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
  • C12N3/00—Spore forming or isolating processes
• • 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

Definitions

• the subject of the present invention is a new process for the production in solid medium of spores and metabolites originating from microorganisms of fungal origin and the use of the spores and metabolites thus obtained in fields such as agriculture, the environment, the environment and the environment. agronomy, agro-food, chemistry and / or pharmacy.
• the spores of microorganisms of fungal origin represent both the form of resistance and the form of reproduction of these microorganisms.
• Various techniques are known to date for the production of spores of microorganisms of fungal origin. The oldest, which is also the most traditional, is to cultivate the microorganism on the surface of an agar medium either in a Petri dish or in Roux vials. This technique causes problems of spore harvesting and handling of a large number of vials when the desired amount of spores is important.
• the production of spores is carried out in trays and uses the culture of the microorganism on plant substrates such as wheat bran, straw and various products or starchy residues arranged in a few centimeters thick layer and placed in incubator ovens.
• plant substrates such as wheat bran, straw and various products or starchy residues arranged in a few centimeters thick layer and placed in incubator ovens.
• Complex automatic devices for loading and unloading trays have been proposed.
• the product obtained consists not of pure spores, but of a mixture of spores, mycelium microorganism and plant residues.
• maintaining aseptic conditions is tricky. Spore harvesting, ambient contamination and body variability are major drawbacks.
• the processes for producing fungal microorganism spores in liquid or solid-state fermenters are performed under high humidity conditions.
• the spores being harvested at the end of these processes by centrifugation, it is necessary to eliminate large volumes of the fermentation juice.
• the moisture of the biomass obtained represents a risk of spore germination.
• dry air is used at a temperature of 40 ° or 50 ° C.
• mold spores are very sensitive to heat, and prolonged exposure to 40 ° C. causes significant spore mortality. .
• the object of the invention is to at least partially overcome these various disadvantages with a process for producing spores of microorganisms of fungal origin on a solid medium.
• Another object of the invention is to provide a fermentation method in solid medium (“FMS”) that does not contaminate the environment.
• FMS solid medium
• the subject of the present invention is a process for producing microorganism spores of fungal origin on a solid medium (solid state fermentation "FMS"), said solid medium comprising a solid absorbent, low density and practically non-fermentable, impregnated support.
• the process of the invention comprises, during the spore production phase, the application of a water stress triggered by aeration with dry air at a temperature ranging from 15 to 30.degree. 0 C, preferably from 18 ° C to 29 ° C, and more preferably still at a temperature of 25 ° C.
• the water stress is advantageously applied at a precise moment of the process that is to say just at the moment when the microorganism sets up its asexual reproduction system to cope with unfavorable external conditions (lack of water, lack of nutrients).
• the method of the present invention more particularly comprises the following steps:
• the support is ground into particles having a diameter preferably of 1 to 5 mm, and moistened with an aqueous nutrient solution containing, for example, inorganic salts.
• the sterilization of the support thus moistened is preferably carried out by a heat treatment.
• step (b) of impregnation of the support the amount of water used in the liquid culture medium is equivalent to about 3 to 5 times the weight of the support.
• the impregnation of the pre-conditioned support with the liquid culture medium is carried out in a mixer to ensure a satisfactory distribution and homogenization of the mixture to be fermented.
• the fermentation step (c) is carried out in an incubator, which will be, for example, a reaction column, a stirred or static incubator equipped with a gaseous effluent analysis device making it possible to monitor the growth of the microorganisms, their respiration, the morphological stages in particular spore germination at the beginning of the FMS and then the start of the spore production phase.
• an incubator which will be, for example, a reaction column, a stirred or static incubator equipped with a gaseous effluent analysis device making it possible to monitor the growth of the microorganisms, their respiration, the morphological stages in particular spore germination at the beginning of the FMS and then the start of the spore production phase.
• an incubator which will be, for example, a reaction column, a stirred or static incubator equipped with a gaseous effluent analysis device making it possible to monitor the growth of the microorganisms, their respiration, the morphological stages in particular spore germination at the beginning
• the choice of the solid support is not related to the nature of the carbon substrate.
• it may be chosen according to specific criteria as defined above (water retention capacity, porosity, resistance to sterilization, etc.).
• the porosity of about 60% of the support facilitates the circulation of dry air and thus evaporates water and effectively maintain water stress.
• the absorbent solid support used in the process of the invention is substantially non-fermentable, which means that after fermentation with the microorganism, the support substantially retains its mechanical properties. In particular, it retains much of its initial porosity.
• solid support of natural origin mention may be made of cereal straw, sawdust, beet pulp, olive pomace, coffee pulp, sugar cane bagasse and / or their mixtures.
• a solid support of synthetic origin there may be mentioned a polymer foam such as polyurethane foam.
• the solid medium culture method of the invention makes it possible in particular to diversify the nature of the usable carbon substrates, to increase the quantity of free water, to provide specific inducers and to maintain favorable conditions during the production stage. spores.
• the triggering of the water stress after the start of the spore production phase advantageously makes it possible to stimulate the production of said spores, to maintain their production and to obtain them in dry form without contamination by the culture medium.
• the carbon substrate included in the culture medium is advantageously chosen from the group comprising simple sugars, polysaccharides, proteins, amino acids, lipids, fatty acids, hydrocarbons, starchy natural substrates, wheat bran and / or mixtures thereof.
• Such a substrate is a soluble substance whose concentration can be modulated.
• the solid support is sugarcane bagasse and the carbonaceous substrate is wheat bran.
• the solid support is used in proportions ranging from 60 to 80% relative to the carbon substrate and the carbon substrate is used in proportions ranging from 40 to 20% relative to to solid support.
• yeast extracts examples include yeast extracts, vitamins, amino acids, trace elements, precursors of metabolites and / or mixtures thereof.
• the process of the invention is further characterized in that the spore suspension included in the culture medium comprises from about 2 to 4. 10 7 spores per gram of total dry matter of the fungal microorganism.
• microorganisms of fungal origin used according to the present process are filamentous fungi. These are in particular nematophages or entomopathogens. These fungi are used in biological control as agents for controlling the population of diseases and / or their vectors.
• filamentous fungi are advantageously chosen from the group comprising the genera Aspergillus, Penicillium, Rhizopus, Geotrichum, Paecilomyces, Metarbizium, Trichoderma, Giberella, Fusarium, Venticillium and Mucor.
• the genera Aspergillus, Penicillium, Rhizopus and Geotrichum will be used more particularly in the food industry, the fungi of the genus Geotrichum being used in biological fight against mold and protecting fruits and grains after harvest.
• the genera Paecilomyces and Metarbizium are used in the field of agronomy as biopesticides, respectively against nematodes and against migratory insects such as locusts.
• the genus Trichoderma is used in the field of environmental biotechnology, the recycling of cellulosic biomasses or the production of secondary metabolites.
• the genus Giberella is used in the field of pharmacy because it allows to produce a phytohormone: gibberellin.
• the method of the invention is characterized in that the aeration with dry air triggering the water stress during the spore production phase is preceded by aeration with air wet, said aeration with moist air being performed from the FMS to promote the growth of fungal microorganisms.
• the aeration with moist air allows more particularly, at the beginning of the FMS, the germination of spores of microorganisms, then finally the start of the spore production phase. Once the production phase has begun, the aeration with moist air can be substituted by aeration with dry air, and this under conditions to obtain the highest number of spores.
• the aeration with dry air is carried out with a flow rate ranging from 50 to 200 ml / min / column of dry air, and preferably from 50 to 100 ml. / min / column of dry air, corresponding to 50 to 20 renewals per hour of the useful volume of the FMS bioreactor.
• the originality of the process of the invention consists in using variable volumes of dry air which, on the one hand, make it possible to maintain the permanent water stress as a means of stimulating the spore production phase, while avoiding a rapid drying out of the spore. culture medium that would stop the growth of microorganisms, and secondly to maintain throughout the process the continuous and massive production of spores. Spore production yields of the order of about 10 10 spores per gram of carrier dry matter are thus obtained.
• the dry air flow allows at the end of the process to obtain a dry fermented product containing the biomass of the fungus mainly in the form of spores, in particular conidiospores.
• the fermented final product containing the conidiospores has less than 3% moisture. It can be stored for several months without loss of viability.
• the method is more particularly characterized in that: the microorganism of fungal origin is a filamentous fungus and is preferably Aspergillus Niger and / or,
• the solid carrier is sugar cane bagasse and / or
• the carbonaceous substrate is wheat bran and / or,
• the mixture solid support / carbon substrate
• namely (sugarcane bagasse / wheat bran) is used in proportions (70/30%).
• Aeration in moist air is carried out at a flow rate of 20 to 40 ml / min / column of moist air, and preferably at a flow rate of 30 ml / min / column of moist air. Under the conditions mentioned above, it will thus be possible to obtain large quantities of spores up to 1. 168 ⁇ 10 10 spores per gram of carrier dry matter after a total duration of FMS of just 6 days.
• the spores obtained according to the process of the invention are in large quantities and are not only dry, but are also viable and easily preserved for several months.
• the method of the invention has other advantages.
• the FMS process of the invention is more particularly carried out in a closed double-walled internal static bioreactor.
• aeration with dry air it promotes the evaporation of water.
• the water has been removed in the form of steam through aeration with dry air and low flow, thus avoiding turbulence and suspension of the spores produced in the air.
• the water stress is exerted with dry air and cold (15 to 30 0 C) which makes it possible to avoid the destruction of spores by the high temperature.
• the product obtained is dry, which avoids treating contaminated liquid effluents from the bioreactor.
• the product is obtained in its packaging ready to be stored long period of time.
• Another advantage of the process of the invention is that it promotes the production of primary and / or secondary, and preferably secondary metabolites.
• p is favored for secondary metabolites since the latter is directly sporulation of the filamentous fungi.
• the production of the secondary moles is directly proportional to the amount of spores produced the more spores produced under the effect of water stress, the more metabolites produced during the process of the invention. As a result, Sf promotes the production of metabolites in general.
• the prc further characterized in that it comprises, at the end of the FMS, a recovery of primary and / or secondary metabolites produced by the sp of enzymes (pectinase , cellulase), exoenzymes, acids and micotoxins, lactones, alkaloids, steroids, gibberell ergotamines, antibiotics (penicillin) and / or mixtures thereof.
• pectinase cellulase
• exoenzymes acids and micotoxins
• lactones lactones
• alkaloids alkaloids
• steroids gibberell ergotamines
• antibiotics penicillin
• the subject of the present invention is also the spores of microo ⁇ of fungal origin and / or their metabolites which can be obtained as defined above.
• Another subject of the present invention relates to the use of microorganisms of fungal origin and / or their metabolites obtained as defined above for application in agronomy and / or agri-food.
• filamentous spi mushrooms are used raw to develop bio-p against insects, nematodes, phytopathogenic fungi.
• spores of fungi as a natural means of control (biological control) against the invasion of the African continent as well as the European continent is an interesting alternative to the use of pesticides traditionally used in agriculture.
• fungi naturally parasite these animals at the same time and thus prevent their proliferation.
• Spores of filamentous fungi can also be used raw to develop weed herbicides.
• the invention therefore also relates to a method of biological control characterized in that an amount effective as a biopesticide and / or as a herbicide, spores of fungal microorganisms and / or their metabolites is used. .
• Another example in the field of agronomy lies in the use of said spores as starter-inoculum ferment to start processes for producing phytohormones (gibberellins) in FMS.
• the spores of certain beneficial molds can thus be used:
• the subject of the invention is also the use of spores of microorganisms of fungal origin and / or their metabolites obtained according to the process as defined above for an application in the field of chemistry.
• the field of biocatalysis where the spores of filamentous fungi can be used as such in unconventional media or can be microencapsulated for use in fine chemistry.
• Another subject of the invention is the use of spores of microorganisms of fungal origin and / or their metabolites obtained according to the process as defined above for an application in the field of pharmacy.
• the spores of filamentous fungi are more particularly used as "starters” for the production of secondary metabolites (mycotoxins, antibiotics) by the pharmaceutical industry.
• FIG. 1 represents the evolution of the humidity of a 70/30% bagasse / wheat bran substrate, at 30 ° C. for different air flows and in FIG. 2 giving the results.
• Dry air flow rates are therefore tested for respectively 5 columns (N ° 11, 12, 13, 14 and 15) of size identical, namely a column of 4 cm in diameter and 20 cm in height. Dry air flow rates are respectively 2, 4, 10, 20 and 40 times the air change of the potential volume for the five columns of the same size used (N 0 11, 12, 13, 14 and 15).
• FIG. 1 represents the kinetic curves of evolution of the moisture of the uninoculated carrier / substrate mixture (70% sugarcane bagasse / 30% wheat bran) at 30 ° C. for the different dry air flow rates (col # 11: 0.5 l / min / neck), (collar # 12: 1 l / min / neck), (collar 13: 2.5 l / min / neck); (Col No. 14: 5 l / min / col), (Col. 15: 10 l / min / col) for 44 hours.
• EXAMPLE 2 Effect of water stress exerted from 30 hours, 3 days and 5 days of FMS.
• the inventors of the present invention have been able to observe that the substitution of wet aeration by dry aeration from 30 hours, 3 days or 5 days of FMS is generally very favorable for the massive production of spores.
• Aspergillus Niger A. niger
• Aspergillus Niger incubated at 30 ° C. on a sugarcane / wheat bran bagasse (70/30%).
• the sporulation index (Is) represents the number of spores produced per gram of dry matter support.
• the humid air aeration carried out at the beginning of the fermentation process in solid medium is carried out at a flow rate of 30 ml / min / column.
• the total duration of the FMS is 10 days and 16 hours whereas from 30 hours the total duration of FMS is approximately 10 days (9 days and 22 hours).
• the sporulation index is increased by 14 times with a sporulation index of 11.68 ⁇ 10 9, unlike dry aeration at from 30 h, where the sporulation index is 7.58 x 10 8 (twice) after 6 days (5 days and 22 h) and 4 and a half days (4 days and 10 h) respectively.
• Dry aeration not only allows the large spore production in A niger but also shortens the total duration of FMS and avoids the phenomenon of the regermination of spores produced by reduction of water activity.
• the availability of water is a paramount parameter for spore germination in filamentous fungi, particularly Aspergillus niger.
• Table 1 Spore production before and after drying at different flow rates in dry air during the cultivation of A nigeren FMS at 30 ° C. for a useful volume of 100 ml.
• the spores of A niger are viable both before and after application of the drying from 3 days of FMS with wet forced aeration. Therefore, drying does not negatively affect the viability of A niger spores.
• Forced ventilation in moist air is generally highly recommended during the solid-state fermentation process as it provides oxygen and water for the growth of the filamentous fungus. However, it negatively influences sporulation and subsequently spore production. The phase of sporulation is generally induced by various factors including the lack of nutrients, the presence of secondary metabolites and also by the low activity of water.
• aeration in dry air has been adopted in order to lower the water activity or the water content (moisture) and to stimulate the sporulation of the fungus concerned.
• the application at a precise moment of the FMS of aeration in dry air makes it possible to significantly increase the sporulation index and avoids the germination of the spores.
• the timing of aeration in dry air is crucial. It allows to calculate the productivity of the process according to the two main variables: the time necessary to have a fermented dry product and a good sporulation index.
• the study showed that aeration at 100 ml / min / neck in dry air after 3 days of moist air FMS allows to have a final product after a total duration of 6 days of FMS with a sporulation index up to at 1,168 x 10 and a very high viability.

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