Mixture of vegetative cells and asci of saccharomyces cerevisiae yeast, methods for preparing same and uses of said mixture

Abstract

The present invention relates to a dry mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast, characterized in that it has a solids content ranging from 95% to 99.9%, and in that said mixture comprises at least 60% asci, preferably at least 70%, and even more preferentially at least 90% asci. The invention also relates to a method for preparing such a dry mixture of vegetative cells and asci, which consists in subjecting to drying a suspension of vegetative cells and asci which comprises a solids content ranging from 1.9% to 2.1%, said drying being carried out using a fluidized bed or by freeze-drying. The invention further relates to the use of the dry mixture of vegetative cells and asci, in particular as a plant biostimulation agent.

Description

Description Title: Mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast, methods of preparation and uses of said mixture. Technical field

[0001] The present application relates to a mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast, the processes for preparing said mixture and the uses of said mixture. Previous technique

[0002] The yeast Saccharomyces cerevisiae is a microscopic, single-celled fungus belonging to the ascomycete family. It can reproduce both asexually, through budding (mitosis), and sexually, through sporulation (meiosis), and can switch between these two modes of reproduction depending on its environment. Thus, if the environment provides favorable conditions, Saccharomyces cerevisiae will bud (asexual reproduction, by mitosis), while if the environment presents unfavorable conditions, the yeast will sporulate (sexual reproduction, by meiosis). Asexual reproduction involves haploid cells that multiply by budding: the mother cell buds from a smaller daughter cell (mitosis), which possesses the same genetic information. One mother cell thus gives rise to 20 to 25 daughter cells.There are haploid cells "a" and haploid cells "a" that correspond to distinct sexual characteristics. The fusion of a haploid cell "a" and a haploid cell "a / a" gives rise to a diploid cell "a / a". As long as the environment is favorable, the diploid cell, which is the vegetative form of the yeast, multiplies by mitosis. If nutrients become scarce, the diploid cell will then multiply by meiosis (reproduction by sporulation) (diploid yeasts sporulate spontaneously in nutrient-deficient environments). Entry into meiosis leads to the formation of asci theoretically containing 4 ascospores. However, there are also asci containing 3 or 2 ascospores if one of the meioses or mitoses has not occurred. Figure 1 illustrates the processes of mitosis and meiosis. Meiosis involves two stages. The first stage of meiosis consists of a first division during which the diploid yeast cell, which contains 2n single-chromatid chromosomes (2n = 32), divides into two haploid daughter cells, each containing n chromosomes (n = 16) with two chromatids. The second stage of meiosis consists of a second division during which each haploid daughter cell divides again to produce two haploid daughter cells, each with n chromosomes (n = 16) but with one chromatid. Through this process, genetic information is preserved, and the diploid cell divides into four haploid daughter cells contained within an ascus: two "a" cells and two "a" cells, which can then restart the cycle. The four haploid daughter cells within an ascus are called ascospores. Ascospores are therefore sexual spores produced from an ascus as a result of meiosis. Each ascus corresponds to one meiosis. The asci can be isolated from the sporulation medium. The sporulation medium is the medium of culture in which the yeast was made to sporulate, in other words the medium in which meiosis took place. Figure 2 illustrates the transformation by meiosis of yeasts and shows that the sporulation culture obtained after meiosis of the yeast (figure on the right) mainly comprises asci containing 4 ascospores but also some vegetative cells, namely yeasts that have not sporulated. The asci can be isolated from the sporulation culture. Similarly, the spores or ascospores can be recovered after enzymatic digestion of the ascus wall. Sporulation is the differentiation process that leads from the vegetative form of yeast (in other words, yeast before sporulation) to the spore. Germination is the reverse transformation.

[0003] A mixture of vegetative cells and yeast asci offers a significant advantage over vegetative cells alone (i.e., yeasts) in its greater resistance to various stresses such as heat, oxidation, humidity, nutrient deficiency, and chemical or enzymatic stresses, because this mixture includes asci and therefore ascospores. This greater resistance of ascospores (compared to yeasts alone) is primarily due to the more elaborate / complex structure of their cell walls compared to those of vegetative cells (yeasts).

[0004] To the Inventors' knowledge, no mixture of vegetative cells and Saccharomyces cerevisiae yeast asci has ever been proposed to date. The term "mixture" in this application means: - a suspension of vegetative cells and asci comprising a dry matter content of 1.9 to 2.1% or, - a dry mixture of vegetative cells and asci, which means that it has a dry matter content of at least 95%. The dry mixture of the invention offers several advantages in addition to its composition, including increased shelf life, and improved ease of storage, handling, and transport. Drying, in particular, ensures good stability of the mixture of vegetative cells and asci, and therefore a good retention of initial viability. Stability is measured by the maintenance of viability over time (shelf life).

[0005] It is to the credit of the Inventors that they have thought not only of proposing a mixture of vegetative cells and yeast asci, and in particular a dry mixture of vegetative cells and asci (namely having a high dry matter content), but also of developing processes to prepare such a mixture, and in particular a dry mixture which retains its viability over time. Summary

[0006] According to a first aspect, the present invention relates to a dry mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast characterized in that it has a dry matter content ranging from 95 to 99.9% and in that said mixture comprises at least 60% asci, preferably at least 70%, and even more preferably at least 90% asci.

[0007] According to a second aspect, the invention relates to a process for preparing the dry mixture of vegetative cells and asci as defined above, characterized in that it consists of subjecting to drying a suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, said suspension comprising at least 60% asci, preferably at least 70%, and more preferably still at least 90% asci, said drying being carried out by fluidized bed or by freeze-drying.

[0008] According to another aspect, the invention also relates to the use of a dry mixture of vegetative cells and asci as defined above, as a plant biostimulation agent.

[0009] According to yet another aspect, the invention relates to a suspension of vegetative cells and asci having a dry matter content ranging from 1.9 to 2.1%. Brief description of the drawings

[0010] Other features, details, and advantages will become apparent upon reading the detailed description below and analyzing the attached drawings, on which: Fig. 1

[0011] [Fig. 1] illustrates the mitotic cycle and the meiotic cycle of the yeast Saccharomyces cerevisiae. Fig. 2

[0012] [Fig. 2] illustrates meiosis / sporulation of the yeast Saccharomyces cerevisiae. The figure on the right represents the sporulation culture after meiosis which mainly comprises asci with two ascospores "a" and two ascospores "a", but also some yeasts that have not sporulated (vegetative form of the yeast). Fig. 3

[0013] [Fig. 3] is a microscopic photograph of the sporulation culture of the yeast Saccharomyces cerevisiae CNCM I-3856 used in the examples of the invention. The figure on the left (3A) is a photograph of the culture medium obtained after sporulation of the yeast CNCM I-3856, which comprises a majority of asci and some vegetative cells, namely the yeast cells that have not sporulated. The figure on the right (3B) is an enlarged view of a portion of Figure 3A showing asci containing four ascospores (represented by the letter A) and the yeast cells that have not sporulated (represented by the letter V). Fig. 4

[0014] [Fig. 4] represents the photos of the dry samples of vegetative cells and asci, as obtained directly after their drying, said drying having been carried out by freeze-drying (figures 4A to 4C) or by fluidized bed (figure 4D). Photos 4A to 4C represent respectively the samples after drying without the addition of maltodextrin (control) (4A), with 12% maltodextrin (4B) and 25% maltodextrin (4C). Fig. 5

[0015] [Fig. 5] is a histogram illustrating the average quantity of live cells in dry samples of vegetative cells and asci obtained after freeze-drying (5A-5C) and fluidized bed (5D). Histograms 5A to 5C are respectively those of the samples after drying without the addition of maltodextrin (control) (5A), with 12% maltodextrin (5B) and 25% maltodextrin (5C). Detailed description

[0016] The ascuses of invention

[0017] The present invention relates to a dry mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast characterized in that it has a dry matter content of 95 to 99.9% and in that said mixture comprises at least 60% asci, preferably at least 70%, and more preferably still at least 90% asci. Yeast vegetative cells refer to vegetative yeast, in other words, yeast. As previously stated, yeast asci result from meiosis / sporulation in yeasts. The yeasts covered by the invention are all Saccharomyces cerevisiae yeasts that sporulate, producing an ascus containing 4 ascospores, or possibly 3 or 2 ascospores. The dry mixture of the invention refers to a mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast which has a dry matter content ranging from 95 to 99.9%. A dry matter content of 95 to 99.9% means that the mixture of vegetative cells and asci comprises 95 to 99.9% dry matter by mass relative to the total mass of the mixture. The dry matter contents given in this application for all vegetative cell and asci samples were determined using a halogen desiccator, the principle of which is as follows. The halogen desiccator records the initial weight of the sample and then gradually dries it using a halogen lamp emitting infrared radiation. The sample weight is recorded regularly until the end of the drying procedure. To complete the measurement, the difference between the initial sample weight and the weight of the dehydrated sample corresponds to its water loss. The proportion of this weight relative to the initial weight corresponds to the moisture content of the sample, which also allows its dry matter content to be deduced. For the purpose of indicating the halogen dryer used in the context of the present invention is that marketed by METTLER Toledo under the name HX204.

[0018] According to an advantageous embodiment, the mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast has a dry matter content of 95 to 98%, and preferably 95 to 96.5%. In this particular case, the mixture of vegetative cells and yeast asci is preferably found in the form of vermicelli-like granules ranging from 0.25 to 1 cm in length and from 0.25 to 0.6 cm in thickness. The measurements were taken using a binocular microscope.

[0019] According to another advantageous embodiment, the mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast has a dry matter content ranging from 98 to 99.9%. In this particular case the mixture of vegetative cells and asci is preferably in the form of a powder, said powder preferably having a particle size ranging from 200 to 315 pm.

[0020] According to yet another advantageous embodiment, the mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast is characterized in that it is viable / stable for a period of 12 to 36 months when stored under vacuum at a temperature of 4°C.

[0021] The stability / viability of the mixture of the invention for a period of 12 to 36 months under vacuum and at 4°C means that the vegetative cells and asci of the mixture exhibit a viability ranging from 7 log CFU / g to 9 log CFU / g, measured according to the standardized method EN 15789. One way to measure the viability of the dry mixture of vegetative cells and asci is to count the number of viable cells (in CFU / g or log CFU / g), that is, cells that are viable and capable of multiplying. The term CFU stands for "colony-forming unit." One CFU corresponds to one colony. Cell viability is measured according to the standardized method EN 15789. For the purposes of this application, stating that a dry mixture of vegetative cells and asci is viable for a period of 12 to 36 months means that the number of live and metabolically active cells in that mixture (CFU / g) does not decrease by more than two log CFU / g over a period of at least 12 months after drying, vacuum sealing, and storage at 4°C. This means, for example, that if the mixture of yeast vegetative cells and asci has a live and metabolically active cell count of 1 x 10 9 CFU / g (equivalent to 9 log CFU / g) at time t0 (t0 being the time when the dry mixture is obtained), then the number of live and metabolically active cells in the dry mixture after a period of at least 12 months, and preferably from 12 to 36 months, is not less than 1x10 7CFU / g (equivalent to 7 log CFU / g). The viability rate of the dry mixture of vegetative cells and asci of the invention is directly related to the stability of this mixture. Thus, if the mixture of vegetative cells and asci is viable after drying, vacuum packaging, and storage at a temperature of 4°C for a period of 12 to 36 months, this means that this mixture is stable after drying, vacuum packaging, and storage at a temperature of 4°C for a period of 12 to 36 months.

[0022] Process for preparing the dry mixture of vegetative cells and asci

[0023] The present invention further relates to a method for preparing the dry mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast as defined above, characterized in that it consists of subjecting to drying a suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, said suspension comprising at least 60% asci, preferably at least 70%, and more preferably still at least 90% asci, said drying being carried out by fluidized bed or by freeze-drying. A dry matter content of 1.9 to 2.1% means that the suspension of vegetative cells and asci comprises 1.9 to 2.1% by mass of dry matter of vegetative cells and asci. relative to the total mass of the suspension. According to an advantageous embodiment, the suspension of ascus having a dry matter content of 1.9 to 2.1% may include an emulsifier.

[0024] Drying is a preservation process that removes all the water from a product to stabilize it and allow it to retain its original properties for an extended period. This process is particularly useful for preserving microorganisms, including bacteria and fungi, which must maintain their viability and metabolic activity over time. Drying keeps microorganisms in a dry state, facilitating their transport and storage. The first step of the drying process of the invention, whether by fluidized bed or by freeze-drying, consists of preparing the starting product, namely the suspension of vegetative cells and asci which has a dry matter content ranging from 1.9 to 2.1%.

[0025] Preparation of the vegetative cell and asci suspension with a dry matter content ranging from 1.9 to 2.1%

[0026] According to an advantageous embodiment of the invention, the suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, used at the start of the drying process of the invention, is prepared according to the process comprising the following steps: - incubation of a fresh culture of Saccharomyces cerevisiae yeast in a sporulation culture medium for 5 days at a temperature of 30°C, in order to induce the sporulation phase of the yeast and to obtain a sporulation culture comprising at least 60% asci, preferably at least 70%, and more preferably still at least 90% asci, - separation of vegetative cells and asci from the sporulation culture, said separation being carried out by centrifugation followed by re-suspension of the vegetative cells and asci in physiological saline, - recovery of a suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, said suspension comprising at least 60% asci, preferably at least 70%, and even more preferably at least 90% asci. The sporulation culture obtained after the sporulation phase of the yeast Saccharomyces cerevisiae consists mainly of asci (at least 60%) but also of vegetative cells, namely yeasts that have not sporulated. According to an advantageous embodiment of the invention, the sporulation culture comprises at least 90% asci.

[0027] The physiological water of the vegetative cell and asci suspension includes 0.9% sodium chloride, which helps to maintain a minimum osmolarity necessary for good cell viability.

[0028] A "sporulation culture containing at least 60% asci" means that the sporulation rate of the culture is 60%. The sporulation rate corresponds to the number of asci formed relative to the total number of cells, namely the yeast cells that have not sporulated as well as the asci that have formed.

[0029] The dry matter content of the vegetative cell and asci suspension is evaluated directly on the suspension, since it is homogeneous.

[0030] According to an advantageous embodiment of the process of the invention, the suspension of vegetative cells and asci obtained, which has a dry matter content ranging from 1.9 to 2.1%, is kept at a temperature of 4°C, under atmospheric pressure, for several weeks.

[0031] Suspension of vegetative cells and asci with a dry matter content ranging from 1.9 to 2.1%

[0032] The present invention also relates to the suspension of vegetative cells and asci having a dry matter content ranging from 1.9 to 2.1%.

[0033] Drying of the vegetative cell and asci suspension with a dry matter content ranging from 1.9 to 2.1%

[0034] One of the problems encountered in the context of the invention was that of developing a drying process for a mixture of vegetative cells and asci, namely more particularly a drying process for a suspension of vegetative cells and asci as defined above, which allows the viability of said mixture to be maintained, not only directly after drying, but also over time.

[0035] Another problem encountered in the context of the invention was the low dry matter content of the vegetative cell and asci suspension used at the start of the drying process of the invention, namely 1.9 to 2.1% dry matter. Indeed, the entire challenge was to lose as little dry matter as possible during the various stages of drying.

[0036] It is therefore to the credit of the Inventors that they have successfully addressed both the problem of preserving the viability of the mixture of vegetative cells and asci, and the low dry matter content of the suspension used at the start of the process of the invention. After extensive research, the Inventors determined the optimal characteristics to be developed in a fluidized bed drying process and a freeze-drying process, respectively, enabling the production of dry vegetative cells and asci whose viability is maintained for several months from a suspension of vegetative cells and asci containing 1.9 to 2.1% dry matter.

[0037] Fluidized bed drying

[0038] Fluidized bed drying, also known as fluidized air bed drying, is a highly efficient method for drying solid particles. The surface of each particle is exposed for drying by suspending it in the airflow, which improves heat transfer and reduces drying time. A consistent temperature and uniform drying of the particles are achieved through continuous control of the inlet and outlet of the drying air. Precise monitoring of the drying air humidity is essential to optimize the drying process. Conditions can vary depending on the humidity and temperature of the inlet air.

[0039] According to an advantageous embodiment of the invention, the process for preparing the dry mixture of vegetative cells and asci as defined above is more particularly characterized in that the fluidized bed drying comprises the following steps: - frontal filtration of the suspension of vegetative cells and asci with a dry matter content of 1.9 to 2.1%, using a filter plate with a pore diameter of 0.6 to 1 pm, in order to obtain a semi-solid form of vegetative cells and asci, also called "vegetative cell and asci cake", with a dry matter content of 29 to 32%; - mixing the semi-solid form of vegetative cells and asci obtained in the previous step with an oil-in-water (O / W) emulsion consisting of water, oil and emulsifier, the quantity of emulsifier ranging from 10 to 14% by weight relative to the total weight of the emulsion, until a homogeneous mixture is obtained which is in a semi-solid form, called "semi-solid mixture of vegetative cells and asci", said semi-solid mixture of vegetative cells and asci comprising 0.7 to 0.9% of emulsifier per gram of dry matter of the semi-solid mixture of vegetative cells and asci; - extrusion of the semi-solid mixture of vegetative cells and asci obtained in the previous step using an extrusion device having an extrusion grid with openings of a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm, in order to obtain an extruded mixture of vegetative cells and asci in the form of filaments called spaghetti; - mechanical / physical fractionation of the filaments of vegetative cells and asci obtained in the previous step in order to obtain vermicelli of vegetative cells and asci called granules; - drying of the vegetative cell granules and asci obtained in the previous step in a fluidized bed dryer where the inlet air temperature is between 45 and 50°C and the relative humidity (RH) is between 13 and 15%, the outlet air temperature is between 36 and 42°C and the relative humidity (RH) is between 10 and 20%, and the fluidization flow rate is 14 to 20 m 3 / h; - recovery of the dry mixture of vegetative cells and asci, said dry mixture having a dry matter content of 95 to 98%, preferably 95 to 96.5%. The yeast vegetative cells and asci obtained after fluidized bed drying are in the form of granules or vermicelli with a length ranging from 0.25 to 1 cm and a thickness ranging from 0.25 to 0.6 cm.

[0040] Frontal filtration is a filtration technique that uses a pressure gradient and involves passing the fluid to be filtered perpendicularly across the filter surface. The particles to be removed or recovered are retained by the filter. The first front filtration stage of the process is preferably carried out using a filter plate with a pore diameter of 0.8 pm, for a duration of 1 to 5 minutes and at a pressure of 1 to 5.5 bars. The front filtration step removes 25 to 35% of the moisture from the vegetative cell and asci suspension, resulting in a semi-solid form of vegetative cells and asci that resembles a pasty cake with a dry matter content ranging from 29 to 32%.

[0041] The resulting paste-like cake of vegetative cells and asci is mixed, preferably with a spatula, with an oil-in-water (O / W) emulsion until a homogeneous mixture is obtained. This mixture has an appearance virtually identical to that of the paste-like cake of vegetative cells and asci obtained after the filtration step. The O / W emulsion protects the material during the extrusion step and also facilitates drying. For the purposes of the invention, an H / W emulsion means a composition consisting of water, oil and an emulsifier, with water being the major component of the composition. According to an advantageous embodiment of the invention, the W / E emulsion used in mixture with the semi-solid form of vegetative cells and asci comprises at least 80% water and at least 10% emulsifier, which means that it comprises at most 10% oil. The emulsifier has a hydrophilic part and a hydrophone part since its function is to help mix the oil and water in the emulsion. As an example of an O / W emulsion, one could cite an emulsion which comprises 81.5% water, preferably demineralized water, 8% sunflower oil and 12.5% ​​sorbitan monostearate as an emulsifier.

[0042] The extrusion of the semi-solid mixture of vegetative cells and asci is preferably carried out using a piston extrusion device with a length of 90 mm and a diameter of 20 mm, which has at its end an extrusion grid with openings with a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm.

[0043] Upon exiting the extrusion grid, the extruded mixture of vegetative cells and asci is in the form of spaghetti-like filaments which will be broken down, physically / mechanically, into granules, for example by simply shaking the container containing the spaghetti of vegetative cells and asci.

[0044] The vegetative cell and asci granules are then placed in the fluidized bed dryer chamber. The inlet air is maintained at, for example, 48°C and 13% relative humidity, while the outlet air is 30°C and 12% relative humidity. As a guideline, the humidity of the drying air is regulated by an air treatment system (between 1 and 4 g of water per kg of dry air). The drying time is preferably 17 to 20 minutes, and could, for example, be 20 minutes. During the drying process, the still-moist granules will gradually decrease in length.

[0045] The dry mixture of vegetative cells and asci obtained at the end of the fluidized bed drying process has a dry matter content ranging from 95 to 98%, and preferably from 95 to 96.5%. The variation in dry matter content depends on the different parameter adjustments of the drying process described above. A dry matter content of 95 to 96.5% is sufficient and quite satisfactory for the mixture of vegetative cells and asci. The final dry matter content of the vegetative cell and asci granules is evaluated using a halogen dryer.

[0046] According to a particularly advantageous embodiment of the invention, the fluidized bed drying process comprises one or all of the following features: - Frontal filtration of the vegetative cell and asci suspension with a dry matter content of 1.9 to 2.1% is carried out using a filter plate with a pore diameter of 0.8 pm, for a duration of 1 to 5 minutes and at a pressure of 1 to 5.5 bars, in order to obtain a semi-solid form of vegetative cells and asci (vegetative cell and asci cake) with a dry matter content of 29 to 32%; - the semi-solid form of vegetative cells and asci obtained at the end of the filtration step is mixed with an oil-in-water (O / W) emulsion consisting of 81.5% water, preferably demineralized water, 8% sunflower oil and 12.5% ​​of the emulsifier sorbitan monostearate (MSS), until a homogeneous mixture is obtained which is in a semi-solid form, the semi-solid mixture of asci thus obtained having 0.9% MSS per gram of dry matter of the semi-solid mixture of vegetative cells and asci; - the semi-solid mixture of vegetative cells and asci obtained at the end of the step of mixing the cake of vegetative cells and asci with the H / W emulsion is extruded using a piston extrusion device, preferably 90 mm long and 20 mm in diameter, said extrusion device having at its end an extrusion grid with openings of a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm, which leads to an extruded mixture of vegetative cells and asci having the form of filaments called spaghetti; - the filaments / spaghetti of vegetative cells and asci are mechanically broken down into vermicelli called granules; - The vegetative cell and asci granules are dried in a fluidized bed dryer where the inlet air has a temperature of 48°C and a relative humidity (RH) of 13%, the outlet air has a temperature of 42°C and an RH of 12%, and the fluidization flow rate of the bed is 16 m 3 / h, said drying being carried out for a period of 17 to 20 minutes, preferably 20 minutes; - the vegetative cell and asci granules obtained after fluidized bed drying have a dry matter content ranging from 95 to 96.5%.

[0047] Freeze-drying

[0048] Freeze-drying stands out from other drying technologies due to its ability to preserve the structure and properties of delicate materials such as pharmaceuticals and certain foods. Freeze-drying involves freezing the product (in liquid, paste, or solid form) and then removing the ice through sublimation under vacuum. During sublimation, the water present in the frozen product passes directly from a solid state (i.e., ice) to a gaseous state. This technique preserves the volume, appearance, and properties of the treated product. It can be carried out in a freeze dryer.

[0049] According to another embodiment of the invention, the process for preparing the ascus is more particularly characterized in that the freeze-drying comprises the following steps: - addition, in the suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, of maltodextrin in an amount of 5 to 25% by weight of maltodextrin relative to the dry matter of the suspension of vegetative cells and asci, and preferably in an amount by weight of 25% of maltodextrin; - freezing the suspension of vegetative cells and asci as defined in the previous step at a temperature ranging from -60 to -80°C for a period of 1 to 4 hours; - sublimation of the suspension of vegetative cells and asci frozen in the previous step by applying a vacuum pressure of 0.05 to 0.5 mbar in order to remove the frozen water, said sublimation step being carried out for a period of 60 to 61 hours and leading to a solid form of vegetative cells and asci, preferably a powdered form; - recovery of the solid form of vegetative cells and asci having a dry matter content of 98 to 99.9%.

[0050] According to an advantageous embodiment of the process of the invention, the solid form of vegetative cells and asci obtained directly after freeze-drying is a powdery solid form. However, this powdery solid form obtained directly after freeze-drying can be further fractionated by a mechanical or physical action, such as grinding with a suitable instrument or simply shaking the container of the freeze-dried solid form. Indeed, fractionating the powdery solid form allows for refining the particle size of the powder obtained directly after freeze-drying. Thus, after fractionation, an asci powder with a particle size ranging from 200 to 315 µm is obtained. Grinding the solid forms of vegetative cells and asci obtained directly after freeze-drying does not affect the dry matter content. Therefore, the dry matter content is the same for solid forms of vegetative cells and asci obtained directly after freeze-drying or after freeze-drying followed by grinding.

[0051] The maltodextrin added to the suspension of vegetative cells and asci of the invention is a cryoprotective agent, which, as its name suggests, protects the asci from drying by freeze-drying.

[0052] The aforementioned freezing step of the freeze-drying process is carried out in a blast freezer at a temperature of -80°C without kinetic monitoring. The suspension of vegetative cells and asci thus frozen can be maintained in a frozen state for up to 4 hours.

[0053] According to an advantageous embodiment, the sublimation step of the frozen vegetative cell and asci suspension is broken down into: - a primary drying stage lasting from 40 to 42 hours during which the vacuum pressure is 0.1 to 0.5 mbar, and - a secondary drying stage lasting from 18 to 20 hours during which the vacuum pressure is 0.05 to 0.2 mbar, which leads to a solid form of vegetative cells and asci, preferably a powdery form.

[0054] According to yet another advantageous embodiment, the freeze-drying process is further characterized in that, at the end of the secondary drying step, the solid form of vegetative cells and asci obtained, preferably in powder form, is further ground or mechanically shaken, which leads to a dry powder of vegetative cells and asci with a particle size ranging from 200 to 315 pm.

[0055] The drying processes developed by the Inventors, whether fluidized bed drying or freeze-drying, are easy to implement on an industrial scale. They advantageously allow for the production of a dry mixture of vegetative cells and Saccharomyces cerevisiae yeast asci with extremely favorable long-term stability and viability. The dry mixture of vegetative cells and yeast asci, in granule or powder form, is easy to store, handle or transport.

[0056] Uses of the mixture of vegetative cells and asci of the invention

[0057] Use of the dry mixture of vegetative cells and asci with a dry matter content ranging from 95 to 99.9%

[0058] The invention also relates to the use of the dry mixture of vegetative cells and asci as defined above or as obtained according to the drying processes as defined above, as a plant biostimulation agent. According to the terms of the invention, plant biostimulation consists of improving the functioning of the plant by stimulating its metabolism or by strengthening its resistance to abiotic stresses.

[0059] Use of the vegetative cell and asci suspension with a dry matter content ranging from 1.9 to 2.1%

[0060] The invention also relates to the use of the suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, for use as an anti-inflammatory agent. Examples

[0061] The following examples refer in particular to Figures 3 to 5.

[0062] Example 1: Preparation of asci by sporulation in liquid medium of a Saccharomyces cerevisiae culture

[0063] Saccharomyces cerevisiae yeast (vegetative cells) spontaneously sporulates in nutrient-deficient media. Entry into meiosis leads to the formation of asci, each theoretically containing four ascospores. However, as previously mentioned, it is also possible to obtain some asci containing only three or two ascospores. Furthermore, the culture medium after meiosis also contains vegetative yeast cells (i.e., yeast cells that have not sporulated).

[0064] The Saccharomyces cerevisiae yeast induced to sporulate is the Saccharomyces cerevisiae strain deposited on October 17, 2007 with the CNCM under number I-3856.

[0065] Reagents used

[0066] The culture, pre-sporulation and sporulation media used in the sporulation phase induction protocol are as defined below. YPD solid culture medium: - Yeast extract (Becton Dickinson and Company, BD 212750) 5 g / L, - Glucose (Sigma Aldrich, CAS 50-99-7) 20 g / L, - Agar (Becton Dickinson and Company, BD 214530) 30 g / L, - Demineralized water qsp 1 L. The YPD solid culture medium was autoclaved for 20 minutes at a temperature of 120°C. YPD liquid culture medium: - Yeast extract (Becton Dickinson and Company, ref. BD 212750) 10 g / L, - Glucose (Sigma Aldrich, CAS 50-99-7) 20 g / L, - Bactopeptone (Becton Dickinson and Company, BD 211677) 20 g / L - Demineralized water qsp 1 L. The YPD liquid culture medium was autoclaved for 20 min at 120°C. Liquid pre-sporulation medium (YPAK): - Yeast extract (Becton Dickinson and Company, ref. BD 212750) 10 g / L, - Bactopeptone (Becton Dickinson and Company ***, BD 211677) 10 g / L, - Potassium acetate (Sigma Aldrich, CAS 127-08-2) 20 g / L, - Demineralized water qsp 1 L. The pH of the pre-sporulation medium is approximately 7 (physiological pH). The pre-sporulation medium is filtered via a polyester sulfone membrane filtration unit and is stored in a polyester bottle at 4°C for a maximum of one month. Liquid sporulation medium (ACK): - Potassium acetate (Sigma Aldrich, CAS 127-08-2) 20 g / L, - Demineralized water qsp 1 L. The pH of the sporulation medium should be 5.5. If necessary, the pH will be adjusted by adding acetic acid. The sporulation medium is filtered via a polyester sulfone membrane filtration unit and is stored in a polyester bottle at 4°C for a maximum of one month.

[0067] Equipment used Agitator-incubator, marketed by Eppendorf under the name "Innova ® S44i" with a 25 mm orbit; Centrifuge marketed under the name "Beckman Coulter Allegra X-15R", Binocular microscope marketed under the name "Olympus BX41".

[0068] Yeast Sporulation Phase Induction Protocol I-3856

[0069] Pre-sporulation in liquid medium

[0070] A fresh culture of I-3856 yeast on YPD solid culture medium is prepared according to the following protocol. The equivalent of one ooze is spread onto a Petri dish using the isolation method. The Petri dish is incubated at 30°C for 24 hours. From a fresh culture of I-3856 yeast on YPD solid culture medium, a 50 mL aliquot of YPD culture medium is inoculated, taking one colony, and transferred to a sterile 250 mL flask with tabs, which is placed in the incubator. The incubation period is 16 to 24 h at a temperature of 30°C with shaking at 150 rpm. After incubation, the entire culture is harvested into a sterile 50 mL tube and then centrifuged for 5 min with a relative centrifugal force (RCF) of 5100 x g. After centrifugation, the supernatant is discarded, and the yeast pellet is resuspended in 10 mL of YPAK pre-sporulation medium and transferred to a sterile 125 mL flask with tabs, which is placed in the incubator. The incubation time is 6 to 8 hours at 30°C and 150 rpm.

[0071] Sporulation in liquid medium

[0072] The incubated sample (10 mL) is transferred into a sterile 50 mL tube and then centrifuged for 5 min (FCR 5100 xg). After removing the supernatant, the resulting pre-sporulation pellet is washed with 10 mL of ACK medium, homogenized and then centrifuged again (5 min, FCR 5100 xg). A volume of 500 pL is added to the pellet, which is then re-homogenized. The resulting sample, which is in creamy form, is divided into three sterile 125 mL flasks with serrated edges, each containing 20 mL of ACK. The sporulation sample was observed under a microscope after 5 days of incubation at 25°C with agitation at 150 rpm. Serial dilutions of the sample were performed in tenfold increments: 100 µL of sample in 900 µL of physiological saline. 5 µL of each dilution were applied to a microscope slide for observation. Photographs and cell counts were taken of the samples to clearly distinguish individual cells. After 5 days of incubation, three sporulation culture samples, samples 1 to 3, were obtained and observed under a microscope. Figure 3 is a photograph of the sporulation medium of sample 1: asci are identified by A, while yeast cells that have not sporulated (vegetative cells) are identified by V.

[0073] Sporulation rate

[0074] The sporulation rate, also called sporulation yield, is determined according to the following mathematical formula:

[0075] [Math] Sporulation rate (%) = [Number of asci / Total number of cells] x 100 where, The number of ascians corresponds to the total number of ascians. The total number of cells corresponds to the number of cells that have not sporulated and the number of asci.

[0076] The number of asci formed in sporulation culture samples after 5 days of incubation at 25°C is counted as follows. The three vials are combined into a single sample. Serial dilutions of 10⁻¹⁰ are performed to obtain a cell concentration sufficient to clearly distinguish individual cells under the microscope. The number of asci formed versus the total number of cells is thus counted (it is easy to distinguish asci from vegetative cells with the naked eye). Three successive experiments are carried out. The results obtained are summarized in Table 1 below.

[0077] [Table 1] Number of asci, Total number of cells, Sporulation rate, Experiment 1, 129, 202, 63.86% Experience 2,131,216, 60.65% Experiment 3 79 180 56.11%

[6078] Table 1 shows that the average sporulation rate across the three experiments is approximately 60%, representing a highly productive sporulation rate. This also means that 40% of the cells present are yeast that did not sporulate.

[0079] Storage of sporulation culture samples Once the sporulation rate has been determined, the sporulation sample obtained after 5 days of incubation at 25°C can be stored in physiological saline. The sporulation sample is then centrifuged for 5 min with an FCR of 5100 x g. The sporulation supernatant is discarded. The pellet is collected and mixed with 20 mL of sterile physiological saline. This yields a suspension of vegetative cells and asci with a dry matter content of 2.04% (content evaluated according to the method previously described with the halogen desiccator). This suspension of vegetative cells and asci is used as a starting product in each of the drying processes of the invention, namely fluidized bed drying (example 2) and freeze-drying (example 3).

[0080] Example 2: Drying of the aqueous suspension of vegetative cells and asci by fluidized bed

[0081] The fluidized bed drying process leads to the obtaining of a mixture of vegetative cells and yeast asci in the form of granules, the dry matter content of which varies from 95 to 96.5%.

[0082] Equipment used - Piston extruder with a length of 90 mm and a diameter of 20 mm, having at its end an extrusion grid with openings of 0.6 mm in diameter. - Fluidized bed dryer “Sherwood Scientific model 501”: laboratory dryer made in a chamber with dimensions of 45 mm in diameter and 230 mm in height. Inlet air humidity regulated by an air treatment process: between 2 and 4 g of water per kg of dry air.

[0083] Steps in the process The starting product is the suspension of vegetative cells and asci as obtained at the end of example 1 which has a dry matter content of 2.04%. Several suspensions of vegetative cells and asci are combined to obtain a sufficient quantity of starting product, namely 286 mL of suspension of vegetative cells and asci with a dry matter content of 2.04%.

[0084] The first step in the fluidized bed drying process consists of performing a frontal filtration of the suspension of vegetative cells and asci. The filtration device used for this first stage is a bell-shaped chamber with an internal diameter of less than 15 cm. It comprises a filter plate with a pore diameter of 0.8 µm, upon which rests a first filter, namely the Beco KDS12, and a second filter, the Whatman® Grade 50, which rests on top of the first. The chamber is connected to compressed air. The plate and filters are pre-moistened with water before filtration begins. A 286 mL sample of the vegetative cell and asci suspension is filtered using the aforementioned filtration device, which includes a filter plate with a 0.8 µm pore diameter. The filtration time is 2 minutes at a pressure of 5 bar. This step removes between 25% and 35% of the moisture from the vegetative cell and asci suspension, resulting in a semi-solid form of vegetative cells and asci (15 grams) with a cake-like consistency and a dry matter content of 32%.

[0085] The semi-solid form of vegetative cells and asci (15 g) obtained after filtration is mixed, by kneading with a spatula, with 0.455 g of oil-in-water (O / W) emulsion consisting of 81.5% demineralized water, 8% sunflower oil, and 12.5% ​​sorbitan monostearate (MSS) emulsifier, until a homogeneous mixture is obtained in a semi-solid form, with a slightly more aerated appearance than the semi-solid form obtained immediately after filtration. Visually, the semi-solid mixture obtained after the addition of the O / W emulsion has almost the same appearance as the semi-solid form of vegetative cells and asci obtained immediately after filtration. The semi-solid mixture of vegetative cells and asci thus obtained contains 0.9% MSS per gram of dry matter of the semi-solid mixture of vegetative cells and asci.

[0086] The semi-solid mixture of vegetative cells and asci obtained in the previous step is then extruded using the aforementioned extrusion device, which features an extrusion grid with 0.6 mm diameter openings, resulting in thin, spaghetti-like filaments that will be transformed, by mechanical / physical fractionation, into small granules. having the shape of small vermicelli. Indeed, it is enough to shake the container containing the spaghetti of the mixture of vegetative cells and asci for the latter to break down into vermicelli / granules of homogeneous size.

[0087] The vegetative cell and asci granules / vermicelli thus obtained are placed in the aforementioned fluidized bed dryer with an air inlet temperature of 48°C and a relative humidity of 13% for 20 min, with a final temperature of approximately 42°C and an outlet relative humidity of 12%.

[0088] At the exit of the dryer, 7.1 g of vegetative cell and asci granules are recovered with a dry matter content of 95.33% (figure 4D).

[0089] Preservation of granules from the mixture of vegetative cells and asci The dry granules of vegetative cells and asci thus obtained are preserved by vacuum packaging in a sealed container, at a temperature of 4°C for several weeks in order to assess their viability over time.

[0090] Example 3: Drying of the aqueous suspension of vegetative cells and asci by lyophilization

[0091] The freeze-drying process leads to the obtaining of a mixture of vegetative cells and yeast asci in powder form, with a dry matter content of 98 to 99.9%.

[0092] Equipment used The freeze-drying device used is the pilot freeze dryer marketed under the name "LYO Vapor L-200" by the company Büchi, which is a standard freeze-drying device.

[0093] Steps in the process

[0094] Preparation of vegetative cell and asci formulations Three formulations of vegetative cells and asci are placed in 20 ml glass pillboxes which will be used directly for lyophilization. The first formulation is the control formulation, which is a suspension of vegetative cells and asci that does not include maltodextrin (cryoprotective agent), unlike the other three formulations, which are suspensions of vegetative cells and asci containing different amounts of the cryoprotective agent maltodextrin. The maltodextrin used is that sold under the name "SOSA Maltodextrin 12DE 500gr". It comes in the form of a fine powder. The three formulations of vegetative cells and asci are prepared from the vegetative cell and asci suspension as defined above, which has a dry matter content of 2.04%. Several vegetative cell and asci suspensions are combined to obtain the quantities required for each formulation. Control formulation of vegetative cells and asci: A quantity of 3 mL of vegetative cell and asci suspension with a dry matter content of 2.04% is injected into a pillbox. Formulation of vegetative cells and asci with 12% maltodextrin: A quantity of 2.90 mL of vegetative cell and asci suspension with a dry matter content of 1.5% is mixed with 0.0505 g of maltodextrin. The resulting formulation, which contains 12% maltodextrin relative to the dry matter of the vegetative cell and asci suspension, is injected into a pillbox. Formulation of vegetative cells and asci with 25% maltodextrin: A 3 mL quantity of vegetative cell and ascus suspension with a dry matter content of 1.5% is mixed with 0.1004 g of maltodextrin. The resulting ascus formulation, which contains 25% maltodextrin relative to the dry matter of the vegetative cell and ascus suspension, is injected into a pillbox. The pillboxes placed in the freeze dryer are fitted with perforated caps to allow the evaporative flow of water to escape during the freeze-drying process.

[0095] Freezing of vegetative cell and asci formulations

[0096] The vegetative cell and asci formulations prepared in the previous step are placed in a freezer at a temperature of -80°C for a period of 3 hours.

[0097] Sublimation of vegetative cell formulations and frozen asci Each formulation of vegetative cells and asci thus frozen is then placed in the freeze dryer to proceed to the sublimation step which is broken down into a primary drying step and a secondary drying step. The primary drying stage is carried out at a vacuum pressure of 0.2 mbar. After primary drying, the formulation of vegetative cells and asci is subjected to secondary drying by applying a vacuum pressure reduced to 0.1 mbar. The vegetative cell and asci samples obtained after secondary drying are in solid form, preferably powdery. They are illustrated in Figure 4 (Fig. 4A: control formulation, Fig. 4B and 4C: formulations with 12% and 25% maltodextrin). The vegetative cell and asci samples obtained by lyophilization of the two formulations with maltodextrin (Fig. 4B and 4C) have the appearance of a fine, white, airy powder that conforms to the shape of the pillbox. After shaking the pillbox by mechanical / physical action or crushing the powder with a spatula, the two vegetative cell and asci samples with maltodextrin are in the form of a powder with a particle size ranging from 200 to 315 µm. The sample of vegetative cells and asci, obtained after freeze-drying the control formulation (without the cryoprotectant maltodextrin, Fig. 4A), has the appearance of a solid but volatile form. The control formulation was indeed affected by pressure variations in the freeze-dryer chamber. Some of this formulation was also found on the freeze-dryer shelf, indicating that it was ejected through the holes in the stopper during pressure changes. The solid form obtained after freeze-drying the control formulation is therefore unsatisfactory, both in terms of its stability and its millability. The presence of a A cryoprotective agent is therefore recommended to obtain a satisfactory solid form of vegetative cells and asci.

[0098] The vegetative cell and asci samples obtained after freeze-drying can be stored in vacuum pillboxes directly within the freeze-drying unit. Thanks to a sealing system integrated into the freeze dryer, the pillboxes remain under vacuum at the end of the freeze-drying cycle. The dry matter contents of the vegetative cell and asci samples obtained after freeze-drying the formulations (control, 12%, and 25% maltodextrin) are evaluated with the halogen desiccator and are reported in Table 2.

[0099] [Table 2] Dry matter content Control sample of vegetative cells and asci 99.98% Sample of vegetative cells and asci with 12% 100% maltodextrin Sample of vegetative cells and asci with 25% 97.03% maltodextrin

[9100] The vegetative cell and asci samples obtained after freeze-drying have a dry matter content ranging from 97 to 100%. It should be noted that the 100% measurement may include a margin of error in the reading because the method is performed on small quantities of samples.

[0101] As noted, the solid forms of vegetative cells and asci obtained directly after freeze-drying can still be ground, for example with a spatula, or simply shaken mechanically, resulting in an asci powder with a particle size ranging from 200 to 315 pm.

[0102] Preservation of vegetative cell and asci powders The dry powders of vegetative cells and asci obtained with or without grinding after freeze-drying are stored by vacuum packaging in an airtight container such as an aluminum bag or a glass pillbox, at a temperature of 4°C for several weeks in order to assess their viability over time.

[0103] Example 4: Study of the viability of the mixture of vegetative cells and asci after drying, vacuum sealing and storage for several weeks

[0104] The viability of the mixture of vegetative cells and asci obtained after drying, vacuum packing and storage at 4°C for 12 weeks is measured by counting the number of viable cells in log CFU / g according to the standardized method EN 15789.

[0105] Sample preparation Samples of the mixture of vegetative cells and asci obtained after drying by fluidized bed air (dry granules) or by lyophilization (dry powder) are resuspended in 10 mL of physiological saline. More specifically, a quantity of 0.01 g of the dry granules or 0.005 g of the dry powder is taken and resuspended in 10 mL of physiological saline. Serial dilutions of each sample are carried out in increments of 10: 100 pL of sample + 900 pL of physiological saline. Respective quantities of 100 pL of the dilution are spread onto Petri dishes containing YPD solid culture medium as described in Example 1 in order to obtain a colony count. After 48 hours of incubation at 30°C in Petri dishes, the number of colonies is counted. This monitoring was carried out immediately after drying up to 12 weeks of storage at 4°C under vacuum.

[0106] The results obtained are described in Table 3 for samples obtained after fluidized bed drying and in Table 4 for samples obtained after freeze-drying. The column "Live cells counted on Petri dish (Log / mL)" in Tables 3 and 4 represents the live cell concentration per millilitre of physiological saline upon resuspension of dry samples. The column "Live cells in dry product (Log / g)" represents the live cell concentration per gram of the resuspended dry sample. The "Actual cell mass (g)" column indicates the actual mass of the resuspended dry sample, attributable solely to vegetative and asci cells. Indeed, in the freeze-drying technique, the sample mass can be high due to the addition of varying amounts of maltodextrin. The column "Quantity of living cells (Log / g)" represents the actual concentration of living vegetative cells and asci. Note that the columns "Live Cells in Dry Product (Log / g)" and "Amount of Live Cells (Log / g)" are identical when the vegetative and asci cells have been dried using the fluidized bed technique, which is not the case when the vegetative and asci cells have been dried by freeze-drying. Indeed, the latter column, "Amount of Live Cells (Log / g)," takes into account the fact that up to 25% maltodextrin is sometimes added to the formulation of vegetative and asci cells. Therefore, 25% of the resuspended weight actually corresponds to the maltodextrin and not to the vegetative and asci cells. This explains why the viability is lower if this correction is not made.

[0107] [Table 3] Living Cehutes Living CelMes Qi;smiié of Tecfm ique of Real Mass Time ie counted in the box in the product; living drying preservation of tests (g) Petri dish (Lag / ml] secflag / g Before drying 9.15 0 weeks 7.16 w 0.0294 9.03 1 week g- 0.98 8.35 0.8107 8.38 2 weeks 7.10 is 0.37 0.8134 8.39 2 weeks 7.49 9.31 0.0130 9.32 4 weeks $7.10 9.07 0.012 9.07 g weeks 7.04 S, 33 0.0113 3.39 12 weeks 7.15 9.02 0.0194 9.03 10 months 7.27 8.98 8.0202 8.97

[0108] With fluidized bed drying, the loss of viability is one log (8.15 log / mL before drying versus 7.16 log / mL after drying). The sample of vegetative cells and asci remains stable under the tested conditions over time. When the quantity of live vegetative cells and asci is expressed as a percentage of the dry cell mass (column "Quantity of live cells (Log / g)"), viability is well maintained at around 9 log / g (approximately 10 9 CFU / g) throughout storage, which is quite satisfactory. The fluidized bed dried mixture of vegetative cells and asci therefore retains its viability well over time.

[0109] [Table 4] Live Celiacs Live Celiacs Quantity se Mass Time Technique réeti® counted on box in te product this ii ni es living drying preservation of estimates igi Petri dish iieg / mOj SOC ilog / gi (tng / gi Before drying 8.15 8 weeks MS 0.52 0.6045 6.53 1 sense ine 6.21 8.56 8.0642 8.55 2 weeks MS 8.81 0.6645 8.51 tyopOyissaticm • 8 weeks 6.12 8.33 6.0680 8.34 ïèmnin 4 weeks 0.34 3.43 0.8645 3.43 8 weeks 8.37 8.53 0.6055 5.54 12 weeks 3.33 8.54 8.0645 8.84 18 months 8.27 8.56 0.6056 8.56 Before drying 8.35 8 weeks 8.28 6.3 0.6037 6.86 1 week 0.08 8.4g 6.0630 8.32 2 weeks 6.16 6.56 6.6046 6.55 lycdhyiisetmn - 3 weeks 6.21 8.53 8.0042 8.38 1298 Maitodestrine 4 weeks 6.34 0.35 0.8035 8.41 8 weeks 6.02 8.34 8.0041 8.40 12 weeks §,23 8.56 0.0643 8.01 18 months 6.23 6.56 0.804 s 3.62 Before drying 6.3.3 6 weeks 6.30 8.50 8.0847 8.53 3 weeks 8.04 8.46 0.8032 8.53 2 weeks 8.11 8.41 6.6038 8.54 lyephylisatien - 3 weeks §,23 8.46 8.6835 8.33 2S% Mahodextrin 4 weeks 6.66 8.35 0.60.35 6.47 5 weeks 6.23 8.37 8.0658 8.48 12 weeks 6.26 8.34 0.0655 8.46 10 months 6.32 8.52 0.8047 8.65

[0110] With freeze-drying, a loss of 2 log is observed for all freeze-drying conditions (8.15 log / mL before drying versus approximately 6.25 log / mL on average for all conditions after drying). As with the fluidized bed technique, the quantities of vegetative cells and live asci remain stable over time, which is quite satisfactory. Therefore, freeze-dried mixtures of vegetative cells and asci retain their viability well over time. However, as shown in Figure 5, a significantly different viability was observed between the control drying conditions (without maltodextrin), 12% maltodextrin, and 25% maltodextrin. The control drying conditions (without maltodextrin), 12% maltodextrin, and 25% maltodextrin were compared to the fluidized bed condition using a Welch's t-test for independent samples. This type of statistical test determines whether there is a significant difference between the means of the two groups. Welch's T-test implements the following mathematical formula:

[0111] [Math2] in which, mA represents the average of group A, ms the average of group B, S 2 A represents the standard deviation of group A, S 2 B represents the standard deviation of group B, nA represents the number of cases in group A, ns represents the number of cases in group B.

[0112] The symbol “****” in Figure 5 represents a significant difference, denoted P, less than 0.0001. The three freeze-drying conditions induce a loss of viability during drying, but allow this viability to be maintained over time during storage at 4°C under vacuum.

[0113] Example 5: Use of the dry mixture of vegetative cells and asci as a plant biostimulant

[0114] The dry mixture of vegetative cells and asci obtained after fluidized bed drying, which has a dry matter content of 95.33% (see example 2), is tested as a plant biostimulant and compared to the yeast Saccharomyces cerevisiae CNCM I-3856 alone.

[0115] Equipment used Hoagland's solution: a phosphorus-free solution that provides plants with the nutrients they need to grow. The composition of this solution is described in Table 5 below.

[0116] [Table 5] Compound Quantity (g / L) Adding an appropriate amount of phosphorus to the Hoagland solution allows for achieving the desired phosphorus concentration. For this experiment, the chosen concentration is 0.5 M monopotassium phosphate, as this concentration is known to be optimal for plant growth.

[0117] Tested plants The dry mixture of vegetative cells and asci, as well as the dry yeast alone Sc I-3856, are respectively tested on corn (Zea mays) seeds of the variety "P0312", produced by PIONEER; and on soybean (Glycine max) seeds "Williams 82".

[0118] Seed processing and plant cultivation

[0119] Corn and soybean seeds are placed in glass tubes containing sterile distilled water (control condition), and Saccharomyces cerevisiae CNCM I-3856 yeast at a concentration of 10 10 CFU / g, or containing the dry mixture of vegetative cells and asci at a concentration of 10 9 CFU / g. The seeds were treated with one gram of the dry mixture per 10 mL of sterile distilled water. To ensure uniform treatment, the seeds were manually agitated in the tube for 30 minutes. The corn and soybean seeds are then planted in plastic pots containing extra-fine vermiculite. Two seeds are planted in each pot and transferred to climate chambers under optimal growing conditions for two weeks. One week after planting, the seedlings are thinned to leave only one plant per pot. Fourteen days after planting, an inorganic phosphorus stress is induced by applying a phosphorus-free Hoagland solution to some plants. For others, an optimal phosphorus supply is applied by applying a Hoagland solution with an optimal phosphorus concentration (0.5M). The Hoagland solution is applied twice a week until the end of the experiment.

[0120] Biostimulation effect on soybean plants under phosphorus stress conditions

[0121] Phenotypic observations of soybean plants at 21, 28, and 35 days post-planting (DPP) are summarized in Table 6 below. These observations were performed on soybean plants grown on phosphorus-free vermiculite and treated with a non-phosphorus-enriched Hoagland solution, either with a dry mixture of vegetative cells and asci or with the yeast form alone. The gray cells represent values ​​that differ significantly from the control plants, i.e., those grown under the same conditions but whose seeds were treated with water only. The "Above-ground length" row represents the total height in centimeters of the plant in its visible, above-ground portion. The "Internodes" line corresponds to the length in centimeters between the different nodes, namely between the soil and the cotyledons, the cotyledons and the single-lobed leaf, the single-lobed leaf and the first leaf layer, between the first and second leaf layers, between the second and third leaf layers, between the third and fourth leaf layers, and the fourth and fifth leaf layers. The "Stem Diameter" line corresponds to the stem diameter at different locations along the stem, namely at the collar, cotyledons, single-lobed leaf, first leaf stage, second leaf stage, third leaf stage, fourth leaf stage, and fifth leaf stage. The line "Weight of aerial part" corresponds to the measurement of the weight (in grams) of the aerial apparatus in the wet state (fresh aerial biomass) and in the dry state (dry aerial biomass). The "Root weight" line corresponds to the measurement of the weight (in grams) of the roots in the wet state (fresh root biomass) and in the dry state (dry root biomass). The line "Aerial / root ratio" corresponds to the ratio between the weights of the aerial part in its dry state and the root part in its dry state.

[0123] Remarks and general conclusion In general, it is observed that soybean plants withstand phosphorus stress better when the seeds have been pretreated with the dry mixture of vegetative cells and asci of the invention than with treatment with vegetative cells alone of Saccharomyces cerevisiae. The plants are larger, resulting in longer internode lengths during the initial observation periods. The stem diameters of plants treated with the dry mixture of vegetative cells and asci are larger compared to untreated control plants, and also compared to plants whose seeds were treated with vegetative cells alone. A larger stem diameter indicates more robust plants. Compared to the vegetative cell treatment, soybean plants have a slightly less developed root system when seeds were treated with the dry mixture of vegetative cells and asci, meaning the plants have less difficulty accessing the necessary nutrients and therefore appear less stressed by phosphorus deficiency.This, coupled with a larger plant, explains a decreased aerial / root ratio for plants whose seeds were treated with the mixture of the invention.

[0124] Similarly, experiments were carried out on corn plants and the results also show that seed treatment helps plants resist phosphorus stress.

[0125] Biostimulation effect on soybean plants under optimal phosphorus concentration conditions

[0126] Phenotypic observations of soybean plants at 21, 28, and 35 days post-planting (DPP) are summarized in Table 7 below. These observations were performed on soybean plants grown on phosphorus-free vermiculite and treated with a 0.5M phosphorus-enriched Hoagland solution. The gray cells represent values ​​that differ significantly from the control plants, i.e., those grown under the same conditions but whose seeds were treated with water only.

[0127] [Table 7]

[0128] Remarks and general conclusion In general, treatment with the dry mixture of vegetative cells and asci of the invention helps soybean plants to develop and grow more significantly than treatment with Saccharomyces cerevisiae vegetative cells alone. The plants are larger, resulting in greater internode lengths. The much higher shoot-to-root ratio reflects the fact that the plants do not need to develop their roots, as they have less difficulty accessing nutrients.

[0129] This disclosure is not limited to the examples described above, which are merely examples, but encompasses all the variations that a person skilled in the art may consider in the context of the protection sought. Reference to deposited biological material

[0130] This application refers to the following biological material and derived variant or mutant strains: - registration number "I-3856" deposited at the National Collection of Microorganism Cultures (CNCM) (25, rue du Docteur Roux, 75724 Paris Cedex 15), in France, on October 17, 2007 by Lesaffre et Compagnie whose address is 41 rue Etienne Marcel, 75009 Paris.

Claims

Demands

1. A dry mixture of vegetative cells and asci of Saccharomyces cerevisiae yeast characterized in that it has a dry matter content of 95 to 99.9% and in that said mixture comprises at least 60% asci, preferably at least 70%, and more preferably at least 90% asci.

2. Dry mixture of vegetative cells and yeast asci according to claim 1, characterized in that it has a dry matter content of 95 to 98%, and preferably 95 to 96.5%.

3. Dry mixture of vegetative cells and asci according to claim 2, characterized in that it is in the form of granules having the shape of vermicelli of a length ranging from 0.25 to 1 cm and of a thickness ranging from 0.25 to 0.6 cm.

4. Dry mixture of vegetative cells and asci according to claim 1, characterized in that it has a dry matter content of 98 to 99.9%.

5. Dry mixture of vegetative cells and asci according to claim 4, characterized in that it is in the form of a powder, said powder preferably having a particle size ranging from 200 to 315 pm.

6. Dry mixture of vegetative cells and asci according to any one of claims 1 to 5, characterized in that it is viable / stable for a period of 12 to 36 months, when stored under vacuum at a temperature of 4°C.

7. Dry mixture of vegetative cells and asci according to claim 6, characterized in that it exhibits a viability ranging from 7 log CFU / g to 9 log CFU / g measured according to the standardized method EN 15789.

8. A method for preparing the dry mixture of vegetative cells and asci according to any one of claims 1 to 7, characterized in that it consists of drying a suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, said suspension comprising at least 60% asci, preferably at least 70%, and more preferably still at least 90% asci, said drying being carried out by fluidized bed or by freeze-drying.

9. A preparation method according to claim 8, characterized in that the suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1% is prepared according to the method comprising the following steps: - incubation of a fresh culture of Saccharomyces cerevisiae yeast in a sporulation culture medium for 5 days at a temperature of 30°C, in order to induce the sporulation phase of the yeast and to obtain a sporulation culture comprising at least 60% asci, preferably at least 70%, and even more preferably at least 90% asci, - separation of vegetative cells and asci from the sporulation culture, said separation being carried out by centrifugation followed by re-suspension of the vegetative cells and asci in physiological saline solution, - recovery of a suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, said suspension comprising at least 60% asci, preferably at least 70%, and even more preferably at least 90% asci.

10. A preparation process according to claim 8, characterized in that the fluidized bed drying comprises the following steps: - frontal filtration of the suspension of vegetative cells and asci with a dry matter content of 1.9 to 2.1%, using a filter plate with a pore diameter of 0.6 to 1 pm, in order to obtain a semi-solid form of vegetative cells and asci, also called "vegetative cell and asci cake", with a dry matter content of 29 to 32%; - mixing the semi-solid form of vegetative cells and asci obtained in the previous step with an oil-in-water (O / W) emulsion consisting of water, oil and emulsifier, the quantity of emulsifier ranging from 10 to 14% by weight relative to the total weight of the emulsion, until a homogeneous mixture is obtained which is in a semi-solid form, called "semi-solid mixture of vegetative cells and asci", said semi-solid mixture of vegetative cells and asci comprising 0.7 to 0.9% of emulsifier per gram of dry matter of the semi-solid mixture of vegetative cells and asci; - extrusion of the semi-solid mixture of vegetative cells and asci obtained in the previous step using an extrusion device having an extrusion grid with openings of a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm, in order to obtain an extruded mixture of vegetative cells and asci in the form of filaments called spaghetti; - mechanical / physical fractionation of the filaments of vegetative cells and asci obtained in the previous step in order to obtain vermicelli of vegetative cells and asci called granules; - drying of the vegetative cell granules and asci obtained in the previous step in a fluidized bed dryer where the inlet air temperature is between 45 and 50°C and the relative humidity (RH) is between 13 and 15%, the outlet air temperature is between 36 and 42°C and the relative humidity (RH) is between 10 and 20%, and the fluidization flow rate is 14 to 20 m 3 / h; - recovery of the dry mixture of vegetative cells and asci, said dry mixture having a dry matter content of 95 to 98%, preferably 95 to 96.5%.

11. A preparation process according to claim 10, characterized in that the fluidized bed drying comprises one or all of the following features: - Frontal filtration of the vegetative cell and asci suspension with a dry matter content of 1.9 to 2.1% is carried out using a filter plate with a pore diameter of 0.8 pm, for a duration of 1 to 5 minutes and at a pressure of 1 to 5.5 bars, in order to obtain a semi-solid form of vegetative cells and asci (vegetative cell and asci cake) with a dry matter content of 29 to 32%; - the semi-solid form of vegetative cells and asci obtained after the filtration step is mixed with an oil-in-water (O / W) emulsion consisting of 81.5% water, preferably water demineralized, 8% sunflower oil and 12.5% ​​of the emulsifier sorbitan monostearate (MSS), until a homogeneous mixture is obtained which is in a semi-solid form, the semi-solid mixture of asci thus obtained having 0.9% MSS per gram of dry matter of the semi-solid mixture of vegetative cells and asci; - the semi-solid mixture of vegetative cells and asci obtained at the end of the step of mixing the cake of vegetative cells and asci with the H / W emulsion is extruded using a piston extrusion device, preferably 90 mm long and 20 mm in diameter, said extrusion device having at its end an extrusion grid with openings of a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm, which leads to an extruded mixture of vegetative cells and asci having the form of filaments called spaghetti; - the filaments / spaghetti of vegetative cells and asci are mechanically broken down into vermicelli called granules; - The vegetative cell and asci granules are dried in a fluidized bed dryer where the inlet air has a temperature of 48°C and a relative humidity (RH) of 13%, the outlet air has a temperature of 42°C and an RH of 12%, and the fluidization flow rate of the bed is 16 m 3 / h, said drying being carried out for a period of 17 to 20 minutes, preferably 20 minutes; - the vegetative cell and asci granules obtained after fluidized bed drying have a dry matter content ranging from 95 to 96.5%.

12. A preparation process according to claim 8, characterized in that the freeze-drying comprises the following steps: - addition, in the suspension of vegetative cells and asci having a dry matter content of 1.9 to 2.1%, of maltodextrin in an amount of 5 to 25% by weight of maltodextrin relative to the dry matter of the suspension of vegetative cells and asci, and preferably in an amount by weight of 25% of maltodextrin; - freezing the suspension of vegetative cells and asci as defined in the previous step at a temperature ranging from -60 to -80°C for a period of 1 to 4 hours; - sublimation of the suspension of vegetative cells and asci frozen in the previous step by applying a vacuum pressure of 0.05 to 0.5 mbar in order to remove the frozen water, said sublimation step being carried out for a period of 60 to 61 hours and leading to a solid form of vegetative cells and asci, preferably a powdered form; - recovery of the solid form of vegetative cells and asci having a dry matter content of 98 to 99.9%.

13. A preparation method according to claim 12, characterized in that the sublimation step of the frozen vegetative cell and asci suspension is broken down into: - a primary drying step lasting from 40 to 42 hours during which the vacuum pressure is 0.1 to 0.5 mbar, and - a secondary drying stage lasting from 18 to 20 hours during which the vacuum pressure is 0.05 to 0.2 mbar, which leads to a solid form of vegetative cells and asci, preferably a powdery form.

14. A preparation method according to claim 13, characterized in that, at the end of the secondary drying step, the solid form of vegetative cells and asci obtained, preferably in powder form, is further ground or mechanically shaken, resulting in a powder form of vegetative cells and asci with a particle size ranging from 200 to 315 pm.

15. Use of the dry mixture of vegetative cells and asci as defined in any one of claims 1 to 7, or as obtained according to the process as defined in any one of claims 8 to 14, as a plant biostimulant.

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