EP3654945A1 - Composition comprising a high concentration of iturin lipopeptides - Google Patents

Abstract

The invention relates to compositions having a high concentration of iturinic lipopeptides. In particular, the compositions according to the invention have an iturinic lipopeptide concentration of between 20 and 150 g/l; they are stable and homogeneous as a result of the presence of surfactants.

Description

 COMPOSITION COMPRISING A HIGH CONCENTRATION

 ITURINIC LIPOPEPTIDES

The invention relates to compositions having a high concentration of iturinic lipopeptides. In particular, the compositions according to the invention have a concentration of iturinic lipopeptides of between 5 and 200 g / l, and are stable and homogeneous thanks to the presence of surfactants and / or hydrotropic molecules.

 Iturinic lipopeptides are molecules produced by different strains of Bacillus sp. and more particularly the strains of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus thermoamylovorans, Bacillus thermocloacae, Bacillus firmus, Bacillus mojavensis, Bacillus velenzensis, Bacillus valissmortis. As an example and in a non-exhaustive manner, the strains Bacillus subtilis ATCC6633, W23, ATCC19659, DSM23117, QST713 or AQ713, FMBJ, 3-10, RB14, BH072 and their derivatives, the strains Bacillus amyloliquefaciens FZB42, KB3, SYBC H47, GAI and their derivatives.

 This family of molecules includes iturin A, AL and C, mojavensin, mycosubtillin and bacillomycins A, B, C, D, F, L and Le. These components are known mainly for their antifungal power but they also have antibacterial properties. These properties come from their amphiphilic nature which allows them to interact with different membrane constituents.

The iturinic molecules can be produced in solution in the fermentation broth by the various strains mentioned above, then they can be recovered according to methods known to those skilled in the art in order to extract, concentrate and purify them. from a culture supernatant. Nevertheless, their behavior in solution is dependent on their concentration. Indeed, it is known that above the critical micellar concentration (situated between 10 and 20 mg / L), iturinic compounds like the other lipopeptide-type molecules form more or less complex micelles. Moreover, the size of these micelles will evolve to form increasingly complex structures when increasing their concentration, from an average size of 10 nm to concentrations below 500 mg / L (Jauregi et al., 2013), to vesicular structures with an average diameter of 150 nm above 1 g / L (Grau et al., 2001), then to lamellar bilayer-type superstructures at a concentration of 10 g / L (Hamley et al., 2013). As revealed by this work on mycosubtiline (Hamley et al., 2013) or iturin A (Grau et al., 2001), these superstructures are not found in other lipopeptide molecules of the surfactin family, for example. Moreover increasing the concentration of iturinic compounds both to increase their interaction with the proteins contained in the culture medium and thus to increase the size of the structures leading to make them insoluble (Jauregi et al., 2013).

 These physicochemical properties of the iturinic compounds thus pose problems of solubility, giving rise to unstable compositions and having a tendency to precipitate or gellate when the concentration of iturinic lipopeptides of the compositions is increased.

State of the art closest to the invention.

 The prior art has few compositions having a concentration of iturinic heptopeptides greater than 5 g / L and even less having a concentration greater than 20 g / L, mainly because it is known to those skilled in the art that iturinic itepopeptides tend to form miceiies at high concentration, making the compositions unstable due to the miceiies of iturine preventing their good solubility. However, it is known in the prior art patent JP2003 128512 (SHOWA DENKO) which has cosmetic compositions, including surfactants, in which are added iurine and surfactin for their antimicrobial properties, but stability and The homogeneity ofiturine in these compositions is not mentioned, since these are not the main compound of the various compositions.

Also known is the patent US 2016/183537 which describes a composition derived from Bacillus amyloidiquesfaciens containingiturine, surfactin and fengycin to improve the growth of plants or protect plants. This document does not mention the concentrations of the molecules contained in this composition, there is no mention of high concentrations of iturine.

The publication of Choukri HBID is also known. have. describing the influence of Iipopeptide extracts on oxygen transfer during fermentation. These extracts corresponding to a mixture iturine / surfactin at concentrations up to 4 g / L the possibility of a higher iturin concentration is not described in the document.

Thus, none of the documents of the prior art encourages the skilled person to make compositions with a high concentration of iturinic hepopeptides, because he knows that they will not be stable and homogeneous and therefore will have little applications. Problems solved by the present invention

 The inventors have shown that it is possible to prepare a stable and homogeneous composition in which the concentration of iturinic lipopeptides is greater than 5 g / L by the addition of molecules having surfactant properties. Indeed, the inventors have demonstrated that the surfactants make it possible to improve the solubilization of iturinic lipopeptides beyond 5 g / l, in particular at 10 g / l and 20 g / l and up to 50, 100, even 200 g / L without appearance of precipitate or gel.

 The present invention finds particular applications in the production of concentrated solutions of biopesticide antifungal, antibacterial or biosurfactant molecules for the phytosanitary industry, but also in the fields of the food, cosmetic, pharmaceutical and petroleum industries.

DETAILED DESCRIPTION OF THE INVENTION

 The general concept of the invention relates to a stable and homogeneous liquid composition comprising a lipopeptide mixture in which the concentration of iturinic lipopeptides is greater than 5 g / l.

 In such a composition, the lipopeptides are associated with molecules having surface-active properties, by virtue of which the concentrated composition of iturinic lipopeptides remains stable and homogeneous.

A first subject of the invention relates to a stable and homogeneous composition comprising a concentration of iturinic lipopeptides greater than 20 g / l and one or more surfactants chosen from the families of anionic surfactants, nonionic surfactants or oils. .

By "stable and homogeneous" composition is meant a composition which does not comprise a precipitate and which remains homogeneous at temperatures of between 4 ° C. and 40 ° C. The absence of caking is also an important criterion, that is to say that the solution is liquid and not in gelled form. The term liquid is understood in the sense of the invention as a fluid composition that is to say a composition whose viscosity (the ratio between the shear stress and the shear rate) is low.

The homogeneous term is understood as a mixture of which the various constituents can not be distinguished with the naked eye after stirring. Homogeneity can be assessed by the uniformity of the concentrations at any point in the mixture and the absence of structures or micro-structures (mass gain or precipitate) visible to the eye. Such homogeneity of the mixture is advantageous because it allows uniform concentrations of iturinic lipopeptides, which can then be diluted in aqueous solution.

The stable term is understood as the fact that the composition remains homogeneous over time. Thus, a stable and homogeneous mixture is a mixture having a uniform concentration which remains constant over time.

According to a preferred embodiment, the composition according to the invention comprises between 20 and 200 g / L of iturinic lipopeptides. It may comprise, for example, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 200 g / L of iturinic lipopeptides. Preferably, the concentration of iturinic lipopeptides of the composition is between 20 and 150 g / l, or between 20 and 100 g / l. Even more preferably, the concentration of iturinic lipopeptides of the composition is greater than 50 g / l, that is to say for example between 50 and 150 g / l, or between 50 and 100 g / l or between 50 and 80 g / L. By "iturin family molecules" is meant iturin A, AL and C, mojavensin, mycosubtilin and bacillomycins A, B, C, D, F, L and Le.

The compositions of the present invention include molecules having surfactant properties.

By "molecules having surfactant properties", also called "surfactants" or simply "surfactants", is meant especially amphiphilic molecules, surfactants, lipopeptides, such as, for example, surfactin, fengycin (or plipastatin), chemical surfactants and biological surfactants, such as rhamnolipids, polysaccharides, etc. These molecules can be used alone or in combination in the compositions of the invention.

 Among the surfactants, mention may be made of heparin, hyaluronic acid, dextran, amylose, chitosan, anionic surfactants derived from amino acids, nonionic surfactants derived from poly-glycosides, surfactants, hydrotropic active agents, lipopeptides such as isomers of surfactin and / or the isomers of fengycin (or plipastatin), rhamnolipids and vegetable oils.

More particularly, the invention relates to anionic surfactants, nonionic surfactants and oils. The surfactants of the family of anionic surfactants are chosen from: surfactins, fengycins, or amino acid derivatives. Surfactin means any molecule of the "family of surfactins", namely surfactins A, B, C, lichenysine and pumilacidine.

By fengycin is meant any molecule of the "fengycin family", is meant fengycins A and B, plipastatin A and B and agrastatin.

The surfactants of the family of nonionic surfactants are chosen from: fatty alcohol axalkylate, pentylene glycol and its derivatives, hydrotropic molecules of alkylpolyglycoside type (alkypolyglycoside and alkylethoxypolyglycoside, polyglycoside texturing agent-type molecules (xanthan gum , gum arabic, gum tragacanth, guar gum, locust bean gum, tamarind gum, pectin, gellan gum, carrageenates, agar-agar, alginates.)

The surfactants of the family of oils are chosen from: oils and extracts of modified oils (acifidies, methylated, esterified), especially derived from: 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, nut, flaxseed, evening primrose, barley, pumpkin seeds, grape seeds, peas, sesame , soya, sunflower. The iturinic lipopeptides will be micro-emulsified in these oily compounds or fats in order to obtain the surfactant effect of the oils and extracts of modified oils.

The compositions of the invention may further contain adjuvants such as lipid sources, salts and solvents. These adjuvants can participate in the solubilization without being surfactants. As examples of solvents, mention may be made of ethanol, methanol, acetonitrile, dimethyl sulfoxide, butanol, pentanol and acetone.

According to a first aspect, the invention relates to a composition comprising between 5 and 50 g / l of iturinic lipopeptides and between 1 and 40 g / l of surfactin.

In a particular composition, iturinic lipopeptides are mycosubtilines. In another particular composition, the amount of iturinic lipopeptides is preferably between 10 g / l and 50 g / l, preferably between 20 and 50 g / l, more preferably between 30 and 50 g / l. According to a second aspect, the invention relates to a composition comprising between 5 and 100 g / l of iturinic lipopeptides, between 1 and 40 g / l of surfactin and between 1 and 100 g / l of fengycin (or plipastatin).

 In a particular composition, the amount of iturinic lipopeptides is preferably between 10 and 80 g / l, preferably between 20 and 80 g / l, most preferably between 30 and 80 g / l.

According to a third aspect, the invention relates to a composition comprising between 5 and 100 g / l of iturinic lipopeptides, between 1 and 40 g / l of surfactin and between 1 and 30 g / l of chemical surfactants.

 In a particular composition, the amount of iturinic lipopeptides is preferably between 10 and 80 g / l, preferably between 20 and 80 g / l, preferably between 30 and 80 g / l. In a particular embodiment, the chemical surfactants may be nonionic surfactants of the family of polyglycosides or anionic surfactants derived from amino acids. Preferably, the chemical surfactants consist of a mixture of anionic surfactants and nonionic surfactants. According to a fourth aspect, the invention relates to a composition comprising between 5 and 100 g / l of iturinic lipopeptides, between 1 and 40 g / l of surfactin, between 1 and 100 g / l of fengycin (or plipastatin) and between 1 and 30 g / l of chemical surfactants.

In a particular composition, the amount of iturinic lipopeptides is preferably between 10 and 80 g / l, preferably between 20 and 80 g / l, preferably between 30 and 80 g / l.

According to a fifth aspect, the invention relates to a composition comprising between 5 and 100 g / L of micro-emulsified iturinic lipopeptides in oily compounds or fatty substances, for example vegetable oil. In such a composition, the oil concentration is between 1% and 100%, preferably between 50% and 100%, particularly preferably between 80% and 100%. Among the vegetable oils that can be used, mention may be made of corn oil, but also modified almond, peanut, argan, avocado, rapeseed, lorenzo, neem, hazelnut, and cashew nut oils and extracts, macadamia nut, olive, pistachio, rice, oleic sunflower, camelina, flax, borage, safflower, hemp, cotton, wheat germ, corn, nut, flaxseed, evening primrose, barley, pumpkin seeds, grape seeds, peas, sesame , soy, and sunflower.

Preferably, the oils used are derived from peanut, olive, oleic sunflower, flax, corn, nuts, soya and sunflower. Such oils present the advantage of being available in large quantities and having a low cost. Even more preferably, the oil used is derived from corn peanuts or sunflower.

In a preferred embodiment, such a composition comprises between 10 and 80 g / l, preferably between 20 and 80 g / l of mycosubtiline, preferably between 30 and 80 g / l and very preferably between 50 and 80 g / l. g / L of mycosubtilin.

Thus, a particular composition according to the invention comprises:

 2 to 8% of iturinic lipopeptides

 - 3% of chemical surfactants consisting of a mixture of nonionic and anionic surfactants up to 1.5% each

 2 to 4% surfactines

 0 to 10% fengycins or plipastatins In a particular embodiment, the composition comprises about 3% of lipopeptides (1.25% of mycosubtilin and 1.85% of surfactin), 1.5% of a non-surfactant. ionic derivative derived from poly-glycosides and 1.5% of anionic surfactant derived from amino acids. In another particular embodiment, the composition comprises about 5% lipopeptides (2.45% mycosubtiline and 2.65% surfactin) and 3-8% fengycin (or plipastatin).

In another particular embodiment, the composition comprises between 30 and 80 g / l of mycosubtiline and 30 to 80 g / l of fengycin (or plipastatin).

In a particular embodiment the composition comprises a concentration of iturinic lipopeptides is between 20 and 100g / l and the surfactants are a vegetable oil extract, derivatives of polyglycosides and surfactin. In another particular embodiment the composition comprises a concentration of iturinic lipopeptides is between 20 and 100g / l and the surfactants are an extract of methylated vegetable seed oil and polyglycerol ester of coconut oil (Synergen OS ™) .

In another particular embodiment, the composition comprises a concentration of iturinic lipopeptides of between 20 and 100 g / l and the surfactants are a mixture of alkylpolyglucoside (Simulsol ™) amino acid derivative (Proteol ™) and surfactin.

In another particular embodiment the composition comprises a concentration of iturinic lipopeptides is between 20 and 100g / l and the surfactants are a mixture of fatty alcohol oxalkylate (Emulsogen ™) and surfactin.

In another particular embodiment the composition comprises a concentration of iturinic lipopeptides is between 20 and 100g / l and the surfactants are a mixture of vegetable oil, xanthan gum and surfactin.

The compositions according to the invention may be ready for use or in concentrated form; concentrated solutions can be diluted before use. A second object according to the invention relates to a dehydrated composition comprising a lipopeptide mixture in which the concentration of iturinic lipopeptides is greater than 0.5%. In a preferred embodiment, the concentration of iturinic lipopeptides is greater than 2%.

 Such dehydrated compositions are in the form of powder or lyophilizate and are "ready to use"; indeed, it is sufficient to rehydrate in 100 ml of solution to obtain a composition whose concentration of iturinic lipopeptides is at least 5 g / l. In a preferred embodiment, such compositions have a concentration of iturinic lipopeptides of at least 20 g / L.

 The ingredients and their relative amounts in these solid compositions are the same as those described above for liquid compositions. In fact, the concentrations by dry weight of the various components of these dehydrated solutions make it possible to obtain liquid compositions whose concentration of iturinic lipopeptides is greater than 5 g / l which are stable and homogeneous in accordance with the invention. In a preferred embodiment, the concentration of iturinic lipopeptides is greater than 20 g / l.

A third subject of the invention relates to a method for preparing a composition comprising a lipopeptide mixture in which the concentration of iturinic lipopeptides is greater than 5 g / l. In a preferred embodiment, the concentration of iturinic lipopeptides in the mixture is 20 g / l.

The iturinic lipopeptides of the composition can be obtained by fermentation of a strain of Bacillus sp. and harvested and concentrated from the culture supernatant. The compositions according to the invention can be prepared using lipopeptide preparations commercially available in powder form. These starting powders contain a variable amount of iturinic lipopeptides which may range from 10 to more than 80%, for example 15% or 75%. These powders may contain a mixture of mycosubtilin and surfactin in a ratio of 30/70 to 70/30, or even 95/5, for example 35/65, 40/60, 45/55 or 50/50. .

To prepare a composition according to the invention, at least one surfactant must be added to a solution containing a high content of iturinic lipopeptides.

The process for preparing such compositions may include an additional dehydration step so as to provide a ready-to-use powder form composition, the characteristics of which are in accordance with the compositions of the present invention when rehydrated.

Thus, the method makes it possible to prepare liquid or solid solutions (in the form of powder or lyophilisate). Such concentrated solutions are particularly suitable for storage and distribution. They are to be diluted before use. As a result, a composition according to the invention can be obtained by dissolving a powder allowing the reconstitution of a liquid composition as defined above.

The compositions according to the invention find their applications in the field of agri-food, phytosanitary and cosmetic as well as in the medical and pharmaceutical field.

The invention is illustrated by the following examples.

EXAMPLES

 A. Preparation of solutions containing lipopeptides from supernatant of cultures of strains Bacillus 1 - Obtaining culture supernatants

The lipopeptides are obtained from an aerobic fermentation process of a Bacillus strain derived from the strain Bacillus subtilis ATCC 6633 for the production of lipopeptides iturinic and strains derived from the Bacillus subtilis 168 strain for the production of surfactin and / or fengycin (or plipastatin). The culture is carried out in a medium containing a source of carbon (glucose, sucrose, etc.), a source of nitrogen (ammonium sulphate, peptone, free amino acids, yeast extract, etc.) and trace elements and salts with stirring at 30 ° C. The pH is maintained at a value of 7. The crop is harvested after 48 to 72 hours. It is then centrifuged or filtered to remove the cells. The culture supernatant is then concentrated.

2 - Preparation of compositions with a high content of iturinic lipopeptides from culture supernatants

To obtain compositions with a high content of iturinic lipopeptides, the culture supernatants can be concentrated. The concentration of the culture supernatant can be obtained by any method known to those skilled in the art, in particular:

 - By tangential ultrafiltration using a membrane whose cutoff threshold can be from lKDa to 300 KDa. For example, 1000L of culture supernatant obtained as described above are concentrated by passage over the ultrafiltration membrane to obtain a retentate with a volume of 10 to 100L. The small molecules are then removed by one or more diafiltration steps. The solution thus obtained can be dried by lyophilization, atomization or used as it is.

 By precipitation at acidic pH: a decrease of the pH in order to selectively precipitate the lipopeptides is carried out. An addition of concentrated sulfuric acid is carried out on the obtained supernatant. After obtaining a final pH around 1, the solution is stirred for 2 to 12 hours. Centrifugation makes it possible to recover a pellet of material containing the lipopeptides. This pellet is then dissolved by addition of water and sodium hydroxide in order to obtain a pH value of between 7 and 8.5. The solution thus obtained can be dried by lyophilization, atomization or used as it is.

 - By evaporation: the supernatant is concentrated by evaporation under vacuum. For example 20L are introduced into a rotavapor and concentrated to 1 to 2L.

The percentage of lipopeptides obtained at the end of these two examples of preparation is of the order of 0.5 to 20% (weight / volume). B-Preparation of compositions concentrated in iturinic lipopeptides

1 - Study of the effect of different surfactants on the solubility of an iturinic lipopeptide and in a composition with a high concentration An iturinic lipopeptide (in this case mycosubtiline), having a purity (on dry matter) of between 45% and 80%, was solubilized alone in water at different concentrations and this control composition was compared with various compositions in which were added different surfactant compounds as described in the application. After storage for 15 days at 21 ° C., only the compositions having a fluid-type rheological behavior and a homogeneous appearance (not exhibiting a mass-formation preventing flow and / or phase shift and / or precipitate) are selected, mixed together. by stirring, then these compositions are centrifuged for 10 minutes at 10,000 g and the supernatant is analyzed by the RP-HPLC reference method. This method makes it possible to check the solubility of the iturinic compounds which are then found in the supernatant after centrifugation.

 The solubility of the iturinic lipopeptide (in this case mycosubtilin) alone in an aqueous solution as a function of its concentration is presented in FIG.

 The maximum concentration of soluble iturinic lipopeptides in the various compositions is shown in FIG.

No surfactant is added in composition 1 which contains only iturinic lipopeptides. The surfactants added in the compositions 2 to 13 are:

10% fengycins (composition 2)

alkylpolyglucoside and amino acid derivatives (Simulsol ™ / Protéo ^M ) in a proportion of 10% each (composition 3)

 soybean lecithin (4%) and poly-glycoside derivatives (0.1%) (Elvis ™ / Xanthan Gum) (composition 4)

 of methyl vegetable seed oil extract and polyglycerol ester derived from coconut oil (0.2%) (Synergen OS ™) (composition 5)

 nonionic surfactant of the fatty alcohol oxalkylate (0.2%) type (Emulsogen ™) (composition 6)

nonionic surfactant pentylene glycol (50%) (composition 7)

 derivatives of 0.4% poly-glycosides (AlkylEthoxyGlycoside = AEG) (composition 8) -4% surfactins (composition 9)

 surfactines at 4% and fengycins at 10% (composition 10)

 vegetable oil extract (30%) and derivatives of poly-glycosides (0.1%) and surfactin (4%) (corn oil / Xanthan Gum / Surfactin) (composition 11)

alkylpolyglucoside and amino acid derivatives (Simulsol ™ / Protéo ^M ) at 10% each and surfactin at 4% (composition 12) 0.2% fatty alcohol oxalylate nonionic surfactant (Emulsogen ™) and 4% surfactin (composition 13)

Results analysis

Figure 1 shows the solubility of iturinic lipopeptides (in this case mycosubtiline) in aqueous solution at room temperature as a function of their concentration

In FIG. 1, it can be observed that the mycosubtilin alone solubilizes very well in an aqueous solution at a maximum concentration of 2 g / l, but beyond this concentration (for example between 5 and 25 g / l) a decant precipitate can be observed and only a small part remains soluble.

FIG. 2 shows the analysis of the solubility of various compositions comprising an iturinic lipopeptide (in this case mycosubtilin) and various compounds making it possible to obtain high and soluble concentrations of iturinic lipopeptide after 15 days of storage at ambient temperature. The measurement is made on the supernatant after a centrifugation step to eliminate the insolubles

In FIG. 2, it can be observed that the addition of different surfactants makes it possible to significantly increase the solubility of the mycosubtiline, in particular with anionic compounds alone or as a mixture with nonionic surfactants. The presence of fengycin or surfactin makes it possible to obtain a solubility of the mycosubtilin close to 30 g / l. In a surprising and original way, the combination of these two anionic lipopeptide compounds makes it possible to achieve a solubility of the iturinic compound which is still superior. It will also be noted that the use of certain surfactants despite a homogeneous and stable composition does not make it possible to obtain a solubilization above 20 g / l. This is the case of soy lecithin coupled with Xanthan gum (max = 1.5g / L), the case of the nonionic compound Emulsogen ™ (Clariant) (max = 11.6 g / L) but also nonionic compounds Simulsol ™ and anionic Protéol ™ (Ceppic) (max = 15.3 g / L). It has been noted for these compositions the presence of a large precipitate after the centrifugation step. On the other hand, when these compositions are supplemented with another anionic lipopeptide compound such as surfactin, the solubility of the mycosubtiline then increases to reach values between 55 and 120 g / l. Other nonionic compounds such as penthylene glycol and alkylethoxyglycoside also make it possible to obtain a solubility of greater than 30 g / l. The composition allowing a maximum solubility of the iturinic compound (112 g / L) is that containing an anionic lipopeptide (surfactin) and a nonionic surfactant (Emulsogen ™). 2 - Preparation of compositions with a high content of iturinic lipopeptides from lipopeptide powders A composition with a high content of iturinic lipopeptides can be prepared from lipopeptide powder, as illustrated in the following two examples of compositions: Composition 1: A composition comprising approximately 3.1% (w / v) of lipopeptides (ie 1.25% mycosubtiline and 1.85% surfactin) was obtained by resuspending a lipopeptide powder comprising a mycosubtilin / surfactin mixture in proportions of 40%. 60 and a purity of about 15% in aqueous phase (pH between 7.5 and 8.5).

 Composition 2: A composition comprising approximately 5.1% (w / v) of lipopeptides (ie 2.45% mycosubtiline and 2.65% surfactin) was obtained by resuspending a lipopeptide powder comprising a mycosubtilin / surfactin mixture in proportions 45/55 and a purity of about 75% in aqueous phase (pH between 7.5 and 8.5).

3 - Preparation of compositions with a high content of iturinic lipopeptides comprising surfactants.

Surfactants were added to compositions 1 and 2 described above so as to evaluate the effect of such molecules on the properties of the compositions.

Composition 1A: Two surfactants were added to the composition 1 each in a proportion of 10% (v / v), namely a nonionic hydrotropic surfactant derived from polyglycosides (Trade name = Simulsol ^™ SL 7C marketed by the company Seppic) and an anionic surfactant derived from amino acids (Trade name = Proteol ^™ APL marketed by Seppic).

 Composition 2A: 3 to 8% (w / v) fengycin (or plipastatin) was added to composition 2.

4 - Characterization of the compositions obtained: homogeneity and stability over time.

Stability test at room temperature

The appearance of the compositions with and without surfactants has been studied at ambient temperature, here at about 21 ° C.

 Composition 1: The addition of the two surfactants made it possible to obtain a homogeneous composition (composition 1A) and to avoid any phenomenon of precipitation or sedimentation. The composition 1A containing surfactants has a cloudy but homogeneous appearance.

 Composition 2: The addition of fengycin (or plipastatin) made it possible to obtain a homogeneous composition and to avoid any phenomenon of setting in mass (which takes place in the absence of fengycin or plipastatin). Composition 2A with fengycin (or plipastatin) has a clear and homogeneous appearance. b. Low temperature stability tests

Concentrated lipopeptide solutions (Compositions 1, 2 and 1A, 2A) were placed at 4 ° C for a period of 1 to 30 days. Only solutions supplemented with surfactants or fengycin (or plipastatin) (namely 1A and 2A) remained homogeneous and no deposit was observed. Without these additions, concentrated lipopeptide solutions are likely to set in bulk or to have a large insoluble deposit.

5 - Characterization of the rheology of the compositions obtained: impact of conservation at ambient temperature and accelerated aging

 at. Analytical methods

 The 2 compositions described above (1 and 2) and those containing adjuvants (1A and 2A) were stored for 15 days at 21 ° C and for 15 days at 54 ° C (to simulate accelerated aging). The preservation of compositions at elevated temperature (54 ° C.) is a method known to those skilled in the art, which makes it possible to mimic accelerated aging of the compositions by increasing the Brownian motion, accelerating the destabilization of the compositions.

At the end of these periods, the rheology of the compositions was studied using a Anton Paar MCR102 modular compact rheometer. The method consisted in monitoring the shear rate when the shear stress gradually increased between 1 and 100 Pa (500 s test duration). Viscosity (the ratio of shear stress to shear rate) was also expressed as a function of shear stress. This allowed to visualize the evolution of the viscosity during the test. These tests were carried out from samples trilplicat.

 Two rheological parameters have been studied on these compositions after storage at 21 ° C. or at 54 ° C., namely:

The minimum viscosity (Pa.s) Shear stress at maximum viscosity (Pa) b. Results

All the samples showed behaviors of non-Newtonian fluids (the viscosity was not constant during the tests). As the shear stress increased, the viscosity decreased as it approached a minimum viscosity. The decrease in viscosity with agitation can be explained by a progressive alignment of the structural units (or molecules) in the direction of flow as the shear rate increases, thus promoting the flow of the different layers of liquids. The fluids are then qualified as rhefluidifiers and can be characterized by their minimum viscosities (in the case of the applied tests it was the viscosity at the end of the test) and by the shear stress at the maximum viscosity (Pa).

Study of the minimum viscosity

FIG. 3 shows the analysis of the minimum viscosity of the various concentrated iturin compositions without or with additions of surfactants and other adjuvants (A), after storage at 21 ° C. or at 54 ° C.

The results presented in FIG. 3 show that the minimum viscosity of the composition 1A after storage at 21 ° C. is significantly different and lower than that of the unformulated composition (1). No significant difference is observed between compositions 2 and 2A after storage at 21 ° C. Nevertheless, during the aging test at 54 ° C. the minimum viscosity of the composition 2 has very markedly increased while that of the composition 2A has remained close to 0. This shows the very important effect of the addition of fengycin (plipastatin ) in the latter to facilitate the solubility of iturinic lipopeptides. Note a similar effect between the composition 1 and 1A after storage at 54 ° C, showing the interest of the formulation of the composition 1A.

Study of shear stress at maximum viscosity

FIG. 4 presents the analysis of the maximum viscosity shear stress of the various lipopeptide compositions concentrated without or with additions of surfactants and other adjuvants (A), after storage at 21 ° C. or at 54 ° C.

The results presented in FIG. 4 show that the shear stresses at maximum viscosity after storage for 15 days at 21 ° C. of composition 1A are lower than to those obtained with the unformulated composition 1. No significant difference is observed between the compositions 2 and 2A. Nevertheless, when studying the impact of accelerated aging at 54 ° C. on this parameter, the results of FIG. 4 show that the shear stress increases very strongly in the non-formulated compositions 1 and 2, whereas those of the compositions Forms (1A and 2A) remained almost identical to those obtained at 21 ° C. These results show the very positive impact of the formulations according to the invention to facilitate the solubilization in time and high concentration of iturinic compounds. In conclusion of these studies, it can be established that:

The composition 1A makes it possible to solubilize the iturinic compounds avoiding the formation of precipitate and / or gel and this is characterized by a significant decrease in the minimum viscosity and the shear stress at maximum viscosity, whether after storage at 21 ° C. or even more importantly when aging. Compositions 1 and 1A are related to non-Newtonian fluids of the Casson type

 The composition 2A makes it possible to solubilize the iturinic compounds avoiding the formation of precipitate and / or gel at the end of a long storage period and this is characterized by a significant decrease in the minimum viscosity and the shear stress at the viscosity. maximum after storage at 54 ° C. Compositions 2 and 2A are related to non-Newtonian fluids of rheofluidifying type initially, but the unformulated composition 2 becomes very thick during aging.

Claims

1. stable and homogeneous composition characterized in that it comprises a concentration of iturinic lipopeptides greater than 20g / L and one or more surfactants selected from families of anionic surfactants, nonionic surfactants or oils. 2. Composition according to claim 1 wherein the concentration of iturinic lipopeptides is between 20 and 150 g / l. 3. Composition according to one of claims 1 or 2 characterized in that said surfactants of the family of anionic surfactants are selected from: molecules of the surfactin family, molecules of the fengycin family, or derivatives of amino acid. 4. Composition according to one of claims 1 or 2 characterized in that said surfactants of the family of nonionic surfactants are selected from fatty alcohol axalkylate, pentylene glycol and its derivatives, hydrotropic alkylpolyglycoside type molecules (alkypolyglycoside and alkylethoxypolyglycoside, polyglycoside texturing agent molecules (xanthan gum, gum arabic, gum tragacanth, guar gum, locust bean gum, tamarind gum, pectin, gellan gum, carrageenan, agar-agar, alginates.) 5. Composition according to one of claims 1 or 2 characterized in that said surfactants of the family of oils are chosen from: oils and extracts of modified oils (acifidies, methylated, esterified) including derived from: 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, but, nut, flax , evening primrose, barley, pumpkin seeds, grape seeds, peas, sesame, soya, sunflower. 6. Composition according to one of the preceding claimswherein the concentration of iturinic lipopeptides is between 20 and 50 g / 1 and the surfactants are surfactin, the concentration of surfactin in the composition being between 1 and 40 g / L. 7. Composition according to one of the preceding claimswherein the concentration of iturinic lipopeptides is between 20 and 50 g / l and the surfactants are fengycin, the concentration of fengycin in the composition being between 1 and 100 g / l. 8. Composition according to one of the preceding claimswherein the concentration of iturinic lipopeptides is between 20 and 100 g / 1 and the surfactants are surfactin and fengycin, the concentration of surfactin being between 1 and 40 g / L and the concentration of fengycin being between 1 and 100 g / l. 9. Composition according to one of the preceding claims wherein the concentration of iturinic lipopeptides is between 20 and 100g / l and the surfactants are a vegetable oil extract, derivatives of polyglycosides and surfactin. 10. Composition according to one of the preceding claims wherein the concentration of iturinic lipopeptides is between 20 and 100g / l and the surfactants are an extract of methylated vegetable seed oil and polyglycerol ester of coconut oil. 11. A method for preparing a composition as defined in one of the preceding claims comprising the steps of preparing a solution with a high content of iturinic lipopeptides and adding surfactants. 12. Preparation process according to claim 11 further comprising a dehydration step. 13. Use of a composition according to one of claims 1 to 10 in agri-food, phytosanitary or cosmetic applications. 14. Composition according to one of claims 1 to 10 for use in the medical or pharmaceutical field.