WO2017144500A1 - Resistant aerial conidia of filamentous fungus strains and method for obtaining same - Google Patents

Resistant aerial conidia of filamentous fungus strains and method for obtaining same Download PDF

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Publication number
WO2017144500A1
WO2017144500A1 PCT/EP2017/053994 EP2017053994W WO2017144500A1 WO 2017144500 A1 WO2017144500 A1 WO 2017144500A1 EP 2017053994 W EP2017053994 W EP 2017053994W WO 2017144500 A1 WO2017144500 A1 WO 2017144500A1
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Prior art keywords
conidia
trichoderma
spores
strains
solid support
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PCT/EP2017/053994
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French (fr)
Inventor
Omran ZAKI
Ahmed Sabri
Philippe Thonart
Frédéric Weekers
Philippe Jacques
Caroline LABE
Richard Belanger
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Artechno Sa
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Priority to EP17710489.0A priority Critical patent/EP3420069A1/en
Publication of WO2017144500A1 publication Critical patent/WO2017144500A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N3/00—Spore forming or isolating processes

Definitions

  • the present invention relates to the field of biological control or bio-fertilization, and relates in particular to conidia or spores, aerial and resistant, strains of filamentous fungi, in particular conidia or spores. Pseudozyma, Trichoderma or Penicillium in powder form, as well as their production process and their applications, as a biological control agent or in the food industry. Technological background on which the invention is based
  • Bio ⁇ control is a known method for the protection of plants against for example pests, crop pests, such as insects, mites or nematodes, protection or treatment against diseases , in particular fungal, bacterial or viral diseases, or a method of controlling weeds, weeds, by the use either of living antagonistic organisms or of biodegradable products constituting these organisms, or of metabolites produced by these organisms, called biological control agents.
  • Pseudozyma flocculosa is a known strain of epiphytic phyllosphere fungus, making part of the class Basidiomycetes and belonging to the order Ustilaginale.
  • This fungus is recognized as a very effective biological control agent against white induced Erysiphe poligoni, several plants including rose and cucumber.
  • Trichoderma is also known to group a group of fungi that are commonly found in the soil, dead wood, plant debris and the aerial part of plants.
  • Trichoderma Many species of Trichoderma have been found effective as biological agents against for example Pythium, Phytophthora cinnamomi, Rhizoctonia solani, Sclerotium rolfsiie and Cryphonectria parasitica. These Pathogenic fungi cause significant damage to fruit, vegetable, greenhouse and ornamental crops.
  • Trichoderma atroviride as a biological control agent is due in part to the production of endochitinase and it has also been shown that this fungus intervenes, according to various mechanisms and at various levels, in the stimulation of root growth by the use of fertilizing minerals and the stimulation of natural plant defenses.
  • Conidia or spores are the main means of protection, and conservation, fungal genomes, thanks to their resistance to conditioning treatments and hostile environmental conditions, such as extreme temperatures, ultraviolet rays and low light levels. 'relative humidity. For example, some of the conidia or spores of Aspergillus niger maintain their germinability after passage through an autoclave for one hour at 120 ° C (Morozova et al 2001).
  • fungi including filamentous
  • liquid media that is to say during a so-called liquid fermentation phase.
  • liquid fermentation is particularly problematic.
  • Hyphomycetes can be caused to develop in various forms, including thin-walled unicellular hyphae, some forms called blastospores, and submerged conidia or spores. These are obtained either directly from blastospores microcycles, by conidiation, or from conidiogenic cells that form on submerged hyphae.
  • the preferred method for the production of conidia or spores on a large scale is the submerged culture because of a part of its good profitability, its short production time, and the availability of all the materials necessary for a liquid fermentation.
  • Paecilomyces fumosoroseus for example, needs light for optimal production of aerial conidia (Wraight et al., 2001), and the efficient production of conidia of this fungal strain therefore requires surface culture, or periodic agitation of the media or particulate solid substrates, to increase the exposure to light.
  • Verticillium conidia or spores in turn, develop into sticky globules produced at relatively low densities on hyphae growth, whereas those of aschersonia must be produced in convolutions, or as pits in a dense stroma. .
  • the patent application WO2009 / 037399 describes a process for producing conidia or spores, and their metabolites, on a support, or substrate, solid by a conventional fermentation, the growth phase is also on such a support.
  • the solid medium must comprise a suitable solid support or substrate, that is to say a support or absorbent substrate of low density and practically non-fermentable, but impregnated with a culture medium suitable for the growth of conidia or spores of fungi .
  • the process involves the application of water stress during the spore production phase.
  • the patent application EP2390345 describes the production of metabolites and other molecules of interest by fermentation in a solid medium on multiple supports or solid substrates, such as plant biomass, in particular. agricultural products such as cereals, by-products of the agri-food industry, pulp and paper industry or sewage sludge.
  • US Pat. No. 4,837,155 describes a method for producing the Trichoderma strain in the liquid phase and under aerobic conditions at temperatures of between about 25 ° C. and about 30 ° C. and at a pH of between about 5.8 and about 7.0, to generate from an inoculant a conidial density of at least 5 x 10 8 per ml, but without any solid support maturation phase.
  • EP0223809 discloses a process for producing conidia or spores of filamentous fungi in a rotary disk fermentor in the liquid phase.
  • Microbiology and Biotechnology, V0.64, No. 2, pages 175-186 (2004) discloses that the best method of producing filamentous fungal spores is a solid state fermentation (SSF) and that it is possible to combine a submerged fermentation technology (SMF) to generate biomass, with a solid support fermentation (SSF) to obtain a large number of spores.
  • SSF solid state fermentation
  • the present invention aims at obtaining filamentous fungi strains of agronomic or agri-food interest, usable in solid form, in particular in the form of a powder, and a novel process for obtaining these strains, in particular in the form of powder and which do not have the drawbacks of the state of the art.
  • the present invention aims to provide strains of filamentous fungi that are easily manipulated, have improved viability, especially after long periods of storage, and possibly high purity, ie strains essentially free of contaminants and / or residues of the culture medium, in order to be able to use them for agronomic or agri-food applications, in particular for the preparation of cheeses or cold meats.
  • Another object of the present invention is to obtain a process for obtaining said filamentous fungi, preferably in the form of powder of conidia or spores or of their primary or secondary metabolites, in particular a process which is rapid, of simple design, which has an efficient efficiency and whose physicochemical parameters of each stage are easily controllable.
  • a purpose of the invention is to obtain a support or solid substrate suitable for the maturation of conidia or spores of filamentous fungi.
  • the present invention relates to a process for obtaining conidia or spores of strains of filamentous fungi comprising the following steps:
  • an inert solid support of organic or non-organic origin such as the sound of a cereal, in particular sound wheat, preferably a tissue in which the sound cells of the cereal forming the inert solid support are free of their fermentable fractions, in particular starch, able to allow only maturation, in particular of cell differentiation, in the absence of cell growth of mycelial biomass into conidia or spores, without causing fermentation or only fermentation of less than 10%, preferably less than 5%, more preferably less than 1% of the cells.
  • Example 4 Preferably, in this process of maturation, in particular in this process of cell differentiation, it is observed as shown in Example 4 below, essentially an absence of cell multiplication and individualization or cell differentiation from biomass. mycelium, as well as a morphological modification of the cells, in particular of their cell wall, which become conidia or spores.
  • the maturation phase (substantially without fermentation) generates a conidia or spores concentration in the range, preferably between 08 and 3x E 5xl0 9 conidia / g for Pseudozyma flocculosa, and between E +09 and 5x10 10 conidia / g, for Trichoderma atroviride and between 5x E +8 and 1x E +10 conidia / g of Trichoderma harzianum conidia.
  • the duration of obtaining conidia or spores is preferably less than three weeks, preferably less than two weeks, more particularly less than one week.
  • the filamentous fungus is preferably of the genus Pseudozyma sp. , in particular Pseudozyma flocculosa, of the genus Tricoderma sp. , in particular Trichoderma harzianum, Trichoderma atroviride, Trichoderma hamatum, and Trichoderma viride, of the genus Penicilum sp. , in particular Penicilium camemberti and Penicilium nalgiovense, of the genus Metarhizium sp. or of the genus Bauveria sp.
  • Another aspect of the invention relates to the conidia or spores of strains of filamentous fungi obtained by the process of the invention, resistant to desiccation and stored in the dry state for a period greater than three months, preferably between three month and twenty-four months, or even more than twenty-four months, at an ambient temperature, that is to say a temperature of between about 15 ° C. and about 30 ° C., more particularly a temperature of between about 20 ° C. and about 30 ° C.
  • the invention also relates to conidia or spores of strains of mature filamentous fungi, of the genus Pseudozyma sp. , in particular Pseudozyma flocculosa, whose cytoplasmic fraction is charged with reserve and enriched with melanin.
  • Another aspect of the invention relates to a powder of conidia or spores of the invention, in particular a powder of conidia or spores obtained by the method of the invention.
  • the invention also relates to food compositions, in particular deli meats and cheeses comprising the conidia, spores or conidia or spore powder of the invention.
  • a final aspect of the invention relates to the use of conidia, spores and conidia powder or spores of the invention, against the proliferation of pests, in particular fungi, bacteria, viruses or viruses. insects, affecting plants or fruits, in particular their use to promote the production yield of said plants or fruits, ie the growth of plants, in particular the growth of plant roots, germination and the growth rate cellular of said plants.
  • this plant or this fruit is chosen from the group constituted by the rose bush, the cucurbits, in particular cucumber, squash or melon, tomato, strawberry, raspberry, currant, grape, grape, apple, apple, pear, pear, plum, plum and potato.
  • a final aspect of the present invention relates to the use of conidia, spores or the powder of conidia or spores of the invention for the preparation of food compositions, in particular sausages and cheeses.
  • FIG. 1 schematically represents the procedure for producing conidial or spore powder according to the invention, advantageously combining a first fermentation stage in the liquid phase generating mycelial biomass followed by a second stage of maturation, called the sporulation phase, in solid phase.
  • Figure 2 in A) is a binocular microscope observation G: 40x and which represents the structure of a wheat bran particle having undergone enzymatic curettage. These cells are emptied of their contents and acquire a honeycomb honeycomb structure highly aerated; in B) and C) are histological sections of about 50 ⁇ m of a fabric impregnated in an epoxy resin, after fixation with glutaraldehyde and osmium tetroxide and a revelation with toluidine blue B) is the cross section of a particle of sound before enzymatic treatment. The cells are loaded with reserves, and C) is the cross section of a particle of the sound derivative forming the inert solid support or substrate of the invention and used in the process of the invention. The cells are emptied of their contents following successive enzymatic treatments, this new structure of the support or inert solid substrate the invention is conducive to a large capacity for absorbing liquids.
  • FIG. 3 represents a phase-contrast optical microscopy photograph of the spores of biofungicide produced in liquid medium (A) and by the invention (B).
  • FIG. 4 represents photos of scanning electron microscopy highlighting the morphological differences of the two types of conidia in Pseudozyma flocculosa.
  • the arrows, at A, show the conidia produced in liquid culture, with their smooth structures without ornamentations and their rounded edges.
  • Figures B and C show the conidia obtained by solid support maturation with concave structures, linked to extensive dehydration, and a verrucous ornamentation conducive to dissemination by air.
  • FIG. 5 includes photos of transmission electron microscopy highlighting the differences in the cytoplasm of the two types of conidia in Pseudozyma flocculosa.
  • a and A ' conidia from the liquid culture are rich in mitochondria (mi) and in free ribosomes (ri).
  • B and B ' those obtained with the new culture procedure have cytoplasms loaded with reserves (res) scattered all along the conidium in the form of various inclusions. The ribosomes are much less present and the mitochondria are undetectable which indicates a slowed metabolism.
  • Figures 6 A, B and C are photos taken by fluorescence microscopy, where it is seen that the conidia produced in liquid medium (A) do not exhibit autofluorescence.
  • Conidia matured on solid support (B and C) have a fluorescent layer on their surfaces.
  • the fluorescent compound appears on the surface of the wall (P) by precipitating the contrast agent osmium tetroxide (E-shaped arrowheads). This compound is almost non-existent in conidia from liquid culture (D).
  • FIG. 7 represents the viability of a Gram- bacterium under different storage conditions after treatment according to the process described in Example 4. Detailed description of the invention
  • the method according to the present invention combines, for the first time, a first so-called growth step (in fermentation) to generate a mycelial biomass in a medium or in a liquid phase and a second so-called maturation stage, preferably differentiation stage ( and essentially without fermentation) in a medium or solid phase on a suitable inert solid support or substrate, which is preferably natural, that is to say obtained from renewable biological material, or which is synthetic, in particular dextran or agarose gel beads or a plastic derivative of petroleum, preferably recyclable, such as expanded polystyrene beads or in a suitable alveolar form.
  • this support or solid substrate must have the following characteristics:
  • honeycomb structure very ventilated propitious at optimal gas exchange and evaporation of water, that is to say a solid substrate or support having empty cells or cells with diameters of between about 2 ⁇ m and about 1000 ⁇ m, preferably between about 10 ⁇ m; and about 500 ⁇ m;
  • liquids in particular water
  • this substrate or inert solid carrier of plant origin essentially comprises tissues, the cells of which have retained their walls, but whose starch and the fractions of the cells most accessible to fermentation by the fungus and bacterial growth (cellulose and hemi-cellulose) were eliminated.
  • This substrate or inert solid support may be in the form of beads or fibers, and be produced from plant waste, such as straw, sawdust, beet pulp, olive grunts, coffee pulp, sugarcane bagasse and their mixtures.
  • this substrate or inert solid support is a derivative of cereals, preferably a derivative of wheat, or rice, more particularly wheat bran or rice, which is devoid of any trace of starch and dough. a large part of its cellulosic fibrous fraction and hemi ⁇ cellulosic, in particular following a sequential enzymatic treatment of the bran, which present the above characteristics and which histological sections are shown in figures 2A to 2C.
  • This inert solid substrate or support of the invention in particular this derivative of wheat bran, is used as the support or inert solid substrate for a second stage of maturation, in the absence of nutrients, of several species of filamentous fungi, in particular conidia or spores of Pseudozyma flocculosa, Trichoderma Atroviride, Trichoderma Hazianum, Penicillium camembert! and Penicillium nalgiovense, Beauveria bassiana and Metarhizium anisopliae, or other species of filamentous fungi.
  • filamentous fungi in particular conidia or spores of Pseudozyma flocculosa, Trichoderma Atroviride, Trichoderma Hazianum, Penicillium camembert! and Penicillium nalgiovense, Beauveria bassiana and Metarhizium anisopliae, or other species of filamentous fungi.
  • the use of the substrate or inert solid support (essentially non-fermentable) according to the invention has the advantage of providing solutions to the major problems experienced by fermentation in a solid medium.
  • the massive inoculation of such a derivative of cereals, especially of such a derivative of wheat bran, and the low water content of the mixture, but especially the fact that this product is preferably natural and derived from cereals, in particular this derivative of wheat bran, also lacking nutrients, including any fermentable part present in plant tissue cells such as starch, can significantly reduce microbial contamination, especially bacterial, in the solid phase.
  • the combination of two modes or steps of successive cultures namely a first phase or liquid fermentation, followed by a second phase of maturation (without fermentation) in solid phase on such a substrate or solid support suitable inert as described above, advantageously controls the growth parameters and maintain them at their optimum values, without generating risks related to excessive growth obtained in the processes of the state of the art and having the drawbacks of generating significant changes in temperature and pH.
  • the first vegetative growth phase generally lasts between about two days. and about four days, generating heat and organic acids, is carried out in a fermenter according to conditions (temperature, pH, choice and quantity of nutrients added, stress induced or not, ...) well known to the man of art and adaptable according to the type of filamentous mushroom produced.
  • the mycelium of the strain of this filamentous fungus massively generates a mycelial biomass 2.
  • the physicochemical parameters followed by the fermenter are preferably as follows:
  • Stirring speed between about 70 rpm and about 250 rpm, with a P02 greater than 30%, preferably greater than 50% and more particularly greater than 80%.
  • the mycelial biomass 2 obtained is then harvested and mixed, as represented by step 3 of FIG. 1, with an inert solid substrate or support, preferably consisting of a wheat bran derivative without starch. to undergo a maturation step, preferably of solid phase differentiation 4, of a duration generally of between about two days and about nine days, but variable depending on the type of fungus filamentous used to generate these conidia or spores from the mycelial biomass 2.
  • a drying step 5 generally lasting between about a day and about eight days, and a step of generating conidia or aerial spores 6, before any final treatment of separation or purification of the final product obtained in the form of a powder of conidia or spores.
  • the maturation phase 4 does not require pH regulation and the little heat produced in the solid phase can be easily controlled by means of ventilation.
  • this method is particularly effective and advantageously generates a large amount of these conidia or spores in a short time, preferably less than three weeks, less than two weeks, less than a week, or even less than one week. some days.
  • the process of the invention may also comprise a step of extraction and purification of primary or secondary metabolites or of any molecule of interest produced by these conidia or spores, in particular enzymes, such as amylases, phosphatases, proteases, vitamins, alkaloids, organic acids, flavors, pigments, lactones, melanin or antibiotics.
  • enzymes such as amylases, phosphatases, proteases, vitamins, alkaloids, organic acids, flavors, pigments, lactones, melanin or antibiotics.
  • the present invention provides a solution to the problems of the substrate culture or solid support, including the possible disadvantage of the heterogeneity of the final product obtained in powder form. Indeed, it is possible that at the end of the drying step 5, one finds oneself with aerial conidia mixed with large quantities of mycelium and solid support residues, for example wheat bran without starch, which are eliminated or even recycled for a new stage of maturation, preferably differentiation, by an additional separation or purification step.
  • Another aspect of the invention relates to the conidia or spores obtained by the method of the invention, in particular conidia or spores of Pseudozyma flocculosa, the cytoplasmic fraction of which is completely transformed and is responsible for storage vesicles, and whose mitochondria and ribosomes are undetectable, which has never been observed before in the liquid culture mode of the same strains of filamentous fungi which are generally enriched in mitochondria and in free ribosomes which are markers of a metabolic activity and intense protein synthesis, no conducive to conservation.
  • Fluorescence microscopy analysis also reveals that by using the method of the invention, the conidia or spores obtained produce a fluorescent substance, it is an accumulation on the surface of the conidium or spore of melanin, which would be a form of protection against ultraviolet rays. This characteristic has never been observed before in conidia or spores produced in liquid culture mode. Conversely, conidia or spores from the submerged medium show a thinning of their wall.
  • Another structural characteristic is the essentially concave appearance of the external shape of the conidium or spore obtained, which is specific to its high degree of maturation, preferably of differentiation, in connection with the mode of production. dissemination of conidia or spores by air.
  • These characteristics have advantages in terms of harvesting, purifying and dispersing conidia or spores.
  • the final step of separating and purifying the final product in powder form has the advantage of considerably reducing the presence of possible contaminants, such as mycelium or residues of the carrier or inert solid substrate used.
  • Pseudozyma flocculosa mycelia from liquid fermentation are matured with the inert solid support or substrate of the invention, which is a derivative of wheat bran.
  • the two-day liquid fermentation step is followed by a condition step on the derivative of the bran for four to eight days, to obtain a final concentration, after ripening, of about 1 E + 09 at 3 ⁇ 10 10 conidia / g, Trichoderma atroviride conidia and 5x E +8 to 1xE + 10 conidia / g Trichoderma harzianum conidia.
  • Example 3 Comparative Study of Spores Produced in a Liquid Medium and on a Solid Support
  • the method of the invention combines a vegetative growth phase, which generates heat and organic acids in liquid culture, which makes it possible to control all the growth parameters and to maintain them at their optimum values; then the mass produced mycelium is then mixed with the solid support suitable to undergo a second phase of sporulation or maturation.
  • This maturation phase is only a phase of cell differentiation without growth that requires very little energy that comes mainly from accumulated reserves in the liquid culture phase. Therefore, this maturation phase without any fermentation does not require pH regulation and the little heat produced can be easily controlled.
  • the spores from the liquid culture have rather rough cytoplasm with many organelles and inclusions that suggest a strong metabolic activity. This is not the case of the spores obtained by the process of the invention, the cytoplasm of which lacks visible inclusions and indicates a lack of metabolic activity.
  • the spores from the submerged culture are much more elongated with an average size around 10 ⁇ m and with rounded edges that form curved hells hiles. Spores obtained by the process of the invention, on the other hand, are smaller by about 3 ⁇ m to about 5 ⁇ m in length. Their edges end with one or two apiculate hiles that look more like a conidial scar.
  • Another aspect of the invention relates to a process for obtaining dried bacteria, preferably GRAM negative bacteria (gram-), in particular of the genus Azospirillum, such as Azospirillum brasilense, which is one of the most studied, because it favors the growth of plants, but which presents a great fragility linked to its structural characteristic of gram - bacterium.
  • gram- preferably GRAM negative bacteria
  • Azospirillum such as Azospirillum brasilense
  • a Gram bacterium - unlike Gram + bacteria does not have a thick wall composed of peptidoglycans and its outer phospholipid membrane does not withstand the physical constraints of drying.
  • the bacterium Aeospirillum brasilense living in the soil, has the ability to influence the growth of many agricultural crops by the excretion of different phytohormones (such as indole acetic acid, also called Auxin) and the ability to fix nitrogen from the air in the soil.
  • This bacterium can be used as a plant growth inoculant, possibly in combination with other plant growth promoting bacteria.
  • the process for obtaining different microorganisms comprises the following steps: to grow said microorganisms during a growth phase in the liquid phase, that is to say by fermentation, to generate a biomass of these microorganisms,
  • this support being advantageously able to partially dehydrate this biomass, without thermal stress and thus reduce or stop the cellular metabolism in order to obtain advantageously an increase in the dry matter content of this biomass, preferably from about 3% to more than 35%, preferably greater than 40%,
  • the inert solid support is preferably the sound of a cereal, in particular wheat bran, that is to say a tissue in which the cells of the cereal bran forming the inert solid support are devoid of their fermentable fractions, in particular starch.
  • this inert solid support may also be a polymeric support as described above.
  • the duration of obtaining said dried microorganisms is advantageously less than 4 weeks or less than 3 weeks, preferably is a duration of between about 1 and 2 weeks, and allows the production microorganisms particularly sensitive and fragile.
  • the invention also relates to microorganisms, preferably bacteria, molds or yeasts, in particular Gram - bacteria, especially of the genus Azospirillum, preferably Azospirillum brasilense, obtained by the process of the invention, which are resistant to desiccation as well as preservation and suitable for use in promoting the growth of agricultural crops, preferably by the excretion of preserved phytohormones, in particular auxin, as well as their ability to fix nitrogen.
  • These microorganisms, preferably these bacteria, yeasts or molds, obtained can be in the form of a powder of said microorganisms, preferably said bacteria, yeasts or molds, obtained by the process of the invention and included in a phytosanitary composition. As shown in Figure 7, these microorganisms in particular these bacteria can be advantageously preserved for long periods, even under extreme conditions of conservation. References
  • Boekhout T., 1995. Pseudozyma Bandoni emend. Boekhout, has genus for yeast-like anamorphs of Ustilaginales. Tea

Abstract

The present invention relates to a method for obtaining conidia or spores of filamentous fungus strains, to the thus obtained conidia or spores, and to their uses in agriculture and the agri-food industry.

Description

Conidies aériennes et résistantes de souches de champignons filamenteux et leur procédé d' obtention Objet de l'invention Aerial and resistant conidia of strains of filamentous fungi and method for obtaining them Object of the invention
[0001] La présente invention se rapporte au domaine de la lutte biologique ou à la bio-fertilisation, et a trait en particulier à des conidies ou des spores, aériennes et résistantes, de souches de champignons filamenteux, en particulier des conidies ou des spores de Pseudozyma , de Trichoderma ou de Pénicillium sous forme de poudre, ainsi qu'à leur procédé d'obtention et leurs applications, en tant qu'agent de lutte biologique ou dans 1 ' agroalimentaire . Arrière-plan technologique à la base de l'invention  The present invention relates to the field of biological control or bio-fertilization, and relates in particular to conidia or spores, aerial and resistant, strains of filamentous fungi, in particular conidia or spores. Pseudozyma, Trichoderma or Penicillium in powder form, as well as their production process and their applications, as a biological control agent or in the food industry. Technological background on which the invention is based
[0002] La lutte biologique, encore appelée bio¬ contrôle, est une méthode connue pour la protection des végétaux contre par exemple des nuisibles, ravageurs de culture, comme les insectes, les acariens ou les nématodes, la protection ou le traitement contre des maladies, en particulier les maladies fongiques, bactériennes ou virales, ou une méthode de lutte contre les mauvaises herbes, plantes adventices, par l'utilisation, soit d'organismes vivants antagonistes, soit de produits biodégradables constitutifs de ces organismes, ou bien de métabolites produits par ces organismes, appelés agents de lutte biologique. [0002] Biological control, also called bio¬¬ control, is a known method for the protection of plants against for example pests, crop pests, such as insects, mites or nematodes, protection or treatment against diseases , in particular fungal, bacterial or viral diseases, or a method of controlling weeds, weeds, by the use either of living antagonistic organisms or of biodegradable products constituting these organisms, or of metabolites produced by these organisms, called biological control agents.
[0003] Comme cette lutte met en jeu des organismes vivants, comme des prédateurs tels les nématodes, arthropodes, vertébrés, ou mollusques, ou à des agents pathogènes, tels des bactéries ou champignons, ou phytophages, ou à des constituants ou métabolites produits par ces organismes vivants, elle présente l'avantage de se faire sans recours à des pesticides chimiques. [0003] As this fight involves living organisms, such as predators such as nematodes, arthropods, vertebrates, or molluscs, or agents. pathogens, such as bacteria or fungi, or phytophages, or to constituents or metabolites produced by these living organisms, it has the advantage of being without the use of chemical pesticides.
[0004] En ce qui concerne la bio-fertilisation, les mêmes types d'organismes sont utilisés, non pas pour leur effet sur les ennemis d'une plante, mais pour leur effet bénéfique sur la croissance d'une plante ou de certaines des parties d'une plante (ex. racines), voire sur la santé écologique de la plante (santé écologique = reproduction x survie) . As regards bio-fertilization, the same types of organisms are used, not for their effect on the enemies of a plant, but for their beneficial effect on the growth of a plant or some of the parts of a plant (eg roots), or even on the ecological health of the plant (ecological health = reproduction x survival).
[0005] En effet, avec toute la problématique provoquée par les pesticides chimiques sur l'environnement, il y a un intérêt croissant à exploiter des nouvelles matières biologiques, ou des nouveaux organismes vivants, tels que par exemple des champignons filamenteux, ou leurs métabolites primaires ou secondaires, pour le contrôle des parasites, des mauvaises herbes et/ou des maladies.  Indeed, with all the problems caused by chemical pesticides on the environment, there is a growing interest in exploiting new biological materials, or new living organisms, such as for example filamentous fungi, or their metabolites. primary or secondary, for the control of pests, weeds and / or diseases.
[0006] De plus, il est à noter que, pour ce qui concerne en particulier la lutte biologique fongique, cette dernière s'articule autour de multiples disciplines, telle que la pathologie de la souche utilisée, l'écologie, la physiologie, la production de masse, la formulation et les stratégies d'application. Cette précision s'applique aussi à la bio-fertilisation. In addition, it should be noted that, particularly with regard to the fungal biological control, the latter is articulated around multiple disciplines, such as the pathology of the strain used, ecology, physiology, mass production, formulation and application strategies. This precision also applies to bio-fertilization.
[0007] Il existe un grand nombre de souches de champignons, en particulier des champignons filamenteux, qui ont un grand intérêt agroalimentaire et agronomique, en particulier comme agents de bio-contrôle, notamment comme fongicides, mais qui ne sont pas encore pleinement exploitées en raison de la difficulté à les faire sporuler en culture liquide .  [0007] There are a large number of fungal strains, in particular filamentous fungi, which have a great agri-food and agronomic interest, in particular as biocontrol agents, especially as fungicides, but which are not yet fully exploited. because of the difficulty to sporulate them in liquid culture.
[0008] Par exemple, Pseudozyma flocculosa est une souche connue de champignon phyllosphère épiphyte, faisant partie de la classe des Basidiomycètes et appartenant à l'ordre des Ustilaginale . For example, Pseudozyma flocculosa is a known strain of epiphytic phyllosphere fungus, making part of the class Basidiomycetes and belonging to the order Ustilaginale.
[0009] Sa classification définitive a été établie en [0009] Its definitive classification has been established in
1995 par Boekhout sur la base d'analyses approfondies sur le plan génétique. Actuellement, ce champignon est classé dans le genre Pseudozyma apparenté aux Ustilaginales du phylum des Basidiomycètes (Boekhout 1995) . La taxonomie complète de ce champignon est consignée dans le tableau 1. Tableau 1. Description taxonomique de Pseudozyma flocculosa (Taxonomy ID: 84751) . 1995 by Boekhout on the basis of in-depth genetic analysis. Currently, this fungus is classified as Pseudozyma related to the Ustilaginales of the phylum Basidiomycetes (Boekhout 1995). The complete taxonomy of this fungus is recorded in Table 1. Table 1. Taxonomic description of Pseudozyma flocculosa (Taxonomy ID: 84751).
Rangs taxonomiques Taxonomic ranks
Domaine Eukaryota  Eukaryota Estate
Règne Fungi  Reign Fungi
Embranchement ou Basidiomycota  Branch or Basidiomycota
phylum  phylum
Sous-embranchement Ustilaginomycotina  Subphylum Ustilaginomycotina
Classe Ustilaginomycetes  Class Ustilaginomycetes
Ordre Ustilaginales  Order Ustilaginales
Famille Ustilaginaceae  Family Ustilaginaceae
Genre Pseudozyma  Pseudozyma genus
Espèce Flocculosa  Flocculosa species
[0010] Ce champignon est reconnu comme agent de lutte biologique très efficace contre le blanc induit par Erysiphe poligoni , de plusieurs plantes dont le rosier et le concombre . This fungus is recognized as a very effective biological control agent against white induced Erysiphe poligoni, several plants including rose and cucumber.
[0011] Par ailleurs, le genre Trichoderma est également connu pour regrouper un ensemble de champignons qui se retrouvent couramment dans le sol, sur le bois mort, les débris végétaux et la partie aérienne des plantes.  Moreover, the genus Trichoderma is also known to group a group of fungi that are commonly found in the soil, dead wood, plant debris and the aerial part of plants.
[0012] De nombreuses espèces de Trichoderma se sont avérés efficaces comme agents biologiques contre par exemple Pythium, Phytophthora cinnamomi , Rhizoctonia solani, Sclerotium rolfsiie et Cryphonectria parasitica. Ces champignons pathogènes causent d'importants dégâts dans les cultures fruitières, légumières, en serre et ornementales. [0012] Many species of Trichoderma have been found effective as biological agents against for example Pythium, Phytophthora cinnamomi, Rhizoctonia solani, Sclerotium rolfsiie and Cryphonectria parasitica. These Pathogenic fungi cause significant damage to fruit, vegetable, greenhouse and ornamental crops.
[0013] Les performances de Trichoderma atroviride comme agent de lutte biologique est due en partie à la production d' endochitinase et il a également été montré que ce champignon intervient, selon divers mécanismes et à divers niveaux, dans la stimulation de la croissance racinaire par l'utilisation de minéraux fertilisants et la stimulation des défenses naturelles des plantes. The performance of Trichoderma atroviride as a biological control agent is due in part to the production of endochitinase and it has also been shown that this fungus intervenes, according to various mechanisms and at various levels, in the stimulation of root growth by the use of fertilizing minerals and the stimulation of natural plant defenses.
[0014] Par ailleurs, il est connu que les champignons des divisions de Zygomycètes, Ascomycètes et Basidiomycètes produisent des spores asexuées appelées conidies, leur permettant une propagation végétative rapide. Par contre, chez les Deutéromycètes , dits aussi champignons imparfaits, le cycle sexuel n'a jamais été observé et la reproduction se fait uniquement par voie asexuée, via la production de conidies . Moreover, it is known that the fungi of the divisions of Zygomycetes, Ascomycetes and Basidiomycetes produce asexual spores called conidia, allowing them rapid vegetative propagation. On the other hand, in Deuteromycetes, also called imperfect fungi, the sexual cycle has never been observed and the reproduction is done only asexually, via the production of conidia.
[0015] Les conidies ou spores sont les principaux moyens de protection, et de conservation, des génomes fongiques, grâce à leur résistance aux traitements de conditionnement et aux conditions environnementales hostiles, telles que les températures extrêmes, les rayons ultraviolets et les faibles valeurs d'humidité relative. A titre d'exemple, une partie des conidies ou spores d' Aspergillus niger maintiennent leur capacité de germination après passage dans un autoclave pendant une heure à 120°C (Morozova et al 2001) .  Conidia or spores are the main means of protection, and conservation, fungal genomes, thanks to their resistance to conditioning treatments and hostile environmental conditions, such as extreme temperatures, ultraviolet rays and low light levels. 'relative humidity. For example, some of the conidia or spores of Aspergillus niger maintain their germinability after passage through an autoclave for one hour at 120 ° C (Morozova et al 2001).
[0016] En outre, la production de ces spores asexuées, ou conidies, est essentielle dans le cycle de vie de nombreux champignons. Elle forme le principal moyen de dispersion pour le génome fongique dans des conditions environnementales défavorables.  In addition, the production of these asexual spores, or conidia, is essential in the life cycle of many fungi. It forms the primary means of dispersal for the fungal genome under adverse environmental conditions.
[0017] De nombreux obstacles restent à surmonter afin de pouvoir commercialiser des agents de lutte biologique fongique à base de champignons filamenteux, notamment du fait de leur faible aptitude à être stocké sur des longues périodes, en particulier à cause de la faible viabilité des conidies ou spores issues d'une fermentation liquide. Many obstacles remain to be overcome in order to market biological control agents. fungal fungi based on filamentous fungi, especially because of their low ability to be stored over long periods, especially because of the low viability of conidia or spores from a liquid fermentation.
[0018] De nombreux champignons, y compris les filamenteux, peuvent être cultivés dans des milieux liquides, c'est-à-dire au cours d'une phase de fermentation dite liquide. Toutefois, la fermentation liquide est particulièrement problématique. Many fungi, including filamentous, can be grown in liquid media, that is to say during a so-called liquid fermentation phase. However, liquid fermentation is particularly problematic.
[0019] Dans cet environnement liquide, selon les espèces, les Hyphomycètes peuvent être amenés à se développer sous des formes diverses, y compris, les organismes hyphes unicellulaires à paroi mince, quelques formes dénommées blastospores , et les conidies ou spores submergées. Ces dernières sont obtenues soit directement à partir de blastospores microcycles, par conidiation, ou soit à partir de cellules conidiogènes qui se forment sur les hyphes submergées . In this liquid environment, depending on the species, Hyphomycetes can be caused to develop in various forms, including thin-walled unicellular hyphae, some forms called blastospores, and submerged conidia or spores. These are obtained either directly from blastospores microcycles, by conidiation, or from conidiogenic cells that form on submerged hyphae.
[0020] En général, la méthode préférée pour la production de conidies ou spores à grande échelle est la culture submergée en raison d'une part de sa bonne rentabilité, sa courte durée de production, et la disponibilité de tous les matériaux nécessaires pour une fermentation liquide.  In general, the preferred method for the production of conidia or spores on a large scale is the submerged culture because of a part of its good profitability, its short production time, and the availability of all the materials necessary for a liquid fermentation.
[0021] Malgré tous ses avantages, les conidies ou spores produites par cette technique ont une trop courte durée de vie dans des conditions environnementales défavorables. C'est pourquoi, la plupart des agents de bio¬ contrôle, à base de conidies ou spores submergées, nécessitent une utilisation immédiate sur les plantes et les terrains infestés par les ravageurs, qu'ils soient fongiques ou d' insectes . Despite all its advantages, the conidia or spores produced by this technique have too short a life in adverse environmental conditions. Therefore, most biocontrol agents, based on conidia or submerged spores, require immediate use on pest - infested plants and grounds, whether they be fungal or insect - borne.
[0022] Il est également possible de produire des conidies par fermentation sur un support ou un substrat solide, selon une production dénommée phase ou fermentation solide . It is also possible to produce conidia by fermentation on a support or a substrate. solid, according to a production called phase or solid fermentation.
[0023] Bien que la fermentation solide soit connue depuis longtemps, elle n'a jamais connu un véritable essor industriel et a souvent été délaissée pour la culture en milieu liquide.  Although solid fermentation has been known for a long time, it has never experienced a real industrial boom and has often been neglected for culture in a liquid medium.
[0024] La fermentation solide présente plusieurs difficultés pour ce qui concerne de nombreuses espèces de champignons. Paecilomyces fumosoroseus, par exemple, a besoin de lumière pour une production optimale de conidies aériennes (Wraight et al. 2001), et la production efficace de conidies de cette souche de champignon nécessite donc une culture en surface, ou une agitation périodique des supports ou substrats solides particulaires , pour augmenter l'exposition à la lumière. Les conidies ou spores de Verticillium se développent, quant à elles, en globules collantes produites à des densités relativement faibles sur une croissance d'hyphes, alors que celles d' aschersonia doivent être produites en circonvolutions, ou sous forme de fosses dans un stroma dense.  Solid fermentation presents several difficulties with regard to many species of fungi. Paecilomyces fumosoroseus, for example, needs light for optimal production of aerial conidia (Wraight et al., 2001), and the efficient production of conidia of this fungal strain therefore requires surface culture, or periodic agitation of the media or particulate solid substrates, to increase the exposure to light. Verticillium conidia or spores, in turn, develop into sticky globules produced at relatively low densities on hyphae growth, whereas those of aschersonia must be produced in convolutions, or as pits in a dense stroma. .
[0025] Par ailleurs, l'augmentation de la température et le changement de pH durant la phase de croissance végétative est un problème critique dans le domaine de fermentation solide (Smits et al. 1998) .  Furthermore, the increase in temperature and the change in pH during the vegetative growth phase is a critical problem in the solid fermentation field (Smits et al., 1998).
[0026] L' intérêt pour la fermentation en milieu solide n'est pas nouveau, pourtant ce mode de culture n'a jamais été développé à cause des difficultés de contrôle des paramètres physico-chimiques de la culture. En effet, la production massive de conidies ou spores doit être initiée à un stade de développement végétatif du mycélium, durant lequel il faut non seulement maintenir les conditions optimales de croissance (température, aération, humidité, pH) , mais également contrôler la différenciation du mycélium et empêcher un phénomène de sporulation précoce qui peut réduire fortement le rendement de production (Raimbault & Durand 1980) . The interest in fermentation in solid medium is not new, yet this mode of cultivation has never been developed because of difficulties in controlling the physicochemical parameters of the crop. In fact, the massive production of conidia or spores must be initiated at a stage of vegetative development of the mycelium, during which it is necessary not only to maintain the optimal growth conditions (temperature, aeration, humidity, pH), but also to control the differentiation of the mycelium and prevent an early sporulation phenomenon that can greatly reduce the production yield (Raimbault & Durand 1980).
[0027] De plus, le problème d'évacuation de la chaleur, produite lors de la fermentation solide, semble particulièrement difficile à résoudre. En effet, la chaleur dégagée par les réactions métaboliques, lors de la phase de croissance végétative, peut être délétère si la température dépasse les seuils limites permis. Il en est de même pour l'évolution du pH qui est quasiment impossible à réguler en l'absence de circulation de liquide (Pandey 2003) .  In addition, the problem of heat removal, produced during the solid fermentation, seems particularly difficult to solve. Indeed, the heat released by the metabolic reactions, during the vegetative growth phase, can be deleterious if the temperature exceeds the allowed thresholds. It is the same for the evolution of the pH which is almost impossible to regulate in the absence of circulation of liquid (Pandey 2003).
[0028] A ces difficultés, liées à la fermentation s'ajoute un autre inconvénient, aussi important, lié au fait qu'en fin de culture on se trouve avec des conidies ou spores mélangées à de grandes quantités de mycélium et de résidus du support ou substrat solide, ce qui peut réduire la qualité du produit final obtenu et les possibilités d'application de celui-ci, notamment dans le domaine agroalimentaire.  To these difficulties, related to the fermentation is added another disadvantage, as important, related to the fact that at the end of culture we are with conidia or spores mixed with large amounts of mycelium and residues of the support or solid substrate, which can reduce the quality of the final product obtained and the application possibilities thereof, particularly in the agri-food field.
Etat de la technique State of the art
[0029] La demande de brevet WO2009/037399 décrit un procédé de production de conidies ou spores, et de leurs métabolites, sur un support, ou substrat, solide par une fermentation classique, la phase de croissance se faisant également sur un tel support. Le milieu solide doit comporter un support ou substrat solide adéquat, c'est-à-dire un support ou substrat absorbant de faible densité et pratiquement non fermentable, mais imprégné d'un milieu de culture approprié à la croissance des conidies ou spores de champignons. Le procédé consiste en l'application d'un stress hydrique durant la phase de production des spores. The patent application WO2009 / 037399 describes a process for producing conidia or spores, and their metabolites, on a support, or substrate, solid by a conventional fermentation, the growth phase is also on such a support. The solid medium must comprise a suitable solid support or substrate, that is to say a support or absorbent substrate of low density and practically non-fermentable, but impregnated with a culture medium suitable for the growth of conidia or spores of fungi . The process involves the application of water stress during the spore production phase.
[0030] La demande de brevet EP2390345 décrit la production de métabolites et autres molécules d' intérêt par fermentation en milieu solide sur de multiples supports ou substrats solides, tels que de la biomasse végétale, en particulier des produits agricoles comme des céréales, des sous-produits de l'industrie agroalimentaire forestière, papetière ou des boues d'épuration. The patent application EP2390345 describes the production of metabolites and other molecules of interest by fermentation in a solid medium on multiple supports or solid substrates, such as plant biomass, in particular. agricultural products such as cereals, by-products of the agri-food industry, pulp and paper industry or sewage sludge.
[0031] Le brevet US 4 837 155 décrit une méthode de production de la souche Trichoderma en phase liquide et en condition aérobie à des températures comprises entre environ 25°C et environ 30°C et à un pH compris entre environ 5,8 et environ 7,0, pour générer à partir d'un inoculant, une densité de conidies d'au moins 5 x 108 par ml, mais sans aucune phase de maturation sur support solide. US Pat. No. 4,837,155 describes a method for producing the Trichoderma strain in the liquid phase and under aerobic conditions at temperatures of between about 25 ° C. and about 30 ° C. and at a pH of between about 5.8 and about 7.0, to generate from an inoculant a conidial density of at least 5 x 10 8 per ml, but without any solid support maturation phase.
[0032] Le document EP0223809 décrit un procédé de production de conidies ou spores de champignons filamenteux dans un fermenteur à disques rotatifs en phase liquide.  EP0223809 discloses a process for producing conidia or spores of filamentous fungi in a rotary disk fermentor in the liquid phase.
[0033] Il est aussi connu d'obtenir un compostage accéléré grâce à une combinaison d'une fermentation liquide et solide générant une grande quantité de biomasse sur un support non inerte fermentable, qui sera dégradé par le développement des souches et qui peut devenir un compost comprenant des conidies de Trichoderma sp. It is also known to obtain an accelerated composting through a combination of a liquid and solid fermentation generating a large amount of biomass on a fermentable non-inert support, which will be degraded by the development of the strains and which can become a compost comprising conidia of Trichoderma sp.
[0034] La publication de U. Hôlker et (Applied[0034] The publication of U. Hölker and (Applied
Microbiology and Biotechnology, V0.64, N°2, pages 175-186 (2004)) décrit que la meilleure méthode de production de spores de champignons filamenteux est une fermentation sur support solide (solid state (substrate) fermentation (SSF) ) et qu' il est possible de combiner une fermentation en phase liquide (submerged fermentation technology (SMF) ) pour générer de la biomasse, avec une fermentation sur support solide (SSF) pour obtenir un grand nombre de spores. Microbiology and Biotechnology, V0.64, No. 2, pages 175-186 (2004)) discloses that the best method of producing filamentous fungal spores is a solid state fermentation (SSF) and that it is possible to combine a submerged fermentation technology (SMF) to generate biomass, with a solid support fermentation (SSF) to obtain a large number of spores.
[0035] La publication de Z . OMRAN et al (Séminaire international des biotechnologies (2015) ) compare les conidies de P. flocculosa produites par fermentation en phase liquide (SMF) et sur support solide (SSF) et compare les différences morphologiques et anatomiques obtenues. [0035] The publication of Z. OMRAN et al (International Seminar on Biotechnology (2015)) compares P. flocculosa conidia produced by liquid phase fermentation (SMF) and solid support (SSF) and compares the morphological and anatomical differences obtained.
[0036] Ainsi, il apparaît que les procédés connus de l'état de technique effectuent toutes les étapes de production de souches de champignons, ou de métabolites issus de ces souches de champignons, soit par fermentation en phase liquide, soit par fermentation en phase solide sur un substrat adéquat. [0036] Thus, it appears that the known methods of the state of the art perform all stages of production of fungal strains, or metabolites derived from these strains of fungi, either by fermentation in the liquid phase, or by solid phase fermentation on a suitable substrate.
Buts de l' invention Goals of the invention
[0037] La présente invention vise à obtenir des souches de champignons filamenteux présentant un intérêt agronomique ou agroalimentaire, utilisables sous forme solide, en particulier sous forme d'une poudre, ainsi qu'un nouveau procédé d'obtention de ces souches, en particulier sous forme de poudre et qui ne présentent pas les inconvénients de l'état de la technique.  The present invention aims at obtaining filamentous fungi strains of agronomic or agri-food interest, usable in solid form, in particular in the form of a powder, and a novel process for obtaining these strains, in particular in the form of powder and which do not have the drawbacks of the state of the art.
[0038] En particulier, la présente invention vise à fournir des souches de champignons filamenteux qui soient aisément manipulables , présentent une viabilité améliorée, en particulier après des longues durées de conservation, et éventuellement d'une haute pureté, c'est dire des souches essentiellement dépourvues de contaminants et/ou de résidus du support de culture, afin de pouvoir les utiliser pour des applications agronomiques ou agroalimentaires, en particulier pour la préparation de fromages ou de charcuteries . In particular, the present invention aims to provide strains of filamentous fungi that are easily manipulated, have improved viability, especially after long periods of storage, and possibly high purity, ie strains essentially free of contaminants and / or residues of the culture medium, in order to be able to use them for agronomic or agri-food applications, in particular for the preparation of cheeses or cold meats.
[0039] Un autre but de la présente invention est d' obtenir un procédé d' obtention desdits champignons filamenteux, de préférence sous forme de poudre de conidies ou spores ou de leurs métabolites primaires ou secondaires, en particulier un procédé qui soit rapide, de conception simple, qui présente un rendement efficace et dont les paramètres physico-chimiques de chaque étape soient aisément contrôlables . Another object of the present invention is to obtain a process for obtaining said filamentous fungi, preferably in the form of powder of conidia or spores or of their primary or secondary metabolites, in particular a process which is rapid, of simple design, which has an efficient efficiency and whose physicochemical parameters of each stage are easily controllable.
[0040] Un denier but de l'invention vise à obtenir un support ou substrat solide adéquat pour la maturation de conidies ou spores de champignons filamenteux. A purpose of the invention is to obtain a support or solid substrate suitable for the maturation of conidia or spores of filamentous fungi.
Eléments caractéristiques de l' invention Characteristic elements of the invention
[0041] La présente invention concerne un procédé d' obtention de conidies ou spores de souches de champignons filamenteux comprenant les étapes suivantes : The present invention relates to a process for obtaining conidia or spores of strains of filamentous fungi comprising the following steps:
croître (par fermentation) des champignons filamenteux durant une première phase de croissance mycélienne (fermentation) en phase liquide, pour générer une biomasse mycélienne,  to grow (by fermentation) the filamentous fungi during a first phase of mycelial growth (fermentation) in the liquid phase, to generate a mycelial biomass,
mélanger ladite biomasse mycélienne obtenue avec un support solide inerte,  mixing said obtained mycelial biomass with an inert solid support,
générer essentiellement sans fermentation des conidies ou spores durant une seconde phase de maturation, en particulier de différentiation cellulaire, de ladite biomasse mycélienne sur ledit support solide inerte, éventuellement sécher les conidies ou les spores obtenues, et  generating, essentially without fermentation, conidia or spores during a second phase of maturation, in particular cell differentiation, of said mycelial biomass on said inert solid support, optionally drying the conidia or spores obtained, and
- éventuellement extraire une ou plusieurs molécules d'intérêt des conidies ou des spores obtenues. optionally extracting one or more molecules of interest from the conidia or spores obtained.
[0042] Dans le procédé de l'invention, on entend parIn the process of the invention, the term
« générer des conidies ou spores durant une phase de maturation, en particulier de différentiation cellulaire, essentiellement sans fermentation » une génération sur un support solide inerte d'origine organique ou non organique, tel que le son d'une céréale, en particulier du son de blé, de préférence un tissu dans lequel les cellules du son de la céréale formant le support solide inerte sont dépourvues de leurs fractions fermentescibles , en particulier d'amidon, aptes à permettre uniquement une maturation, en particulier de différentiation cellulaire, en absence de croissance cellulaire de la biomasse mycélienne en des conidies ou spores, sans provoquer de fermentation ou seulement une fermentation de moins de 10%, de préférence moins de 5%, plus préférentiellement moins de 1% des cellules. De préférence, dans ce processus de maturation, en particulier dans ce processus de différentiation cellulaire, on observe tel que présenté dans l'exemple 4 ci-dessous, essentiellement une absence de la multiplication cellulaire et une individualisation ou différentiation cellulaire à partir de la biomasse mycélienne, ainsi qu'une modification morphologique des cellules, en particulier de leur paroi cellulaire et qui deviennent des conidies ou spores. "To generate conidia or spores during a maturation phase, in particular cell differentiation, essentially without fermentation" a generation on an inert solid support of organic or non-organic origin, such as the sound of a cereal, in particular sound wheat, preferably a tissue in which the sound cells of the cereal forming the inert solid support are free of their fermentable fractions, in particular starch, able to allow only maturation, in particular of cell differentiation, in the absence of cell growth of mycelial biomass into conidia or spores, without causing fermentation or only fermentation of less than 10%, preferably less than 5%, more preferably less than 1% of the cells. Preferably, in this process of maturation, in particular in this process of cell differentiation, it is observed as shown in Example 4 below, essentially an absence of cell multiplication and individualization or cell differentiation from biomass. mycelium, as well as a morphological modification of the cells, in particular of their cell wall, which become conidia or spores.
[0043] Dans le procédé de l'invention, la phase de maturation (essentiellement sans fermentation) génère une concentration en conidies ou spores comprise, de préférence entre 3xE+08 et 5xl09 conidies/g pour Pseudozyma flocculosa, et entre lE+09 et 5xl010 conidies/g, pour Trichoderma atroviride et entre 5xE+8 et lxE+10 conidies/g de conidies de Trichoderma harzianum . [0043] In the method of the invention, the maturation phase (substantially without fermentation) generates a conidia or spores concentration in the range, preferably between 08 and 3x E 5xl0 9 conidia / g for Pseudozyma flocculosa, and between E +09 and 5x10 10 conidia / g, for Trichoderma atroviride and between 5x E +8 and 1x E +10 conidia / g of Trichoderma harzianum conidia.
[0044] Dans le procédé de l'invention, la durée d' obtention des conidies ou des spores est avantageusement inférieure à trois semaines, de préférence inférieure à deux semaines, plus particulièrement inférieure à une semaine.  In the process of the invention, the duration of obtaining conidia or spores is preferably less than three weeks, preferably less than two weeks, more particularly less than one week.
[0045] Selon l'invention, le champignon filamenteux est de préférence du genre Pseudozyma sp . , en particulier Pseudozyma flocculosa, du genre Tricoderma sp . , en particulier Trichoderma harzianum, Trichoderma atroviride, Trichoderma hamatum, et Trichoderma viride, du genre Penicilum sp . , en particulier Penicilium camemberti et Penicilium nalgiovense, du genre Metarhizium sp . ou du genre Bauveria sp . According to the invention, the filamentous fungus is preferably of the genus Pseudozyma sp. , in particular Pseudozyma flocculosa, of the genus Tricoderma sp. , in particular Trichoderma harzianum, Trichoderma atroviride, Trichoderma hamatum, and Trichoderma viride, of the genus Penicilum sp. , in particular Penicilium camemberti and Penicilium nalgiovense, of the genus Metarhizium sp. or of the genus Bauveria sp.
[0046] Un autre aspect de l'invention concerne les conidies ou les spores de souches de champignons filamenteux obtenues par le procédé de l'invention, résistantes à la dessiccation et se conservant à l'état sec durant une période supérieure à trois mois, de préférence comprise entre trois mois et vingt-quatre mois, voire plus de vingt-quatre mois, à une température ambiante, c'est à dire une température comprise entre environ 15°c et environ 30°C, plus particulièrement une température comprise entre environ 20 °C et environ 30°C. Another aspect of the invention relates to the conidia or spores of strains of filamentous fungi obtained by the process of the invention, resistant to desiccation and stored in the dry state for a period greater than three months, preferably between three month and twenty-four months, or even more than twenty-four months, at an ambient temperature, that is to say a temperature of between about 15 ° C. and about 30 ° C., more particularly a temperature of between about 20 ° C. and about 30 ° C.
[0047] L'invention concerne aussi les conidies ou des spores de souches de champignons filamenteux matures, du genre Pseudozyma sp . , en particulier Pseudozyma flocculosa, dont la fraction cytoplasmique est chargée de réserve et enrichie en mélanine.  The invention also relates to conidia or spores of strains of mature filamentous fungi, of the genus Pseudozyma sp. , in particular Pseudozyma flocculosa, whose cytoplasmic fraction is charged with reserve and enriched with melanin.
[0048] Un autre aspect de l'invention concerne une poudre de conidies ou spores de l'invention, en particulier une poudre de conidies ou de spores obtenue par le procédé de l'invention.  Another aspect of the invention relates to a powder of conidia or spores of the invention, in particular a powder of conidia or spores obtained by the method of the invention.
[0049] L' invention concerne aussi des compositions alimentaires, en particulier des charcuteries et des fromages comprenant les conidies, les spores ou la poudre de conidies ou de spores de l'invention. The invention also relates to food compositions, in particular deli meats and cheeses comprising the conidia, spores or conidia or spore powder of the invention.
[0050] Un dernier aspect de l'invention concerne l'utilisation des conidies, spores et de la poudre de conidies ou spores de l'invention, contre la prolifération de nuisibles, en particulier des champignons, des bactéries, des virus ou d'insectes, affectant des plantes ou des fruits, en particulier leur utilisation pour favoriser le rendement de production desdites plantes ou fruits, c'est à dire la croissance des plantes, en particulier la croissance des racines de plantes, la germination et le taux de croissance cellulaire desdites plantes. De préférence, cette plante ou ce fruit est choisi parmi le groupe constitué par le rosier, les cucurbitacées , en particulier le concombre, la courge ou le melon, la tomate, la fraise, la framboise, la groseille, la vigne, le raisin, la pomme, le pommier, la poire, le poirier, la prune, le prunier et la pomme de terre. [0051] Un dernier aspect de la présente invention concerne l'utilisation des conidies, des spores ou la poudre de conidies ou de spores de l'invention pour la préparation de compositions alimentaires, en particulier des charcuteries et des fromages. A final aspect of the invention relates to the use of conidia, spores and conidia powder or spores of the invention, against the proliferation of pests, in particular fungi, bacteria, viruses or viruses. insects, affecting plants or fruits, in particular their use to promote the production yield of said plants or fruits, ie the growth of plants, in particular the growth of plant roots, germination and the growth rate cellular of said plants. Preferably, this plant or this fruit is chosen from the group constituted by the rose bush, the cucurbits, in particular cucumber, squash or melon, tomato, strawberry, raspberry, currant, grape, grape, apple, apple, pear, pear, plum, plum and potato. A final aspect of the present invention relates to the use of conidia, spores or the powder of conidia or spores of the invention for the preparation of food compositions, in particular sausages and cheeses.
[0052] La présente invention sera décrite dans la description détaillée ci-dessous en référence aux figures annexées et présentées à titre d' illustrations non limitatives de l'invention.  The present invention will be described in the detailed description below with reference to the accompanying figures and presented as non-limiting illustrations of the invention.
Brève description des figures Brief description of the figures
[0053] La figure 1 représente schématiquement la procédure de production de poudre de conidies ou spores selon l'invention combinant avantageusement une première étape fermentation en phase liquide générant de la biomasse mycélienne suivie d'une seconde étape de maturation, dite phase de sporulation, en phase solide.  FIG. 1 schematically represents the procedure for producing conidial or spore powder according to the invention, advantageously combining a first fermentation stage in the liquid phase generating mycelial biomass followed by a second stage of maturation, called the sporulation phase, in solid phase.
[0054] La figure 2 en A) est une observation au microscope binoculaire G:40x et qui représente la structure d'une particule de son de blé ayant subi un curetage enzymatique. Ces cellules sont vidées de leurs contenus et acquièrent une structure alvéolaire en nid d'abeilles fortement aérée ; en B) et C) sont des coupes histologiques d'environ 50 ym d'un tissu imprégné dans une résine époxy, après une fixation au glutaraldehyde et au tétroxyde d' osmium et une révélation au bleu de toluidine B) est la coupe transversale d'une particule de son avant traitement enzymatique. Les cellules sont chargées de réserves, et C) est la coupe transversale d'une particule du dérivé du son formant le support ou substrat solide inerte de l'invention et utilisé dans le procédé de l'invention. Les cellules sont vidées de leurs contenus suite aux traitements enzymatiques successifs, cette nouvelle structure du support ou substrat solide inerte l'invention est propice à une grande capacité d'absorption de liquides. Figure 2 in A) is a binocular microscope observation G: 40x and which represents the structure of a wheat bran particle having undergone enzymatic curettage. These cells are emptied of their contents and acquire a honeycomb honeycomb structure highly aerated; in B) and C) are histological sections of about 50 μm of a fabric impregnated in an epoxy resin, after fixation with glutaraldehyde and osmium tetroxide and a revelation with toluidine blue B) is the cross section of a particle of sound before enzymatic treatment. The cells are loaded with reserves, and C) is the cross section of a particle of the sound derivative forming the inert solid support or substrate of the invention and used in the process of the invention. The cells are emptied of their contents following successive enzymatic treatments, this new structure of the support or inert solid substrate the invention is conducive to a large capacity for absorbing liquids.
[0055] La figure 3 représente une photographie en microscopie optique à contraste de phase des spores de bio- fongicide produite en milieu liquide (A) et par l'invention (B) .  FIG. 3 represents a phase-contrast optical microscopy photograph of the spores of biofungicide produced in liquid medium (A) and by the invention (B).
[0056] La figure 4 représente des photos de la microscopie électronique à balayage mettant en évidence les différences morphologiques des deux types de conidies chez Pseudozyma flocculosa. Les flèches, en A, montrent les conidies produites en culture liquide, avec leurs structures lisses sans ornementations et leurs bords arrondis. Les figures B et C montrent les conidies obtenues par maturation sur support solide avec des structures concaves, liées à une déshydratation poussée, et une ornementation verruqueuse propice à une dissémination par voie aérienne.  [0056] FIG. 4 represents photos of scanning electron microscopy highlighting the morphological differences of the two types of conidia in Pseudozyma flocculosa. The arrows, at A, show the conidia produced in liquid culture, with their smooth structures without ornamentations and their rounded edges. Figures B and C show the conidia obtained by solid support maturation with concave structures, linked to extensive dehydration, and a verrucous ornamentation conducive to dissemination by air.
[0057] La figure 5 inclut des photos de la microscopie électronique à transmission mettant en évidence les différences au niveau des cytoplasmes des deux types de conidies chez Pseudozyma flocculosa. A et A' : conidies issues de la culture liquide sont riches en mitochondrie (mi) et en ribosomes libres (ri) . B et B' : celles obtenues avec la nouvelle procédure de culture ont des cytoplasmes chargés de réserves (res) dispersées tout le long de la conidie sous forme d'inclusions diverses. Les ribosomes sont beaucoup moins présents et les mitochondries sont indétectables ce qui témoigne d'un métabolisme ralenti.  FIG. 5 includes photos of transmission electron microscopy highlighting the differences in the cytoplasm of the two types of conidia in Pseudozyma flocculosa. A and A ': conidia from the liquid culture are rich in mitochondria (mi) and in free ribosomes (ri). B and B ': those obtained with the new culture procedure have cytoplasms loaded with reserves (res) scattered all along the conidium in the form of various inclusions. The ribosomes are much less present and the mitochondria are undetectable which indicates a slowed metabolism.
[0058] Les figures 6 A, B et C sont photos prises en microscopie à fluorescence, où on voit que les conidies produites en milieu liquide (A) ne présentent pas d'auto-fluorescence. Les conidies ayant subi une maturation sur support solide (B et C) présentent une couche fluorescente à leurs surfaces. En microscopie électronique à transmission, le composé fluorescent se manifeste, à la surface de la paroi (P) , en précipitant l'agent de contraste le tétroxyde d'osmium (têtes de flèches en E) . Ce composé est quasi inexistant chez les conidies issues de la culture liquide (D) . Figures 6 A, B and C are photos taken by fluorescence microscopy, where it is seen that the conidia produced in liquid medium (A) do not exhibit autofluorescence. Conidia matured on solid support (B and C) have a fluorescent layer on their surfaces. In electron microscopy With transmission, the fluorescent compound appears on the surface of the wall (P) by precipitating the contrast agent osmium tetroxide (E-shaped arrowheads). This compound is almost non-existent in conidia from liquid culture (D).
[0059] La figure 7 représente la viabilité d'une bactérie Gram- dans différentes conditions de conservation après traitement selon le procédé décrit dans l'exemple 4. Description détaillée de l'invention  FIG. 7 represents the viability of a Gram- bacterium under different storage conditions after treatment according to the process described in Example 4. Detailed description of the invention
[0060] De nombreuses souches de champignons ne sporulent que faiblement, de façon aléatoire et non stable dans le temps en culture liquide, ce qui représente un frein majeur à leur commercialisation et leurs applications.  Many strains of fungi sporulate only weakly, randomly and not stable over time in liquid culture, which represents a major brake on their marketing and their applications.
[0061] Le procédé selon la présente invention combine, pour la première fois, une première étape dite de croissance (en fermentation) pour générer une biomasse mycélienne en milieu ou en phase liquide et une seconde étape dite de maturation, de préférence de différentiation (et essentiellement sans fermentation) en milieu ou en phase solide sur un support ou substrat solide inerte adéquat, qui soit de préférence naturel, c'est-à-dire obtenu à partir de matière biologique renouvelable, ou qui soit de synthèse, en particulier des billes de gel de dextrane ou d' agarose ou un dérivé plastique du pétrole, de préférence recyclable, tel que du polystyrène expansé sous forme de billes ou sous une forme alvéolaire adéquate. The method according to the present invention combines, for the first time, a first so-called growth step (in fermentation) to generate a mycelial biomass in a medium or in a liquid phase and a second so-called maturation stage, preferably differentiation stage ( and essentially without fermentation) in a medium or solid phase on a suitable inert solid support or substrate, which is preferably natural, that is to say obtained from renewable biological material, or which is synthetic, in particular dextran or agarose gel beads or a plastic derivative of petroleum, preferably recyclable, such as expanded polystyrene beads or in a suitable alveolar form.
[0062] En effet, ce support ou substrat solide doit présenter les caractéristiques suivantes :  Indeed, this support or solid substrate must have the following characteristics:
- être inerte, non dégradable et sans nutriments, c'est- à-dire non ou peu fermentable et donc non ou peu accessible au développement du mycélium et à la dégradation bactérienne ; - be inert, non-degradable and nutrient-free, that is to say, no or little fermentable and therefore not or hardly accessible to the development of mycelium and bacterial degradation;
présenter une structure alvéolaire, très aérée propice à des échanges gazeux optimaux et à 1 ' évaporation de l'eau, c'est à dire un substrat ou support solide ayant des cellules ou alvéoles vides avec des diamètres compris entre environ 2 ym et environ 1000 ym, de préférence entre environ 10 ym et environ 500 ym ; have a honeycomb structure, very ventilated propitious at optimal gas exchange and evaporation of water, that is to say a solid substrate or support having empty cells or cells with diameters of between about 2 μm and about 1000 μm, preferably between about 10 μm; and about 500 μm;
présenter une grande capacité d' absorption et de rétention de liquides, en particulier de l'eau ; c'est à dire avoir la capacité d'absorber entre environ 5 fois et environ 15 fois, de préférence entre environ 8 fois et environ 10 fois, plus particulièrement environ 9 fois son propre poids en ces liquides.  have a high capacity for absorption and retention of liquids, in particular water; that is, having the capacity to absorb between about 5 times and about 15 times, preferably between about 8 times and about 10 times, more particularly about 9 times its own weight in these liquids.
[0063] De préférence, ce substrat ou support solide inerte d'origine végétale, comprend essentiellement des tissus, dont les cellules ont conservées leurs parois, mais dont l'amidon et les fractions des cellules les plus accessibles à une fermentation par le champignon et au développement bactérien (cellulose et hémi-cellulose) ont été éliminés. Ce substrat ou support solide inerte peut se présenter sous forme de billes ou de fibres, et être produit à partir de déchets végétaux, tels que de la paille, de la sciure de bois, de la pulpe de betterave, de grognons d'olives, de la pulpe de café, de la bagasse de canne à sucre et leur mélanges. Preferably, this substrate or inert solid carrier of plant origin, essentially comprises tissues, the cells of which have retained their walls, but whose starch and the fractions of the cells most accessible to fermentation by the fungus and bacterial growth (cellulose and hemi-cellulose) were eliminated. This substrate or inert solid support may be in the form of beads or fibers, and be produced from plant waste, such as straw, sawdust, beet pulp, olive grunts, coffee pulp, sugarcane bagasse and their mixtures.
[0064] Avantageusement, ce substrat ou support solide inerte est un dérivé de céréales, de préférence un dérivé du blé, ou de riz, plus particulièrement de son de blé ou de riz, qui se trouve dépourvu de toute trace d'amidon et d'une grande partie de sa fraction fibreuse cellulosique et hémi¬ cellulosique, en particulier suite à un traitement enzymatique séquentiel de ce son de blé qui présentera les caractéristiques susmentionnées et dont des coupes histologiques sont représentées dans les figures 2A à 2C. Ce substrat ou support solide inerte de l'invention, en particulier ce dérivé du son de blé, est utilisé comme support ou substrat solide inerte pour une seconde étape dite de maturation, en l'absence de nutriments, de plusieurs espèces des champignons filamenteux, en particulier des conidies ou spores de Pseudozyma flocculosa , de Trichoderma Atroviride, Trichoderma Hazianum, Pénicillium camembert! et Pénicillium nalgiovense, Beauveria bassiana et Metarhizium anisopliae, ou d'autres espèces de champignons filamenteux. Advantageously, this substrate or inert solid support is a derivative of cereals, preferably a derivative of wheat, or rice, more particularly wheat bran or rice, which is devoid of any trace of starch and dough. a large part of its cellulosic fibrous fraction and hemi ¬ cellulosic, in particular following a sequential enzymatic treatment of the bran, which present the above characteristics and which histological sections are shown in figures 2A to 2C. This inert solid substrate or support of the invention, in particular this derivative of wheat bran, is used as the support or inert solid substrate for a second stage of maturation, in the absence of nutrients, of several species of filamentous fungi, in particular conidia or spores of Pseudozyma flocculosa, Trichoderma Atroviride, Trichoderma Hazianum, Penicillium camembert! and Penicillium nalgiovense, Beauveria bassiana and Metarhizium anisopliae, or other species of filamentous fungi.
[0065] L'utilisation du substrat ou support solide inerte (essentiellement non fermentable) selon l'invention présente l'avantage d'apporter des solutions aux problèmes majeurs que connaît la fermentation en milieu solide. L'inoculation massive d'un tel dérivé de céréales, en particulier d'un tel dérivé de son de blé, et la faible teneur en eau du mélange, mais surtout le fait que ce produit de préférence naturel et dérivé de céréales, en particulier ce dérivé du son de blé, soit aussi dépourvu de nutriments, y compris de toute partie fermentescible présente dans les cellules du tissu végétal comme de l'amidon, permet de réduire considérablement les contaminations microbiennes, en particulier bactérienne, en phase solide. The use of the substrate or inert solid support (essentially non-fermentable) according to the invention has the advantage of providing solutions to the major problems experienced by fermentation in a solid medium. The massive inoculation of such a derivative of cereals, especially of such a derivative of wheat bran, and the low water content of the mixture, but especially the fact that this product is preferably natural and derived from cereals, in particular this derivative of wheat bran, also lacking nutrients, including any fermentable part present in plant tissue cells such as starch, can significantly reduce microbial contamination, especially bacterial, in the solid phase.
[0066] Selon l'invention, la combinaison de deux modes ou étapes de cultures successifs, à savoir une première phase ou fermentation liquide, suivie d'une seconde phase de maturation (sans fermentation) en phase solide sur un tel substrat ou support solide inerte adéquat tel que sus-décrit, permet de contrôler avantageusement les paramètres de croissance et de les maintenir à leurs valeurs optimales, sans générer de risques liés aux croissances excessives obtenues dans les procédés de l'état de la technique et présentant les inconvénients de générer des modifications importantes de la température et du pH.  According to the invention, the combination of two modes or steps of successive cultures, namely a first phase or liquid fermentation, followed by a second phase of maturation (without fermentation) in solid phase on such a substrate or solid support suitable inert as described above, advantageously controls the growth parameters and maintain them at their optimum values, without generating risks related to excessive growth obtained in the processes of the state of the art and having the drawbacks of generating significant changes in temperature and pH.
[0067] En effet, tel que décrit dans la figure 1, la première phase de croissance végétative, dite phase liquide 1, d'une durée généralement comprise entre environ deux jours et environ quatre jours, génératrice de chaleur et d'acides organiques, est réalisée en un fermenteur selon des conditions (température, pH, choix et quantité des nutriments ajoutés, stress induit ou non, ...) bien connues de l'homme de l'art et adaptables en fonction du type de champignon filamenteux produit. Le mycélium de la souche de ce champignon filamenteux génère massivement une biomasse mycélienne 2. Les paramètres physicochimiques suivis du fermenteur sont de préférence les suivants: Indeed, as described in FIG. 1, the first vegetative growth phase, called the liquid phase 1, generally lasts between about two days. and about four days, generating heat and organic acids, is carried out in a fermenter according to conditions (temperature, pH, choice and quantity of nutrients added, stress induced or not, ...) well known to the man of art and adaptable according to the type of filamentous mushroom produced. The mycelium of the strain of this filamentous fungus massively generates a mycelial biomass 2. The physicochemical parameters followed by the fermenter are preferably as follows:
- Température : maintenue entre environ 20 °C et environ - Temperature: maintained between about 20 ° C and about
40°C. 40 ° C.
- Vitesse d'agitation : comprise entre environ 70 rpm et environ 250 rpm, avec une P02 supérieur à 30%, de préférence supérieur à 50% et plus particulièrement supérieur à 80%.  Stirring speed: between about 70 rpm and about 250 rpm, with a P02 greater than 30%, preferably greater than 50% and more particularly greater than 80%.
- pH : maintenu entre environ 4 et environ 7.8 (pas de régulation de pH nécessaire dans cette production) . - pH: maintained between about 4 and about 7.8 (no pH regulation necessary in this production).
- Pas de stress induit durant la fermentation. - No stress induced during fermentation.
- Plusieurs milieux de culture connus de l'homme de l'art peuvent être utilisés comme:  Several culture media known to those skilled in the art can be used as:
- MAI (extrait de malt 10 g) . - MAY (malt extract 10 g).
- MA2 (Extrait de malt 20 g) .  - MA2 (Malt extract 20 g).
- MEA (Extrait de malt 20 g, Extrait de levure 2 g) - MEA (malted extract 20 g, yeast extract 2 g)
- YMPD (Extrait de malt 5 g, Extrait de levure 3 g, Dextrose 15 g, peptone de soja 6 g) - YMPD (malt extract 5 g, yeast extract 3 g, Dextrose 15 g, soy peptone 6 g)
[0068] La biomasse mycélienne 2 obtenue est ensuite récoltée et mélangée, comme représenté par l'étape 3 de la figure 1, avec un substrat ou support solide inerte, de préférence constitué d'un dérivé de son de blé dépourvu d'amidon, pour subir une étape de maturation, de préférence de différentiation 4 en phase solide, d'une durée généralement comprise entre environ deux jours et environ neuf jours, mais variable en fonction du type de champignon filamenteux utilisé, afin de générer ces conidies ou ces spores à partir de la biomasse mycélienne 2. Ces conidies ou spores peuvent ensuite avantageusement être soumis à une étape de séchage 5, généralement d'une durée comprise entre environ un jour et environ huit jours, et une étape de génération des conidies ou spores aériennes 6, avant un éventuel traitement final de séparation ou purification du produit final obtenu sous forme d'une poudre de conidies ou de spores. Comme le substrat ou le support solide inerte selon l'invention est avantageusement non fermentescible, la phase de maturation 4 ne nécessite pas de régulation de pH et le peu de chaleur produite en phase solide peut être facilement contrôlée, via une ventilation. En outre, ce procédé est particulièrement efficace et génère de manière avantageuse une grande quantité de ces conidies ou ces spores dans des délais courts, de préférence de moins de trois semaines, de moins de deux semaines, de moins d'une semaine, voire de quelques jours. De manière optionnelle, le procédé de l'invention peut aussi comporter une étape d'extraction et de purification de métabolites primaires ou secondaires ou de toute molécule d' intérêt produits par ces conidies ou spores, en particulier des enzymes, telles que des amylases, des phosphatases , des protéases, des vitamines, des alcaloïdes, des acides organiques, des arômes, des pigments, des lactones, de la mélanine ou des antibiotiques. The mycelial biomass 2 obtained is then harvested and mixed, as represented by step 3 of FIG. 1, with an inert solid substrate or support, preferably consisting of a wheat bran derivative without starch. to undergo a maturation step, preferably of solid phase differentiation 4, of a duration generally of between about two days and about nine days, but variable depending on the type of fungus filamentous used to generate these conidia or spores from the mycelial biomass 2. These conidia or spores can then advantageously be subjected to a drying step 5, generally lasting between about a day and about eight days, and a step of generating conidia or aerial spores 6, before any final treatment of separation or purification of the final product obtained in the form of a powder of conidia or spores. Since the substrate or the inert solid support according to the invention is advantageously non-fermentable, the maturation phase 4 does not require pH regulation and the little heat produced in the solid phase can be easily controlled by means of ventilation. In addition, this method is particularly effective and advantageously generates a large amount of these conidia or spores in a short time, preferably less than three weeks, less than two weeks, less than a week, or even less than one week. some days. Optionally, the process of the invention may also comprise a step of extraction and purification of primary or secondary metabolites or of any molecule of interest produced by these conidia or spores, in particular enzymes, such as amylases, phosphatases, proteases, vitamins, alkaloids, organic acids, flavors, pigments, lactones, melanin or antibiotics.
[0069] Par conséquent, la présente invention apporte une solution aux problèmes de la culture sur substrat ou support solide, y compris à l'inconvénient éventuel de l'hétérogénéité du produit final obtenu sous forme de poudre. En effet, il est possible qu'en fin d'étape de séchage 5, on se retrouve avec des conidies aériennes mélangées à de grandes quantités de mycélium et de résidus de support solide, par exemple le son de blé dépourvu d'amidon, qui sont éliminés, voire recyclés pour une nouvelle étape de maturation, de préférence de différentiation, par une étape de séparation ou de purification supplémentaire. Therefore, the present invention provides a solution to the problems of the substrate culture or solid support, including the possible disadvantage of the heterogeneity of the final product obtained in powder form. Indeed, it is possible that at the end of the drying step 5, one finds oneself with aerial conidia mixed with large quantities of mycelium and solid support residues, for example wheat bran without starch, which are eliminated or even recycled for a new stage of maturation, preferably differentiation, by an additional separation or purification step.
[0070] Un autre aspect de l'invention concerne les conidies ou spores obtenues par le procédé de l'invention, en particulier des conidies ou spores de Pseudozyma flocculosa , dont la fraction cytoplasmique est complètement transformée et se charge de vésicules de réserve, et dont les mitochondries et ribosomes sont non détectables ce qui n'a jamais été observé auparavant en mode de culture liquide des mêmes souches de champignons filamenteux qui sont généralement enrichies en mitochondries et en ribosomes libres marqueurs d'une activité métabolique et synthèse protéique intense, non propice à une conservation. Another aspect of the invention relates to the conidia or spores obtained by the method of the invention, in particular conidia or spores of Pseudozyma flocculosa, the cytoplasmic fraction of which is completely transformed and is responsible for storage vesicles, and whose mitochondria and ribosomes are undetectable, which has never been observed before in the liquid culture mode of the same strains of filamentous fungi which are generally enriched in mitochondria and in free ribosomes which are markers of a metabolic activity and intense protein synthesis, no conducive to conservation.
[0071] L'analyse par microscopie à fluorescence révèle également qu'en employant le procédé de l'invention, les conidies ou spores obtenues produisent une substance fluorescente, il s'agit d'une accumulation à la surface de la conidie ou spore de mélanine, ce qui serait un mode de protection contre les rayons ultraviolets. Cette caractéristique n'a jamais été observée auparavant chez les conidies ou spores produites en mode de culture liquide. Au contraire, les conidies ou spores issues du milieu submergé présentent un amincissement de leur paroi. Fluorescence microscopy analysis also reveals that by using the method of the invention, the conidia or spores obtained produce a fluorescent substance, it is an accumulation on the surface of the conidium or spore of melanin, which would be a form of protection against ultraviolet rays. This characteristic has never been observed before in conidia or spores produced in liquid culture mode. Conversely, conidia or spores from the submerged medium show a thinning of their wall.
[0072] Par ailleurs, une autre caractéristique structurale est l'aspect essentiellement concave de la forme extérieure de la conidie ou de la spore obtenue, qui est spécifique à son haut degré de maturation, de préférence de différentiation, en lien avec le mode de dissémination des conidies ou spores par voie aérienne. Ces caractéristiques présentent des avantages en termes de récolte, de purification et de dispersion des conidies ou spores. En particulier, l'étape finale de séparation et de purification du produit final sous forme de poudre, présente l'avantage de réduire considérablement la présence de possibles contaminants, tels que du mycélium ou des résidus du support ou substrat solide inerte utilisé. Furthermore, another structural characteristic is the essentially concave appearance of the external shape of the conidium or spore obtained, which is specific to its high degree of maturation, preferably of differentiation, in connection with the mode of production. dissemination of conidia or spores by air. These characteristics have advantages in terms of harvesting, purifying and dispersing conidia or spores. In particular, the final step of separating and purifying the final product in powder form has the advantage of considerably reducing the presence of possible contaminants, such as mycelium or residues of the carrier or inert solid substrate used.
Exemples Examples
Exemple 1: Production de conidies de Pseudozyma flocculosa Example 1 Production of Conidia of Pseudozyma flocculosa
[0073] Pour la production de conidies ou spores deFor the production of conidia or spores of
Pseudozyma flocculosa, des mycéliums issus de la fermentation liquide sont mis à maturation avec le support ou substrat solide inerte de l'invention, qui est un dérivé du son de blé. Pseudozyma flocculosa, mycelia from liquid fermentation are matured with the inert solid support or substrate of the invention, which is a derivative of wheat bran.
[0074] L'étape de maturation ou de conidiation, c'est-à-dire d'obtention de conidies, ou de sporulation, d'une durée de deux jours à sept jours est suivie d'une étape de séchage pour obtenir une concentration finale de conidies ou spores de Pseudozyma flocculosa après maturation d'environ de 3xE+08 à 5xl09 conidies/g. Des tests de protection de plantes ou de lutte biologique contre des nuisibles de plantes, effectués avec ces conidies ou spores ont montré une efficacité similaire à celle obtenue avec des conidies ou spores obtenues par les procédés de l'état de la technique . Exemple 2: Production de Trichoderma harzianum et Trichderma a troviride The stage of maturation or conidiation, that is to say to obtain conidia, or sporulation, lasting from two days to seven days is followed by a drying step to obtain a final concentration of conidia or spores of Pseudozyma flocculosa after maturation from about 3x E + 08 to 5x10 9 conidia / g. Plant protection or biological pest control tests carried out with these conidia or spores have shown an efficiency similar to that obtained with conidia or spores obtained by the methods of the state of the art. Example 2: Production of Trichoderma harzianum and Trichderma a troviride
[0075] L'étape de fermentation liquide de deux jours est suivie d'une étape de condition sur le dérivé du son pendant quatre à huit jours, pour obtenir une concentration finale, après maturation, d'environ lE+09 à 3xl010 conidies/g, de conidies de Trichoderma atroviride et de 5xE+8 à lxE+10 conidies/g de conidies de Trichoderma harzianum . Exemple 3 : Etude comparative des spores produites en milieu liquide et sur support solide The two-day liquid fermentation step is followed by a condition step on the derivative of the bran for four to eight days, to obtain a final concentration, after ripening, of about 1 E + 09 at 3 × 10 10 conidia / g, Trichoderma atroviride conidia and 5x E +8 to 1xE + 10 conidia / g Trichoderma harzianum conidia. Example 3 Comparative Study of Spores Produced in a Liquid Medium and on a Solid Support
[0076] Chez la plupart des champignons filamenteux, la production massive de conidies ou de spores commence d'abord par un stade de développement végétatif du mycélium. Lors de cette phase, il faut non seulement maintenir les conditions optimales de croissance (température, aération, humidité, pH) , mais également contrôler la différenciation du mycélium et empêcher le phénomène de sporulation précoce qui réduit fortement les rendements. In most filamentous fungi, the massive production of conidia or spores first begins with a stage of vegetative development of the mycelium. During this phase, it is necessary not only to maintain the optimal growth conditions (temperature, aeration, humidity, pH), but also to control the differentiation of the mycelium and to prevent the early sporulation phenomenon which strongly reduces the yields.
[0077] Le problème d'évacuation des calories semble particulièrement difficile à résoudre. En effet, la chaleur dégagée par les réactions métaboliques lors de la phase de croissance végétative peut atteindre des niveaux tels que la température s'élève rapidement au-dessus des températures permissives. Il en est de même pour l'évolution du pH qui est quasi impossible à réguler en l'absence de circulation de liquide.  The problem of evacuation of calories seems particularly difficult to solve. Indeed, the heat released by metabolic reactions during the vegetative growth phase can reach levels such that the temperature rises rapidly above permissive temperatures. It is the same for the evolution of the pH which is almost impossible to regulate in the absence of circulation of liquid.
[0078] A ces difficultés liées à la fermentation s'ajoute un autre inconvénient, aussi important, lié au fait qu'en fin de culture, on se trouve avec des conidies mélangées à de grandes quantités de mycélium et de résidus du support. Ceci réduit fortement la qualité du produit et les possibilités de son utilisation. To these difficulties related to the fermentation is added another disadvantage, as important, related to the fact that at the end of culture, we are with conidia mixed with large amounts of mycelium and residues of the support. This greatly reduces the quality of the product and the possibilities of its use.
[0079] Le procédé de l'invention combine une phase de croissance végétative, génératrice de chaleur et d'acides organiques en culture liquide, ce qui permet de contrôler tous les paramètres de croissance et de les maintenir à leurs valeurs optimales ; ensuite le mycélium produit massivement est ensuite mélangé au support solide adéquat pour subir une seconde phase de sporulation ou de maturation. Cette phase de maturation est uniquement une phase de différentiation cellulaire sans croissance qui ne nécessite que très peu d'énergie qui provient essentiellement des réserves accumulées dans la phase de culture liquide. Par conséquent, cette phase de maturation sans aucune fermentation ne nécessite pas de régulation de pH et le peu de chaleur produit peut être facilement contrôlé. The method of the invention combines a vegetative growth phase, which generates heat and organic acids in liquid culture, which makes it possible to control all the growth parameters and to maintain them at their optimum values; then the mass produced mycelium is then mixed with the solid support suitable to undergo a second phase of sporulation or maturation. This maturation phase is only a phase of cell differentiation without growth that requires very little energy that comes mainly from accumulated reserves in the liquid culture phase. Therefore, this maturation phase without any fermentation does not require pH regulation and the little heat produced can be easily controlled.
Anatomie et morphologie Anatomy and morphology
[0080] Comme étude préliminaire, les inventeurs ont réalisé une analyse en microscopie optique à contraste de phases et en microscopie électronique à balayage. Les figures suivantes (3 et 4) montrent des différences morphologiques et anatomiques observées.  As a preliminary study, the inventors carried out a phase contrast optical microscopy analysis and scanning electron microscopy. The following figures (3 and 4) show morphological and anatomical differences observed.
[0081] Au niveau anatomique, on constate des différences majeures entre les deux types de spores. Les spores issues de la culture liquide ont des cytoplasmes assez rugueux avec beaucoup d' organites et d'inclusions qui laissent penser à une forte activité métabolique. Ceci n'est pas le cas des spores obtenues par le procédé de l'invention dont les cytoplasmes sont dépourvus d' inclusions visibles et témoignent d'une absence d'activité métabolique.  At the anatomical level, there are major differences between the two types of spores. The spores from the liquid culture have rather rough cytoplasm with many organelles and inclusions that suggest a strong metabolic activity. This is not the case of the spores obtained by the process of the invention, the cytoplasm of which lacks visible inclusions and indicates a lack of metabolic activity.
[0082] Au niveau morphologique on voit clairement une différence importante dans la taille. Les spores issues de la culture submergée sont beaucoup plus allongées avec une moyenne de taille aux environs de 10 ym et avec des bords arrondis qui forment des hiles cicatrisés bombés. Les spores obtenues par le procédé de l'invention, par contre, sont plus petites d'environ 3 ym à environ 5 ym de long. Leurs bords se terminent avec un ou deux hiles apiculés qui ressemblent plus à une cicatrice conidienne.  At the morphological level we clearly see a significant difference in size. The spores from the submerged culture are much more elongated with an average size around 10 μm and with rounded edges that form curved hells hiles. Spores obtained by the process of the invention, on the other hand, are smaller by about 3 μm to about 5 μm in length. Their edges end with one or two apiculate hiles that look more like a conidial scar.
[0083] La présence de ces hiles témoigne que la conidiogénèse s'est déroulée dans des conditions optimales et représente une bonne indication sur le mode de conidiation de ce champignon. Les conidies obtenues en culture liquide ne présentent pas ces caractéristiques, ce qui témoigne d'une absence de maturation. The presence of these hiles testifies that the conidiogenesis was conducted under optimal conditions and is a good indication of the conidiation mode of this fungus. Conidia obtained in liquid culture do not have these characteristics, which indicates a lack of maturation.
Cytologie Cytology
[0084] L'une des différences les plus spectaculaires, qui distingue les conidies obtenues par le procédé de l'invention, concerne les fractions cytoplasmiques . En effet, on observe dans les figures 4 à 6, que le cytoplasme des conidies issues de la culture liquide montre des signes d'une forte activité métabolique liée à l'abondance d' organites cellulaires (mitochondrie et ribosomes) , celui des conidies obtenues par une fermentation sur support solide, semble avoir une activité ralentie comme en témoigne l'absence d' organites et la richesse en réserves sous forme d' inclusions dispersées dans toute la fraction cytoplasmique . L'abondance de ces réserves témoigne d'une excellente maturation et garantit une bonne longévité à la conservation et un bon taux de germination lorsque les conditions deviennent favorables. One of the most spectacular differences, which distinguishes the conidia obtained by the process of the invention, concerns the cytoplasmic fractions. Indeed, it is observed in FIGS. 4 to 6 that the cytoplasm of the conidia resulting from the liquid culture shows signs of a strong metabolic activity linked to the abundance of cellular organelles (mitochondria and ribosomes), that of the conidia obtained. by a solid support fermentation, seems to have a slowed down activity as evidenced by the absence of organelles and the richness of reserves in the form of inclusions dispersed throughout the cytoplasmic fraction. The abundance of these reserves indicates excellent ripening and guarantees a good longevity in the conservation and a good rate of germination when the conditions become favorable.
Fluorescence Fluorescence
[0085] Cette analyse montre également Dans la figure This analysis also shows In the figure
6, une différence majeure entre les cellules obtenues par le procédé de l'invention et selon le procédé de l'état de la technique. Alors que les cellules issues de la culture submergée ne fluorescent que très faiblement, celles produites par le procédé de l'invention sont très fluorescentes. Cette auto-fluorescence se localise autour de la spore, vraisemblablement au niveau de membrane et/ou de la paroi (têtes de flèches en E) . Elle se concentre particulièrement au niveau de la cicatrice conidienne (flèches en E) . 6, a major difference between the cells obtained by the process of the invention and according to the method of the state of the art. While the cells from the submerged culture fluoresce only very slightly, those produced by the process of the invention are highly fluorescent. This auto-fluorescence is located around the spore, probably at the membrane and / or the wall (E-shaped arrowheads). It is particularly concentrated at the level of the conidial scar (arrows in E).
[0086] Ce phénomène d'auto-fluorescence est le plus souvent observé au niveau des matrices extracellulaires à cause de la présence certaines molécules protectrices. Ici, il s'agit de la protéine mélanine qui est impliquée dans la résistance des conidies aux conditions de conservation. Exemple 4 : Production d' Azospirillum Brasilense This phenomenon of auto-fluorescence is most often observed at the level of the extracellular matrices at because of the presence some protective molecules. Here, it is the melanin protein that is involved in the resistance of conidia to the conditions of conservation. Example 4: Production of Azospirillum Brasilense
[0087] Un autre aspect de l'invention concerne un procédé d'obtention de bactéries séchées, de préférence de bactéries GRAM négatif (gram-) , en particulier du genre Azospirillum, tel qu' Azospirillum brasilense, qui est une des bactéries les plus étudiées, car elle favorise la croissance des plantes, mais qui présente une grande fragilité liée à sa caractéristique structurale de bactérie gram -. En effet, une bactérie gram - contrairement aux bactéries gram + ne comporte pas une épaisse paroi composée de peptidoglycanes et sa membrane phospolipidique externe ne résiste pas aux contraintes physiques de séchage. [0087] Another aspect of the invention relates to a process for obtaining dried bacteria, preferably GRAM negative bacteria (gram-), in particular of the genus Azospirillum, such as Azospirillum brasilense, which is one of the most studied, because it favors the growth of plants, but which presents a great fragility linked to its structural characteristic of gram - bacterium. Indeed, a Gram bacterium - unlike Gram + bacteria does not have a thick wall composed of peptidoglycans and its outer phospholipid membrane does not withstand the physical constraints of drying.
[0088] La bactérie Aeospirillum brasilense, vivant dans le sol, a la capacité d'influencer la croissance de nombreuses cultures agricoles par l'excrétion de différentes phytohormones (telles que l'acide indole acétique, dénommé également Auxine) et la capacité de fixer l'azote de l'air dans le sol. Cette bactérie peut être utilisée comme inoculant de croissance des plantes, éventuellement en combinaison avec d'autres bactéries favorisant la croissance des plantes. The bacterium Aeospirillum brasilense, living in the soil, has the ability to influence the growth of many agricultural crops by the excretion of different phytohormones (such as indole acetic acid, also called Auxin) and the ability to fix nitrogen from the air in the soil. This bacterium can be used as a plant growth inoculant, possibly in combination with other plant growth promoting bacteria.
[0089] De manière avantageuse, le procédé d'obtention de différents microorganismes, tels que des bactéries, des moisissures ou des levures, voire des virus, en particulier de bactéries gram -, de préférence des bactéries du genre Azospirillum, plus particulièrement des bactéries Azospirillum brasilense, selon l'invention comprend les étapes suivantes : croître les dits microorganismes pendant une phase de croissance en phase liquide, c'est-à-dire par fermentation, pour générer une biomasse de ces microorganismes , Advantageously, the process for obtaining different microorganisms, such as bacteria, molds or yeasts, or even viruses, in particular gram-like bacteria, preferably bacteria of the genus Azospirillum, more particularly bacteria. Azospirillum brasilense, according to the invention comprises the following steps: to grow said microorganisms during a growth phase in the liquid phase, that is to say by fermentation, to generate a biomass of these microorganisms,
mélangez la dite biomasse des microorganismes obtenue avec un support solide inerte, en particulier le support décrit ci-dessus (pour la maturation des conidies ou spores de souches de champignons filamenteux) , mixing the said biomass of the microorganisms obtained with an inert solid support, in particular the support described above (for the maturation of conidia or spores of strains of filamentous fungi),
générer ces microorganismes séchés durant une seconde phase de séchage, essentiellement sans fermentation de la dite biomasse sur ledit support solide inerte, ce support étant avantageusement apte à déshydrater partiellement cette biomasse, sans contrainte thermique et donc réduire ou stopper le métabolisme cellulaire afin d' obtenir avantageusement une augmentation du taux de matière sèche de cette biomasse, de préférence d'environ 3% à plus de 35%, de préférence supérieur à 40%, generate these dried microorganisms during a second drying phase, essentially without fermentation of said biomass on said inert solid support, this support being advantageously able to partially dehydrate this biomass, without thermal stress and thus reduce or stop the cellular metabolism in order to obtain advantageously an increase in the dry matter content of this biomass, preferably from about 3% to more than 35%, preferably greater than 40%,
éventuellement poursuivre cette déshydratation de ladite biomasse des microorganismes à une température ambiante, de préférence sur un lit fluidisé ou fixe présent à la surface de plateaux aérés et avantageusement conservés dans une enceinte ou un local à taux d'humidité contrôlé, pour obtenir à nouveau un taux de matière sèche de cette biomasse, de préférence à un taux supérieur à 85% de préférence d' environ 88~6, après moins de 3 semaines, de préférence entre environ 15 jours et 20 jours et optionally continuing this dehydration of said biomass of the microorganisms at an ambient temperature, preferably on a fluidized or fixed bed present on the surface of aerated trays and advantageously preserved in a chamber or room with a controlled humidity level, to obtain again a dry matter content of this biomass, preferably at a level greater than 85%, preferably about 88 ~ 6, after less than 3 weeks, preferably between about 15 days and 20 days and
éventuellement extraire une ou plusieurs molécules d'intérêt, en particulier des phytohormones , telle que de l'auxine (acide indole acétique) des microorganismes obtenus . [0090] Dans le procédé de l'invention, le support solide inerte est de préférence le son d'une céréale, en particulier le son de blé, c'est-à-dire un tissu dans lequel les cellules du son de la céréale, formant le support solide inerte sont dépourvues de leurs fractions fermentescibles en particulier d'amidon. Cependant, ce support solide inerte peut également être un support polymérique tel que décrit ci-dessus. optionally extracting one or more molecules of interest, in particular phytohormones, such as auxin (indole acetic acid) microorganisms obtained. In the process of the invention, the inert solid support is preferably the sound of a cereal, in particular wheat bran, that is to say a tissue in which the cells of the cereal bran forming the inert solid support are devoid of their fermentable fractions, in particular starch. However, this inert solid support may also be a polymeric support as described above.
[0091] Dans ce procédé d'obtention de la biomasse, la durée d'obtention des dits microorganismes séchés est avantageusement inférieure à 4 semaines ou inférieure 3 semaines, de préférence est une durée comprise entre environ 1 et 2 semaines, et permet la production de microorganismes particulièrement sensibles et fragiles.  In this process for obtaining the biomass, the duration of obtaining said dried microorganisms is advantageously less than 4 weeks or less than 3 weeks, preferably is a duration of between about 1 and 2 weeks, and allows the production microorganisms particularly sensitive and fragile.
[0092] L' invention concerne également les microorganismes, de préférence les bactéries, les moisissures ou les levures, en particulier les bactéries gram -, notamment du genre Azospirillum, de préférence Azospirillum brasilense, obtenus par le procédé de l'invention, résistantes à la dessiccation ainsi qu'à la conservation et aptes à être utilisées pour favoriser la croissance des cultures agricoles, de préférence par l'excrétion des phytohormones conservées, en particulier l'auxine, ainsi que pour leur capacité de fixation d'azote. Ces microorganismes, de préférence ces bactéries, levures ou moisissures, obtenus peuvent être sous forme d'une poudre desdits microorganismes, de préférence desdites bactéries, levures ou moisissures, obtenus par le procédé de l'invention et incluse à une composition phytosanitaire . Comme représenté à la figure 7, ces microorganismes en particulier ces bactéries peuvent être avantageusement conservées pendant des longues durées, même dans des conditions de conservation extrêmes. Références The invention also relates to microorganisms, preferably bacteria, molds or yeasts, in particular Gram - bacteria, especially of the genus Azospirillum, preferably Azospirillum brasilense, obtained by the process of the invention, which are resistant to desiccation as well as preservation and suitable for use in promoting the growth of agricultural crops, preferably by the excretion of preserved phytohormones, in particular auxin, as well as their ability to fix nitrogen. These microorganisms, preferably these bacteria, yeasts or molds, obtained can be in the form of a powder of said microorganisms, preferably said bacteria, yeasts or molds, obtained by the process of the invention and included in a phytosanitary composition. As shown in Figure 7, these microorganisms in particular these bacteria can be advantageously preserved for long periods, even under extreme conditions of conservation. References
Boekhout, T., 1995. Pseudozyma Bandoni emend. Boekhout, a genus for yeast-like anamorphs of Ustilaginales . TheBoekhout, T., 1995. Pseudozyma Bandoni emend. Boekhout, has genus for yeast-like anamorphs of Ustilaginales. Tea
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Claims

REVENDICATIONS
1. Procédé d'obtention de conidies de souches de champignons filamenteux comprenant les étapes suivantes : - croître des champignons filamenteux durant une première phase de croissance mycélienne en phase liquide, pour générer une biomasse mycélienne, 1. Process for obtaining conidia of strains of filamentous fungi comprising the following steps: - to grow filamentous fungi during a first phase of mycelial growth in the liquid phase, to generate a mycelial biomass,
mélanger ladite biomasse mycélienne obtenue avec un support solide inerte, et  mixing said obtained mycelial biomass with an inert solid support, and
- générer essentiellement sans fermentation des conidies et/ou des spores durant une seconde phase de maturation de ladite biomasse mycélienne sur ledit support solide inerte. generating, essentially without fermentation, conidia and / or spores during a second phase of maturation of said mycelial biomass on said inert solid support.
2. Le procédé selon la revendication 1, comprenant en outre une étape de séchage des conidies.  2. The method of claim 1, further comprising a step of drying the conidia.
3. Le procédé selon la revendication 1 ou 2, comprenant en outre une étape d'extraction d'une ou de plusieurs molécule (s) d'intérêt des conidies.  3. The method of claim 1 or 2, further comprising a step of extracting one or more molecule (s) of interest conidia.
4. Le procédé selon 1 xune quelconque des revendications précédentes, dans lequel le support solide inerte est du son d'une céréale. 4. The method of 1 x any one of the preceding claims, wherein the inert solid support is the sound of a cereal.
5. Le procédé selon la revendication 4 dans lequel la céréale est du blé.  5. The method of claim 4 wherein the cereal is wheat.
6. Le procédé selon la revendication 4 ou la revendication 5, dans lequel les cellules du son de la céréale formant le support solide inerte, sont dépourvues de leur fraction fermentescible, en particulier d'amidon.  6. The method of claim 4 or claim 5, wherein the sound cells of the cereal forming the inert solid support, are free of their fermentable fraction, in particular starch.
7. Le procédé selon l'une quelconque des revendications précédentes, dans lequel la phase de maturation génère une concentration en conidies comprise entre 3xE+08 conidies/g et 5xl09 conidies/g pour Pseudozyma flocculosa, et entre lE+09 conidies/g et 5xl010 conidies/g, pour Trichoderma atroviride et entre 5xE+8 conidies/g et lxE+10 conidies/g de conidies de Trichoderma harzianum . 7. The method according to any preceding claim, wherein the ripening phase generates a conidia concentration of between 3x E 08 conidia / g and 5xl0 9 conidia / g for Pseudozyma flocculosa, and between E 09 conidia / g and 5x10 10 conidia / g, for Trichoderma atroviride and between 5x E + 8 conidia / g and 1x E +10 conidia / g of Trichoderma harzianum conidia.
8. Le procédé selon l'une quelconque des revendications précédentes, dont la durée d'obtention des conidies est inférieure à 3 semaines, de préférence moins de deux semaines, plus particulièrement moins de 1 semaine. 8. The method according to any one of the preceding claims, the duration of conidia obtaining is less than 3 weeks, preferably less than two weeks, more particularly less than 1 week.
9. Le procédé selon l'une quelconque des revendications précédentes, dans lequel le champignon filamenteux est du genre Pseudozyma sp . , en particulier Pseudozyma flocculosa.  9. The method according to any one of the preceding claims, wherein the filamentous fungus is of the genus Pseudozyma sp. , in particular Pseudozyma flocculosa.
10. Le procédé selon l'une quelconque des revendications précédentes 1 à 8, dans lequel le champignon filamenteux est du genre Trichoderma sp . , en particulier Trichoderma harzianum, Trichoderma atroviride, Trichoderma hamatum et Trichoderma viride  The method of any one of the preceding claims 1 to 8, wherein the filamentous fungus is of the genus Trichoderma sp. , in particular Trichoderma harzianum, Trichoderma atroviride, Trichoderma hamatum and Trichoderma viride
11. Le procédé selon l'une quelconque des revendications précédentes 1 à 8, dans lequel le champignon filamenteux est du genre Pénicillium sp. , en particulier Penicilium camembert! et Penicilium nalgiovense .  11. The method according to any one of the preceding claims 1 to 8, wherein the filamentous fungus is of the genus Penicillium sp. , especially Penicilium camembert! and Penicilium nalgiovense.
12. Le procédé selon l'une quelconque des revendications précédentes 1 à 8, dans lequel le champignon filamenteux est choisi parmi le groupe constitué par Metarhizium sp . et Bauveria sp .  The process according to any one of the preceding claims 1 to 8, wherein the filamentous fungus is selected from the group consisting of Metarhizium sp. and Bauveria sp.
13. Conidies de souches de champignons filamenteux obtenues par le procédé selon l'une quelconque des revendications précédentes, résistantes à la dessiccation et se conservant à l'état sec à température ambiante, durant une période supérieure à un mois ou deux mois, de préférence durant une période comprise entre trois mois et vingt-quatre mois.  13. Conidia of strains of filamentous fungi obtained by the method according to any one of the preceding claims, resistant to drying and stored in a dry state at room temperature, for a period greater than one month or two months, preferably during a period of between three months and twenty-four months.
14. Conidies de souches de champignons filamenteux matures du genre Pseudozyma sp . , en particulier de Pseudozyma flocculosa, dont la fraction cytoplasmique est chargée de réserves, en particulier en mélanine et dont la cicatrice conidiennne est auto-fluorescente. 14. Conidia of mature filamentous fungus strains of the genus Pseudozyma sp. , in particular of Pseudozyma flocculosa, whose cytoplasmic fraction is charged with reserves, in particular with melanin and whose conidial scar is auto-fluorescent.
15. Conidies de souches de champignons filamenteux selon la revendication 14, présentant une taille comprise entre 3 microns et 15 microns, présentant de préférence un ou plusieurs hile(s) apiculé(s) et de préférence obtenues par le procédé selon l'une quelconque des revendications 1 à 12. 15. Conidia of strains of filamentous fungi according to claim 14, having a size between 3 microns and 15 microns, preferably having one or more hile (s) apiculé (s) and preferably obtained by the method according to any one Claims 1 to 12.
16. Poudre de conidies et/ou des spores selon l'une quelconque des revendications précédentes 13 à 15.  16. Conidia powder and / or spores according to any one of the preceding claims 13 to 15.
17. Composition alimentaire comprenant les conidies ou la poudre de conidies selon l'une quelconque des revendications précédentes 13 à 16.  17. A food composition comprising the conidia or the conidia powder according to any one of the preceding claims 13 to 16.
18. Composition alimentaire selon la revendication 17, caractérisée en ce qu'elle est choisie parmi le groupe constitué par les charcuteries et les fromages.  18. Food composition according to claim 17, characterized in that it is chosen from the group consisting of meats and cheeses.
19. Utilisation de conidies ou d'une poudre de conidies selon l'une quelconque des revendications précédentes 13 à 16, contre la prolifération de nuisibles de plantes ou de fruits.  19. Use of conidia or a conidia powder according to any one of the preceding claims 13 to 16, against the proliferation of pests of plants or fruits.
20. Utilisation de conidies ou d'une poudre de conidies selon l'une quelconque des revendications précédentes 13 à 16, pour favoriser la croissance de plantes.  20. Use of conidia or a conidia powder according to any one of the preceding claims 13 to 16, for promoting the growth of plants.
21. Utilisation selon la revendication 20, pour favoriser la croissance des plantes, en particulier la germination et le taux de croissance cellulaire et racinaire de plantes.  21. Use according to claim 20 for promoting plant growth, in particular germination and the rate of cell and root growth of plants.
22. Utilisation selon l'une quelconque des revendications précédentes 19 à 21, dans laquelle la plante ou le fruit est choisi parmi le groupe constitué par le rosier, les cucurbitacées , en particulier, le concombre, la courge ou le melon, la tomate, la fraise, la framboise, la groseille, la vigne, le raisin, la pomme, le pommier, la poire, le poirier, la prune, le prunier et la pomme-de-terre. 22. Use according to any one of the preceding claims 19 to 21, wherein the plant or the fruit is chosen from the group consisting of the rose bush, the cucurbits, in particular, the cucumber, the squash or the melon, the tomato, strawberry, raspberry, currant, grape, grape, apple, apple, pear, pear, plum, plum and potato.
23. Utilisation de conidies ou d'une poudre de conidies selon l'une quelconque des revendications précédentes 13 à 16 pour la préparation d'une composition alimentaire . 23. Use of conidia or a conidia powder according to any one of the preceding claims 13 to 16 for the preparation of a food composition.
24. Utilisation selon la revendication 23, dans laquelle la composition alimentaire est choisie parmi le groupe constitué par les charcuteries et les fromages.  24. Use according to claim 23, wherein the food composition is selected from the group consisting of sausages and cheeses.
25. Utilisation du son de céréales comme support solide inerte de maturation de conidies de souches de champignons filamenteux.  25. Use of cereal bran as an inert solid support for the maturation of conidia of filamentous fungi strains.
26. L'utilisation du son de céréales selon la revendication 25, dans laquelle des cellules du son de céréales, en particulier de son de blé, formant le support solide inerte, sont dépourvues de leur fraction fermentescible, en particulier d'amidon.  26. The use of cereal bran according to claim 25, wherein cereal bran cells, in particular wheat bran, forming the inert solid support, are devoid of their fermentable fraction, in particular starch.
27. L'utilisation selon les revendications 25 ou 26, dans laquelle le champignon filamenteux est choisi parmi le groupe constitué par les souches de Pseudozyma sp . , de Trichoderma sp . , de Penicilium sp . , de Metharizium sp . et de Beauveria sp .  27. The use according to claim 25 or 26, wherein the filamentous fungus is selected from the group consisting of strains of Pseudozyma sp. , of Trichoderma sp. of Penicilium sp. , of Metharizium sp. and Beauveria sp.
28. L'utilisation selon l'une quelconque des revendications 25 à 27, dans laquelle le champignon filamenteux est choisi parmi le groupe constitué par les souches de Pseudozyma flocculosa, de Trichoderma harzianum, de Trichoderma atroviride, de Trichoderma conidia , de trichoderma , de Trichoderma hamatum, de Trichoderma viride, de Penicilium camenberti et de Peniciluim nalgiovensie .  28. The use according to any one of claims 25 to 27, wherein the filamentous fungus is selected from the group consisting of strains of Pseudozyma flocculosa, Trichoderma harzianum, Trichoderma atroviride, Trichoderma conidia, Trichoderma, Trichoderma hamatum, Trichoderma viride, Penicilium camenberti and Peniciluim nalgiovensie.
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