Classifications

• • 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

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