FR3029741A1 - METHOD FOR PRESERVING AMBIENT TEMPERATURE MICROORGANISMS AND COMPOSITION - Google Patents

Abstract

The invention relates to a method for the preservation of microorganisms at ambient temperature, in which: a composition, called a preservation composition, comprising the microorganisms to be preserved and a preservation medium for said microorganisms, is prepared, characterized in that: The preservation medium comprises at least one compound, said preservative compound, selected from the group consisting of compounds for which there is at least a predetermined inhibitory proportion of the preservative compound in the preservative composition which is: ▪ inhibiting the growth of said micro -organisms in the room temperature preservation composition, and; Non-lethal for said microorganisms, and in that; Said preservative compound is in the preservative composition in a proportion greater than said predetermined inhibitory proportion and in a non-lethal proportion for said microorganisms; and wherein the storage composition is placed at room temperature for the purpose of preserving said microorganisms. In particular, the invention relates to such a process in which the preservative compound is a glycerol carbonic ester.

Description

The invention relates to a method for the in vitro and at ambient preservation of living microorganisms and a composition for the preservation of living microorganisms at ambient temperature for the purpose of setting up microorganisms at ambient temperature. of such a process. The invention is also directed to a method of treating plants in which a preservation composition comprising living microorganisms useful for plants is used. Cell preservation methods are known in which glycerol and / or dimethylsulfoxide (DMSO) are used as additive preserving the cells with respect to freezing / thawing during their storage at low temperature (-20 ° C. , -80 ° C or -176 ° C in liquid nitrogen). Such a method of cell preservation is complex in its implementation and requires the use of specific devices for storing cells at a temperature below 0 ° C., in particular below -20 ° C. There is also known a method of preserving microorganisms in the at least partially dehydrated state. Such dehydration is generally carried out by freezing the cells at low temperature and then sublimation of the water at low pressure by lyophilization. On the one hand, many cell types can not be preserved by lyophilization, this dehydration by lyophilization not being compatible with the maintenance of the integrity of the cells. On the other hand, such a method requires the use, for freeze-drying, of a specific freezing device which may be a freezer or a bath of liquid nitrogen. The maintenance of such a specific freezing device is complex and expensive. The use of living microorganisms to combat fungi and pathogenic bacteria of plants and diseases caused by these fungi and pathogenic bacteria is known, for example WO 2012/016140, WO 2009/156688 or WO 2009 / 156687. These documents do not deal with the conservation of microorganisms for use in agriculture for the treatment of plants.

Indeed, such use requires a farmer to have a composition - for example, a liquid composition - comprising such living microorganisms ready to be applied to plants. Such a liquid composition comprising live microorganisms in the growth phase can not be conserved sustainably by the farmer. Indeed, such a composition comprising bacteria in exponential growth phase is likely to quickly enter the stationary phase and then decay phase leading to the loss of bacterial culture. Such a composition must therefore be prepared extemporaneously by the farmer or made available to the farmer - by the distributor or the manufacturer - at the moment when his use is envisaged. Another solution is to be able to keep the liquid composition comprising living microorganisms at a low temperature, at + 4 ° C. or at -20 ° C. or at -80 ° C. in a freezer or at -176 ° C. in the nitrogen. liquid. Such storage conditions can not in practice be implemented by the farmer. The invention aims to overcome the disadvantages mentioned above by providing a method for the preservation of microorganisms at room temperature and a composition for conserving microorganisms that do not require storage of said microorganisms at low temperature.

The object of the invention is thus to propose a process for the preservation of microorganisms at ambient temperature, that is to say a process which does not require placing microorganisms in a specific cooling device. The object of the invention is therefore to propose a process for the storage of microorganisms at ambient temperature which is applicable in any technical field in which it is necessary to conserve microorganisms. The object of the invention is to propose such a process which is particularly applicable in the agricultural field in which micro-organisms are likely to have to be applied to plants, in particular to stimulate their growth and / or their natural defenses (SDN). .

The invention also aims to provide a preservation method and composition that preserve farmers' work habits, are easy to use, and involve little manipulation in their implementation.

The invention also aims at providing a preservation method and composition in which compounds which can be obtained from renewable plant resources are used. Another object of the invention is to provide such a method and such a conservation composition which respect the environment, in particular during the application of said composition for preserving microorganisms on plants. To this end, the invention relates to a method for preserving microorganisms at room temperature, in which: a composition, called a preservation composition, is prepared, comprising the microorganisms to be preserved and a medium for conserving said microorganisms; organisms, characterized in that: the preservation medium comprises at least one compound, said preservative compound, selected from the group consisting of compounds for which there is at least a predetermined inhibitory proportion of the preservative compound in the preservation composition which is inhibiting the growth of said microorganisms in the storage composition at room temperature, and; not lethal to said microorganisms, and in that; Wherein said at least one preservative compound is in the preservative composition in a proportion greater than said predetermined inhibitory proportion and in a non-lethal proportion for said microorganisms; and in which the storage composition is placed at room temperature, particularly at a temperature greater than or equal to + 4 ° C., for the purpose of preserving said microorganisms.

In the following, the term "conservation of microorganisms at ambient temperature" means the conservation of microorganisms which does not require any specific means for controlling and adjusting the temperature of the composition comprising the microorganisms. .

The invention therefore consists in providing a method for the preservation of microorganisms in vitro and at room temperature in which a preservation medium comprising at least one preservative compound admitting at least a predetermined proportion which is inhibiting the growth of at least one microorganism and not lethal to said at least one microorganism, said at least one preservative compound being in the preservation medium in a proportion greater than its predetermined inhibitory proportion. The proportion of preservative compound in the preservation composition is chosen to be able to reversibly inhibit the growth of microorganisms, but without being lethal to said microorganisms. In particular, the process according to the invention makes it possible, in a first conservation phase, to inhibit the growth of microorganisms at room temperature, but also, after appropriate dilution of the preservation composition, to allow the growth of the microorganisms in a growth composition obtained by dilution of the preservation composition.

The preservation medium is chosen to maintain the culture of microorganisms in a stationary phase of growth-without degeneration of said culture-at room temperature, while preserving the potential of said microorganism culture to resume later exponential phase of growth. The microorganisms stored at room temperature in the preservation composition are therefore not in the decay phase. The proportion of preservative compound greater than the predetermined inhibitory proportion in the preservation composition is therefore not lethal for the microorganisms. The inventors have indeed found that it is possible to keep microorganisms at room temperature-that is to say to maintain microorganisms in the latency phase (or dormancy) by inhibiting their growth. preserving their potential for subsequent growth by placing these microorganisms in a suitable preservative composition comprising a preservative compound in a proportion selected to be growth inhibiting but not lethal for these microorganisms. A preservative compound 5 according to the invention therefore has no antibiotic property-that is to say, it does not lead to the death of microorganisms-but only a reversible inhibitory property of the growth of microorganisms. . In a process according to the invention, at least one preservative compound is selected from the group consisting of compounds admitting at least a proportion in the preservation medium which is inhibiting the growth of microorganisms, but not lethal to these microorganisms. organizations. The predetermined inhibitory proportion of a preservative compound in the preservative composition is the value of the proportion of preservative compound in the preservative composition resulting in the complete cessation of growth of a microorganism without being lethal to the microorganisms. organizations. It is determined that a proportion of preservative compound greater than the predetermined inhibitory proportion is non-lethal by diluting the preservative composition-so that the proportion of preservative compound is less than the predetermined inhibitory proportion-and by noting the growth of microorganisms. organisms in the growth composition obtained by dilution of the preservation composition. The minimum inhibitory proportion of a preservative compound is predetermined by methods for analyzing the growth of microorganisms known per se.

Advantageously, the preservative compound used in a process according to the invention in a proportion greater than the predetermined inhibitory proportion makes it possible to inhibit the growth of microorganisms for a period of at least 30 days, without being lethal to said microorganisms. In a process according to the invention, the dilution of the preservation medium - especially in a culture medium adapted for the growth of the microorganisms - makes it possible in fact to lift the inhibition of the growth of the microorganisms by the preservative compound. The invention therefore relates to a process in which the preservation medium and the preservation composition comprise a preservative compound capable of inhibiting the growth of microorganisms for a proportion of preservative compound greater than a predetermined inhibitory proportion value, said proportion of however, the preserving compound is non-lethal to microorganisms and capable of permitting the growth of microorganisms at a proportion of preservative compound less than said predetermined inhibitory proportion value. Advantageously and according to the invention, at least one preservative compound is selected from the group consisting of undecylenic acid, esters of undecylenic acid and glycerol carbon esters. Throughout the text, the term "glycerol carbonic ester" means any compound comprising at least one carboxylic ester chemical function formed between a group derived from carbonic acid and a group derived from glycerol; "carbonic acid-derived" group, any group of the following formula: ## STR2 ## in which the hydrogen atoms of the carbonic acid are substituted with an organic group which is therefore distinct from the group hydrogen, and by; "glycerol derivative" group means any group of the following formula: in which at least one hydrogen atom of the hydroxyl groups of the glycerol is substituted with an organic group that is distinct from hydrogen.

Advantageously and according to the invention, at least one preservative compound is selected from the group consisting of glycerol carbonate (cyclic), esters of glycerol (cyclic) carbonate and linear carboxy esters of glycerol.

The glycerol carboxy esters, in particular glycerol carbonate, glycerol carbonate (cyclic) esters and glycerol, linear carboxylic esters, undecylenic acid and undecylenic acid esters are suitable for application on plants and meet the standards of protection and preservation of the environment. They do not in themselves exhibit toxicity to human or animal health. They are biodegradable and do not present a risk of accumulating in the soil and contaminating or damaging the environment, especially when applied to plants and / or when introduced into the soil. They do not present a risk of contaminating the environment when they are applied in the open field and are likely to be carried away by runoff with rainwater or watering. They can be "biobased", that is to say obtained from natural resources - in particular plant resources - available and renewable. In a variant of a process according to the invention, at least one preservative compound is glycerol carbonate. By glycerol carbonate is meant the five-membered (4- (hydroxymethyl) -1,3-dioxolan-2-one) glycerol cyclic carbonate of the following formula (I): (3C / x / CH-CH 2 CH 2 OH ( I), referenced under the number CAS 931-40-8 and of which a synthesis method is described for example in FR 2 778 182.

In another variant of a process according to the invention, at least one preservative compound is an ester of glycerol carbonate (or acylated glycerol carbonate). In this variant, at least one preservative compound is a 5-membered α / α'-acylated glycerol carbonate (cyclic) ester of formula (II) below: (II), in which R 1 is a grouping aliphatic hydrocarbon -saturated, unsaturated, linear or branched-5 comprising between 1 and 30 carbon atoms. The group R 1 can be chosen from the group formed by saturated aliphatic hydrocarbon groups. The group R 1 can be methyl (-CH 3), ethyl (-CH 2 -CH 3), n-propyl (-CH 2 -CH 2 -CH 3), iso-propyl (-CH (CH 3) 2), n-propyl butyl (-CH 2 -CH 2 -CH 2 -CH 3), iso-butyl (-CH 2 -CH (CH 3) 2), tert-butyl (-C (CH 3) 3), n-pentyl (- (CH 2) 4-CH3), n-hexyl (- (CH2) 5-CH3), n-heptyl (- (CH2) 6-CH3), n-octyl (- (CH2) 7-CH3), n- nonyl (- (CH2) 8-CH3), n-decyl (- (CH2) 9-CH3), n-undecyl (- (CH2) 10 -CH3), n-dodecyl (- (CH2) 11- CH3), n-tridecyl (- (CH2) 12-CH3), n-tetradecyl (- (CH2) 13-CH3), n-pentadecyl (- (CH2) 14-CH3), n-hexadecyl (- (CH2) 15-CH3), n-heptadecyl (- (CH2) 16-CH3), n-octadecyl (- (CH2) 17-CH3), n-nonadecyl (- (CH2) 18 -CH3 ), n-dodecadecyl (- (CH2) 19-CH3). The group R 1 can be chosen from the group formed by unsaturated aliphatic hydrocarbon groups. The group R 1 may be an aliphatic hydrocarbon group having at least one ethylenic double bond. The group R 1 may be an aliphatic hydrocarbon group having 1, 2, 3, 4 or 5 ethylenic double bonds, conjugated or otherwise. Advantageously, the group R 1 can also be chosen from the group formed: 9-ene-decyl of formula CH 2 = CH- (CH 2) 7 -CH 2 -. At least one preservative compound is the (cyclic) carbonate ester of glycerol and undecylenic acid; ## STR2 ## ## STR1 ## ## STR1 ## ## STR1 ## ## STR2 ## At least one preservative compound is the (cyclic) carbonate ester of glycerol and palmitic acid; 8-ene-heptadecyl CH3- (CH2) 7-CH = CH- (CH2) 7-. At least one preservative compound is the (cyclic) carbonate ester of glycerol and oleic acid; the 12-ene-heneicosyl group CH 3 - (CH 2) 7 -CH = CH- (CH 2) i-. At least one preservative compound is the (cyclic) carbonate ester of glycerol and docosaenoic acid.

In a particular embodiment, it is possible to choose at least one preservative compound from the group formed: esters of the (cyclic) carbonate of glycerol and a hydrocarbon acid with an even number of carbon atoms - or acid hydrocarbon-pair, and; linear carbonic esters of glycerol and at least one hydrocarbon acid with an even number of carbon atoms or even hydrocarbon acid. In another embodiment, advantageously and according to the invention, at least one preservative compound is selected from the group consisting of: esters of the (cyclic) carbonate of glycerol and of an odd number of carbon atoms hydrocarbon acid or odd hydrocarbon acid, and; linear carbonic esters of glycerol and at least one hydrocarbon acid with an odd number of carbon atoms or an odd hydrocarbon-based acid. Advantageously and according to the invention, R 1 is a linear aliphatic hydrocarbon group - unsaturated or unsaturated - having a main carbon chain with an even number of carbon atoms. The acyl group (R1-00-) of the cyclic carboxylic ester of glycerol α / α'-acylated is therefore a group having an carbon chain main carbon odd number of carbon atoms. In particular, R 1 is selected from the group consisting of n-hexyl (- (CH 2) 5 -CH 3), n-octyl (- (CH 2) 7 -CH 3) and a 9-ene-decyl (- (CH 2 ) 8-CH = CH2). At least one preservative compound may be: the ester of glycerol carbonate and heptanoic acid (ECG-C7) of the following formula (III): (III); the ester of glycerol carbonate and nonanoic acid (ECG-C9) of the following formula (IV): (IV); - The ester of glycerol carbonate and undecylenic acid (ECGC11,1) of formula (V) below: (V).

In another variant of a process according to the invention, at least one preservative compound may be a 6-membered cyclic glycerol carbonic ester. In another variant of a process according to the invention, at least one preservative compound is a linear carboxy ester of glycerol having at least one group of atoms of general formula (VI) below: O (VI) in which Go is selected from the group consisting of: - propyl a / a'-oxyacyl groups of the following general formula (VII): ## STR2 ## f 3 -oxyacylated propyl groups of formula (VIII) - a-hydroxy-substituted propyl group of the following formula (IX): H2C-OH 5 -CHFH (IX) - propyl group f3- hydroxyl of the following formula (X): embedded image in which R 1 and R 2 are chosen independently of each other from the group consisting of saturated, unsaturated, linear or branched aliphatic hydrocarbon-containing groups; 1 and 30 carbon atoms. The R2 group of a linear carbonic ester of glycerol can be chosen, independently of R1, in the group formed by saturated aliphatic hydrocarbon groups. The R2 group may be methyl (-CH3), ethyl (-CH2-CH3), n-propyl (-CH2-CH2-CH3), iso-propyl (-CH (CH3) 2), n -butyl (-CH2-CH2-CH2-CH3), iso-butyl (-CH2-CH (CH3) 2), tert-butyl (-C (CH3) 3), n-pentyl (- (CH2) 4-CH3), n-hexyl (- (CH2) 5-CH3), n-heptyl (- (CH2) 6-CH3), n-octyl (- (CH2) 7-CH3), n- nonyl (- (CH2) 8-CH3), n-decyl (- (CH2) 9-CH3), n-undecyl (- (CH2) 10 -CH3), n-dodecyl (- (CH2) CH3), n-tridecyl (- (CH2) 12-CH3), n-tetradecyl (- (CH2) 13-CH3), n-pentadecyl (- (CH2) 14 -CH3), n-hexadecyl (- (CH2) 15-CH3), n-heptadecyl (- (CH2) 16-CH3), n-octadecyl (- (CH2) 17-CH3), n-nonadecyl (- (CH2) 18 -CH3 ), n-dodecadecyl (- (CH2) 19-CH3). The R2 group of a linear glycerol carbonic ester can be selected, independently of R 1, from the group consisting of unsaturated aliphatic hydrocarbon groups. The R2 group may be an aliphatic hydrocarbon group having at least one ethylenic double bond. The R2 group may be an aliphatic hydrocarbon group having 1, 2, 3, 4 or 5 ethylenic double bonds, at least two of which may or may not be conjugated. Advantageously, the group R2 may also be chosen from the group formed by: 5-9-ene-decyl of formula CH 2 = CH- (CH 2) 8-; 8-butene-pentadecyl CH3- (CH2) 5-CH = CH- (CH2) 7-; 8-ene-heptadecyl CH3- (CH2) 7-CH = CH- (CH2) 7-, and; 12-ene-heneicosyl CH3- (CH2) 7-CH = CH- (CH2) 11-. A linear carboxy ester of glycerol having at least one group of atoms of formula (VI) may be chosen from the group consisting of the linear carboxy esters of glycerol of general formula (VI '): MO-C-O-GO- Q1 (VI ') I in which: G1 is selected from the group consisting of: - propyl a / a'-oxyacyl groups of formula (VII); f 3 -oxyacyl propyl groups of formula (VIII); - the propyl a / a'-hydroxyl group of formula (IX); f 3 -hydroxy propyl group of formula (X); Q 1 is selected from the group consisting of hydrogen (H) and organic groups consisting of at least two atoms linked by covalent bonds and belonging to the group consisting of carbon (C) and hydrogen (H) and optionally oxygen (O) -, and; M1 represents an organic group formed by at least two atoms linked by covalent bonds and belonging to the group formed by carbon (C) and hydrogen (H) and optionally oxygen (O) -. In this variant of a process according to the invention, the preservative compound is a linear carbyl ester of acylated glycerol of the following general formula (XI): ## STR2 ## wherein: x is an integer equal to 0 or 1 which can vary in the formula (XI) according to each group of formula (XI-a): C 0 * G 2 O XHO (XI-a) 5 x not always being zero ; n is an integer from 1 to 20, in particular from 1 to 10, inclusive; Q 2 is selected from the group consisting of hydrogen (H) and organic groups consisting of at least two atoms linked by covalent bonds, said atoms belonging to the group consisting of carbon (C) and hydrogen ( H) and optionally oxygen (O) -, and; M2 represents an organic group formed by at least two atoms linked by covalent bonds, said atoms belonging to the group formed by carbon (C) and hydrogen (H) and optionally oxygen (O) -, and; G 2 represents a group of atoms chosen from the group formed: propyl α / α'-oxyacyl groups of general formula (VII), and f 3-oxyacyl propyl groups of general formula (VIII). Advantageously and according to the invention, at least one preservative compound is an acylated oligocarbonate / oligoglycerol of formula (XII) below: ## STR1 ## M3 is an organic group selected from the group consisting of the following groups of formulas: (XIIA); ## STR1 ## H 11 (XIIc), and; R-C-O-CitCH-O-C- (XIID); Wherein R is a hydrocarbon group, especially a saturated hydrocarbon group, an unsaturated hydrocarbon group, or a branched hydrocarbon group; and wherein R is a hydrocarbon group, especially a saturated hydrocarbon group, an unsaturated hydrocarbon group, or a branched hydrocarbon group. comprising 1 to 21 carbon atoms, and G11 is selected from the group consisting of hydroxylated propyl groups of the following general formulas (XIIE) and (IIF): OH 1 - CHICH-CHF (XIIE), and; CH 2H -CHICH-10 (XIIF); G12 and G13 are α, '-oxyacylated propyl groups of the following general formula (VII): II (VII), and CH-O-C-R1 2 -CI-ICH Q 3 is selected from the group consisting of hydrogen and organic groups consisting of at least two atoms linked by covalent bonds and belonging to the group consisting of carbon (C), hydrogen (H) and oxygen (0); n is a natural number less than 9-that is, of the interval [0; 8] - such that if n = 0 and m 0, then M1 is chosen from the group formed of and m, and m is a natural number less than 5 -not of the interval [0; 4], and; p is a natural number less than 4 -not of the interval [0; 3] - In the following, the G11 groups of formula (XIIF), G12 and G13 of formula (VII) may be present in the oligomer according to one and / or both of their two directions. possible insertion into the oligomer. Advantageously and according to the invention, the proportion of preservative compound in the preservation medium is chosen to be a volume proportion of between 0.01% and 100%. It is possible that the preservative compound is in the pure state in the preservation medium. Advantageously and according to the invention, the proportion of preservative compound in the preservation medium is a volume proportion of between 0.01% and 10%, preferably between 0.01% and 1%, more preferably between 0, 01% and 0.10%, in particular between 0.03% and 0.07%, in particular of the order of 0.05% -. Advantageously, the process according to the invention is a method for preserving microorganisms in vitro. Advantageously and according to the invention, the preservative compound and its proportion in the preservative composition are selected to allow storage of microorganisms at room temperature for a shelf life of at least 30 days. By "ambient temperature" is meant a temperature, particularly a temperature of the preservation composition, which is achieved without requiring the use of heating and / or cooling means. In particular, the term "ambient temperature" means a temperature greater than or equal to + 4 ° C, in particular between 15 ° C and 60 ° C, in particular between 20 ° C and 40 ° C. The method of preserving microorganisms at room temperature is therefore a method of preserving microorganisms at a temperature of at least + 4 ° C. Advantageously and according to the invention, the preservation medium is an aqueous medium. Advantageously, the microorganism preservation medium is an aqueous medium which does not in itself allow the growth and development of microorganisms. Advantageously and according to the invention, the preservation medium is chosen from the group consisting of water and aqueous saline media.

Advantageously and according to a variant of the invention, the preservation medium is in the liquid state at room temperature. It may be an essentially aqueous preservation medium comprising at least one preservative compound. The microorganism is therefore stored in a liquid medium that can be further diluted in a dilution liquid, especially in water, or in any culture medium of the microorganism for its further development. However, according to another variant of the invention, the preservation medium may be in the solid state at room temperature. It may be a composition comprising a proportion of at least one gelling agent such as for example agar-agar. The preservation medium is thus an agar-containing preservation medium comprising at least one preservative compound and which is in the solid state at room temperature. Advantageously and according to the invention, the preservation medium comprises microorganisms selected from the group consisting of bacteria, unicellular fungi, yeasts and protozoa. The invention also relates to a method of treating plants in which a preservation medium according to the invention is used. The invention also extends to a method of treating plants in which: - a preservation composition is prepared by a process according to the invention, then: - said storage composition is stored at ambient temperature, then: said preservative composition is diluted so that the proportion of preservative compound in the diluted composition is lower than the predetermined inhibitory proportion and allows growth of the microorganisms, and - said diluted composition is applied to plants. The invention therefore also relates to a method of treating plants in which a microorganism preservation composition is prepared which enables the farmer to have a preservation composition comprising concentrated and stable microorganisms. which is capable of being diluted-especially in water-for the application of said diluted composition on plants. Advantageously and according to the invention, the preservation composition is preserved for a period of at least 30 days. The storage composition is stored at room temperature without significant variation in the number of live microorganisms in the preservation composition. Advantageously and according to the invention, at least one microorganism is chosen from the group formed by micro-organisms that are useful for the growth and development of plants. Advantageously and according to the invention, at least one microorganism to be preserved is selected from the group consisting of Gram-positive bacteria, in particular of the genus Streptomyces.

In particular, advantageously and according to the invention, at least one microorganism to be preserved is a bacterium of the genus Streptomyces conforming to the strain deposited and registered at the CNCM under number I-4667. At least one microorganism is a Streptomyces bacterium conforming to the strain deposited and registered on April 7, 2011 under No. I-4467 with the National Collection of Microorganism Cultures (CNCM) of the Institut Pasteur (of which the address is 25, rue du Docteur Roux, 75724 Paris cedex 15) having the status of international depositary authority according to the Budapest Treaty. Advantageously and according to the invention, at least one microorganism is chosen from the group formed by microorganisms capable of stimulating natural defenses of plants. The diluted composition is adapted to stimulate the natural defenses of plants in culture. In particular, the application of a diluted composition according to the invention has a stimulating activity of the natural defenses of plants, that is to say, it makes it possible to activate the expression in plants of defense genes - for example PR-1 to plant pathogens. The invention also extends to a composition comprising: at least one microorganism; a preservation medium of at least one microorganism, and at least one compound, said preservative compound, selected from the group consisting of compounds for which there is a predetermined inhibitory proportion of the preservative compound in the preservation medium which is: inhibiting the growth of at least one microorganism in the storage medium at room temperature, and non-lethal for said at least one microorganism, said preservation composition being adapted to retain said at least one microorganism at room temperature. A preservation composition according to the invention means: a composition which comprises microorganisms in the latent state-that is to say in which the preservative compound has a higher proportion in the composition than predetermined minimum inhibitory proportion and inhibits the growth of microorganisms and is non-lethal for said microorganisms; or - a composition which comprises microorganisms in the growth phase-that is, in which the preserving compound has a lower proportion in the composition than the predetermined minimum inhibitory proportion and allows the growth of microorganisms. The invention therefore relates in particular to a microorganism preservation composition which makes it possible to conserve living microorganisms, said preservation composition being capable of being diluted - in particular in water - with a view to the application of said composition diluted on plants. Advantageously and according to the invention, at least one preservative compound is selected from the group consisting of undecylenic acid, esters of undecylenic acid and glycerol carbon esters. Advantageously and according to the invention, at least one micro-organism is of the genus Streptomyces and conforms to the strain deposited and registered at the CNCM under number I-4667.

The invention also relates to a method for preserving microorganisms, a plant treatment method and a composition for implementing at least one of these processes characterized in combination by all or some of the characteristics mentioned. above or below.

Other objects, features and advantages of the invention will appear on reading the following description and nonlimiting examples which refer to the appended figures, and in which: FIG. 1 is a graphical representation of the viability chart (PFU / mL) of the Streptomyces I-4467 bacterium at the beginning (D + 0) of preservation (solid / grayed histogram) and after 28 days of preservation (hatched histogram) at room temperature in absence (H2O) or in the presence of conservative compound (ECG-C7, ECG-C9, ECG-C11: 1, OECG-C7, OECG-C9 and OECG-C11: 1) in a mass ratio of 0.05% according to the invention, - Figure 2 is a graphical representation of the viability (PFU / mL) of the Streptomyces I-4467 bacterium at the beginning of conservation (solid / gray histogram) and after 28 days of preservation (hatched histogram) at room temperature in absence (H2O) or in presence of preservative compound (ECG-C7, ECG-C9, ECG-C11: 1, OECG-C7, OECG-C9 and OECG-C11: 1) in a weight ratio of 1%, Figure 3 is a graphical representation of the viability (PFU / mL) of Streptomyces I-4467 at the beginning of preservation (open histogram) and after 28 days of storage (hatched histogram) at room temperature in the presence of glycerol, glycerol carbonate or undecylenic acid in a mass proportion of 0.05%, FIG. 4 is a graphical representation. growth curves of the yeast Saccharomyces cerevisiae in the presence of ECG-C7 as a preservative compound according to the invention in a proportion of 0.01%; 0.05% and 0.10% in water; FIG. 5 is a graphical representation of growth curves of the yeast Saccharomyces cerevisiae in the presence of ECG-C9 as a preservative compound according to the invention; in proportion of 0.01%; 0.05% and 0.10% in water; FIG. 6 is a graphical representation of growth curves of the yeast Saccharomyces cerevisiae in the presence of ECG-C11: 1 as a preservative compound according to the invention; in proportion of 0.01%; 0.05% and 0.10% in water; FIG. 7 is a graphical representation of growth curves of the yeast Saccharomyces cerevisiae in the presence of OECG-C7 as a preservative compound according to the invention in proportion to 0.01%; 0.05% and 0.10% in water; FIG. 8 is a graphical representation of growth curves of the yeast Saccharomyces cerevisiae in the presence of OECG-C9 as a preservative compound according to the invention in proportion 0.01%; 0.05% and 0.10% in water.

A preservation composition according to the invention comprises - microorganisms; a preservation medium for said microorganisms comprising at least one compound, said preservative compound, chosen to: inhibit the growth of at least one microorganism in vitro and at room temperature, and; o be non-lethal to said at least one microorganism, in a proportion of the preservative compound in the composition greater than a predetermined minimum inhibitory proportion, and to allow the growth of at least one microorganism, in a proportion of the preservative compound in the composition lower than said predetermined minimum inhibitory proportion. Such a preservation composition makes it possible to preserve microorganisms in vitro at room temperature. Such a composition according to the invention, when brought into contact with microorganisms, makes it possible to limit the growth of said microorganisms at room temperature, without significant losses of microorganism and without depleting the culture medium.

A preservation medium, when placed in contact with microorganisms in a preservation composition, enables the microorganisms to resume their growth as soon as the proportion of preservative compound in the preservation composition becomes lower. for example by dilution of the preservative composition to the predetermined minimum inhibitory proportion of the preservative compound. A composition according to the invention may comprise glycerol carbonate. Glycerol carbonate is obtained by a process described in FR 2 778 1821 in which urea and glycerol are reacted in the presence of a solid catalyst in the form of a powder consisting of a metal salt or a mixture of salts. metallic, containing Lewis acid sites. A composition according to the invention may also comprise an α / α'-acylated glycerol carbonate ester (5-membered) or a 13'-acylated (6-membered) cyclic glycerol carbonic ester. Such α / α'-acylated glycerol (cyclic) carboating esters are obtained by synthetic methods as described in Examples 1, 2 and 3 below. EXAMPLE 1 Synthesis of esters of glycerol carbonate (glycerol carbonate a / a'-acylated) The synthesis of esters of glycerol carbonate 20 selected from the group consisting of: - the ester of glycerol carbonate and heptanoic acid (ECG-C7), and; ester of glycerol carbonate and nonanoic acid (ECG-C9), and Ester of glycerol carbonate and undecylenic acid (ECG-C11: 1), and; cyclic carbonic ester of glycerol and oleic acid (ECG-C18: 1) by esterification of the glycerol carbonate (cyclic α / α-hydroxylated) with the corresponding fatty acid. In a 500 mL reactor equipped with a mechanical stirring device, a pressurizing device and a "Dean-Stark" water removal device formed, 1 64 moles of fatty acid and 0.0078 moles of 4-methylbenzenesulfonic acid (CAS No. 6192-52-5, para-toluenesulfonic acid, ApTs). The temperature of the mixture is raised to a temperature of 110 ° C. under a pressure of 800 hPa for a period of 15 minutes. 0.84 mol of glycerol carbonate with mechanical stirring is then added dropwise to the reactor at 800 rotations per minute (rpm) for 15 minutes. The reactor is placed in an oil bath heated to 110 ° C. and mechanically stirred (800 rpm) for 3 hours.

EXAMPLE 2 - Purification of esters of glycerol carbonate The reaction medium is diluted in 150 ml of ethyl ether and the mixture obtained is placed in a 1 L separating funnel. The mixture is washed successively with 4 volumes of saturated NaCl water until the aqueous phase is neutral. . The washed organic phase is dried over magnesium sulfate and is separated from the magnesium sulfate hydrate by filtration. The ether of the organic phase is removed by evaporation under reduced pressure. A mass of dry product of 277 g is obtained. The ester of the glycerol carbonate is separated from the excess fatty acids by thin-film distillation under reduced pressure at a temperature below the boiling point of the fatty acid under this reduced pressure and below 155 ° C. The ester of glycerol carbonate is obtained, the purity evaluated by gas chromatography is between 85% and 95%. EXAMPLE 3 Synthesis of α / α'-acetylated glycerol carbonate (ECG-C2) Synthesis of the α / α'-acetylated glycerol carbonate of formula below: ## EQU1 ## Wherein R 1 is a methyl group. ## STR2 ## wherein R 1 is a methyl group. In a 2L glass three-neck flask equipped with a mechanical stirrer and a "Dean-Stark" device for removing water formed comprising a condenser and placed in an oil bath, 472 g of Cyclic glycerol carbonate (4- (hydroxymethyl) -1,3-dioxolan-2-one, CAS 931-40-8) and 4 g of Lewatit K2431 resin. 6 moles of acetic anhydride are added dropwise to the reactor so as to control and maintain the temperature of the reactor at 150 ° C. with mechanical stirring of 800 rpm for 4 hours.

The excess acetic anhydride was removed by evaporation at a temperature of 60 ° C and a reduced pressure of 55 hPa. The α / α'-acetylated linear glycerol carbonic ester was purified by the thin film technique conducted in an evaporator / separator at a temperature of 170 ° C and a reduced pressure of 0.33 hPa. The α, a-acetylated glycerol carboxy ester is obtained, the purity of which is evaluated by gas chromatography between 98% and 99%. The degree of purity of the esters of glycerol carbonate obtained in Examples 1, 2 and 3 and the molecular ion (m / z) analyzed by mass spectrometry are given in Table 1 below. Purity,% Mass spectrometry, m / z ECG-C2 98 160.1 ECG-C7 94 230.2 ECG-C9 95 258.3 ECG-C11: 1 85 284.3 ECG-C18: 1 96 382.5 Table A composition according to the invention may also comprise linear carboxy esters of glycerol. Such linear carbonyl esters of glycerol are obtained by condensation / oligomerization from cyclic carbonyl esters of glycerol, in particular cyclic carbon esters of α / '-acylated glycerol, in the presence of an organic initiator - for example glycerol and a metal catalyst, for example zinc sulphate or zinc stearate as described in Examples 4 and 5. EXAMPLE 4 - Oligomerisation of α / α'-acetylated cyclic glycerol carbonate (ECG-C2) . The oligomerization of the α, β-acetylated glycerol carbonic acid ester (ECG-C2) obtained in Example 3 is carried out in the presence of a metal catalyst, of glycerol as organic initiator, and in the conditions described in Table 2 below. Mass Catalyst Metal Primer Glycerol, TC Conditions,% Mass ECG-C2, gg molar in number Nature Mass, mg 8.5 Zn (C18H3502) 1.5 160 ° C, 95 970, 688, 2 Pat., 2h 352, 274, 202, 190 21.25 Zn (C 18 H 35 O 2) 125 3.75 160 ° C, 84 714, 336, 2 Pat., 2h 206, 139, 92 160 ° C, 431, 160, 9 ZnS 04 50 1.7 Table 2 The TC (%) value indicates the conversion rate of the starting α / α'-acetyl carboxy ester of glycerol. The molar mass values of the number of products of the reaction are obtained by analysis of the reaction medium in gel permeation chromatography on a PL-GelTM 3 ium MIX-E column. The linear α / α'-acetylated glycerol carboxylic esters are detected at the column outlet by refractometry and the molecular weights are determined by comparison with polystyrene standards. The mass spectrum produced on a reaction medium obtained by carrying out an oligomerization process of the ester of α / α'-acetylated glycerol carbonate (ECG-C2) as described in the present example comprises signals corresponding to molecular ions and fragments of m / z values between 180.9 and 761.4 and corresponding to oligomers of the following formulas (A), (B) and (C): oo H3C 00 oo C Wherein c may be 1, 2 or 4 and d may be 1, 2, 3 or 4; o H3C 00 0 b CH3 OH OH (B); Wherein b may be 1, 2, or 3; Wherein (a) is 1, 2, 3, 4, 5, 6, 7 or 8, and Wherein g can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

EXAMPLE 5 Synthesis of linear α-acylated glycerol carboxylic esters (OECG-C2, OECG-C7, OECG-C9 and OECG-Cita) from glycerol carbonate esters (cyclic) a / a -acylated (ECG-C2, ECG-C7, ECG-C9 and ECG-Cita). Oligomerization of esters of glycerol carbonate (cyclic α-acyl) according to the invention under the conditions described in Table 3 below.

3029741 26 ECG, Metallic catalyst Primer Glycerol, TC conditions,% Mass masses g molar number Nature Weight, mg ECG-C2, Zn (C18H3502) 125 3.75 160 ° C, Pat., 98 714, 335, 21.25 206, 139, 92 ECG-C7, 200 ° C, Patm, 639, 420, ZnSO4 113 2.42 64 g 252, 96 ECG-C9, 180 ° C, Patm, 992, 541, ZnSO4 110 2.15 88 20 g 2h 303, 90 ECG-ZnSO4 86 1.3 190 ° C, Pat., 98 7632, C11: 1, 2h 2113.1084 10g, 750, 486 Table 3 The TC (%) value indicates the conversion rate of the starting glycerol carbonate ester. The apparent molecular weight values are obtained by analysis of the reaction medium by gel permeation chromatography. Oligomers of apparent molecular weight up to 7600 Da are obtained. EXAMPLE 6 - Streptomyces strain I-4467 The Streptomyces strain, called Streptomyces I-4467, which is the subject of the present example, was deposited and registered on April 7, 2011 under number I-4467 with the National Collection of Microorganism Cultures ( CNCM) of the Institut Pasteur (whose address is 25, rue du Docteur Roux, 75724 Paris cedex 15) having the status of international depositary authority according to the Budapest Treaty. The strain of Streptomyces I-4467 has a DNA sequence, called DNA, 16S, coding for the ribosomal RNA 16S which has SEQ ID_NO1.

15 SEQ ID_NO1 sequence is described below: tagtggcgaa cgggtgagta acacgtgggc aatctgccct gcactctggg acaagccctg 60 gaaacggggt ctaataccgg atatgacacg ctcccgcatg ggatgcgtgt ggaaagctcc 120 ggcggtgcag gatgagcccg cggcctatca gcttgttggt ggggtgatgg cctaccaagg 180 3029741 27 cgacgacggg tagccggcct gagagggcga ccggccacac tgggactgag acacggccca 240 gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgaaagcc tgatgcagcg 300 acgccgcgtg agggatgacg gccttcgggt tgtaaacctc tttcagcagg gaagaagcga 360 gagtgacggt acctgcagaa gaagcgccgg ctaactacgt gccagcagcc gcggtaatac 420 5 gtagggcgca agcgttgtcc ggaattattg ggcgtaaaga gctcgtaggc ggcttgtcgc 480 gtcggatgtg aaagcccggg gcttaacccc gggtctgcat tcgatacggg caggctagag 540 ttcggtaggg gagatcggaa ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa 600 caccggtggc gaaggcggat ctctgggccg atactgacgc tgaggagcga aagcgtgggg 660 agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgttgggaac taggtgtggg 720 10 cgacattcca cgtcgtccgc gccgcagcta acgcattaag ttccccgcct ggggagtacg 780 gccgcaaggc taaaactcaa a ggaattgac ggggggcccg cacaagcggc ggagcatgtg 840 gcttaattcg acgcaacgcg aagaacctta ccaaggcttg acatacaccc ggaaacctct 900 ggagacaggg gccccccttg tggtcggtgt acaggtggtg catggcttgt cgtcagctcg 960 tgtcgtgaga tgttgggtta agtccccgca acgagcgcaa cccttgttct gtgttgccag 1020 15 catgcctttc gggggntgat ggggacttnc acaggagact gccggggtca actcggagga 1080 aggtggggac gacgtcaagt catcatgccc cttatgtctt gggctgcaca cgtgctacaa 1140 tggccggtac aatgagctgc gaagccgtga ggtggagcga atctcaaaaa gccggtctca 1200 gttcggattg gggtctgcaa in the sequence SEQ ID_NO1 above, the symbol "n" in positions 1036 and 1049 of the sequence SEQ ID_NO1 designates, according to the IUPAC ("International"), in the sequence SEQ ID_NO1 above, the symbol "n" in positions 1036 and 1049 of the sequence SEQ ID_NO1 designates, according to IUPAC ("International Union of Pure and Applied Chemistry "), any of the four nucleotides a, t, c or g. Thus, nucleotide n at position 1036 is selected from the group consisting of nucleotide "a", nucleotide "t", nucleotide "g" and nucleotide "c", and nucleotide n at position 1049 is selected independently nucleotide at position 1036, in the group consisting of nucleotide "a", nucleotide "t", nucleotide "g" and nucleotide "c". Streptomyces strain I-4467 is a Gram-positive bacterium.

The strain Streptomyces I-4467 can be used for the protection of plants and the stimulation of their vitality by natural means and processes (biocontrol), aimed in particular at controlling the balance between the plants and the populations of aggressors rather than to eradicate these populations of aggressors. In particular, the strain Streptomyces I-4467 is able to: - promote the solubilization of solid nutritional elements - in particular phosphorus - and its provision of plants. The strain Streptomyces I-4467 makes it possible to promote the dissolution in the rhizosphere of plants of solid fertilization products which can not be assimilated by said plants and to improve the nutrition of plants; - stimulate the growth of plants - in particular the growth of aerial parts of plants - for example such as sunflower and corn. To do this, a liquid composition according to the invention comprising between 1 and 2 g (mass of wet bacteria) of bacteria of the strain Streptomyces I-4467 per liter is laminated onto sunflower seeds and maize seeds. decomposition. Seeds of sunflower and maize treated with the liquid composition according to the invention are sown and as controls untreated sunflower seeds and maize. Initial growth stimulation of sunflower and maize plants compared to untreated sunflower and maize plants is observed; - slow down growth: o certain bacteria such as Micrococcus luteus and Bacillus subtilis; o certain phyto-pathogenic target microorganisms such as Botrytis cinerea, Streptomyces scabies, Botrytis cinerae, Fusarium culmorum, Pythium ultimum, Phaeomoniella chlamydospora, Phaeomoniella aelophilum, Eutypa tata, Fomitiporia mediterranea and Botryosphaeria obtusa; - stimulate the natural defenses of plants in culture. The application of a treatment composition according to the invention has a stimulating activity of the natural defenses of the plants, that is to say, it makes it possible to activate the expression in plants of defense genes. for example PR-1 with respect to plant pathogenic organisms. The strain Streptomyces I-4467 may be cultured in an aqueous liquid culture medium, in particular in a medium selected from group 5 consisting of complete media and rich media comprising all the mineral elements and organic precursors necessary for the growth of bacteria according to the invention, in particular a carbon source, a nitrogen source, a source of phosphorus, vitamins and trace elements. Such a complete medium may comprise D-glucose, yeast extract, dibasic potassium phosphate (K2HPO4), ammonium sulfate ((NH4) 2SO4), potassium chloride (KCl) and glycerol at pH 2. Streptomyces strain I-4467, when cultured on ISP-2 medium or solid Bennett medium, forms bacterial clusters or "colonies" having: a mycelium, so-called substrate mycelium, branched developing in the thickness of the solid medium of color varying from yellow-brown to gray-brown depending on the composition of the solid medium, and - aerial mycelium developing at the air / solid interface which is white in color.

The optimum growth temperature of Streptomyces strain I-4467 is between 12 ° C and 37 ° C. It is preferably between 28 ° C and 30 ° C. The optimum storage temperature of the strain Streptomyces I-4467 in its culture medium is + 4 ° C. EXAMPLE 7 - Storage of Streptomyces I-4467 at room temperature (28 ° C.) A suspension of Streptomyces I-4467 bacterium comprising 800 CFU (Colony Forming Unit) per mL of water is prepared. 0.05% (v / v) of a preserving compound selected from the group consisting of esters of glycerol carbonate (ECG-C7, ECG-C9, ECG-Cita) and linear carbon esters of 30 glycerol (OECG-C7, OECG-C9, and OECG-Cii, i). The preservation composition thus obtained is placed at a temperature of 28 ° C for 28 days. As a control, a suspension of Streptomyces I-4467 bacterium comprising 800 CFU per ml of culture medium was prepared and 0.05% water was added. At the end of the 28-day period, the viability of the bacteria in each of the preservation compositions was evaluated by spreading 200 μl of the composition or serial dilutions of the composition on a solid Bennett culture medium (10 g of glucose, 1 g of yeast extract, 1 g of meat extract, 2 g of bactopeptone, 18 g of agar in 1 L of osmosis water), incubation at 28 ° C and enumeration of bacterial colonies formed after 7 days. The results are shown in FIG. 1, in which the solid histograms (greyed out) represent the number of bacteria (CFU / ml) seeded at day 0 and the hatched histograms represent the number of bacteria (CFU / ml) measured at day 28. . In comparison with the control (H2O), the growth of Streptomyces strain I-4467 is strongly limited or prevented by the ECG-C9, ECG-Cita, OECG-C7 and OECG-Cita preservative compounds and, to a lesser extent with preservative compounds ECG-C7 and OECG-C9. Preservative compounds can keep Streptomyces I-4467 bacteria for 28 days by inhibiting their growth while maintaining their subsequent growth potential. They make it possible to preserve the Streptomyces I-4467 bacteria for 28 days without requiring dilution and regular subcultures of the strain Streptomyces I-4467 and without depleting the culture medium. As a control, a similar test is carried out with a proportion of the same preservative compounds of the order of 1% (v / v) and suspensions of Streptomyces I-4467 bacterium comprising 400 CFU per ml of culture medium. The results are shown in FIG. 2 in which the solid histograms (greyed out) represent the number of bacteria (CFU / mL) seeded at OJ and the hatched histograms represent the number of bacteria (CFU / mL) measured at D + 28. At a level of 1% in the medium, the preservative compounds ECG-C7, ECG-C9, ECG-C18, ECG-C7, OECG-C9 and OECG-Cita exhibit bactericidal behavior.

EXAMPLE 8 - Storage of Streptomyces strain I-4467 at room temperature (28 ° C.) The procedure is as in Example 7 using glycerol carbonate and undecylenic acid as preservative compounds. A suspension of Streptomyces I-4467 bacterium comprising about 400 CFU (Colony Forming Unit) per ml of water is prepared. 0.05% (v / v) of preservative compound selected from glycerol carbonate or undecylenic acid is added. The preservation composition thus obtained is placed at a temperature of 28 ° C for 28 days. As a control, a suspension of Streptomyces I-4467 bacterium comprising 400 CFU per ml of culture medium is prepared and 0.05% glycerol is added. At the end of the 28-day period, the viability of the bacteria in each of the preservation compositions is evaluated by spreading 200 μl of the composition or a serial dilution of the composition on a solid Bennet culture medium (10 g). of glucose, 1 g of yeast extract, 1 g of meat extract, 2 g of bactopeptone, 18 g of agar-agar in 1 L of osmosis water), incubation of the dishes at 28 ° C. and enumeration bacterial colonies formed after 7 days. The results are shown in FIG. 3 in which the solid histograms (greyed out) represent the number of bacteria (CFU / ml) seeded at day 0 and the hatched histograms represent the number of bacteria (CFU / ml) measured at day 28. . In comparison with the control (glycerol), the growth of the strain Streptomyces I-4467 is strongly limited by preservative compounds of glycerol carbonate and undecylenic acid. These preservative compounds maintain Streptomyces I-4467 bacteria for 28 days by inhibiting their growth while maintaining their subsequent growth potential. They make it possible to preserve the Streptomyces I-4467 bacteria for 28 days without the need for dilution and regular transplanting of the strain Streptomyces I-4467 and without depleting the culture medium EXAMPLE 9 Growth curve of the yeast Saccharomyces cerevisiae in a liquid medium at room temperature (28 ° C.) in the presence of ester of glycerol carbonate and heptanoic acid (ECG-C7). Precipitation of pure Saccharomyces cerevisiae yeast is carried out in 3 ml of YPD medium (comprising 2 g. % bactopeptone, 1% yeast extract, 2% glucose and osmosis water qsp) with stirring (220 rpm) at 28 ° C for 12 hours. The optical density (OD at 600 nm) of the preculture is in the range of 0.4 to 0.5. 100 μl of this preculture is taken and introduced into a tube containing 5 ml of fresh medium for control. 100 μl of this preculture are also introduced into tubes containing 5 ml of fresh medium and a volume proportion of 0.01, 0.05 and 0.1% of cyclic glycerol cyclic ester ECG-C7. The tubes are placed at 28 ° C. and the evolution of the optical density at 595 nm of the culture is measured over time. The results are shown in FIG. 4. An inhibition of the growth of the yeast Saccharomyces cerevisiae is observed from 0.1% of ECG-C7. EXAMPLE 10 Growth curve of the yeast Saccharomyces cerevisiae in a liquid medium at room temperature (28 ° C.) in the presence of ester of glycerol carbonate and of nonanoic acid (ECG-C9) The procedure is identical to EXAMPLE 9 100 Lat of preculture is introduced into tubes containing 5 ml of fresh medium and a volume proportion of 0.01, 0.05 and 0.1% of ECG-C9 glycerol carbonate ester. The tubes are placed at 28 ° C. and the evolution of the optical density of the culture is measured over time. The results are shown in FIG. 5. An inhibition of the growth of the yeast Saccharomyces cerevisiae is observed from 0.1% of ECG-C9. EXAMPLE 11 Growth curve of the yeast Saccharomyces cerevisiae in a liquid medium at ambient temperature (28 ° C.) in the presence of ester of glycerol carbonate and of undecylenic acid (ECG-C12: 1) The procedure is identical in Example 9. 100 Lat of pre-culture are introduced into tubes containing 5 ml of fresh medium and a volume proportion of 0.01, 0.05 and 0.1% of glycerol carbonate ester ECG-Ci Li. The tubes are placed at 28 ° C. and the evolution of the optical density of the culture is measured over time. The results are shown in FIG. 6. An inhibition of the growth of the yeast Saccharomyces cerevisiae is observed from 0.05% of ECG-Cita. EXAMPLE 12 - Growth curve of the yeast Saccharomyces cerevisiae in a liquid medium at room temperature (28 ° C.) in the presence of linear carbonic ester of glycerol and of nonanoic acid (OECG-C9). The procedure is identical to Example 9. 100 Lat of pre-culture are introduced into tubes containing 5 ml of fresh medium and a proportion by volume of 0.01, 0.05 and 0.1% of OECG-C9. . The tubes are placed at 28 ° C. and the evolution of the optical density of the culture is measured over time. The results are shown in FIG. 7. An inhibition of the growth of the yeast Saccharomyces cerevisiae is observed starting from 0.05% of OECG-C9. EXAMPLE XIII - Growth curve of the yeast Saccharomyces cerevisiae in a liquid medium at room temperature (28 ° C.) in the presence of linear carbonic ester of glycerol and heptanoic acid (OECG-C7) The procedure is identical to EXAMPLE 9 100 Lat of pre-culture is introduced into tubes containing 5 ml of fresh medium and a volume proportion of 0.01, 0.05 and 0.1% of OECG-C7. The tubes are placed at 28 ° C. and the evolution of the optical density of the culture is measured over time. The results are shown in FIG. 8. An inhibition of the growth of the yeast Saccharomyces cerevisiae is observed starting from 0.05% of OECG-C7. It goes without saying that the invention can be the subject of many variants and applications. In particular, a composition, a method of preserving microorganism and a method of treating plants in which such a composition is used are subject to an infinity of variations both in the formulation of the composition and in the modes of implementation. processes.

Claims (2)

CLAIMS1 / A method for storing microorganisms at ambient temperature, in which: - a composition, called a preservation composition, comprising the microorganisms to be conserved and a preservation medium for said microorganisms, characterized in that: the preservation medium comprises at least one compound, referred to as a preservative compound, selected from the group consisting of compounds for which there is at least a predetermined inhibitory proportion of the preservative compound in the preservative composition which is inhibiting the growth of said microorganisms. organisms in the storage composition at room temperature, and; Non-lethal for said microorganisms, and in that; said at least one preservative compound is in the preservative composition in a proportion greater than said predetermined inhibitory proportion and in a non-lethal proportion for said microorganisms; and wherein the storage composition is placed at room temperature for the purpose of preserving said microorganisms. 2 / A method according to claim 1, characterized in that one chooses at least one preservative compound in the group consisting of undecylenic acid, esters of undecylenic acid and glycerol carbon esters. 3 / A method according to one of claims 1 or 2, characterized in that at least one preservative is selected from the group consisting of glycerol carbonate, esters of glycerol carbonate and linear carboxy esters of glycerol. 4 / A method according to one of claims 1 to 3, characterized in that one chooses at least one preservative compound in the group formed: esters of glycerol carbonate and an odd number of hydrocarbon acid d carbon atoms, and; linear carbonic esters of glycerol and at least one hydrocarbon acid with an odd number of carbon atoms. 5 / A method according to one of claims 1 to 4, characterized in that the proportion of preservative compound in the preservative composition is chosen to be a volume proportion of between 0.01% and 100%. 6 / A method according to one of claims 1 to 5, characterized in that the preservative compound and proportion in the preservative composition are selected to allow storage of microorganisms at room temperature for a shelf life at least equal at 30 days. 7 / A method according to one of claims 1 to 6, characterized in that the preservation medium is an aqueous medium. 8 / A method of treating plants in which: - a preservation composition is prepared by a method according to one of claims 1 to 7, then: - said storage composition is stored at ambient temperature, then: - diluted said composition so that the proportion of preservative compound in the diluted composition is lower than the predetermined inhibitory proportion and allows growth of the microorganisms, and - said diluted composition is applied to plants. 9 / A method according to claim 8, characterized in that at least one microorganism is selected from the group consisting of microorganisms capable of stimulating natural defenses of plants. 10 / A method according to one of claims 8 or 9, characterized in that at least one microorganism to be preserved is selected from the group consisting of Gram-positive bacteria. 11. The method as claimed in one of claims 8 to 10, characterized in that at least one microorganism to be preserved is a bacterium of the Streptomyces genus conforming to the strain deposited and registered at the CNCM under number I. 4667. 12 / Composition comprising - at least one microorganism; A preservation medium of at least one microorganism, and at least one compound, said preservative compound, selected from the group consisting of compounds for which there is a predetermined inhibitory proportion of the preservative compound in the preservative composition which is inhibiting the growth of at least one microorganism in the storage composition at room temperature, and; o non-lethal for said at least one microorganism, the preservative compound having in the composition a proportion greater than the predetermined inhibitory proportion, said preservation composition being adapted to retain said at least one microorganism at room temperature. 13. The composition of claim 12 wherein at least one preservative compound is selected from the group consisting of undecylenic acid, undecylenic acid esters and glycerol carboxy esters. 14 / Composition according to one of claims 12 or 13, characterized in that at least one microorganism is of the genus Streptomyces and conforms to the strain deposited and registered at the CNCM under the number I-4667. 25