Method for manufacturing a microbial support, associated product and its use

Abstract

The present invention relates to a method for manufacturing a microbial support. The method according to the invention aims to produce supports on which microorganisms are immobilized, said supports are intended to be used in various biological applications and make it possible to maintain cell viability over time. Figure for the abstract: [FIG 1]

Description

Title of the invention: Method for manufacturing a microbial support, associated product and its use

[0001] The present invention relates to a method for manufacturing a microbial support. The invention also relates to a product resulting from this method, namely the support on which microorganisms are immobilized, as well as its use.

[0002] The method according to the invention aims to produce supports on which microorganisms are immobilized, said supports being intended to be used in various biological applications.

[0003] Such applications are based on the use of microorganisms, particularly in the fields of agriculture, oenology, water treatment, soil decontamination, surface treatment, or even veterinary medicine.

[0004] More generally, it is appropriate to distinguish the use of free microorganisms or in planktonic form as opposed to the use of microorganisms in non-free form, namely fixed or immobilized.

[0005] Indeed, it is known that non-free forms of microorganisms can be more resistant and more persistent over time for the same species. Sometimes, this aggregation is likely to pose a problem when it relates to pathogenic microorganisms, but it can however prove to be beneficial with non-pathogenic microorganisms that are described as beneficial or effectors.

[0006] This benefit then takes on its full meaning in the context of agriculture as well as in the development of new phytosanitary techniques or even in that of biocontrol in plants or animals in a key context which is that of ecology and respect for the environment.

[0007] There is a need to rethink sustainable agriculture and treatments using fewer pesticides and chemical fertilizers, by limiting soil inputs, but we must also reposition ourselves on biocontrol to limit the use of chemical pest control in plants or animals. In this respect, we know of microorganisms that have proven effects on parasites or that act as a real growth or protection agent in plants.

[0008] In the phytosanitary field, we find bioinoculant or biofertilizer type products in powder or liquid form intended for agricultural spreading. However, these products require repeated applications over time, which leads to increased costs and more frequent mobilization of resources. In addition, these uses are not entirely satisfactory because the viability of microorganisms poses a problem and constitutes a major obstacle before and after use.

[0009] Indeed, the population of microorganisms tends to fall drastically after use, several hypotheses can explain this.

[0010] Concerning liquid forms, there is a certain competition between the indigenous microbiota of the soil and the exogenous microorganisms, brought during the treatment. We can add to this the stress undergone by the latter, which must adapt to a new environment to survive under different constraints such as hygrometry, ultraviolet (UV) rays, pH or pollution, we then speak of the need for acclimatization.

[0011] Concerning the powder forms of bioinoculants, the microorganisms are mixed with mineral or inorganic powders, for example based on talc, clays or aluminosilicate. After being cultivated in a humid or liquid environment, the latter are often subjected to desiccation or lyophilization before mixing with mineral powders and then spreading. Here again, cell viability is contested, current techniques being too aggressive to be able to claim an economical and stable biological product over time.

[0012] The development of better formulations to ensure survival and activity in the field on the one hand, and compatibility with chemical and biological treatments on the other hand, is receiving increasing attention. Approaches include optimizing growth conditions prior to formulation and developing improved support and application technology. As such, maintaining the stability, efficacy and viability of effector strains is a real challenge that must be done in compliance with the standards associated with bioinoculants.

[0013] Techniques for immobilizing microorganisms on supports can be a response in various applications. Thus, it is understood within the meaning of the present application that the method according to the invention has no limiting character concerning its field of application.

[0014] The development of formulations or products must take into account the change of scale, the lifespan as well as the compatibility with current practices.

[0015] Among the known means using microorganisms on a support, there are known methods of encapsulation or inclusion of microorganisms with mineral powders containing nutrient medium for the development of microorganisms after spreading. But the stability over time remains less than 5 months, which is not satisfactory.

[0016] In agriculture, we also find means aimed at preserving microorganisms in the rhizosphere, in particular with the use of alginate in the form of micro-beads. The alginate matrix covering the seed must ensure the protection and release of microbes in the field, near the seed or the rhizosphere of the plant.

[0017] Encapsulated formulations provide physical protection against harsh environments as well as a three-dimensional structure to which cells can adhere, but these do not last over time. All of these methods and processes are intended for seed coating. Unfortunately, there are many disadvantages to this.

[0018] Indeed, when applying directly to the seed or to the seeds, the phytosanitary constraint imposes strict compliance with certain health standards regarding microorganisms. The handling of microorganisms on seeds concerning foodstuffs must be able to be done in the presence of an antifungal agent, the latter does not promote the development of all microorganisms, which can constitute a hindrance in certain applications.

[0019] It can also be added that working on seeds is not without risk for the development of the plant itself, germination problems are frequently encountered among coated or treated grains. There is thus a need for a means of preparing a product based on microorganisms, capable of responding to the disadvantages cited, respectful of the environment, which can respond to a problem of viability and stability over time of said microorganisms.

[0020] An aim of the invention is thus to provide a method for manufacturing a solid microbial support allowing better stability and viability of microorganisms over time. The invention also relates to the product obtained by this method and its use in improving the survival of microorganisms over time. The microbial support of the invention is defined by a solid support, which after the different steps of the method, will serve as a microbial vector.

[0021] By microbial vector is meant the solid support obtained from the manufacturing process according to the invention, which will allow the supply of a quantity of microorganisms in various biological applications, in particular agriculture.

[0022] The method for manufacturing the microbial support proposed by the applicant makes it possible to obtain a product which overcomes the previous obstacles, and whose applications are multiple. It is understood that the use of the product resulting from this method is not limited to the agricultural field.

[0023] The method for manufacturing a solid microbial support according to the invention comprises the following steps: a. Preparation of the solid support b. Immobilization of microorganisms on the support c. Drying

[0024] Immobilization is understood to mean an adhesion of microorganisms to the support, similar to a biofilm, and acting as a stable microbial coating. It is suitable within the meaning of this application to define immobilization as an adsorption or absorption of microorganisms on the support which allows a stable and effective fixation of the latter over time. By stable, within the meaning of this application, is meant the capacity of a microorganism to preserve or renew itself over time.

[0025] The solid supports according to the invention may be porous materials, capable of increasing in volume when brought into contact with a liquid and which have good tolerance to desiccation. The latter must be easily accessible, physically and chemically stable, non-toxic to use, non-toxic to microorganisms, free of pollutants, easy to process, and possibly have a high moisture retention capacity. In certain applications according to the invention, the support is chosen for its capacity to retain water. Advantageously, the solid supports of the invention have a water retention capacity greater than or equal to 0.8 times their weight.

[0026] The solid supports can be chosen from organic materials, fibrous materials, biodegradable polymers as well as materials of plant origin, and any other biosourced materials having a good specific surface area.

[0027] Good specific surface area means a ratio of the actual surface area of the material to the apparent volume of the object, sufficient to allow surface phenomena such as adsorption or absorption of microorganisms. Ideally, the support has good surface roughness which can be defined as the capacity of the material to be a support for microorganisms while being porous.

[0028] In one embodiment, the solid supports used in the method according to the invention are therefore preferably in the form of granules or chips whose particle size is between 1500 μm and 5000 μm, preferably between 2000 μm and 4500 μm and even more preferably between 2500 μm and 3800 μm.

[0029] In a variant, the supports may also be membrane-type materials, particularly in the context of supports immersed in a liquid, or even materials resulting from three-dimensional printing, said materials being able to be bio-sourced.

[0030] In another embodiment, the solid supports have a density of between 100 kg / m3 and 1200 kg / m3, or between 300 kg / m3 and 500 kg / m3, preferably between 360 kg / m3 and 460 kg / m3.

[0031] In a preferred embodiment, the solid support is a bio-sourced and biodegradable material.

[0032] In a most preferred embodiment, the solid support is corn cob.

[0033] Microorganisms are understood to mean organisms belonging to the category of bacteria, fungi, microalgae or even yeasts. The supports according to the invention can thus be used to immobilize these categories of microorganisms taken individually or in the form of a consortium. Thus, the method according to the invention can allow the immobilization of several species, or categories, on the same support.

[0034] The bacteria capable of being immobilized according to the invention are chosen from the genera Bacillus, Pseudomonas, Rhizobium, Azospirillum, Nitrosomonas, Nitrobacter, Oenococcus, Lactobacillus, Lactococcus, Leuconostoc, Arthrospira, Limnospira, Aphanizomenon or their mixture. The preferred bacteria according to the invention are chosen from Bacillus Thuringiensis, Oenococcus oeni, Pseudomonas fluorescens, Rhizobium leguminorosarum or their mixture.

[0035] The fungi capable of being immobilized according to the invention are chosen from the genera Aspergillus or Trichoderma, preferably Aspergillus bra-siliensis and Trichoderma harzianum or their mixture.

[0036] The microalgae capable of being immobilized according to the invention are chosen from the genera Chlorella, Duneliella, Odontella, Haematococcus, Scenedesmus, Porphyridium, preferentially Chlorella vulgaris and Haematococcus pluvialis or their mixture.

[0037] The yeasts capable of being immobilized according to the method of the invention are chosen from yeasts, such as the genus Saccharomyces, Kluyveromyces, Pichia, Rhodotorula, Yarrowia, Candida, preferentially Saccharomyces cerevisiae and Pichia pastoris or their mixture.

[0038] In addition to the microorganisms mentioned above, the method according to the invention does not exclude the use of probiotic strains known to those skilled in the art.

[0039] One embodiment of the invention relates to a method for manufacturing a microbial support on which bacteria, fungi, yeasts, microalgae or a mixture thereof can be immobilized. The support is a solid support, preferably corn cob.

[0040] In a preferred variant, the invention relates to such a method involving bacteria or fungi on corn cob.

[0041] The first step of the method according to the invention is that of preparing the support. Indeed, it is possible that spores of undesirable microorganisms are still present on the supports. However, this sporulating form is more resistant and therefore more difficult to eliminate. In order to guarantee the elimination of these spores, the applicant has included a germination step in this first step of preparing the supports. Germination is defined as the fact that a microorganism potentially present in the form of a spore on the initial support comes out of its sporulation state.

[0042] Said germination step consists, after rinsing the supports with water and a first sanitization, in placing the wet supports for 6 h to 24 h at a temperature between 20°C and 40°C in a suitable closed container, which the person skilled in the art will know how to choose and adapt. The purpose of this germination is to germinate any spores of undesirable microorganisms which will then be able to be eliminated during sanitization.

[0043] It is understood within the meaning of the invention that the means for sanitizing the supports are conventional means commonly used by those skilled in the art, such as the use of dry or wet thermal sterilization means, such as an autoclave or any other equivalent means known to those skilled in the art.

[0044] The preparation step of the method according to the invention comprises at least one sanitization step after germination. In a variant, there is a pre-germination sanitization step and a post-germination sanitization step, said germination being an intermediate step between two sanitization phases.

[0045] The second step of the method according to the invention is a step of immobilizing the microorganisms on a solid support. This immobilization step is divided into two successive and complementary phases, namely inoculation and incubation of the supports in a seeding solution. Incubation also allows the growth and fixation of the cells on the support.

[0046] Inoculation, within the meaning of the invention, is understood to mean the action which consists of bringing a previously prepared seeding solution into contact with the supports obtained during the first stage of the process, namely that of preparing the support.

[0047] By contacting is meant the immersion of the support in an adequate volume of a seeding solution.

[0048] In order to ensure optimal immobilization of the microorganisms, the applicant found that a preferential ratio of 2.5:10, i.e. 2.5 g of supports for 10 ml of seeding solution, allowed lasting immobilization of the microorganisms on said supports.

[0049] Said seeding solution consists of a mixture of a microorganism suspension and a depleted minimum medium. This seeding solution can be defined and understood as serving for immobilization according to the method of the invention.

[0050] The seeding solution can thus be constituted by a mixture of an impoverished minimum medium with a suspension of microorganism. In a variant, the seeding solution can also be constituted by a mixture of an impoverished minimum medium with a culture of microorganism.

[0051] By microorganism suspension is meant a mixture of microorganisms with an isotonic aqueous diluent known to those skilled in the art such as a tryptone-salt type diluent, or a mixture of microorganisms with a culture medium or any other equivalent diluent known to those skilled in the art.

[0052] A minimum medium depleted is understood to mean a minimum medium known to those skilled in the art, in which the applicant has reduced the total quantity of carbon and possibly the quantity of trace elements. This is a minimum medium depleted in nutrients.

[0053] Advantageously, the use of this medium makes it possible to cause beneficial stress among the microorganisms used in the present process; the latter will in fact tend to modify their behavior and their metabolism in order to survive.

[0054] These non-limiting variants allow the method according to the invention to be applied to a wide variety of microorganisms.

[0055] Since microorganisms are sensitive to environmental conditions, the applicant has observed that the pH tends to decrease with the addition of a bio-sourced support, in particular corn cob. To counteract the acidity associated with the use of such solid supports, the applicant therefore proposes inoculating said supports with a seeding solution containing carbonate or carbonated species in a concentration preferably between 4 and 15 mmol / L.

[0056] The carbonates and carbonate species usable in the invention can be chosen from carbonate ions (CO32), hydrogen carbonate ions (HCO3), carbonic acid (H2CO3) or their mixture.

[0057] Sodium hydrogen carbonate (NaHCO3) or calcium carbonate (CaCO3) are the preferred alkali carbonates within the meaning of the invention.

[0058] In a preferred embodiment, the immobilization of the microorganisms on the supports is carried out with a seeding solution consisting of a mixture containing a suspension of microorganisms, a minimum medium depleted in a respective volume equivalent ratio of 5:95 (v / v) as well as the addition of 0.067% by weight of this mixture of sodium carbonate (NaHCO3). It is understood within the meaning of the invention that this ratio is adjustable depending on the quantity and nature of the support.

[0059] In order to finalize the immobilization step, it is appropriate to incubate the mixture consisting of the supports and the seeding solution. This step will allow the adhesion of the microorganisms to the support by adsorption and / or absorption as well as their growth.

[0060] Incubation means maintaining the microorganisms in favorable growth conditions. The incubation conditions within the meaning of the invention comprise an incubation temperature of between 20°C and 37°C, an incubation time of between 8 h and 10 d with stirring or recirculation, if necessary.

[0061] The incubation times can be between 8 h and 240 h, between 8 h and 216 h, between 8 a.m. and 7:00 p.m., between 8 a.m. and 4:00 p.m., between 8 a.m. and 144 a.m., between 8 a.m. and 120 a.m., between 8 a.m. and 96 a.m., between 8 a.m. and 72 a.m., preferably between 8 a.m. and 48 a.m. and even more preferably between 24 a.m. and 48 a.m.

[0062] It is understood that these conditions apply to living microorganisms, and that it will be a question of modulating them according to the microorganism concerned.

[0063] For the purposes of the invention, it is defined in an undifferentiated manner that the immobilized supports are considered to be supports obtained at the end of the incubation.

[0064] The third step of the method according to the invention concerns drying. The immobilized supports must be dried in order to be packaged. This is a critical step which avoids freeze-drying and other processes which are detrimental to the survival of microorganisms. Indeed, freeze-drying and known processes pose problems with regard to the survival of microorganisms. In the context of freeze-drying, a significant drop in the percentage of survival of microorganisms is observed during drying and storage in freeze-dried form which is a means conventionally used to preserve them before use.

[0065] Advantageously, the method according to the invention makes it possible to maintain an average survival percentage at least twice that obtained with freeze-dried microorganisms after one year.

[0066] Drying can be carried out using a vacuum oven or any other equivalent and suitable drying means that a person skilled in the art will be able to use.

[0067] Thus, the applicant was able to develop drying conditions allowing the production of a solid support having a residual water content of between 7% and 25% by total weight of support, preferably between 15 and 23%. Surprisingly, the applicant found that residual moisture was necessary to ensure cell viability.

[0068] This residual water content ensures good maintenance of the adhesion of microorganisms to the support and their survival during storage periods.

[0069] It is understood within the meaning of the invention that the drying parameters are dependent on the equipment and strains used.

[0070] Good results were obtained when the drying conditions included a temperature between 0°C and 45°C, a drying time between 2 h and 12 h, and a pressure between 15 mbar and 100 mbar.

[0071] The applicant was able to demonstrate that the best results were obtained when the drying conditions cumulatively combined the following parameters: a temperature of 35°C, a drying time of 4 h and a pressure of 30 mbar. The average residual water content obtained after drying under these conditions is 21%.

[0072] The measurement of the residual water content is carried out after obtaining the supports according to the process, by drying said supports at 105°C until a stable mass is obtained.

[0073] Thanks to this process, the applicant was able to develop an effective means of preserving the cellular viability of microorganisms, particularly with regard to bacteria inoculated onto corn cobs.

[0074] The method according to the invention allows homogeneous and lasting immobilization which results in maintaining at least 50% of the survival of the microorganisms after storage for 12 months.

[0075] The method of manufacturing microbial support as well as said microbial support will be better understood through the examples and the figures.

[0076] Example 1: Carrying out the process according to the invention with the P.fluorescens strain on corn cobs

[0077] 1. Preparation of supports

[0078] The solid support used for this test is corn cob marketed under the name Eu-Grits 6 / 8 by Eurocob®.

[0079] A 500 ml glass bottle is filled to 1 / 3 of its volume with the stalk rinsed with water before autoclaving for 25 min at 121 °C. This bottle is then closed and incubated for 12 h at 30 °C, to germinate any unwanted spores potentially contained on the support.

[0080] 250 ml Erlenmeyer flasks are filled with 25 g of corn cob and autoclaved again. The Erlenmeyer flasks with supports are then left open under microbiological safety cabinet for 15 min.

[0081] Before proceeding with immobilization, a P.fluorescens seeding solution is then prepared by subculturing the strain on a Petri dish containing TSA agar medium and incubated for 24 h at 30°C. The colonies are then collected and suspended in 20 ml of Tryptone-Salt broth.

[0082] 2. Inoculation and incubation of supports

[0083] The supports are brought into contact with a mixture containing 95 ml of depleted minimum medium, 0.067% sodium carbonate (NaHCO3) and 5 ml of the previously prepared P.fluorescens bacterial suspension, this mixture constituting the seeding solution.

[0084] The whole is then incubated for 48 hours at 30°C with agitation of 150 rpm and an eccentricity of 5 cm.

[0085] 3. Drying

[0086] 1 g of inoculated support is taken from the Erlenmeyer flasks, then the supports are placed in a Petri dish with a diameter of 55 mm. These are left to dry in a vacuum oven for 4 hours at 35°C under a pressure of 30 mbar.

[0087] Example 2: Comparative study of the survival of microorganisms from lyophilization- lization and those resulting from the process according to the invention

[0088] In order to compare the effectiveness of the method of the invention in maintaining the survival of microorganisms over time, a strain of Pseudomonas fluorescens bacteria immobilized according to the method of the invention was compared with this same strain marketed in lyophilized form.

[0089] After drying, each dried 1 g sample is stored in a sterile polypropylene (PP) bottle placed in the dark. At each stability time, two 1 g samples are analyzed. These samples are then placed in 9 ml of tryptone salt and then subjected to a vortex for 1 min before being placed in an ultrasonic bath for 10 min. The suspension obtained is diluted in cascade and the dilutions are counted on the surface on TSA agar medium, then incubated at a temperature between 30 and 32 °C for 24 h.

[0090] After incubation, the colonies are counted to deduce the survival percentage. Finally, the average of the bacterial concentrations of the two samples analyzed is taken.

[0091] These results show that when the bacteria are freeze-dried, the survival rate drops drastically during the first 10 weeks, with a loss of more than 50% of bacterial survival. In contrast, when the bacteria are immobilized, the 50% loss of bacterial survival appears only after 30 weeks.

[0092] Over a full year, or approximately 52 weeks, these results are all the more convincing regarding the effectiveness of the applicant's process in maintaining the survival of bacteria.

[0093] Indeed, the average survival remains around 50% after one year for immobilization while it continues to decrease for freeze-drying to reach 10% after one year.

[0094] These results thus show that the applicant's process allows the survival of microorganisms over time to be maintained.

[0095] [Fig. 1] shows a comparison of the percentage of survival of the Pseudomonas fluorescens bacterium immobilized on the corn cob according to the manufacturing process of the microbial support of the invention and of this same strain in freeze-dried form.

[0096] The invention relates to a method for manufacturing a solid microbial support comprising the following steps:

[0097] - Preparation of a solid support, in which a germination step is carried out and which includes at least one sanitization phase;

[0098] - Immobilization of microorganisms by inoculation then incubation of the support at using a seeding solution comprising an impoverished minimum medium;

[0099] - Drying, wherein the temperature is between 0°C and 45°C, the duration of drying between 2 h and 12 h and in which the pressure is between 15 mbar and 100 mbar.

[0100] In a variant, during the support preparation step, germination is carried out at a temperature of 30°C for 12 h to 24 h, said germination being an intermediate step between two sanitization phases.

[0101] In the immobilization step, the seeding solution consists of a mixture of a depleted minimum medium and a microorganism suspension. In a variant of this step, the seeding solution consists of a mixture of a depleted minimum medium and a microorganism culture.

[0102] In one embodiment of the method, the supports are inoculated with the seeding solution in a volume proportional to the weight of supports equivalent to a respective ratio of 2.5:10 (w / v).

[0103] In a variant of this embodiment, the seeding solution of the inoculation step further comprises carbonates or carbonate species.

[0104] In another embodiment, the seeding solution is a mixture containing a suspension of microorganisms, a minimum medium depleted in a respective volume equivalent ratio of 5:95 (v / v) as well as 0.067% by total weight of said mixture (w / v) in alkali carbonate.

[0105] In an alternative embodiment, the incubation of the immobilization step takes place after the inoculation and is carried out at a temperature between 20°C and 37°C, over a period of between 8 h and 240 h.

[0106] In another variant of the process, the drying step is carried out at a temperature of 35°C, for a drying time of 4 h and at a pressure of 30 mbar.

[0107] According to one embodiment, the solid support is a biosourced and biodegradable material with an average particle size of between 1500 μm and 5000 μm.

[0108] In one variant, the solid support is corn cob.

[0109] The microorganisms capable of being used in the process of the invention are chosen from bacteria, fungi, yeasts, microalgae, probiotics or their mixture.

[0110] The invention also relates to the solid supports obtained during the process, and their use for improving the survival of microorganisms over time.

Claims

Claims

1. Method for manufacturing a solid microbial support comprising the following steps: - Preparation of a solid support, in which a step of germination of the spores of undesirable microorganisms is carried out comprising at least one sanitization phase; - Immobilization of the microorganisms by inoculation then incubation of the support using a seeding solution consisting of a mixture of an impoverished minimum medium and a suspension of microorganism, the inoculation consisting of immersing said support with said seeding solution; - Drying, in which the temperature is between 0 °C and 45 °C, the drying time is between 2 h and 12 h and in which the pressure is between 15 mbar and 100 mbar.

2. Method according to claim 1, wherein during the step of preparing the support, the germination is carried out at a temperature of 30°C for 12 h to 24 h, said germination being an intermediate step between two sanitization phases.

3. A method according to claim 1 or 2, wherein during the immobilization step, the seeding solution consists of a mixture of an impoverished minimum medium and a microorganism culture.

4. A method according to claim 1 or 3, wherein the supports are inoculated with the seeding solution with a volume proportional to the weight of supports equivalent to a respective ratio of 2.5:10 (w / v).

5. The method of claim 4, wherein the seed solution of the inoculation step further comprises carbonates or carbonate species.

6. A method according to claims 1 to 5, wherein the seeding solution is a mixture containing a suspension of microorganisms and a depleted minimum medium in a respective volume equivalent ratio of 5:95 (v / v) as well as 0.067% by total weight of said

7.

8.

9.

10.

11. mixture (w / v) in alkali carbonate. Method according to claims 1 to 6, in which the incubation of the immobilization step is carried out at a temperature between 20°C and 37°C, for a period between 8 h and 240 h. Method according to claims 1 to 7, wherein during the drying step the temperature is 35°C, the drying time is 4 h and the pressure is 30 mbar. Method according to one of the preceding claims, in which the solid support is a bio-sourced and biodegradable material with an average particle size of between 1500 pm and 5000 pm. The method of claim 9, wherein the solid support is corn cob. Method according to claims 1, 9 or 10, in which the microorganisms are chosen from bacteria, fungi, yeasts, microalgae, probiotics or their mixture.