 1-100 g / L of fengycin (or plipastatin), and 1-30 g / L of a chemical surfactant.

[0043] In certain compositions, the amount of iturin-based lipopeptide is preferably 10-80 g / L, preferably 20-80 g / L, preferably 30-80 g / L.

[0044] According to a fifth aspect, the present invention relates to a composition comprising 5-100 g / L of microemulsified iturin-based lipopeptides in an oily or fatty compound such as a vegetable oil. In such a composition, the concentration of the oil is 1-100%, preferably 50-100%, particularly preferably 80-100%. Usable vegetable oils include corn oil, as well as oils and modified oil extracts of almond, peanut, argan, avocado, rapeseed, Lorenzo, neem, hazelnut, cashew, macadamia nut, olive, pistachio, rice, oleic sunflower, Camelina, flax, borage, safflower, hemp, cotton, wheat germ, maize, walnut, poppy, evening primrose, barley, pumpkin seed, grape seed, pea, sesame, soybean, sunflower.

[0045] Preferably, the oil used is from peanut, olive, oleic sunflower, flax, corn, walnut, soybean and sunflower. Such oils have the advantage of being available in large quantities and at low cost. Even more preferably, the oil used is from corn or sunflower seeds.

[0046] In a preferred embodiment, such a composition comprises 10-80 g / L, preferably 20-80 g / L of mycosubtilin, preferably 30-80 g / L, very preferably 50-80 g / L of mycosubtilin.

[0047] Thus, a particular composition according to the invention comprises: -2-8% Iturin-based lipopeptides - 3% chemical surfactants, consisting of 1.5% each of nonionic and anionic surfactants -2~4% surfactant -0-10% fengycin or plipastatin

[0048] In a particular embodiment, the composition comprises about 3% lipopeptides (1.25% mycosubtilin and 1.85% surfactin), 1.5% non-ionic surfactant derived from polyglycosides and 1.5% anionic surfactant derived from amino acids.

[0049] In another specific embodiment, the composition comprises about 5% lipopeptides (2.45% mycosubtilin and 2.65% surfactin) and 3-8% fengycin (or plipastatin).

[0050] In another particular embodiment, the composition comprises 30-80 g / L of mycosubtilin, and and 30-80 g / L of fengycin (or plipastatin).

[0051] In a particular embodiment, the composition comprises an iturin-based lipopeptide at a concentration of 20-100 g / L, and the surfactant is a plant extract oil, a polyglycoside derivative and surfactin.

[0052] In another particular embodiment, the composition comprises an iturin-based lipopeptide at a concentration of 20-100 g / L, and the surfactant is a methylated plant seed extract oil, and a polyglycerol coconut oil ester (Synergen OS®).

[0053] In another particular embodiment, the composition comprises an iturin-based lipopeptide at a concentration of 20-100 g / L and the surfactant is a mixture of an alkyl polyglycoside (Simulsol®), an amino acid derivative (Proteol®) and surfactin.

[0054] In another particular embodiment, the composition comprises an iturin-based lipopeptide at a concentration of 20-100 g / L and the surfactant is a mixture of aliphatic alcohol oxyalkylate (Emulsogensol®) and surfactin.

[0055] In another particular embodiment, the composition comprises an iturin-based lipopeptide at a concentration of 20-100 g / L and the surfactant is a mixture of vegetable oil, xanthan gum and surfactin.

[0056] The compositions according to the invention may be prepared in a ready-to-use or concentrated form, the concentrated solutions being dilutable prior to use.

[0057] A second object of the invention relates to a dry composition comprising a mixture of lipopeptides, the concentration of which is greater than 0.5%. In a preferred embodiment, the concentration of iturin-based lipopeptides is greater than 2%.

[0058] Such a dry composition is in powder or lyophilized form and is ready for use; in fact, its rehydration with 100 mL of solution is sufficient to obtain a composition with a concentration of iturin-based lipopeptide of at least 5 g / L. In a preferred embodiment, such a composition has a concentration of iturin-based lipopeptide of at least 20 g / L.

[0059] The components and the relative amounts of components in these solid compositions are the same as those described above for the liquid compositions. In fact, the concentration by dry weight of the various components of these dehydrated solutions makes it possible to obtain a stable and homogeneous liquid composition according to the invention with a concentration of 5 g / L of iturin-based lipopeptide. According to a preferred embodiment, the concentration of iturin-based lipopeptide is greater than 20 g / L.

[0060] A third object of the present invention relates to a method for preparing a composition comprising a mixture of lipopeptides, the concentration of which is greater than 5 g / L. In a preferred embodiment, the concentration of iturin-based lipopeptide is greater than 20 g / L.

[0061] The iturin-based lipopeptide of the composition can be obtained by fermentation of a strain of Bacillus species and can be harvested and concentrated from the culture supernatant.

[0062] The compositions of the present invention can be prepared using commercially available lipopeptide preparations in powder form. These commercially available powders contain various amounts of iturin-based lipopeptides, ranging from 10 to 80% or more, for example 15% or 75%. A mixture of mycosubtilin and surfactin is Ratios ranging from 30 / 70 to 70 / 30 or 95 / 5, for example 35 / 65, 40 / 60, 45 / 55, or 50 / 50.

[0063] To prepare the composition according to the invention, at least one surfactant must be added to a solution containing a high content of iturin-based lipopeptide.

[0064] The method of preparing such a composition may include an additional dehydration step to provide a "ready to use" composition in powder form, the properties of which upon rehydration correspond to the compositions of the present invention.

[0065] The method thus makes it possible to prepare liquid or solid solutions (in the form of powders or lyophilisates). Such concentrated solutions are particularly suitable for storage and distribution. They must be diluted before use.

[0066] Thus, the composition according to the invention can also be obtained by dissolving a powder which allows the reconstitution of the liquid composition described above.

[0067] The compositions according to the invention find application in the food, plant protection and cosmetics sectors as well as in the medical and pharmaceutical sectors.

[0068] The invention will now be illustrated by the following examples. [Brief description of the drawings]

[0069] [Figure 1] FIG. 1 is an explanatory diagram showing the solubility of an iturin-based lipopeptide (here, mycosablity) alone in an aqueous solution in relation to its concentration. [Diagram 2] FIG. 1 is an illustration showing the maximum soluble concentrations of iturin-based lipopeptides in various compositions. [Diagram 3] FIG. 1 shows the analysis of the minimum viscosity of different concentrated iturin-based compositions with and without the addition of surfactant and other additives A after storage at 21° C. or 54° C. [Figure 4] FIG. 1 shows the analysis of maximum viscosity of different concentrated lipopeptide compositions with and without surfactant and other additive A after storage at 21° C. or 54° C.

[0070] Working Example A. Preparation of lipopeptide-containing solutions from the supernatants of Bacillus strain cultures

[0071] 1- Obtaining culture supernatant Lipopeptides are obtained from the aerobic fermentation process of Bacillus strains derived from Bacillus subtilis ATCC 6633 for the production of iturin-series lipopeptides, and of Bacillus subtilis 168-derived strains for the production of surfactin and / or fengycin (or plipastatin). The cultivation is carried out at 30 °C in a stirred medium containing a carbon source (glucose, sucrose, etc.), a nitrogen source (ammonium sulfate, peptone, etc.) and trace elements. The PH value is maintained at 7. The culture is harvested after 48-72 hours. It is then centrifuged or filtered to remove bacteria. The culture supernatant is then concentrated.

[0072] 2- Preparation of a composition containing a high content of iturin-based lipopeptides from culture supernatant To obtain a composition containing a high content of iturin-type lipopeptides, the culture supernatant can be concentrated. The concentration of the culture supernatant can be obtained by any method known to the person skilled in the art, in particular by the following method: -By tangential ultrafiltration using membranes with a cut-off threshold between 1 KDa and 300 KDa. For example, 1000 L of culture supernatant obtained as described above is concentrated by passing through a membrane to obtain a retentate with a volume of 10-100 L. Small molecules are then removed in one or more diafiltration steps. The solution thus obtained can be dried by lyophilization, atomization or used as is. - Precipitation at acidic pH. The pH value is lowered in order to selectively precipitate the lipopeptides. Additional concentrated sulfuric acid is added to the resulting supernatant. Once a final pH value of about 1 is obtained, the solution is stirred for 2-12 hours. By centrifugation, a pellet of material containing the lipopeptides can be removed. This pellet is then dissolved by adding water and a sodium compound to obtain a pH value of 7-8.5. The solution thus obtained can be dried by lyophilization, atomization or can be used as it is. - Evaporation method Concentrate the supernatant by vacuum evaporation, e.g., place 20 L in a rotary evaporator and concentrate to 1-2 L.

[0073] The proportion of lipopeptide at the end of one of these two examples of the preparation is 0.5-20% (weight / volume).

[0074] B. Preparation of concentrated iturin-based lipopeptide solutions 1-Study on the effects of various surfactants on the solubility and high concentration composition of iturin-based lipopeptides An iturin-based lipopeptide (here mycosablityl) with a purity (on a dry matter basis) of 45-80% was dissolved alone in water at various concentrations and this control composition was compared with various compositions to which the various surfactants mentioned in the application section had been added. After 15 days of storage at 21°C, only compositions with a fluid type and rheological behavior with a homogenous appearance (no solidification and / or phase shift and / or precipitation that would impede flow) were selected and mixed by vortexing, after which these compositions were centrifuged at 10,000G for 10 minutes and the supernatants were analyzed using RP-HPLC comparative method. This method makes it possible to examine the solubility of the iturin-based compounds found in the supernatant after centrifugation.

[0075] The solubility of the iturin-based lipopeptide (here, mycosablity) alone in aqueous solution is shown in FIG. 1 in relation to its concentration.

[0076] The maximum soluble concentrations of iturin-based lipopeptides in various compositions are shown in FIG.

[0077] Composition 1 contains only iturin-based lipopeptides and no added surfactant.

[0078] Compositions 2 to 13 had surfactants added as follows: -10% Fengycin (Composition 2) - alkylpolyglucosides and alkypolyglycosides in proportions of 10% each Amino acid derivatives (Simulsol® / Proteol®) (Composition 3) -Soy lecithin (4%) and polyglycoside derivatives (0.1%) (Elvis® ) / xanthan gum) (Composition 4) -Methylated plant seed extract oil and polyglycerol ester from coconut oil (0.2%) (Synergen OS®) (Composition 5) - non-ionic surfactant of fatty alcohol oxalkylate type (0.2%) (Emulsogensol®) (Composition 6) - Pentylene glycol type nonionic surfactant (50%) (Composition 7) - 0.4% polyglycoside derivatives (alkylethoxyglycosides = AEG) (composition 8) -4% Surfactin (composition 9) - 4% surfactin and 10% fengycin (composition 10) -Plant extract oil (30%) and polyglycoside derivative (0.1%) and surfactin (4%) (corn oil / xanthan gum / surfactin) (composition 11) - alkyl polyglucosides and amino acid derivatives (Simulsol® / Proteol®) in proportions of 10% each and surfactin at 4% (composition 12) - 0.2% of a non-ionic surfactant of the fatty alcohol oxaalkylated type (Emulsogen®) and 4% Surfactin (Composition 13)

[0079] Interpreting the results FIG. 1 shows the solubility of iturin-based lipopeptides (here mycosablity) in aqueous solutions at room temperature as a function of their concentration.

[0080] In Figure 1, it can be seen that mycosublicin alone is very well solubilized in aqueous solution at a maximum concentration of 2 g / L, but above this concentration (for example between 5 g / L and 25 g / L) a precipitate can be observed and only a portion of it is soluble.

[0081] Figure 2 shows the solubility analysis (solubility analysis) of different compositions containing an iturin-based lipopeptide (here mycosablity) and different compounds to obtain a high soluble concentration of the iturin-based lipopeptide after 15 days of storage at room temperature. The measurements are carried out on the supernatant after a centrifugation step to remove the insoluble material.

[0082] In figure 2 it can be observed that the addition of different surfactants, especially anionic compounds alone or in combination with non-ionic surfactants, significantly increases the solubility of mycosubritin. The presence of fengycin or surfactin makes it possible to obtain a solubility of mycosubritin close to 30 g / L. In a surprising and original way, the combination of these two anionic lipopeptide compounds makes it possible to obtain a higher solubility of the iturin-based compound. It should also be noted that, despite the homogeneous and stable compositions, some surfactants do not achieve a solubility above 20 g / L. This is the case for soy lecithin combined with xanthan gum (up to 1.5 g / L), for the non-ionic surfactant (Emulsogensol®) (Clariant) (up to 11.6 g / L), but also for the non-ionic compounds (Simulsol®) and the anionic (Proteol®) (Seppic) (up to 15.3 g / L). For these compositions, significant precipitation was observed after the centrifugation step. On the other hand, when these compositions are supplemented with another anionic lipopeptide compound, such as surfactin, the solubility of mycosablitzin increases to values ​​between 55 and 120 g / L. Other non-ionic compounds such as pentylene glycol and alkyl ethoxy glycosides also provide solubilities of over 30 g / L. The composition that allows the highest solubility of iturin-based compounds (112 g / L) is one that includes an anionic lipopeptide (surfactin) and a non-ionic surfactant (Emulsogen®).

[0083] Preparation of a composition containing a high content of iturin-based lipopeptides from 2-lipopeptide powder Compositions containing high contents of iturin-based lipopeptides can be prepared from lipopeptide powders as shown in the following two composition examples. Composition 1: A composition containing about 3.1% (m / v) lipopeptides (1.25% mycosublicin and 1.85% surfactin), containing a mixture of mycosublicin and surfactin in a ratio of 40:60, with a purity of about 15% lipopeptides in the aqueous phase (pH value 7.5-8.5). The peptide powder was obtained by resuspending. Composition 2: A composition containing about 5.1% (m / v) lipopeptides (2.45% mycosublicin and 2.65% surfactin) was obtained by resuspending lipopeptide powder of about 75% purity in an aqueous phase (pH value 7.5-8.5) containing a mixture of mycosublicin and surfactin in a ratio of 45:55.

[0084] 3- Preparation of a composition containing a surfactant and a high content of iturin-based lipopeptides Surfactants were added to compositions 1 and 2 above to evaluate the effect of the surfactant molecules on the properties of the compositions. Composition 1A: Two surfactants, a nonionic hydrotropic surfactant derived from polyglycosides (trade name Simulsol, available from Seppic under the trade name SL 7C) and an anionic surfactant derived from amino acids (trade name Proteol, available from Seppic under the trade name AP L) were added to composition 1 at a ratio of 10% (v / v). Composition 2A: 3-8% (m / v) fengycin (or plipastatin) was added to composition 2.

[0085] 4- Characterization of the obtained composition: homogeneity and stability over time a. Stability test at room temperature The appearance of the compositions with and without surfactant was examined at room temperature, here about 21°C. Composition 1: The addition of the two surfactants described above makes it possible to obtain a homogeneous composition (composition 1A), avoiding precipitation and settling phenomena. Composition 1A, including surfactants, has a cloudy but homogeneous appearance. Composition 2: The addition of fengycin (or plipastatin) makes it possible to obtain a homogeneous composition and to avoid the phenomenon of solidification (which occurs in the absence of fengycin (or plipastatin)). Composition 2A with fengycin (or plipastatin) has a transparent and homogeneous appearance.

[0086] b. Stability test at low temperature The concentrated lipopeptide solutions (Compositions 1, 2 and 1A, 2A) were placed at 4° C. for 1 to 30 days. Only the solutions with added surfactant or fengycin (or plipastatin) (i.e., 1A and 2A) remained homogeneous and no deposits were observed. In the absence of these additions, the concentrated lipopeptide solutions would likely have caked or developed significant insoluble deposits.

[0087] 5-Rheological characterization of the obtained compositions: effect of room temperature storage and accelerated aging a. Analysis method The two compositions 1 and 2 above and compositions containing additives 1A and 2A were stored for 15 days at 21° C. and (to simulate accelerated aging) for 15 days at 54° C. Storage of compositions at elevated temperatures (54° C.) is a method known to those skilled in the art and can mimic accelerated aging of the compositions by increasing Brownian motion and accelerating the destabilization of the compositions.

[0088] At the end of these periods, the rheology of the compositions was investigated using an Anton Paar modular compact rheometer MCR102. The method consists of monitoring the shear rate as the shear stress is gradually increased between 1 and 100 Pa (test duration 500 s). The viscosity (shear stress / shear rate ratio) is also expressed as a function of the shear stress. This makes it possible to visualize the evolution of the viscosity during the test period. Each of these tests was carried out on triplicate samples.

[0089] After storage of these compositions at 21°C or 54°C, two rheological parameters were measured: That is, the shear stress (Pa) at the minimum viscosity (Pa·s) and maximum viscosity were investigated.

[0090] b.Results All samples showed non-Newtonian fluid behavior (viscosity was not constant during the test period). As the shear stress increases, the viscosity decreases and approaches a minimum viscosity. The decrease in viscosity under agitation can be explained by a gradual alignment of structural units (or molecules) in the direction of flow, thus facilitating the flow of the different liquid layers as the shear rate increases. The fluids are then called shear-thinners and are characterized by the shear stress at which the viscosity is minimum (the viscosity at the end of the test in the case of the applied tests) and maximum viscosity (Pa).

[0091] -Investigation of minimum viscosity FIG. 3 shows the analysis of the minimum viscosity of different concentrated iturin-based compositions with and without the addition of surfactants and other additives A after storage at 21° C. or 54° C.

[0092] The results shown in FIG. 3 show that the minimum viscosity of composition 1A after storage at 21° C. is significantly different and lower than that of unformulated composition 1. No significant differences were observed between compositions 2 and 2A after storage at 21° C. Nevertheless, in the aging test at 54° C., the minimum viscosity of composition 2 increases very significantly while that of composition 2A remains close to zero. This shows the very important effect of adding fengycin (plipastatin) to the latter to promote the solubility of the iturin-based lipopeptide. A similar effect can be observed between compositions 1 and 1A after storage at 54° C., showing the benefit of the formulation of composition 1A.

[0093] -Investigation of shear stress at maximum viscosity FIG. 4 shows the analysis of maximum viscosity of different concentrated lipopeptide compositions with and without the addition of surfactant and other additives A after storage at 21° C. or 54° C.

[0094] The results shown in Figure 4 show that the maximum viscosity of composition 1A after 15 days of storage at 21°C is lower than that of unformulated composition 1. No significant differences were observed for compositions 2 and 2A. Nevertheless, when investigating the effect of accelerated aging at 54°C on this parameter, the results in Figure 4 show that the shear stress increases very significantly for unformulated compositions 1 and 2, while formulated compositions 1A and 2A remain almost in agreement with the results obtained at 21°C. These results show the very favorable impact of the formulation according to the invention on promoting the solubility over time of high concentrations of iturin-based compounds.

[0095] As a conclusion of these studies it was possible to establish the following: - Composition 1A makes it possible to solubilize iturin-based compounds while avoiding precipitation and / or gel formation, which is characterized by a significant decrease in the minimum viscosity and in the shear stress at the maximum viscosity at 21 ° C or upon their aging. Compositions 1 and 1A relate to non-Newtonian cussion type fluids. Composition 2A makes it possible to solubilize the iturin-based compounds after prolonged storage while avoiding precipitation and / or gel formation. This is characterized by a significant decrease in the shear stress at the minimum and maximum viscosity after storage at 54° C. Compositions 2 and 2A initially relate to non-Newtonian fluids of the shear-thinning type, whereas unformulated composition 2 thickens significantly after its aging.

Claims

1. A stable and homogenous aqueous composition for antifungal, antibacterial or biopesticide use in plant protection, comprising an iturin-based lipopeptide at a concentration of more than 20 g / L and one or more surfactants, wherein the iturin-based lipopeptide is a mycosubtilin and the surfactant is selected from the group consisting of surfactin, fengycin and alkylethoxyglycoside, wherein the concentration of the surfactin is between 1 and 40 g / L and the concentration of the fengycin is between 1 and 100 g / L.

2. The composition of claim 1, wherein the concentration of said mycosubtilin is from 20 to 150 g / L.

3. 2. The composition according to claim 1, characterized in that the concentration of said mycosubtilin is 20-50 g / L and said surfactant is surfactin.

4. 2. The composition of claim 1, wherein the concentration of said mycosubtilin is 20-50 g / L and said surfactant is fengycin.

5. The composition according to claim 1, characterized in that the concentration of said mycosubtilin is 20-100 g / L and said surfactants are surfactin and fengycin.

6. A method for preparing the composition according to any one of claims 1 to 5, comprising the steps of preparing an aqueous solution containing a high content of iturin-based lipopeptide and adding a surfactant to the prepared solution.

7. The process according to claim 6, further comprising a dehydration step.

Images