Ascospores of the yeast Saccharomyces cerevisiae, in dry or suspended form, their preparation methods and uses

Abstract

The present invention relates to Saccharomyces cerevisiae yeast ascospores in dry or suspension form for use as a medicinal product, and in particular for use as an anti-inflammatory, antiviral, or antitumor agent. The present invention also relates to Saccharomyces cerevisiae yeast ascospores characterized in that they are in dry form and have a dry matter content of 95 to 99.9%. It further relates to a method for preparing Saccharomyces cerevisiae yeast ascospores in dry or suspension form. The method for preparing ascospores in dry form consists of drying a suspension of ascospores having a dry matter content of 1.5 to 5%, said drying being carried out by fluidized bed or freeze-drying. [No abstract figure]

Description

Title of the invention: Ascospores of Saccharomyces cerevisiae yeast, in dry or suspended form, their preparation methods and their uses. Technical field

[0001] This application relates to ascospores of the yeast Saccharomyces cerevisiae, in dry or suspended form, their preparation methods, and their uses. In particular, the application describes yeast ascospores for use as an anti-inflammatory, antiviral, or antitumor agent. Prior art

[0002] The yeast Saccharomyces cerevisiae is a microscopic, unicellular fungus of the ascomycete family, which has the ability to multiply both by asymmetric cell division, namely by budding (the process of mitosis), and by sexual reproduction, namely by sporulation (the process of meiosis), and to switch between these two types of reproduction depending on the environment in which it is found. Thus, if the environment provides favorable conditions, the yeast Saccharomyces cerevisiae will bud (mitotic reproduction), while if the environment presents unfavorable conditions, the yeast will sporulate (sexual reproduction, by meiosis). Reproduction by budding occurs through the following process: the parent cell buds from a smaller daughter cell (mitosis), which possesses the same genetic information. A single parent cell thus gives rise to 20 to 25 daughter cells. There are haploid "a" cells and haploid "a" cells, which correspond to distinct sexual characteristics. The fusion between a haploid "a" cell and a haploid "a" cell results in a diploid "a / a" cell. As long as the environment is favorable, the diploid cell, which is the stable vegetative form of 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 four ascospores. However, asci containing three or two ascospores can also occur if one of the meiotic divisions has not been completed successfully. Figure 1 illustrates the cellular processes of mitosis and meiosis. The process of meiosis has two main stages. The first stage of meiosis consists of a first division during which the diploid yeast cell, comprising 2n single-chromatid chromosomes (2n = 32), divides into two haploid daughter cells, each comprising n chromosomes (n = 16) with two chromatids. The second stage of meiosis consists of a second division during which each haploid daughter cell will divide again into two to give two haploid daughter cells 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 one by one from the sporulation medium. The sporulation medium is the culture medium in which the yeast has been conditioned to sporulate, in other words, the medium to induce meiosis. Figure 2 illustrates the transformation by meiosis of yeasts and shows that the sporulation culture obtained after meiosis (figure on the right) mainly comprises asci with 4 ascospores but also some vegetative cells, namely yeasts that have not sporulated. As with the asci which can be isolated, 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] Yeast ascospores have the advantage, compared to the vegetative form of yeast, of being more resistant to various stresses such as heat, oxidation, humidity, nutrient deficiency, and chemical or enzymatic stresses. This greater resistance of ascospores (compared to yeasts) is due in particular to the structure of the ascospore cell wall, which is more complex than that of the vegetative cell wall (vegetative yeasts). For illustrative purposes, the cell wall structure of yeasts (vegetative) and that of yeast ascospores is shown in [Fig. 3] (Neiman AM, Budding Yeast Saccharomyces cerevisiae, Genetics, Vol. 189, Issue 3, 2011). There are several prior art studies that compare yeasts (vegetative) and ascospores, particularly with regard to their resistance to various stresses. Thus, in the publication by Elham A. Milani et al., Vol. 206, 2015, entitled "Thermal resistance of Saccharomyces yeast ascospores", it is shown that yeast ascospores are at least four times more heat resistant than vegetative cells in a beer with 4% alcohol by volume. In the publication by Alison E. Coluccio et al., 2008, entitled “The Yeast Spore Wall Enables Spores to Survive Passage through the Digestive Tract of Drosophila” It has been shown that Saccharomyces cerevisiae spores survive passage through the gut of Drosophila melanogaster, and that the spore wall components, which distinguish it from the vegetative cell wall, are necessary for this resistance. Document PCT / SE99 / 00231 describes a probiotic composition that includes spores of one or more strains of the yeast Endomyces fibuliger. This composition is specifically described for combating gastrointestinal disorders.

[0004] However, to the Inventors' knowledge, Saccharomyces cerevisiae yeast ascospores, in dry or suspended form, have never been proposed for pharmaceutical use, and in particular as an anti-inflammatory agent. Similarly, to the Inventors' knowledge, Saccharomyces cerevisiae yeast ascospores in dry form, i.e., with a dry matter content of at least 95%, have never been proposed. Having dried ascospores offers several advantages, such as increased shelf life, and easier storage, handling, and transport. Drying, in particular, ensures good stability of the ascospores and therefore a good retention of their initial viability. Stability is measured by the maintenance of viability over time (shelf life).

[0005] The Inventors deserve credit for having determined that yeast ascospores, whether in dry form or in suspension, possess, in particular, anti-inflammatory activity. The Inventors also deserve credit for having conceived not only the preparation of dry yeast ascospores, namely yeast ascospores with a high dry matter content, but also for having developed processes for their preparation, such yeast ascospores thus prepared retaining their viability and activity over time. Summary

[0006] According to a first aspect, the present invention relates to Saccharomyces cerevisiae yeast ascospores in dry or suspended form, for use as a medicinal product, and in particular as an anti-inflammatory, antiviral or antitumor agent.

[0007] According to another aspect, the present invention relates to Saccharomyces cerevisiae yeast ascospores characterized in that they are in dry form and have a dry matter content ranging from 95 to 99.9%.

[0008] According to yet another aspect, the invention relates to a process for preparing yeast ascospores in dry form as defined above, characterized in that it consists of subjecting a suspension of ascospores having a dry matter content of 1.5 to 5.5% to drying, said drying being carried out by fluidized bed or by freeze-drying.

[0009] According to yet another aspect, the invention also relates to a method for preparing a suspension of Saccharomyces cerevisiae yeast ascospores having a dry matter content ranging from 1.5 to 5.5%. Brief description of the drawings

[0010] Other features, details and advantages will become apparent from reading the detailed description below and from analyzing the accompanying drawings, in 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 post-meiotic sporulation culture, which mainly comprises asci with two "a" ascospores and two "a" ascospores, but also some yeast cells that have not sporulated (vegetative form of the yeast). Fig. 3

[0013] [Fig.3] is a diagram showing the structure of the cell walls of vegetative yeast and of the ascospore walls. Fig. 4

[0014] [Fig.4] is a photograph observed under a microscope of the yeast sporulation culture Saccharomyces cerevisiae CNCM 1-3856 used in the examples of the invention. The figure on the left (4A) is a photograph of the culture medium obtained after sporulation of the yeast CNCM 1-3856, which comprises a majority of asci and some vegetative cells (namely the yeasts that have not sporulated) and the figure on the right (4B) is an enlarged view of a part of figure 4A which shows asci comprising 4 ascospores (represented by the letter A) and the yeasts that have not sporulated (represented by the letter V). Fig. 5

[0015] [Fig. 5] is a microscopic photograph of the ascospore suspension showing a dry matter content ranging from 1.5 to 5.5% obtained after enzymatic digestion of the ascus wall using Zymolyase® solution. Fig. 6

[0016] [Fig. 6] shows photographs of the ascospore sample obtained at different stages fluidized bed drying. Figure 6A is a photograph of the semi-solid form of ascospores obtained after frontal filtration of an ascospore suspension containing 1.5% dry matter, said semi-solid form being called an ascospore cake. Figure 6B is a photo of the ascospore sample obtained after extrusion, which have the shape of filaments called "spaghetti". Figure 6C shows the ascospore sample which this time has the form of vermicelli called granules, the said ascospore granules being obtained by mechanical / physical fractionation of the ascospore spaghetti. Figure 6D shows the ascospore sample in granule form during drying in the fluidized bed. Figure 6E shows dry ascospore granules placed in sterile, airtight flasks under vacuum. Fig. 7

[0017] [Fig.7] shows photographs of the ascospore samples obtained at the end of the freeze-drying. The photo in Figure 7A is a photo of an ascospore sample obtained after lyophilization of an ascospore suspension without maltodextrin (control). The photos in figures 7B, 7C and 7D show the ascospore samples obtained after freeze-drying the ascospore suspensions with 12, 25 and 75% maltodextrin respectively. Fig. 8

[0018] [Fig-8] is a histogram illustrating the average quantity of live ascospores measured in ascospore samples obtained after freeze-drying and fluidization. Histogram 8A is that of the ascospore sample obtained after lyophilization of an ascospore suspension without maltodextrin (control). Histograms 8B, 8C and 8D are those of ascospore samples obtained after lyophilization of ascospore suspensions with 12, 25 and 75% maltodextrin respectively. Histogram 8E is that of the ascospore sample obtained after fluidization. Fig. 9

[0019] [Fig.9] is a scatter plot measuring the quantity of interleukin 17A (IL-17A) secreted by CD4+ T lymphocyte immune cells into their cellular environment. The scatter plot 9A represents unstimulated CD4+ T lymphocytes. The scatter plot 9B represents CD4+ T lymphocytes stimulated by anti-CD3 and anti-CD28 antibodies. The scatter plot 9C represents CD4+ T lymphocytes treated with cyclosporine A. The scatter plots 9D and 9E are those of CD4+ T lymphocytes treated with the ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively. The point clouds 9F and 9G are those of CD4+ T lymphocytes treated with the ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively, heated for 15 minutes at 100°C. Fig. 10

[0020] [Fig. 10] is a scatter plot graph measuring the amount of interleukin 10 (IL-10) secreted by CD4+ T lymphocyte immune cells into their cellular environment. The scatter plot 9A represents unstimulated CD4+ T lymphocytes. The scatter plot 9B represents CD4+ T lymphocytes stimulated by anti-CD3 and anti-CD28 antibodies. The scatter plot 9C represents CD4+ T lymphocytes treated with cyclosporine A. Scatter plots 9D and 9E represent CD4+ T lymphocytes treated with ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively. The point clouds 9F and 9G are those of CD4+ T lymphocytes treated with the ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively, heated for 15 minutes at 100°C. Fig. 11

[0021] [Fig. 11] is a scatter plot measuring the amount of interferon gamma (IFN-y) secreted by CD4+ T lymphocyte immune cells into their cellular medium for D2 and D3 donors. The scatter plot 11A represents unstimulated CD4+ T lymphocytes. The scatter plot 1 IB is that of CD4+ T lymphocytes stimulated by anti-CD3 and anti-CD28 antibodies. The scatter plot 1 IC is that of CD4+ T lymphocytes treated with cyclosporine A. The scatter plots 11D and 11E are those of CD4+ T lymphocytes treated with ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively. The point clouds 11F and 1 IG are those of CD4+ T lymphocytes treated with the ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively, heated for 15 minutes at 100°C. Fig. 12

[0022] [Fig. 12] is a scatter plot measuring the amount of interferon gamma (IFN-y) secreted by CD4+ T lymphocyte immune cells into their cellular environment for DI and D4 donors. The scatter plot 12A represents unstimulated CD4+ T lymphocytes. The scatter plot 12B represents CD4+ T lymphocytes stimulated by anti-CD3 and anti-CD28 antibodies. The scatter plot 12C is that of CD4+ T lymphocytes treated with cyclosporine A. The scatter plots 12D and 12E are those of CD4+ T lymphocytes treated with ascospore suspension at 1.5% dry matter at 3x0+5 and 3x0+6 cells / mL respectively. The point clouds 12F and 12G are those of CD4+ T lymphocytes treated with the ascospore suspension at 1.5% dry matter at 3xl0+5 and 3xl0+6 cells / mL respectively, heated for 15 minutes at 100°C. Fig. 13

[0023] [Fig. 13] is a diagram measuring the amount of CXCL10 chemokines secreted by THPl-Blue lymphocyte macrophage immune cells in their cellular environment. Diagram 13A is that of unstimulated THPl-Blue macrophages. Diagram 13B is that of THPl-Blue macrophages stimulated with LPS. Diagram 13C is that of THPl-Blue macrophages stimulated with LPS and treated with hydrocortisone. Diagram 13D is that of THPl-Blue macrophages stimulated with LPS and treated with sodium butyrate. Diagram 13E is that of THPi-Blue macrophages treated with physiological saline mixed with 0.05% Tween 80. Diagrams 13F, 13G and 13H are those of THPl-Blue macrophages treated with the ascospore suspension at 1.5% dry matter at respectively IxlO44, lxl0+6 and lxl0+7 cells / mL. Fig. 14

[0024] [Fig. 14] is a diagram measuring the amount of IL-8 chemokines secreted by THPl-Blue lymphocyte macrophage immune cells in their cellular environment. Diagram 14A is that of unstimulated THPl-Blue macrophages. Diagram 14B is that of THPl-Blue macrophages stimulated with LPS. Diagram 14C is that of THPl-Blue macrophages stimulated with LPS and treated with hydrocortisone. Diagram 14D is that of THPl-Blue macrophages stimulated with LPS and treated with sodium butyrate. Diagram 14E is that of THPi-Blue macrophages treated with physiological saline mixed with 0.05% Tween 80. Diagrams 14F, 14G and 14H are those of THPl-Blue macrophages treated with the ascospore suspension at 1.5% dry matter at respectively IxlO44, lxl0+6 and lxl0+7 cells / mL. Fig. 15

[0025] [Fig.15] is a diagram measuring the amount of MCP-1 chemokines secreted by the immune cells lymphocytes macrophages THPl-Blue in their cellular environment. Diagram 15A is that of unstimulated THPl-Blue macrophages. Diagram 15B is that of THPl-Blue macrophages stimulated with LPS. Diagram 15C is that of THPl-Blue macrophages stimulated with LPS and treated with hydrocortisone. Diagram 15D is that of THPl-Blue macrophages stimulated with LPS and treated with sodium butyrate. Diagram 15E is that of THPi-Blue macrophages treated with physiological saline mixed with 0.05% Tween 80. Diagram 15F, 15G and 15H is that of THPl-Blue macrophages treated with the ascospore suspension at 1.5% dry matter at respectively lxl0+4, lxl0+6 and lxl0+7 cells / mL. Detailed description

[0026] The ascospores of the invention The present invention relates to Saccharomyces cerevisiae yeast ascospores in dry or suspended form for use as a medicinal product. In this application, yeast ascospores may also be referred to as "spores" and designate the cells contained within an ascus produced as a result of meiosis in the yeast Saccharomyces cerevisiae. The Saccharomyces cerevisiae yeasts covered by the invention are all Saccharomyces cerevisiae yeasts that sporulate, resulting in an ascus comprising 4 ascospores, or possibly 3 or 2 ascospores. According to an advantageous embodiment of the invention, the drug is an anti-inflammatory agent, an antiviral agent or an antitumor agent. According to a particularly advantageous embodiment, the invention relates to Saccharomyces cerevisiae yeast ascospores in dry or suspended form for use in the prevention and / or treatment of diseases Inflammatory diseases. Examples of inflammatory diseases include Crohn's disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, asthma, ankylosing spondylitis, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In a further particularly advantageous embodiment, the invention relates to Saccharomyces cerevisiae yeast ascospores in dry or suspended form for use in the prevention and / or treatment of inflammatory skin diseases. Examples of inflammatory skin diseases include psoriasis, atopic dermatitis, and hidradenitis suppurativa. According to an advantageous embodiment of the invention, yeast ascospores in dry form refer to ascospores having a dry matter content of at least 95%, and preferably from 95% to 99.9%. The terms "ascospores in dry form" and "dry ascospores" may be used interchangeably in the application. The dry matter content of 95% to 99.9% means that the ascospores of the invention comprise 95% to 99.9% dry matter by mass relative to the total mass of the ascospores. According to another advantageous embodiment of the invention, yeast ascospores in suspension refers to a suspension of ascospores having a dry matter content of 1.5 to 5.5%. More particularly, the ascospore suspension refers to an aqueous suspension of ascospores, namely a dispersion of ascospores in water, preferably physiological saline. A dry matter content of 1.5 to 5.5% means that the ascospore suspension comprises 1.5 to 5.5% ascospore dry matter in proportion to the total mass of the suspension.

[0027] The dry matter contents given in this application for all the ascospore samples were determined using a halogen desiccator, the principle of which is as follows. The halogen desiccator records the initial weight of the sample, then gradually dries it using a halogen lamp that emits infrared radiation. The weight of the sample is recorded regularly until the end of the drying procedure. To complete the measurement, the difference between the initial weight of the sample and the weight of the sample after dehydration 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 the company Mettler Toledo under the name halogen dryer HX204.

[0028] The present invention also relates to Saccharomyces cerevisiae yeast ascospores characterized in that they are in dry form and have a dry matter content ranging from 95 to 99.9%.

[0029] According to an advantageous embodiment, the yeast ascospores of the invention have a dry matter content ranging from 95 to 98%, and preferably from 95 to 96.5%. In this particular case, the yeast ascospores preferentially appear as 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.

[0030] According to another advantageous embodiment, the yeast ascospores of the invention have a dry matter content ranging from 98 to 99.9%. In this particular case, yeast ascospores are preferably in the form of a powder, said powder preferably having a particle size ranging from 200 to 315 pm.

[0031] According to yet another advantageous embodiment, the yeast ascospores according to the invention are characterized in that they are viable / stable, for a period of 12 to 36 months, when stored under vacuum at a temperature of 4°C.

[0032] The stability / viability of the yeast ascospores of the invention for a period of 12 to 36 months under vacuum and at 4°C means that the ascospores of the invention have a viability ranging from 7 log CFU / g to 9 log CFU / g measured according to the standard method EN 15789. One way to measure the viability of dried yeast spore cells 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 was measured according to the EN15789 standard. For the purposes of this application, stating that dried ascospores are viable for a period of 12 to 36 months means that the number of live and metabolically active cells of dried ascospores (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 dried yeast ascospores have a live and metabolically active cell count of 1109 CFU / g (equivalent to 9 log CFU / g) at time t0 (t0 being the time when the dried ascospores have just been obtained), then the dried yeast ascospores will, after a period of at least 12 months, and preferably from 12 to 36 months, have a number of living and metabolically active cells which will not be less than 1xO7UFC / g (equivalent to 7 log UFC / g). The viability rate of the dried ascospores of the invention is directly related to the stability of the ascospores. Thus, if the ascospores of the invention are viable after drying, vacuum packaging, and storage at a temperature of 4°C for a period of 12 to 36 months, this also means that the ascospores of the invention are stable after drying, vacuum packaging, and storage at a temperature of 4°C for a period of 12 to 36 months.

[0033] Process for preparing ascospores

[0034] The present invention further relates to a process for preparing yeast ascospores having a dry matter content of 95 to 99.9%, said process being characterized in that it consists of subjecting to drying a suspension of ascospores having a dry matter content of 1.5 to 5.5%, said drying being carried out by fluidized bed or by lyophilization. As an indication, an ascospore suspension with a dry matter content of 1.5 to 5.5% corresponds to an aqueous suspension of ascospores comprising 10⁸ to 10⁹ ascospores per millilitre of suspension. According to an advantageous embodiment, the ascospore suspension having a dry matter content of 1.5 to 5.5% may include an emulsifier.

[0035] Drying is a preservation process that involves removing all the water from a product to stabilize it and allow it to retain its original properties for a long 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, which facilitates 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 ascospore suspension which has a dry matter content ranging from 1.5 to 5.5%.

[0036] Preparation of the asco s pores suspension having a dry matter content ranging from 1.5 to 5.5%

[0037] According to an advantageous embodiment of the invention, the ascospore suspension having a dry matter content of 1.5 to 5.5%, 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 yeast sporulation phase and to obtain a sporulation culture comprising at least 60% 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, - addition of an enzyme to the sporulation culture medium to enzymatically digest the vegetative cell walls, resulting in their degradation and elimination, as well as enzymatically digest the asci to release the ascospores, - density gradient centrifugation of the sporulation culture to separate ascospores from cellular debris resulting from enzymatic digestion, - recovery of ascospores in the centrifugation pellet and suspension of the ascospores in physiological water, possibly with added emulsifier, in order to obtain an ascospore suspension with a dry matter content ranging from 1.5 to 5.5%. 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.

[0038] The physiological saline solution of the ascospore suspension contains 0.9% sodium chloride, which maintains the minimum osmolarity necessary for the proper viability of the ascospores. It is pH alkaline to better solubilize the ascospores. The emulsifier further enhances the solubilization of the ascospores, which are hydrophobic and tend to aggregate.

[0039] A "sporulation culture comprising 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.

[0040] The dry matter content of the ascospore suspension is determined using the halogen desiccator as defined above. The ascospore suspension is first centrifuged using a conical centrifuge tube. After centrifugation, the pellet (which includes the ascospores, i.e., the dry mass of the suspension) is separated from the supernatant. The pellet and the supernatant are weighed, and their dry matter content is determined using the halogen desiccator.

[0041] According to an advantageous embodiment of the process of the invention, the ascospore suspension obtained, which has a dry matter content ranging from 1.5 to 5.5%, is kept at a temperature of 4°C, under atmospheric pressure, for several weeks. The measurement of the viability of the ascospore suspension after 16 months showed that the number of live and metabolically active cells did not decrease by more than 1 log CFU / g.

[0042] One of the problems encountered in the context of the invention was that of developing a process for drying ascospores, namely more particularly a process for drying a suspension of ascospores, which allows their viability to be maintained, not only directly after drying, but also over time.

[0043] Another problem encountered in the context of the invention was the low dry matter content of the ascospore suspension used at the start of the drying process of the invention, namely 1.5 to 5.5% dry matter. Indeed, the whole point was to lose as little dry matter as possible during the various stages of drying.

[0044] It is therefore to the credit of the Inventors that they have succeeded in addressing both the problem of preserving the viability of ascospores and their low dry matter content within the suspension. After extensive research, the Inventors have thus determined the optimal characteristics to be developed in a fluidized bed drying process and in a freeze-drying process, respectively, allowing the production of dried ascospores whose viability is maintained for several months from an ascospore suspension containing 1.5 to 5.5% dry matter.

[0045] Fluidized bed drying

[0046] Fluidized bed or 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. Homogeneous temperature and uniform drying of the particles are possible through continuous control of the inlet and outlet of the drying air. Precise monitoring of the humidity of the drying air is necessary to optimize the drying process. Conditions can vary depending on the humidity and temperature of the inlet air.

[0047] According to an advantageous embodiment of the invention, the ascospore preparation process as defined above is more particularly characterized in that the fluidized bed drying comprises the following steps: - frontal filtration of the ascospore suspension having a dry matter content of 1.5 to 5.5%, using a filter plate having a pore diameter of 0.6 to 1 pm, in order to obtain a semi-solid form of ascospores, also called "ascospore cake", having a dry matter content of 29 to 32%; - mixing the semi-solid form of ascospores 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 ascospore mixture", said semi-solid ascospore mixture comprising 0.7 to 0.9% of emulsifier per gram of dry matter of the semi-solid ascospore mixture; - extrusion of the semi-solid mixture of ascospores 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 ascospores extruded in the form of filaments called spaghetti; - mechanical / physical fractionation of the ascospore filaments obtained in the previous step in order to obtain ascospore vermicelli called granules; - drying of the ascospore granules obtained in the previous step in a fluidized bed dryer where the inlet air has a temperature of 45 to 50°C and a relative humidity (RH) of 5 to 8%, the outlet air has a temperature of 36 to 40°C and a relative humidity (RH) of 4 to 7%, and where the fluidization flow rate is 14 to 20 m3 / h; - recovery of ascospores with a dry matter content of 95 to 98%, preferably 95 to 96.5%. The yeast ascospores 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.

[0048] Frontal filtration is a filtration technique that uses a pressure gradient and consists of passing the fluid to be filtered perpendicularly to the surface of the filter. 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 10 seconds to 1 minute and at a pressure of 1 to 3.5 bars. The front filtration step removes 25 to 35% of the moisture from the ascospore suspension and yields a semi-solid form of ascospores that looks like a pasty cake with a dry matter content ranging from 29 to 32%.

[0049] The resulting ascospore paste cake is mixed, preferably with a spatula, with an oil-in-water (O / W) emulsion until a homogeneous mixture is obtained, which, to the naked eye, has almost the same appearance as the ascospore paste cake obtained after the filtration step. The O / W emulsion protects the material to be dried 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 oil-in-water emulsion used in mixture with the semi-solid form of ascospores comprises at least 80% water and at least 10% emulsifier, meaning that it comprises a maximum of 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.

[0050] The extrusion of the semi-solid mixture of ascospores 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.

[0051] At the exit of the extrusion grid, the extruded ascospore mixture 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 ascospore spaghetti.

[0052] The ascospore granules are then placed in the chamber of the fluidized bed dryer, the inlet air of which has, for example, a temperature of 48°C and a relative humidity of 6%, and the outlet air a temperature of 36°C and a relative humidity of 4%. As an example, the humidity of the drying air is regulated by an air treatment process (between 1 and 2 g of water per kg of dry air). The drying time is preferably 7 to 12 minutes, and could, for example, be 9 minutes. During the drying process, the still-damp granules will gradually decrease in length.

[0053] The ascospores obtained after the fluidized bed drying process have 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 a sufficient and entirely satisfactory value for ascospores. The final dry matter content of the ascospore granules is evaluated using a halogen dryer.

[0054] 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 ascospore suspension with a dry matter content of 1.5 to 5.5% is carried out using a filter plate with a pore diameter of 0.8 pm, for a duration of 10 seconds to 1 minute and at a pressure of 1 to 3.5 bars, in order to obtain a semi-solid form of ascospores (ascospore cake) with a dry matter content of 29 to 32%; - the semi-solid form of ascospores 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 ascospores thus obtained having 0.9% MSS per gram of dry matter of the semi-solid mixture of ascospores; - the semi-solid mixture of ascospores obtained at the end of the step of mixing the ascospore cake with the H / W emulsion is extruded using a piston extrusion device, preferably with a length of 90 mm and a diameter of 20 mm, said extrusion device having at its end an extrusion grid with openings with a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm, which leads to extruded ascospores having the form of filaments called spaghetti; - the ascospore filaments / spaghetti are mechanically broken down into vermicelli called granules; - the ascospore granules are dried in a fluidized bed dryer where the inlet air has a temperature of 48°C, a relative humidity (RH) of 6%, the outlet air has a temperature of 36°C, an RH of 4%, and the fluidization flow rate is 14 m3 / h, said drying being carried out for a period of 7 to 12 minutes, preferably 9 minutes; - the ascospore granules obtained after fluidized bed drying have a dry matter content ranging from 95 to 96.5%.

[0055] Freeze-drying

[0056] Freeze-drying differs from other drying technologies in 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 by 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 take place in a freeze dryer.

[0057] According to another embodiment of the invention, the ascospore preparation process is more particularly characterized in that freeze-drying comprises the following steps: - addition, in the ascospore suspension having a dry matter content of 1.5 to 5.5%, of maltodextrin in an amount of 5 to 75% by weight of maltodextrin relative to the dry matter of the ascospore suspension, and preferably in an amount by weight of 75% maltodextrin; - freezing of the ascospore suspension as defined in the previous step by cooling the suspension to a negative temperature ranging from -50 to -55°C, said freezing step being carried out for a period of 5 to 7 hours, - sublimation of the ascospore suspension frozen in the previous step by applying a vacuum pressure ranging from 0.05 to 0.5 mbar in order to remove the frozen water, said sublimation step being carried out for a period of 43 to 53 hours and leading to a solid form of ascospores, preferably a powdery form; - recovery of the solid form of ascospores with a dry matter content ranging from 98 to 99.9%.

[0058] According to an advantageous embodiment of the process of the invention, the solid form of ascospores 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 solid form obtained after freeze-drying. Indeed, fractionating the powdery solid form will refine the particle size of the powder obtained directly after freeze-drying. Thus, after fractionation, an ascospore powder with a particle size ranging from 200 to 315 µm is obtained. Grinding the solid ascospore forms obtained directly after freeze-drying does not affect the dry matter content. Therefore, the dry matter content is the same for solid ascospore forms obtained directly after freeze-drying as for solid ascospore forms (powders) obtained after grinding the solid forms obtained directly after freeze-drying.

[0059] The maltodextrin added to the ascospore suspension of the invention is a cryoprotectant agent, which, as its name indicates, protects the ascospores from drying by lyophilization.

[0060] According to an advantageous embodiment, the freezing step of the freeze-drying process is carried out by decreasing the temperature of the ascospore suspension in several stages, namely a decrease: - from ambient temperature, namely ranging from 18 to 21°C, to a temperature of 12°C for a period of 5 to 15 minutes, - from a temperature of 12°C to a temperature of -1 to -2°C, for a period of 30 to 50 minutes, - from a temperature of -1 to -2°C to a temperature of -50 to -55°C, for a period of 5 to 6 hours. The frozen ascospore suspension can be kept in a frozen state for up to 5 hours. It should be noted that all the temperatures given in the freeze-drying process may vary by a few degrees (from 0.5 to 5°C) while remaining within the scope of the process of the invention.

[0061] According to an advantageous embodiment, the sublimation step of the frozen ascospore suspension at a temperature of -50 to -55°C is broken down into: - a primary drying stage lasting from 21 to 24 hours during which the vacuum pressure is 0.1 to 0.5 mbar, and - a secondary drying stage lasting from 18 to 21 hours during which the vacuum pressure is 0.05 to 0.2 mbar, which leads to a solid form of ascospores, preferably a powdery form.

[0062] According to yet another advantageous embodiment of the invention, the primary drying step is carried out by increasing the temperature of the frozen ascospore suspension in several stages, namely an increase: - from a temperature of -55 to -50°C to a temperature of -48°C, for a period of 20 to 40 minutes; - of the temperature of -48 to -5°C, for a period of 9 to 10 hours; - a temperature of -5 to 6°C, for a period of 1 to 2 hours; - at a temperature of 6 to 24°C, for a period of 10 to 11 hours, resulting in a solid form of ascospores, preferably a powdery form.

[0063] Finally, the secondary drying step may advantageously be carried out by maintaining the solid form of ascospores obtained after primary drying at a temperature of 24 to 25°C for a period of 18 to 21 hours.

[0064] Finally, the freeze-drying process is further characterized in that, at the end of the secondary drying step, the solid form of ascospores obtained, preferably in powder form, is further ground or mechanically shaken, which leads to an ascospore powder with a particle size ranging from 200 to 315 pm.

[0065] The drying processes developed by the Inventors, whether fluidized bed drying or freeze-drying, are easy to implement at on an industrial scale. They advantageously allow the production of yeast ascospores exhibiting extremely advantageous stability (i.e., the preservation of viability) over time. The dry yeast ascospores of the invention, by virtue of their granule or powder form, are easy to store, handle or transport.

[0066] Uses of the ascospores of the invention

[0067] The invention also relates to ascospores as defined above or as obtained according to the processes as defined above, for use as a medicinal product, and in particular as an anti-inflammatory agent or as an antiviral agent. Examples

[0068] The following examples refer in particular to Figures 4 to 15.

[0069] Example 1: preparation of asci by sporulation in liquid medium of a Saccharomyces cerevisiae culture

[0070] Saccharomyces cerevisiae yeast (vegetative cells) spontaneously sporulates in nutrient-deficient media. Entry into meiosis leads to the formation of asci, each ascus theoretically containing 4 ascospores. However, as already mentioned, it is also possible to obtain some asci containing only 3 or 2 ascospores. Furthermore, the culture medium after meiosis also includes vegetative yeast cells (i.e., yeast cells that have not sporulated).

[0071] The Saccharomyces cerevisiae yeast induced to sporulate is the Saccharomyces cerevisiae yeast filed on October 17, 2007 with the CNCM under number 1-3856.

[0072] Reagents used

[0073] The culture, pre-sporulation and sporulation media used in the sporulation phase induction protocol are as defined below. YEG 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 YEG solid culture medium was autoclaved for 20 minutes at a temperature of 120°C. YPG 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 YPG 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.

[0074] Material 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".

[0075] Protocol for inducing the sporulation phase of yeast L47

[0076] Pre-sporulation in liquid medium

[0077] A fresh culture of CNCM 1-3856 yeast on YEG 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 the fresh culture of the yeast on YEG solid culture medium, 50 mL of YPG culture medium is inoculated and transferred to a sterile 250 mL flask with tabs, which is placed in the incubator. The incubation period is 16 to 24 hours at a temperature of 37°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 4100 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 period is 6 to 8 hours at 30°C and 150 rpm.

[0078] Sporulation in liquid medium

[0079] The sample thus incubated (10mL) is transferred into a sterile 50 mL tube and then centrifuged for 5 min (FCR 4100 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 4100 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 is observed under a microscope after 5 days of incubation at a temperature of 25°C and agitation at 150 rpm. Serial dilutions of the sample are carried out in increments of 10: 100 pL of sample in 900 pL of physiological saline. 5 µL of several dilutions are placed on a microscope slide for observation. Photographs and cell counts are performed on the samples, allowing for the clear distinction of individual cells. After 5 days of incubation, three sporulation culture samples, samples 1 to 3, were obtained and observed under a microscope. Figure 4 shows a photograph of the sporulation medium of sample 1: asci are labeled A, while non-sporulating yeast cells (vegetative cells) are labeled V. All sporulation structures are usable for the next step of isolating ascospores / asci spores.

[0080] Sporulation rate

[0081] The sporulation rate, also called sporulation yield, is determined according to the following mathematical formula:

[0082] [Math.l] Sporulation rate (%) = [Number of asci / Total number of cells] x 100 in which, The number of asci corresponds to the total number of asci, the total number of cells corresponds to the number of cells that have not sporulated and the number of asci.

[0083] The counting of the number of asci formed in the sporulation culture samples after 5 days of incubation at 25°C is carried out 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 isolated 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.

[0084] [Tables 1] Number of asci, Total number of cells, Sporulation rate: Experiment 1: 129,202, 63.86%; Experiment 2: 131,216, 60.65%; Experiment 3: 79,180, 56.11%

[0085] Table 1 shows that the average sporulation rate of the 3 experiments is approximately 60%, which represents a very productive sporulation rate. This also means that 40% of the cells present are yeasts that did not sporulate.

[0086] 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 4100 x g. The sporulation supernatant is discarded. The pellet is collected and mixed with 20 mL of sterile physiological saline.

[0087] Example 2: Isolation of ascospores from sporulation cultures

[0088] Ascospores are isolated from the sporulation culture sample obtained in Example 1 by enzymatic digestion of the asci wall. The enzyme lyses the asci but also the yeasts because the wall structures are very similar (see [Fig. 5]).

[0089] Materials and reagents used Microscope: the same as in example 1. Sonicator marketed under the name "Ultrasonic processor 120 Watt 20 kHz".

[0090] The product used to perform the enzymatic digestion of the ascus wall is that marketed under the name "Zymolyase® 20T (20,000 U / g)" (T = 1 unit lytic activity) and is in powder form. It comprises the enzyme [3-1,3-glucan-laminaripenta-hydrolase] which has strong lytic activity on the cell walls of living cells from a multitude of yeast strains.

[0091] Ascospore isolation protocol Zymolyase solution (25 mg / mL) is added to the sporulation culture sample stored in physiological saline obtained in Example 1, at a rate of 1 mL of Zymolyase solution per 100 mL of sporulation culture sample. The sporulation culture sample is transferred to baffle flasks and incubated for 30 min at 35°C with agitation at 220 rpm. The baffle flask provides mechanical agitation that creates shocks, thus breaking down the walls of the asci weakened by the enzyme (and also by vegetative yeasts). After incubation, the sample is transferred to a sterile 50 mL tube and centrifuged for 5 min, FCR 1000 x g. The supernatant is discarded and the pellet obtained is washed with 40 mL of physiological saline at pH 11 and 0.05% of the emulsifier polysorbate 80 marketed under the name Tween® 80. The physiological saline suspension thus obtained is centrifuged for 5 min, FCR 1000 x g. The supernatant is discarded and the resulting ascospore pellet is washed with 40 mL of physiological saline at pH 11 and 0.05% of Tween® 80. The resulting ascospore suspension is centrifuged again for 5 min, FCR 1000 x g. The supernatant is discarded and the pellet is washed with 10 mL of physiological saline at pH 11 and 0.05% Tween® 80. Cellular debris (namely the asci wall or debris from the lysis of vegetative yeasts) is separated and eliminated during the various washes and centrifugations: it remains in suspension while the ascospores are found in the centrifugation pellet. The resulting ascospore suspension (10 mL) is visualized under the microscope (see [Fig.5]). The resulting ascospore suspension is stored in a sterile container at 4°C.

[0092] Ascospore count

[0093] The device used to count the number of ascospores in the ascospore suspension obtained in the previous step is the one marketed under the name Nexcelom Cellometer ® K2.

[0094] Sonication Because spores tend to clump together, sonication is performed beforehand to separate them and improve counting. The sonicator's settings are as follows: 5 min, 10 sec ON / 10 sec OFF, amplitude 50%. These settings allow for cycles of 10 seconds of sonication followed by 10 seconds without sonication, for a total duration of 5 minutes, with a wave power of 50%. Tests were conducted to ensure that sonication does not alter spore viability. Serial dilutions of the sample were prepared before and after sonication. These dilutions were spread onto Petri dishes containing YEG medium and incubated at 37°C for 48 hours. After counting the colonies on the dishes, the viability of the sample after sonication should not differ by more than one log CFU / mL from the viability of the sample before sonication. These conditions were met in the tests performed.

[0095] Ascospore counting A 20 µl sample of the ascospore suspension after sonication is taken and placed on a Cellometer® counting slide. After waiting 1 minute to allow the suspension to stabilize, the slide is inserted into the Cellometer. The "cell counting" mode is selected, and the software view is adjusted so that the spores are clearly visible. Finally, the spore count is recorded. The results obtained are illustrated in Table 2 below.

[0096] Evaluation of the dry matter content of ascospore suspensions The dry matter content of the ascospore suspension is evaluated as previously described. The results obtained are also illustrated in Table 2.

[0097] [Tables2] Number of ascospores Dry matter content Sample of ascospores from example 1 109 ascospores / mL 1.5%

[0098] The ascospore suspension as defined in Table 2 is used as a starting product in each of the drying processes of the invention, namely fluidized bed drying (example 3) and freeze-drying (example 4).

[0099] Example 3: Drying of the aqueous suspension of ascospores by fluidized bed

[0100] The fluidized bed drying process leads to the obtaining of yeast ascospores in the form of granules, the dry matter content of which varies from 95 to 96.5%.

[0101] Material used - Piston extruder with a length of 90 mm, 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 1 and 2 g of water per kg of dry air.

[0102] Process steps The starting product is the ascospore suspension as defined in Table 2, which comprises 109 ascospores per ml of suspension and a dry matter content of 1.5%. Several ascospore suspensions with the characteristics of Table 2 were combined to obtain a sufficient quantity of starting material, namely 180 mL of ascospore suspension with a dry matter content of 1.5%.

[0103] The first step of the fluidized bed drying process consists of performing a front filtration of the ascospore suspension. The filtration device used for this first stage is a bell-shaped structure 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 bell is connected to a compressed air supply. The plate and filters are pre-moistened with water before filtration begins. A quantity of 180 mL of the ascospore suspension is filtered using the aforementioned filtration device, which includes a filter plate with a pore diameter of 0.8 µm. The filtration time is less than 1 minute at a pressure of 3 bar. This step removes between 25 and 35% of the moisture from the ascospore suspension, resulting in a semi-solid form of ascospores (2 grams) with a pasty cake-like consistency (see [Fig. 6]A), and a dry matter content of 32%.

[0104] The semi-solid form of ascospores (2 g) obtained after filtration is mixed, by stirring 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% ​​of the emulsifier sorbitan monostearate (MSS), 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 form of ascospores obtained after mixing with the O / W emulsion has virtually the same appearance as the semi-solid form of ascospores obtained immediately after filtration. The semi-solid mixture of ascospores thus obtained contains 0.9% MSS per gram of dry matter of the semi-solid mixture of ascospores.

[0105] The semi-solid mixture of ascospores obtained in the previous step is then extruded using the aforementioned extrusion device, which has an extrusion grid with 0.6 mm diameter openings, producing thin filaments shaped like spaghetti (Figure 6B) which are then transformed, by mechanical / physical fractionation, into small granules shaped like tiny vermicelli (Figure 6C). Indeed, simply shaking the container containing the ascospore spaghetti causes them to break into uniformly sized ascospore strands.

[0106] The ascospore granules thus obtained (1.4 g) are placed in the aforementioned fluidized bed dryer (Figure 6D) with an air inlet temperature of 48°C and a relative humidity of 6% for 9 min, with a final temperature of approximately 36°C and an outlet relative humidity of 4%.

[0107] At the outlet of the dryer, 0.6 g of ascospore granules with a dry matter content of 96.5% are recovered, which are distributed and placed under vacuum in 3 pillboxes (figure 6E) which allows them to be kept without breaking the vacuum of the pillbox at each use.

[0108] Preservation of ascospore granules The dry ascospore granules thus obtained are stored by vacuum packaging in a sealed container, at a temperature of 4°C for several weeks in order to assess their viability over time.

[0109] Example 4: Drying of the aqueous suspension of ascospores by freeze-drying

[0110] The freeze-drying process leads to the obtaining of yeast ascospores in powder form, the dry matter content of which is 98 to 99.9%. [YES] Equipment used The freeze-drying device used is the pilot freeze dryer marketed under the name "VirTis Genesis 35L" by SP Scientific Products, which is a standard freeze-drying device.

[0112] Process steps

[0113] Preparation of ascospore formulations Four different ascospore formulations are placed in 20 ml glass pill bottles which will be used directly for freeze-drying. The first formulation is the control formulation, which is an ascospore suspension that does not include maltodextrin (cryoprotective agent), unlike the other three formulations, which are ascospore suspensions including different amounts of the cryoprotective agent maltodextrin. The maltodextrin used is that sold under the name "SOSA Maltodextrin 12DE 500g". It is in the form of a fine powder. The four ascospore formulations are prepared from the ascospore suspension as defined in Table 2 of Example 2, which has a dry matter content of 1.5%. Several ascospore suspensions with the characteristics of Table 2 were combined to obtain the quantities required for each formulation. Asco spore control formulation: A 3 mL quantity of ascospore suspension with a dry matter content of 1.5% is injected into a pillbox. A further 10 mL of physiological saline is added to the pillbox to ensure sufficient height and proper freeze-drying. The height of the suspension in the pillbox will influence the rate of water evaporation during the freeze-drying cycle. Ascospore formulation with 12% maltodextrin: A quantity of 2.90 mL of ascospore suspension with a dry matter content of 1.5% is mixed with 0.0508 g of maltodextrin. The resulting ascospore formulation, which contains 12% maltodextrin relative to the dry matter of the ascospore suspension, is injected into a pillbox and 10 mL of physiological saline is added. Ascospore formulation with 25% maltodextrin: A quantity of 3 mL of ascospore suspension with a dry matter content of 1.5% is mixed with 0.1199 g of maltodextrin. The resulting ascospore formulation, which contains 25% maltodextrin relative to the dry matter of the ascospore suspension, is injected into a pillbox and 10 mL of physiological saline is added. Ascospore formulation with 7.5% maltodextrin: A quantity of 0.5 mL of ascospore suspension with a dry matter content of 1.5% is mixed with 0.1602 g of maltodextrin. The resulting ascospore formulation, which comprises 75% maltodextrin relative to the dry matter of the ascospore suspension, is injected into a pillbox and 10 mL of physiological saline is added.

[0114] 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. Ascospore formulations obtained with maltodextrin can be considered asscospore suspensions containing maltodextrin. Therefore, the expressions "ascospore formulation containing maltodextrin" and "ascospore suspension containing maltodextrin" may be used interchangeably.

[0115] Freezing of ascospore formulations The first step in the freeze-drying process is to freeze each of the 4 ascospore formulations defined above, by cooling the ascospore formulation from room temperature to a negative temperature of -52°C. The ambient temperature is that of the laboratory and can vary from 18 to 21°C. Before placing each ascospore formulation in the freeze dryer, the freeze dryer shelves are cooled to a temperature of 4°C to prevent the freeze dryer from heating up. According to an advantageous embodiment of the invention, each of the 4 ascospore formulations is cooled: - from ambient temperature (18 to 21°C) to a temperature of 12°C in a time ranging from 5 to 15 min, which corresponds to the time required to place the formulations on the shelves of the freeze dryer and to start the freeze-drying cycle; - from a temperature of 12°C to a temperature of -1.8°C, at a rate of 0.35°C per minute and therefore for a duration of 40 minutes, - from the temperature of -1.8 to -52.1°C at a rate of 0.16°C per minute and therefore for a duration of 5h20. Thus, the cooling from ambient temperature to -52°C is carried out over a period of 6 to 6h 15. The frozen ascospore formulation is maintained at a temperature of -52°C for 2 hours.

[0116] Sublimation of frozen ascospore formulations Each frozen ascospore formulation is then subjected to a sublimation step, the first step of which consists of primary drying, which breaks down into the following sub-steps: - a first drying stage lasting 30 minutes, during which the temperature of the frozen ascospore formulation is raised from -52.1 to -48.3°C; - a second drying stage lasting 9h50, during which the temperature of the ascospore formulation is raised from -48.3 to -4.9°C; - a third drying stage lasting 1h50, during which the temperature of the ascospore formulation is raised from -4.9 to 6.4°C; - a fourth drying stage lasting 100 minutes, during which the temperature of the ascospore formulation is raised from 6.4 to 24.2°C. The vacuum pressure applied during the primary drying period is 0.2 mbar. At the end of the primary drying, the ascospore samples obtained are in solid form, preferably a powdery form.

[0117] After primary drying, the ascospore formulation is subjected to secondary drying by maintaining the temperature of the ascospore formulation at 24.2°C for 19h50 and by applying a vacuum pressure reduced to 0.1 mbar. The ascospore samples obtained after secondary drying are illustrated in [Fig. 7]. The ascospore samples obtained by freeze-drying the three formulations with maltodextrin (photos in figures 7B, 7C and 7D) have the appearance of a beautiful white, airy powder that conforms to the shape of the pillbox. After shaking the pillbox mechanically / physically, or crushing the powder with a spatula, the three ascospore samples obtained by freeze-drying the three formulations with maltodextrin were primarily in the form of a powder with a particle size ranging from 200 to 315 µm. The ascospore sample obtained after freeze-drying the control formulation, without the cryoprotectant maltodextrin (photo 7A), had a solid appearance, somewhat resembling a meringue, as it was hard on the outside with a vacuum inside. The control formulation was indeed affected by pressure variations within 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 the pressure changes. The solid form obtained after freeze-drying the control formulation is therefore not satisfactory, neither in terms of its stability nor in terms of its ability to be ground. The presence of a cryoprotectant is therefore recommended to obtain a satisfactory solid form of ascospores.

[0118] The ascospore samples obtained after freeze-drying can be stored in vacuum pillboxes directly within the freeze-drying device. Indeed, 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 ascospore samples obtained after freeze-drying the formulations (control, 12.5%, 25% and 75% maltodextrin) are evaluated with the halogen desiccator and are reported in Table 3.

[0119] [Tables3] Dry matter content. Control ascospore sample 100%. Ascospore sample with 12% maltodextrin formulation 98.04%. Ascospore sample with 25% maltodextrin formulation 100%. Ascospore sample with 75% maltodextrin formulation 99.02%.

[0120] Ascospore samples obtained after freeze-drying have a dry matter content ranging from 98 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.

[0121] As indicated, the solid forms of ascospores obtained directly after freeze-drying can still be ground, for example with a spatula, or simply shaken mechanically, which leads to an ascospore powder with a particle size ranging from 200 to 315 pm.

[0122] Preservation of ascospore powders The dry ascospore powders 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.

[0123] Example 5: Study of the viability of ascospores after drying, vacuum sealing, and storage for several weeks

[0124] The viability of ascospores 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.

[0125] Sample preparation Ascospore samples obtained after drying by fluidized bed (dry granules) or by lyophilization (dry powder) are resuspended in 10 mL of physiological saline. 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 performed 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 YEG 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 was counted. This monitoring was carried out immediately after drying and continued for up to 12 weeks of storage at 4°C under vacuum.

[0126] The results obtained are described in Table 4 for samples obtained after fluidized bed drying and in Table 5 for samples obtained after freeze-drying. The column "Live ascopsores counted on Petri dish (Log / mL)" in Tables 4 and 5 represents the live cell concentration per millilitre of physiological saline upon resuspension of dry samples. The column "Live ascospores in dry product (Log / g)" represents the live cell concentration per gram of the resuspended dry sample. The column "Actual mass of ascospores (g)" indicates the actual mass of the resuspended dry sample attributable solely to ascospores. 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 live ascospores (Log / g)" represents the actual concentration of live ascospores. Note that the columns "Live ascospores in dry product (Log / g)" and "Quantity of live ascospores (Log / g)" are identical when the ascospores have been dried using the fluidized bed technique, which is not the case when the ascospores have been dried by freeze-drying. Indeed, the latter column, "Quantity of live ascospores (Log / g)," takes into account the fact that up to 75% maltodextrin is sometimes added to the ascospore formulation. Therefore, 75% of the resuspended weight actually corresponds to maltodextrin and not to ascospores. This explains why the viability is lower if this correction is not made.

[0127] [Tables4] Drying technique Storage time Live ascospores counted on Petsi plate (Log / mQ Live ascospores in dry product (Log / 8) Actual mass of ascospore (g) Quantity of live ascospores (Log / g) Fluidized air flow Before drying 7.16 G weeks 6.17 8.09 0.0119 8.10 1 week 6.11 7.97 0.0139 7.97 2 weeks 6.19 8.15 0.0109 8.16 3 weeks 6.18 7.98 0.0159 7.98 4 weeks. 6.29 8.09 0.0159 8.09 8 weeks 6.09 8.01 0.0119 8.01 12 weeks 6.10 7.92 0.0149 7.92 16 months 6.01 8.00 0.0149 8.00

[0128] With fluidized bed drying, the loss of viability is one log (7.16 log / mL before drying versus 6.17 log / mL after drying). The ascospore sample remains stable under the tested conditions over time. When the quantity of live ascospores is expressed as a percentage of the mass of dry ascospores (column "Quantity of live ascospores (Log / g)"), viability is well maintained at around 8 log / g (approximately 10⁸ CFU / g) throughout storage, which is quite satisfactory. Fluidized bed dried ascospores therefore retain their viability well over time.

[0129] [Tables5] Drying technique Shelf life Live ascospores counted on Petri dish Live ascospores in dry product (Log / g) Ascospore mass Ï8; Quantity of live ascospores UegA; Freeze-drying ■■ Control Â' / in* drying age 7.16 0 weeks 5.68 7.51 0.0148 7.51, 1 -zemarne 5.53 7.45 8.0129 7.45 2 weeks 5.36 7.75 2.6139 7.75 3 weeks 5.92 7.81 8.2223 7.81 4 weeks 6.66 7.68 5.6149 7.8 weeks 5.85 7.54 8.0155 7.54 12 weeks Pius asset d'&hantiiiori 16 months LyGphyiisstscn -12% Maitodextrin. Before drying 7.16 6 weeks 5.79 7.71 8.8186 7.77 1 week 5.22 7.1 2.6114 7.15 2 weeks 5.33 6.96 8.0106 7.22. 3 weeks. 5.73 7.59 2.8897 7.75 4 weeks ' 91 7.71 65141 5 weeks 7.79 8.8124 7.35 22 weeks 7.87 5.6897 7.13 16 months 4.76 756 8.0816 7.&1 Freeze-Dried -25¾ Maitsdextrin Before drying 7.16 6 weeks 5.82 7.52 0.012G 7.74 1 week 5.57 7.35 8.0298 758 2 weeks 1.32 7.15 2.6113 7.27 3 weeks 5.15 7.56 8.0185 7.12 4 weeks 5.42 7.21 5.8120 7.34 8 weeks 1.46 £,-2S 3.6886 7.41 13 weeks 5.59 7.51 8.6396- 7.4 £ 16 months 5.48 7.72 2.6848 7.S4 IVsphyiisBrion -75:4 Maitcdeidnne- Before drying 7.16 5 weeks 4.72 5.68 8.8828 728 1 week 5.64 7.53 2.6633 €13 2 weeks 5.95 7.78 S,SS4S 8.38 3 weeks 5.38 7 22 5.8830 7...32 4 weeks 5.88 0.0S2E 8.44 5 weeks 5.91 7.83 8.8036 12 weeks 5.98 7.68 2.6838 8.40 16 months 5.26 7.84 8.0026 8.44

[0130] With freeze-drying, a loss of 2 log is observed for all freeze-drying conditions (7.16 log / mL before drying versus approximately 5.5 log / mL on average for all conditions after drying). Similar to the fluidized bed technique, the quantities of live ascospores remain stable over time. Ascospores dried by freeze-drying therefore retain their viability well over time. In contrast, as shown in [Fig. 8], a significantly different viability difference was observed between the control drying conditions (without maltodextrin), 12% maltodextrin, 25% maltodextrin, and the 75% maltodextrin drying condition. The control drying conditions (without maltodextrin), 12% maltodextrin, and 25% maltodextrin were compared to the 75% maltodextrin condition using a Welch's t-test for samples. independent. This type of statistical test makes it possible to determine if there is a significant difference between the means of the two groups considered. Welch's T-test implements the following mathematical formula:

[0131] [Math.2] in which, mA represents the mean of group A, mB the mean of group B, S2a represents the standard deviation of group A, S2b represents the standard deviation of group B, nA represents the number of cases in group A, nB represents the number of cases in group B.

[0132] The symbol “*” in [Fig. 8] represents a significant difference, denoted P, less than 0.05. The symbol “**” represents a significant difference less than 0.01. The lower this P value, the more different the samples are considered to be. The ascospore sample obtained after freeze-drying the 75% maltodextrin ascospore formulation is therefore the one in which ascospore viability was best preserved under all the freeze-drying conditions used. The sample obtained by fluidization (Figure 8E) is not statistically different from the 75% maltodextrin ascospore formulation.

[0133] Example 6: Use of 1.5% dry matter ascospore suspensions as an anti-inflammatory agent

[0134] The ascospore suspension as defined in Table 2 is tested for its anti-inflammatory activity.

[0135] Material used BD Biosciences “CBA Flex Set” cytokine detection kit: reference “560383” for interleukin 17A (IL-17A), “558269” for interferon gamma (IFN-y) and “558274” for interleukin 10 (IL-10).

[0136] Inactivation of suspended ascospores To compare the effect of live and dead ascospores on immune cells, a 1.5% dry matter ascospore suspension is heated for 15 minutes at 100°C. The inactivation efficiency is assessed by spreading 100 µL of the 1.5% dry matter ascospore suspension heated for 15 minutes at 100°C onto a Petri dish containing YEG medium. The Petri dish is incubated at 30°C.

[0137]

[0138]

[0139]

[0140] for 5 days. The absence of colonies developed on the Petri dish after 5 days of incubation proves that the inactivation was effective, and that the ascospores present in the suspension were indeed killed by the inactivation. Isolation of CD4+ T lymphocyte immune cells from blood CD4+ T lymphocyte immune cells are isolated from verified blood bags obtained from the French National Blood Service (EFS) using the magnetic bead technique. Magnetic beads coated with anti-CD4 antibodies are mixed with the cell population. The magnetic beads bind to the CD4-positive cells, which can then be separated from the general cell population by applying a magnetic field. Four (4) donors are used in the following experiments. The sex and age of the donors are described in Table 6. [Tables] I Donor Sex Age 9 liliii IIIM Cf llliilil Cf 66 years Evaluation of the effect of ascospore suspension on cytokine production by CD4+ T lymphocyte immune cells To perform the experiment, CD4+ T lymphocyte immune cells were seeded in a 96-well plate containing RPMI (Roswell Park Memorial Institute) 1640 culture medium, supplemented with 10% fetal wish serum added to the RPMI base medium, and incubated at 37°C and 5% CO2 for 30 minutes. A 1.5% dry matter ascospore suspension was added to some wells of the plate, and the cells were left in contact with the ascospore suspension for 1 hour. Cyclosporine A, a known immune response inhibitor at a concentration of 1 pM, was used concurrently in other wells of the plate to mimic a control for immune pathway blockade efficacy. In parallel, a 96-well plate is coated with an anti-CD3 antibody at a concentration of 10 pg / L.After the pre-incubation period, CD4+ T lymphocyte immune cells are transferred to the plate coated with anti-CD3 antibody. RPMI cell culture medium is added again to the cell-containing wells, to which an anti-CD28 antibody at a concentration of 3 pg / L is added. The CD4+ T lymphocyte immune cells are incubated for 72 hours at 37°C, 5% CO2. At the end of the 72-hour incubation period, the cell culture medium is retrieved, and cytokine levels are measured using the Flex Bead Array Cytometry (BAC) system and cytokine detection kits. Cytokines were supplied by BD Bioscience. All experiments were performed in triplicate with the same donor. Statistical analyses were performed using a t-test with GraphPad Prism version 10.2.3.

[0141] The results obtained are illustrated in figures 9 to 12. The symbols (*), (**), (***) and (****) represent a value of p less than respectively 0.05; 0.01; 0.001 and 0.0001.

[0142] [Fig.9] Without stimulation, CD4+ T lymphocytes do not secrete interleukin IL-17A into their medium (< 5 pg / mL) (9A). Upon costimulation with anti-CD3 and anti-CD28 antibodies, CD4+ T lymphocytes secrete an average of 1944.7 pg / mL of interleukin-17A into their medium (9B), indicating that the cells are indeed capable of responding to stimulation. This stimulation can be inhibited by the presence of an immunosuppressant, cyclosporine A (< 5 pg / mL) (9C). Contact with ascospores at a concentration of 3 x 10⁺⁵ cells / mL induces a significant decrease in IL-17A production by CD4+ T lymphocytes (747.3 pg / mL on average) (9D), representing a decrease of approximately 62% in secretion.This effect of decreasing IL-17A secretion by CD4+ T lymphocytes is even more pronounced when the 1.5% dry matter ascospore suspension is used at 3 x 10+6 cells / mL (9E): the average concentration is 68.66 pg / mL compared to 1944.7 pg / mL for the stimulated control (9B), representing a decrease of approximately 96.5% in cytokine secretion. Heat inactivation of the ascospores partially eliminates their inhibitory effect on immune cells, as the inactivated ascospores used at the 1 x 10+6 cells / mL concentration do not induce significant inhibition of IL-17A secretion by CD4+ T lymphocytes (9F). When inactivated ascospores are used at a concentration of 3xl0+6 cells / mL, they result in a decrease in IL-17A secretion of approximately 26.7% compared to the activation control (9G) (1424.67 pg / mL vs. 1944.7 pg / mL).

[0143] [Fig. 10] . Inhibition of 1TL-17A secretion by CD4+ T lymphocytes is accompanied by inhibition of interleukin-10 (IL-10) secretion in live ascospores as well (10D and 10E). Without stimulation, CD4+ T lymphocytes do not produce IL-10 (< 5 pg / mL) (10A). When stimulated with anti-CD3 and anti-CD28 antibodies, the cells produce approximately 6,893 pg / mL of IL-10 (10B). Cyclosporine A inhibits IL-10 production by CD4+ T lymphocytes (< 5 pg / mL) (10C). Ascospores in a 1.5% dry matter suspension used at a concentration of 3 x 10⁵ cells / mL induce a decrease in IL-10 secretion by CD4+ T lymphocytes, but this decrease is not statistically significant. (4025.67 pg / mL) (10D). This inhibition becomes statistically significant when the ascospore suspension is used at a concentration of 3 x 10⁶ cells / mL (996 pg / mL) (10E), representing an approximately 85.5% decrease in secretion. Inactivation of the ascospores destroys this ability to inhibit IL-10 secretion by CD4+ T lymphocytes (9F and 10G).

[0144] Figures 11 and 12 Since interferon-gamma (IFN-γ) secretion appeared to be highly donor-dependent, the donors were divided into two distinct groups. Identical conclusions can be drawn for both groups: unstimulated CD4+ T lymphocytes do not produce IFN-γ (1 IA and 12A). Activation of CD4+ T lymphocytes by anti-CD3 and anti-CD28 antibodies induces IFN-γ secretion (188,573 pg / mL) (11B) (54,369.5 pg / mL) (12B). This secretion can be inhibited by cyclosporine A (37.5 pg / mL) (11C), (246.5 pg / mL) (12C). The use of the ascospore suspension at 1.5% dry matter at the concentration of 3xl0+6 cells / mL induces a drastic decrease in IFN-γ secretion for the 4 donors (11E and 12E), going from 188,573 pg / mL to 30,108.5 pg / mL on average ([Fig. 11]) and from 54,369.5 pg / mL to 3,796.5 pg / mL ([Fig. 12]); i.e., decreases of 84% and 92.7% respectively.

[0145] These results demonstrate the ability of suspended ascospores to inhibit the secretion of several cytokines by T lymphocytes. This means that yeast ascospores could be used alone or in combination with other compounds for anti-inflammatory therapeutic purposes in various pathologies where a dysregulation of the immune balance is observed in favor of pro-inflammatory cytokines.

[0146] Example 7 j. Use of 1.5% dry matter ascospore suspensions as an anti-inflammatory agent

[0147] The ascospore suspension as defined in Table 2 is also tested for its anti-inflammatory activity in this example. The results obtained are illustrated in figures 13 to 15.

[0148] Materials used Thermo Fisher Scientific's "Luminex® multiplex" chemokine detection kit allows for the measurement of CXCL10, MCP-1 and IL-8 chemokines.

[0149] Cell culture Caco-2 cells are seeded in 24-well semi-permeable inserts. The cells are cultured in DMEM (Dulbecco's Modified Eagle Medium) containing glucose and glutamine, and supplemented with HEPES and 20% defibrinated fetal wish serum. The cells are cultured for 14 days in order to to obtain a monolayer of cells, with 3 changes of culture media per week, until a monolayer of cells with so-called "classical" transepithelial resistance is obtained. In parallel, THPl-Blue cells are cultured in RPMI-1640 medium containing glucose and glutamine, supplemented with HEPES and 10% fetal wish serum. The THPl-Blue cells are seeded into 24-well plates and treated to induce cell differentiation into macrophages capable of adhering to and responding to TLR (Toll-Like Receptor) stimulation. All cells are cultured in a humid atmosphere incubator at 37°C, 5% CO2.

[0150] Cell co-culture The inserts containing Caco-2 cells are placed over the wells containing THP-1 Blue cells differentiated into macrophages. The 1.5% dry matter ascospore suspension is placed in the insert (apical compartment), diluted in the culture medium. A control with the diluent of the 1.5% dry matter ascospore suspension, namely 0.9% NaCl + 0.05% Tween 80, is also diluted in the culture medium at the same concentration as that used for the 1.5% dry matter ascospore suspension (13E, 14E, and 15E). The Caco-2 cells are also treated with the positive controls of sodium butyrate (13D, 14D, and 15D).

[0151] The supernatant in the basolateral compartment is replaced with complete DMEM medium containing ultrapure LPS (Escherichia coli K12, InvivoGen) (13B, 14B, and 15B). Cells are also stimulated basolaterally with LPS combined with hydrocortisone (13C, 14C, and 15C) as a positive control, or with LPS-free medium as a negative control (13A, 14A, and 15A). After LPS stimulation, the supernatant is collected for chemokine assay using a multiplex kit (CXCL10 in [Fig. 13], IL-8 in [Fig. 14], and MCP-1 in [Fig. 15]) according to the manufacturer's instructions. All treatments are performed in triplicate. All cells are cultured in a humid incubator at 37°C, 5% CO2.

[0152] Evaluation of the effect of ascospore suspension on chemokine production by THPl-Blue macrophages

[0153] THPl-Blue macrophages produce only small amounts of CXCL10 (35.80 pg / mL) (13A), IL-8 (86.59 pg / mL) (14A), and MCP-1 (2361.96 pg / mL) (15A). Stimulation of THPl-Blue macrophages with LPS induces a high production of CXCL10 (516.56 pg / mL) (13B), IL-8 (360.66 pg / mL) (14B), and MCP-1 (6652.07 pg / mL) (15B). These productions are counteracted by treatment with hydrocortisone (128.29 pg / mL) (13C); (164.44 pg / mL) (14C); (2555.95 pg / mL) (15C) and by treatment with sodium butyrate (85.99 pg / mL) (13D); (312.05 pg / mL) (14D); (1854.75 pg / mL) (15D).

[0154] Treatment of cells with the diluent of the ascospore suspension at 1.5% dry matter, namely physiological water with 0.05% Tween 80, does not induce a decrease in chemokine production by itself (302.87 pg / mL) (13E); (336.43 pg / mL) (14E); (6036.45 pg / mL) (15E).

[0155] Treating cells with a 1.5% dry matter ascospore suspension at increasing product concentrations (11xO4 cells / mL, 11xO6 cells / mL, 11xO7 cells / mL) induces a progressively greater decrease in CXCL10 chemokine secretion, specifically 430.85 pg / mL (13F), 358.18 pg / mL (13G), and 241.36 pg / mL (13H). This decrease is significant for product concentrations of 11xO6 cells / mL (13G) and 11xO7 cells / mL (13H).

[0156] Treatment of cells with the 1.5% dry matter ascospore suspension at increasing product concentrations (1100 cells / mL, 1100 cells / mL, 1100 cells / mL) induces a progressively greater decrease in IL-8 chemokine secretion, respectively 339.42 pg / mL (14F), 271.52 pg / mL (14G), and 231.03 pg / mL (14H). This decrease is significant for the product concentrations of 1100 cells / mL (14G) and 1100 cells / mL (14H).

[0157] Treatment of cells with the 1.5% dry matter ascospore suspension with increasing concentrations of product (1xlO4 cells / mL, 1xlO6 cells / mL, 1xlO7 cells / mL) induces an increasingly sharp decrease in the secretion of MCP-1 chemokines, respectively 6039.78 pg / mL (15F), 4989.81 pg / mL (15G) and 4409.44 pg / mL (15H).

[0158] These results demonstrate the ability of suspended ascospores to inhibit the secretion of several chemokines by THPl-Blue macrophages. This means that yeast ascospores could be used alone or in combination with other compounds for anti-inflammatory therapeutic purposes in various pathologies where a dysregulation of the immune balance is observed in favor of pro-inflammatory chemokines.

[0159] Example 8: Use of 1.5% dry matter ascospore suspensions as an antiviral or antitumor agent

[0160] The ascospore suspension as defined in Table 2, heated for 15 minutes at 100°C, is tested for its antiviral activity.

[0161] The evaluation of interferon gamma (IFN-γ) secretion by CD4+ T lymphocyte cells is evaluated according to the method described in Example 6. The results obtained are illustrated in figures 11 and 12.

[0162] Since the secretion of interferon gamma (IFN-γ) appeared to be highly dependent on the donor studied, the donors were divided into two distinct groups based on their age. Identical conclusions can, however, be drawn for both groups: Unstimulated CD4+ T lymphocytes do not produce IFN-γ (1 IA and 12A). Activation of CD4+ T lymphocytes by anti-CD3 and anti-CD28 antibodies induces IFN-γ secretion (188,573 pg / mL) (11B); (54,369.5 pg / mL) (12B). Use of the ascospore suspension at a concentration of 3 x 10+5 cells / mL induces an increase in IFN-γ secretion of approximately 289.9% (546,602 pg / mL) (11D) and 131.3% (71,376.5 pg / mL) (12D). Heated ascospore suspension induces an increase in IFN-γ secretion when used at 1x0+6 cells / mL of approximately 442.9% (835,168 pg / mL) (11F) and 124.5% (67,670.5 pg / mL) (12F). Heated ascospore suspension induces an increase in IFN-γ secretion when used at 3x0+6 cells / mL of approximately 189.4% (357,196 pg / mL) (11G) and 126.4% (68,695 pg / mL) (12G).

[0163] These results demonstrate the ability of suspended ascospores to overactivate IFN-γ secretion by T lymphocytes. This means that yeast ascospores could be used alone or in combination with other compounds for therapeutic purposes to enhance the immune response, particularly in the context of viral infection. Since overexpression of the IFN-γ response is associated with improved survival in certain cancers, yeast ascospores could be used alone or in combination with other compounds for antitumor therapeutic purposes.

[0164] 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

[0165] In this application, reference is made to the following biological material and to the derived variant or mutant strains: - registration number "1-3856" 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. List of documents cited Patent documents

[0166] For all intents and purposes, the following patent document is cited: - patcitl: PCT / SE99 / 00231 (filing number). Non-patent literature

[0167] For the avoidance of doubt, the following non-patent elements are cited: - nplcitl: Neiman AM, Sporulation in the Budding Yeast Saccharomyces cerevisiae, Genetics, Volume 189, Issue 3, 1 November 2011, Pages 737-765; - nplcit2 : Elham A. Milani et al., International Journal of Food Microbiology, Volume 206, Aug. 2015, pp. 75-80 ; - nplcit3 : Alison E. Coluccio, 2008 Aug 6. doi: 10.1371 / journal.pone.0002873.

Claims

Demands

1. Ascospores of Saccharomyces cerevisiae yeast in dry form or in suspension for use as a medicinal product.

2. Yeast ascospores for use according to claim 1, wherein the drug is an anti-inflammatory agent, an antiviral agent or an antitumor agent.

3. Yeast ascospores for use according to claim 1 or 2, wherein yeast ascospores in dry form means ascospores having a dry matter content of 95 to 99.9%.

4. Yeast ascospores for use according to claim 1 or 2, wherein yeast ascospores in suspension means a suspension of ascospores having a dry matter content of 1.5 to 5.5%.

5. Ascospores of Saccharomyces cerevisiae yeast characterized in that they are in dry form and have a dry matter content ranging from 95 to 99.9%.

6. Yeast ascospores according to claim 5 or for use according to claim 3, characterized in that they have a dry matter content of 95 to 98%, and preferably 95 to 96.5%.

7. Yeast ascospores according to claim 6 or for use according to claim 3, characterized in that they are in the form of vermicelli-like granules with a length ranging from 0.25 to 1 cm and a thickness ranging from 0.25 to 0.6

8. VIII. Yeast ascospores according to claim 5 or for use according to claim 3, characterized in that they have a dry matter content of 98 to 99.9%.

9. Yeast ascospores according to claim 8 or for use according to claim 3, characterized in that they are in the form of a powder, said powder preferably having a particle size ranging from 200 to 315 pm.

10. Yeast ascospores according to any one of claims 5 to 9 or for use according to claim 3, characterized in that they are viable / stable for a period of 12 to 36 months, when stored under vacuum at a temperature of 4°C.

11. A process for preparing yeast ascospores according to any one of claims 5 to 10, characterized in that it consists of drying a suspension of ascospores having a dry matter content of 1.5 to 5.5%, said drying being carried out by fluidized bed or by freeze-drying.

12. A preparation method according to claim 11, characterized in that the ascospore suspension having a dry matter content of 1.5 to 5.5% 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, - separation of the vegetative cells and asci from the sporulation culture, said separation being carried out by centrifugation and then by resuspension of the vegetative cells and asci in physiological saline, - addition of an enzyme to the sporulation culture in order to carry out the enzymatic digestion of the cell walls of the vegetative cells, resulting in their degradation and elimination,as well as to the enzymatic digestion of the asci allowing the release of the ascospores, - density gradient centrifugation of the sporulation culture in order to separate the ascospores from the cellular debris resulting from the enzymatic digestion, - recovery of the ascospores from the centrifuge pellet and suspension of the ascospores in physiological saline, possibly with the addition of an emulsifier, in order to obtain an aqueous suspension of ascospores with a dry matter content ranging from 1.5 to 5.5%.

13. A preparation method according to claim 11, characterized in that the fluidized bed drying comprises the following steps: - front filtration of the ascospore suspension having a dry matter content of 1.5 to 5.5%, using a filter plate having a pore diameter of 0.6 to 1 pm, in order to obtain a semi-solid form of ascospores, also called "ascospore cake", having a dry matter content of 29 to 32%;

14. - mixing the semi-solid form of ascospores 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 ascospore mixture", said semi-solid ascospore mixture comprising 0.7 to 0.9% of emulsifier per gram of dry matter of the semi-solid ascospore mixture; - extrusion of the semi-solid mixture of ascospores 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 ascospores extruded in the form of filaments called spaghetti; - mechanical / physical fractionation of the ascospore filaments obtained in the previous step in order to obtain ascospore vermicelli called granules; - drying of the ascospore granules obtained in the previous step in a fluidized bed dryer where the inlet air has a temperature of 45 to 50°C and a relative humidity (RH) of 5 to 8%, the outlet air has a temperature of 36 to 40°C and a relative humidity (RH) of 4 to 7%, and the fluidization flow rate is 14 to 20 m3 / h; - recovery of ascospores with a dry matter content of 95 to 98%, preferably 95 to 96.5%. A preparation process according to claim 13, characterized in that the fluidized bed drying comprises one or all of the following features: - Frontal filtration of the ascospore suspension with a dry matter content of 1.5 to 5.5% is carried out using a filter plate with a pore diameter of 0.8 pm, for a duration of 10 seconds to 1 minute and at a pressure of 1 to 3.5 bars, in order to obtain a semi-solid form of ascospores (ascospore cake) with a dry matter content of 29 to 32%; - The semi-solid form of ascospores obtained after 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% ​​monostearate emulsifier

15. of sorbitan (MSS), until a homogeneous mixture is obtained which is in a semi-solid form, the semi-solid mixture of ascospores thus obtained having 0.9% MSS per gram of dry matter of the semi-solid mixture of ascospores; - the semi-solid mixture of ascospores obtained at the end of the step of mixing the ascospore cake with the H / W emulsion is extruded using a piston extrusion device, preferably with a length of 90 mm and a diameter of 20 mm, said extrusion device having at its end an extrusion grid with openings with a diameter ranging from 0.5 to 0.7 mm, preferably 0.6 mm, which leads to extruded ascospores having the form of filaments called spaghetti; - the ascospore filaments / spaghetti are mechanically broken down into vermicelli called granules; - the ascospore granules are dried in a fluidized bed dryer where the inlet air has a temperature of 48°C, a relative humidity (RH) of 6%, the outlet air has a temperature of 36°C, an RH of 4%, and the fluidization flow rate is 14 m3 / h, said drying being carried out for a period of 7 to 12 minutes, preferably 9 minutes; - the ascospore granules obtained after fluidized bed drying have a dry matter content of 95 to 96.5%. Preparation process according to claim 11, characterized in that the freeze-drying comprises the following steps: - addition, in the ascospore suspension having a dry matter content of 1.5 to 5.5%, of the cryoprotectant maltodextrin in an amount of 5 to 75% by weight of maltodextrin relative to the dry matter of the ascospore suspension, and preferably in an amount of 75% by weight of maltodextrin; - freezing of the ascospore suspension as defined in the previous step by cooling the suspension to a negative temperature ranging from -50°C to -55°C, said freezing step being carried out for a period of 6 to 7 hours; - sublimation of the frozen ascospore suspension from the previous step by applying a vacuum pressure ranging from 0.05 to 0.5 mbar in order to remove the frozen water, said sublimation step being carried out for a period ranging from 43 to 53 hours and leading to a solid form of ascospores, preferably a powdery form; - recovery of the solid form of ascospores, preferably a powdery form, having a dry matter content of 98 to 99.9%.

16. A preparation method according to claim 15, characterized in that the freezing step is carried out by a decrease in the temperature of the ascospore suspension in several stages, namely a decrease: - from the ambient temperature, namely from 18 to 21°C, to the temperature of 12°C for a period of 5 to 20 minutes, - from the temperature of 12°C to the temperature of -1 to -2°C, for a period of 30 to 50 minutes, - from the temperature of -1 to -2°C to the temperature of -50 to -55°C, for a period of 5 to 6 hours.

17. A preparation process according to claim 15, characterized in that the sublimation step of the frozen ascospore suspension at a temperature of -50°C to -55°C is decomposed into: - a primary drying step of a duration of 21 to 24 hours during which the vacuum pressure is 0.1 to 0.5 mbar, and - a secondary drying step of a duration of 18 to 21 hours during which the vacuum pressure is 0.05 to 0.2 mbar, which leads to a solid form of ascospores, preferably a powdery form.

18. A preparation method according to claim 17, characterized in that the primary drying step is carried out by increasing the temperature of the frozen ascospore suspension in several stages, namely an increase: - from the temperature from -55°C to -50°C to a temperature of -48°C, for a period of 20 to 40 minutes; - from the temperature from -48°C to -5°C, for a period of 9 to 10 hours; - from the temperature from -5°C to 6°C, for a period of 1 to 2 hours; - from the temperature from 6°C to 24°C, for a period of 10 to 11 hours, which leads to a solid form of ascospores, preferably a powdery form.

19. A preparation process according to claim 17, characterized in that the secondary drying step consists of maintaining the solid form of ascospores, obtained at the end of the primary drying, at a temperature of 24 to 25 °C for a period of 18 to 21 hours.

20. A preparation process according to claim 19, characterized in that, at the end of the secondary drying step, the solid form of ascospores obtained, preferably in powder form, is further ground or mechanically shaken, which leads to a powder form of ascospores having a particle size ranging from 200 to 315 pm.

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