Mixture of at least one bacteriophage and at least one yeast and method for drying same

Abstract

The present application relates to a method for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said mixture being in the form of a solid entity, each solid entity consisting of at least one yeast and / or yeast derivative, at least one bacteriophage and optionally at least one dry excipient, characterized in that the method is carried out by mixing in suspension at least one yeast and / or yeast derivative and at least one bacteriophage and drying the mixture.The present application also relates to a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said dry mixture being in the form of a solid entity, each solid entity consisting of at least one yeast and / or yeast derivative, at least one bacteriophage and optionally at least one dry excipient.

Description

[Technical field]

[0001] The present invention relates to a method for producing a dry mixture of at least one bacteriophage and at least one yeast and / or yeast derivative, a mixture comprising solid entities each consisting of at least one bacteriophage and at least one yeast and / or yeast derivative, and various uses of such a mixture. [Background technology]

[0002] Both humans and animals or plants can act as hosts for bacteria that cause bacterial-type infections. The use of bacteriophages has been chosen to treat diseases or digestive disorders of bacterial origin. For example, US Pat. No. 5,999,333 describes a method for treating or preventing gastrointestinal inflammation or pain in humans, comprising the oral administration of a composition comprising one or more bacteriophages selected from bacteriophages from the Siphoviridae or Myoviridae families. In particular, the one or more bacteriophages are selected from the LH01-Myoviridae, LL5-Siphoviridae, T4D-Myoviridae and LL12-Myoviridae families. Thus, bacteriophages can act as prebiotics that help protect the gastrointestinal flora.

[0003] (2003) deals with a bacteriophage cocktail administered to human patients to eliminate Salmonella strains from the intestine without disrupting the natural microflora, which also serves to prevent the risk of invasion of the intestinal epithelium by Salmonella.

[0004] Non-Patent Document 2 used the same bacteriophage cocktail against E. coli O157 / H7 strain. As a result of this study, the researchers concluded that such a cocktail has an antibiotic effect similar to that of ampicillin, without showing the deleterious effects on the intestinal microflora like ampicillin.

[0005] Moreover, yeast is known to play a beneficial role in both human nutrition and health, and animal or plant nutrition and health. For example, some strains of Saccharomyces cerevisiae are considered as probiotic yeasts that promote intestinal health. As seen above, bacteriophages can be used in their part to prevent or treat bacterial infections in humans, animals or plants. Therefore, it is appropriate to combine at least one bacteriophage with at least one yeast in order to prevent or treat bacterial infections, while also allowing yeasts to develop and play their nutritional, protective and stimulating role in human, animal or plant health.

[0006] An attractive innovative concept consists of combining probiotic yeasts and phages in a single product to combine their respective effects during application (antibacterial effect of the phages and protection offered by the yeasts). For example, US Pat. No. 5,399,433 deals with a composition comprising at least one type of bacteriophage as a prebiotic agent and at least one probiotic agent that may be selected from Saccharomyces boulardii or Saccharomyces cerevisiae. The bacteriophage has the role of promoting the development of beneficial bacteria by reducing the population of harmful bacteria and by releasing nutrients in its environment that are intended to be used by the beneficial bacteria of the individual's digestive system. The listed prebiotic and probiotic agents are added separately in the composition. According to this patent, each bacteriophage is specific for one undesirable bacterium. The bacteriophage therefore has no direct effect on other organisms in the digestive tract nor on the probiotics. Thus, the particular undesirable bacteria are destroyed and their cellular material is available as nutrients for the probiotic or endogenous organisms. Furthermore, by weakening the population of the particular undesirable bacteria, the probiotic organisms may successfully compete and establish colonies that produce an environment suitable for them but unsuitable for the undesirable organisms.

[0007] Presentation of the microorganism in a dry form facilitates handling, stability and long term storage, the possibility of being used in gel capsules or other presentation forms and dosages appropriate for the particular application (animal feed, food, etc.).

[0008] Currently, various methods can be used to dry living microorganisms, but not all of them make it possible to obtain a satisfactory final viability. Existing methods include, for example, freeze-drying, spray-drying and fluidized bed drying.

[0009] The Applicant has extensive knowledge of methods for obtaining yeast in dry form (quick-frozen intermediate moisture yeast, active dry yeast (ADY) and instant dry yeast (IDY)).

[0010] An example of a method for drying live microorganisms is given in US Pat. No. 5,399,633, where bacteria are co-dried with yeast. This co-drying is possible because bacteria have complex walls that are insensitive to degradation by yeast proteases.

[0011] Bacteriophages are viruses that infect bacteria. They have a wall made up only of proteins, which is thinner and more fragile than the bacterial wall. Therefore, when it involves drying a suspension of bacteriophages (one family or a mixture of phages), it is almost always necessary to carry out a support to enhance survival and / or to obtain a final dry product with the required properties (shape, granule size distribution, dry extract, porosity, solubilization or instantaneous properties, compressibility, etc.). In fact, drying in this condition is not possible, since the amount of dry matter in the suspension (originating from the dissolved bacterial culture or saline solution suitable for the preservation of the phages) is generally very low (<5%). Drying under these conditions is economically unattractive and too harmful, especially for the phages.

[0012] The support de sechage is therefore closely related to the formulation concept which involves the introduction of one or more components or supports or excipients together with the microorganism in such a way that drying is possible and easy.

[0013] Moreover, provided that the bacteriophage is in powder form, the mixture of yeast and bacteriophage in the finished industrial product needs to overcome several difficulties, such as ensuring the homogeneity of the mixed powder or filling it into gel capsules or other forms for presentation and administration in a limited volume. To avoid the problems associated with the powder form, it is possible to imagine the mixture of phage and yeast in the form of yeast cream or compressed yeast. However, yeast cream and compressed yeast are acidic (pH 5.8) and may be associated with protease activity, which may alter both the phage titer and the lytic activity of the phage against the bacterial target.

[0014] Finally, phages are biological entities that require protection against stresses commonly encountered during storage and depending on their use (application on inert or biological supports, in a protected atmosphere or in the open air, ingestion by humans or animals, etc.).

[0015] Thus, prevention of bacterial infections by bacteriophages can be complicated by the difficult conditions encountered in the animal or human stomach (pH about 2-3) or by exposure to bile and digestive enzymes in the gastrointestinal tract that render the phages inactive. Similarly, the short persistence of phages in various plant environments remains a major concern in biological control using phages directed against plant pathogens. Indeed, UV irradiation from sunlight can inactivate phages during storage, hindering their potential application as biological control agents.

[0016] It is also known that fresh yeast-based fermented foods and beverages can also be subject to bacterial contamination. As seen above, bacteriophages can be used to combat these bacteria. Among these foods and beverages, mention can be made of bakery products, wine or beer type fermented beverages.

[0017] Similarly, during the production of bioethanol, more specifically first generation bioethanol, it is necessary to control the natural flora of lactic acid bacteria, whose proliferation can have a negative effect on the production yield. The use of bacteriophages can therefore help to control the lactic acid flora. Bioethanol corresponds to ethanol produced by fermentation of agricultural products containing fermentable sugars.

[0018] There is therefore a need for a yeast and bacteriophage mixture that is resistant to pH changes and UV radiation while ensuring the activity of the yeast or yeast derivative and the lytic activity of the bacteriophage.

[0019] Phages are viruses that infect bacteria. According to the present invention, the terms "phage" and "bacteriophage" are interchangeable. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] US Patent Application Publication No. 2019 / 0255122 [Patent Document 2] US Patent Application Publication No. 2018 / 0161382 [Patent Document 3] French Patent No. 2,708,621 [Non-patent literature]

[0021] [Non-Patent Document 1] Moye et al. (A Bacteriophage Cocktail Eliminates Salmonella typhimurium from the Human Colonic Microbiome while Preserving Cytokine Signaling and Preventing Attachment to and Invasion of Human Cells by Salmonella In Vitro. J Food Prot. 2019 Aug;82(8):1336-1349) [Non-Patent Document 2] Dissanayake et al. (Bacteriophages Reduce Pathogenic Escherichia coli Counts in Mice Without Distorting Gut Microbiota. Front Microbiol. 2019 Sep 10;10:1984) Summary of the Invention

[0022] The present invention aims to improve the situation.

[0023] According to a first aspect, the object of the present invention is a method for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said mixture being in the form of a solid entity, each solid entity consisting of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and optionally at least one dry excipient, characterized in that said method is carried out by mixing in suspension the at least one yeast and / or yeast derivative and the at least one bacteriophage and drying this mixture.

[0024] According to a second aspect, the object of the invention is a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said dry mixture being in the form of a solid entity, characterised in that each solid entity consists of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and optionally at least one dry excipient.

[0025] Preferably, said mixture is obtainable by the process according to the first aspect.

[0026] According to a third aspect, the object of the invention is the use of a dry mix according to the second aspect or a dry mix obtained from the process according to the first aspect, as a medicament in a composition intended to reduce the acidity of gastric juices.

[0027] According to a fourth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the method according to the first aspect in a composition for the stimulation, protection, biocontrol and / or nutrition of plants.

[0028] According to a fifth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the process according to the first aspect in a food composition or a food supplement.

[0029] According to a sixth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the method according to the first aspect in beer brewing and / or wine brewing.

[0030] According to a seventh aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the process according to the first aspect in bread making.

[0031] According to an eighth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the method according to the first aspect in the production of bioethanol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Thus, according to a first aspect, the object of the present invention is a method for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, characterized in that said method is carried out by mixing in suspension at least one yeast and / or yeast derivative and at least one bacteriophage and by drying this mixture.

[0033] The mixture is in the form of solid entities, each solid entity being composed of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and, optionally, at least one dry excipient.

[0034] The term "compose de" is to be read as "consistant en."

[0035] Thus, according to a first aspect, the object of the present invention is a method for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, the mixture being in the form of a solid entity, each solid entity consisting of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and optionally at least one dry excipient, said method being characterized in that it is carried out by the steps of mixing the at least one yeast and / or yeast derivative with the at least one bacteriophage and drying this mixture.

[0036] Such methods allow the production of stable formulations containing the phages, optimally protecting the phages and ensuring their passage to the site of action, such as the gastrointestinal tract or the phyllosphere (aerial parts) of the plant. Dry forms are preferred due to their ease of handling and long-term storage stability, e.g. at ambient temperature, thus avoiding the need for a cold chain for storage.

[0037] Yeasts or yeast derivatives used as probiotics in animal, human and plant health have been characterized for their resistance and tolerance to several stress factors related both to production methods (upstream processes, e.g. drying, storage) and to intestinal stress conditions (gastric pH, bile and digestive enzymes).

[0038] The combination of yeast and / or yeast derivatives with phages constitutes an alternative to protect the phages by yeast or its derivatives and transport them to the target site. Thus, the yeast (or its derivatives) has a dual role: on the one hand, it plays a probiotic role, and on the other hand, it acts as a support during the drying step, as shown by the following examples. This solution helps to maximize the beneficial effect of each of the components of the combination.

[0039] Furthermore, especially in the case of use for plant protection, the mixture resulting from the process of co-drying phages with yeast and / or yeast derivatives serves to improve the protection of the phages against environmental conditions and UV effects (in vitro conditions), while also preserving the beneficial activities and properties of the yeast.

[0040] Such a mixture helps to reduce the costs associated with the use of a composition comprising both yeast or yeast derivatives and bacteriophage by optimizing the management of its stock and supply. It is therefore ready to use, reduces the risk of losses and does not result in extra costs associated with the mixing of dry yeast or yeast derivatives with bacteriophage, while avoiding the risk of handling errors in the management of microorganisms, for example dosage errors. In fact, these errors may occur during the preparation of the yeast powder mixture, especially when the user must add ferment in a specific weight ratio. The use of this mixture makes it easier to prepare a composition comprising the use of yeast or yeast derivatives.

[0041] Another notable advantage of this method compared to conventional dry / dry mixtures between at least one yeast and one bacteriophage is the homogeneity of the mixture obtained. Thus, the risk of loss of homogeneity is greatly minimized. This is because each solid entity constituting the dry mixture obtained from the method according to the invention simultaneously contains yeast or yeast derivatives and bacteriophage. This dry mixture also serves to limit the handling of the powdered product and therefore the health risks. In other words, a single dry mixture is used.

[0042] This new approach of mixing yeast with bacteriophage before drying therefore improves performance, eliminates the risk of handling errors associated with the use of several powders or microorganisms in a single location, improves practicality and simplification, reduces costs and helps to significantly limit losses following order changes and market fluctuations.

[0043] The method according to the invention can be carried out by using active yeast and / or yeast derivatives.

[0044] The term "active yeast" is synonymous with "live yeast" or "fresh yeast" and refers to a population of yeast cells that are metabolically active. When using yeast that is referred to as "fresh," activity refers to its viability.

[0045] Yeast derivatives according to the invention are defined as fractions obtained during the decomposition of yeast by physical or chemical action, for example by yeast plasmolysis, hydrolysis or autolysis. Yeast derivatives are all products that can be obtained from whole yeast cells or cells fractionated by physical or chemical action. In particular, they include yeast extracts or autolysates obtained by autolysis, yeast shells, mannoproteins, inactivated yeast. They are most often obtained in the form of a more or less fine powder after grinding or in suspension in a rehydration medium, in yeast cream or compressed yeast form. Advantageously, the yeast derivative is yeast shell or yeast extract. Even more advantageously, the yeast derivative is yeast shell.

[0046] The shells can be obtained or prepared according to techniques known to those skilled in the art, in particular by enzymatic or mechanical dissolution (separation, concentration, etc.).

[0047] In one embodiment, the shell is produced by enzymatic lysis of yeast cells (autolysis or heterolysis with their own proteolytic enzymes) followed by separation of the soluble and insoluble parts by physical means, e.g., centrifugation, and recovery of the insoluble part. The insoluble part is typically recovered by removal of the soluble part by centrifugation. The insoluble part corresponds to the yeast shell. The soluble part obtained from this method, which has a light color and low turbidity, is called yeast extract.

[0048] Preferably, the process for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage comprises the steps of: providing in a suspension at least one yeast and / or yeast derivative, preferably in cream form, and at least one bacteriophage, - mixing said yeast and / or said yeast derivative with at least one bacteriophage to form a mixture; performing a drying step of the mixture to form a dry mixture; recovering the dry mixture; The present invention is characterized by comprising:

[0049] Preferably, the bacteriophage is in suspension in an aqueous solution, preferably saline. Those skilled in the art will know how to adapt such a solution according to their needs, for example by choosing a buffered saline solution.

[0050] According to some embodiments, the drying step is carried out by freeze-drying or spray-drying or on a fluidized bed.

[0051] Preferably, the dry matter content of yeast and / or yeast derivative in the mixture prior to the drying step of the at least one yeast or one yeast derivative and at least one bacteriophage is between 20 and 60% relative to the total amount of dry matter in the mixture to be dried.

[0052] Preferably, the drying step may be preceded by a dehydration step which increases the dry matter content. This dehydration step is then followed by the actual drying step to obtain the final dry mixture to be recovered. In other words, the drying step may be carried out in two stages.

[0053] Preferably, the drying step may be followed by a step in which the dry mixture is further divided, for example by grinding.

[0054] Freeze drying serves to obtain a lyophilisate that can be ground into paillettes or into a fine powder, whereas spray drying serves to obtain a finely divided dried product.

[0055] The principle of spray drying is the dehydration of droplets in a stream of hot gas (e.g. air) circulating in a drying tower. The liquid to be dried (solution or suspension or mixture with drying extract adapted not to be too viscous) is sprayed in the form of fine droplets using a spraying device (nozzle or turbine) usually located at the top of the tower. This involves drying by entrainment, where the droplets are almost instantly converted into solid particles which, depending on the configuration of the dryer (simple, double or multiple effect), are separated from the air at the end of drying, resulting in a fine powder or atomized powder.

[0056] The air inlet temperature is generally high, for example 100-300°C, preferably 120-250°C, while the outlet air temperature, and especially the temperature of the product inside the tower, is several tens of degrees lower, since the particles surrounded by the water film cool during the state change from liquid water to steam. The skilled person knows how to adapt these temperatures based on his needs.

[0057] Nevertheless, even at lower product temperatures, this technique can be destructive for the dehydration of living products such as microorganisms. However, it is possible to partially preserve the viability by utilizing appropriate milder operating conditions (especially the addition of drying supports or additives in the formulation of the initial mixture and the choice of temperature scale). Furthermore, the transit time of the particles in the drying tower can also affect the viability and must be minimized by taking care to target a final moisture content of the powder that is compatible with the desired lifespan of the product (storage).

[0058] It is not possible to spray-dry a suspension of phages alone as they are produced (bacterial lysate) since the dry extract would be too weak, an operation that is not only economically unattractive but can also be harmful to the microorganism (use of high air temperatures). To increase the dry extract, according to the invention, yeasts and / or yeast derivatives are used, possibly with secondary components or excipients that have a specific effect (e.g. protective).

[0059] Particular attention is paid to the dry extract targeted by the preparation to be dried: the liquid itself must not exceed a certain viscosity so that it can be pumped by the spray device and converted into fine droplets.

[0060] The mixture is prepared until the components or supports are completely dissolved or dispersed, and it is dried in a spray tower as quickly as possible to avoid any product destruction or microbial growth.Preferably, the mixture is kept at a low temperature throughout the drying time.Those skilled in the art will know how to select the required temperature.

[0061] The drying parameters are adapted depending on the tower and sprayer configuration as well as the mixture properties (viscosity, dry extract), with the aim to obtain a fine powder with a final moisture content of max. 10%, preferably max. 8%, more preferably 6%.

[0062] In addition to yeast and / or yeast derivatives, additional dry excipients such as maltodextrin, natural starch, trehalose and L-leucine can also be listed.

[0063] Generally, lyophilization (freeze-drying in English) is a drying technique that allows drying under vacuum of previously frozen liquid or semi-paste products. This technique is often used for fragile products that do not tolerate direct drying. It thus makes it possible to guarantee the stability of perishable products, to stop the metabolism of biological products and to obtain powder products that can be easily rehydrated. Lyophilized products therefore have a high affinity for the solvent they contain, generally water.

[0064] From a practical point of view, this procedure involves three main steps and can therefore be referred to as a freeze-drying cycle.

[0065] The first step is the freezing of the product, which serves to solidify the matrix and in particular to crystallize the water it contains in the form of ice. For this purpose, the temperature of the product must be sufficiently lower than the temperature at which it completely solidifies. The skilled person will know how to select the required temperature.

[0066] The second stage is the primary drying or sublimation step. During this stage, the pressure in the freeze-drying chamber needs to be reduced, thus creating a high vacuum. The pressure must therefore be below the vapor pressure of ice at the temperature under consideration. In addition, the temperature of the product needs to be kept below the initial melting temperature.

[0067] The third stage is the secondary drying step, which serves to complete the dehydration by removing the final traces of water by desorption. It is characterized by the lowest possible pressure in the chamber and a high product temperature that remains below its denaturation temperature.

[0068] At this final stage it is therefore possible to obtain a dry product with a very low residual moisture content (eg <1%).

[0069] Finally, the relevant operations of the freeze-drying cycle are as follows: The preparation of the product to be lyophilized generally consists of combining it with a mixture of excipients or carriers having a protective and cryoprotective effect; Protection of the final lyophilized product, which is often unstable due to its highly porous structure and which can quickly absorb the solvents contained in it (hygroscopic in the case of aqueous products). This involves isolating the lyophilisate from the external environment and packaging it in an appropriate manner. · The phage suspension is formulated with adjuvants or excipients that have the role of support and / or cryoprotectant. The dry extract (for example about 25-30%) needs to be increased in order to concentrate the phage suspension and thus reduce the amount of water excluded. According to the invention, this increase in dry matter is made possible by mixing with yeast and / or yeast derivatives. The skilled person will know how to select the "bacteriophage / yeast and / or yeast derivative" ratio in order to obtain the best survival rate of the phage after drying. The preparation of the mixture is carried out until complete dissolution or dispersion of the excipients and / or yeast and / or yeast derivatives.

[0070] In addition to yeast and yeast derivatives, other dry excipients such as maltodextrin, natural starch, trehalose and L-leucine can be mentioned.

[0071] During this step, the mixture is thoroughly cooled (e.g. <8°C), distributed into trays or vials while maintaining a specific bed height (e.g. 15mm), and then frozen below -20°C in a freezer or deep freezer.

[0072] After checking the solidification of the mixture, which should be complete (water has completely crystallized), the container or vial is placed on the shelf of the freeze-dryer, which has been pre-cooled (e.g., to −55° C.) by starting the cold trap.

[0073] A person skilled in the art will know how to perform the drying step and adjust the temperature accordingly.

[0074] At the end of the freeze-drying cycle, the freeze-dried material generally has the appearance of a porous meringue. Optionally, the meringue is reduced to a fine powder by soft milling, preferably in a moisture-controlled enclosure (to avoid reintroducing water) before being quickly packaged under vacuum or inert atmosphere.

[0075] Preferably, the method according to the present invention further comprises the step of extruding the dewatered mixture to form an extruded mixture, and the drying step is carried out by extruding the extruded mixture in a fluidized bed to form a dry mixture.

[0076] Drying in a fluidized bed makes it possible to obtain a dry product in the form of granules or vermicelles.

[0077] Generally, the principle of drying in a fluidized bed is to dehydrate moist solid particles in a hot air stream. In this case, these solid moist particles are obtained after a granulation step that serves to generate a solid with a shape and density suitable for fluidization in air. Thus, the particles (also called granules) are found suspended in the hot air without contact, and it is the entire contact surface with the air that can be dried uniformly. The dried granules are characterized by a residual moisture content of less than 10%, preferably less than 8%, also preferably less than 5%, which provides good stability (storage) of the product over time.

[0078] The advantage of this technique is that it allows moderate drying at low temperatures, which makes it the method of choice for drying live microorganisms or fragile biological products. The size of the particles dried after the granulation step is important: the smaller the size, the faster the product will dry. The exposure time of the product to heat is also reduced, which favors a better viability level after drying.

[0079] From an application point of view, this technique is usually used to dry baker's yeast, in which case the flash dried yeast is obtained in the form of porous granules that can be very quickly rehydrated in water or in powder mixtures (flour) to which water is added.

[0080] Granulation, also called extrusion, is a step that precedes drying in a fluidized bed and can only be carried out on pasty or semi-pasty products that have a dry extract that is compatible with this operation by passing through an extruder. For example, compressed yeast with this characteristic is obtained by filtering yeast cream.

[0081] In fact, the mass to be dried is shaped in an extruder, which produces continuous thin filaments that are then broken into short vermicelli to obtain granules. If the mass to be extruded is too wet, the filaments tend to stick to each other after extrusion and it is no longer possible to dry the filaments in individualized form. At the end of drying, large agglomerates result which retain a certain amount of moisture.

[0082] A person skilled in the art will know how to adapt the moisture content of the extruded mass according to need.

[0083] For example, a way to combat this difficulty is the addition of dry excipients, preferably selected from maltodextrin, native starch, trehalose and L-leucine.

[0084] Preferably, the drying step is carried out in the presence of a drying excipient, preferably maltodextrin.

[0085] The yeast may be derived from a strain selected from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably the yeast is the Saccharomyces cerevisiae strain deposited on October 17, 2007 under number CNCM I-3856, the Saccharomyces cerevisiae strain deposited on March 22, 2018 under number CNCM I-5298, the Saccharomyces cerevisiae strain deposited on August 21, 2007 under number CNCM I-3799, the Saccharomyces cerevisiae strain deposited on August 31, 2016 under number CNCM I-5129, the Saccharomyces cerevisiae strain deposited on August 31, 2016 under number CNCM I-5130, or the Saccharomyces cerevisiae strain deposited on February 9, 2011 under number CNCM I-5131. It is derived from a strain selected from the Saccharomyces cerevisiae strain deposited under I-4444.

[0086] Preferably, the yeast is derived from a strain selected from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably the yeast is derived from a strain selected from the Saccharomyces cerevisiae strain deposited on October 17, 2007 under number CNCM I-3856, the Saccharomyces cerevisiae strain deposited on March 22, 2018 under number CNCM I-5298, and the Saccharomyces boulardii strain deposited on August 21, 2007 under number CNCM I-3799.

[0087] Preferably, the one or more bacteriophages are selected from those having antibacterial activity against bacterial strains selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or Salmonella strains, and against lactic acid bacteria, which may be selected from Lactobacillus fermentum (new classification: Limosilactobacillus fermentum), Lb. delbrueckii, Lb. Reuteri (Limosilacobacillus Reuteri), Lb. Casei (Lacticaseibacillus Casei), Lb. Brevis (Levilactobacillus Brevis), Lb. Perolens (Schleiferilactobacillus perolen) and L. amylovorus. Antibacterial activity is understood to mean the lytic activity by the bacteriophage against bacteria after bacterial infection.

[0088] "Lactic acid bacteria" is understood to mean gram-positive bacteria, which are anaerobic bacteria that are partially tolerant to oxygen and are capable of fermenting sugars to lactic acid.

[0089] Known bacteriophages that may be used are selected from T4, sold by DSMZ under catalog number DSM4505 and belonging to the Myoviridae family, T5, sold by DSMZ under catalog number DSM16353 and belonging to the Siphoviridae family, T7, sold by DSMZ under catalog number DSM4623 and belonging to the Podoviridae family, or mixtures thereof, or from the SalmoFresh™ or FOP™ phage mixture sold by Intralytix Inc. Bacteriophages T4, T5 and T7 have antibacterial activity against Escherichia coli. A cocktail of six bacteriophages belonging to the Myoviridae family, sold under the name SalmoFresh™, has antibacterial activity against pathogenic strains of the Salmonella genus, such as Salmonella enterica or even Salmonella typhimurium, Salmonella Heidelberg, Salmonella Newport, Salmonella Kentucky, Salmonella infantis. FOP™ is a unique and exclusive mixture of 15 individual lytic phages that provides broad-spectrum protection against pathogenic strains of Salmonella enterica, Escherichia coli and Listeria monocytogenes.

[0090] According to a second aspect, the present invention relates to a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, in the form of a solid entity, each solid entity being composed of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and optionally at least one dry excipient.

[0091] The term "compose de" is to be read as "consistant en."

[0092] Thus, according to a second aspect, the present invention relates to a dry mixture of at least one yeast and / or at least one yeast derivative and at least one bacteriophage, which dry mixture is in the form of a solid entity, each solid entity consisting of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and optionally at least one dry excipient.

[0093] Preferably, the mixture is obtained according to the method according to the first aspect.

[0094] Preferably the yeast derivative is yeast shell.

[0095] Preferably, the dry mixture is divided into a powder form comprising the solid ingredients and, optionally, dry excipients.

[0096] Preferably, the solid entities have the form of flakes, grains, vermicelli or granules.

[0097] Preferably, the dry excipient is selected from maltodextrin, native starch, trehalose and L-leucine.

[0098] Preferably, the yeast and bacteriophage are selected from those mentioned above.

[0099] According to a third aspect, the object of the invention is the use of a dry mix according to the second aspect or a dry mix obtained from the process according to the first aspect, as a medicament in a composition intended to reduce the acidity of gastric juices.

[0100] According to a fourth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the method according to the first aspect in a composition for the stimulation, protection, biocontrol and / or nutrition of plants.

[0101] More specifically, it is for treating or protecting plants against diseases produced or induced by pathogens, in particular fungi, bacteria or viruses, and for inducing or stimulating the natural defenses in the plant against pathogens.

[0102] According to a fifth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the process according to the first aspect in a food composition or a food supplement.

[0103] According to a sixth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the method according to the first aspect in beer brewing and / or wine brewing.

[0104] According to a seventh aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the process according to the first aspect in bread making.

[0105] According to an eighth aspect, the object of the present invention is the use of a dry mix according to the second aspect or a dry mix obtained from the method according to the first aspect in the production of bioethanol.

[0106] Materials and Methods T4 bacteriophage or T4 phage belonging to the Myoviridae family with a long contractile tail T5 bacteriophage or T5 phage belonging to the family Siphoviridae with a long noncontractile tail T7 bacteriophage or T7 phage belonging to the family Podoviridae with a short noncontractile tail

[0107] These three phages are lytic phages that infect E. coli.

[0108] SalmoFresh™ is a unique and exclusive mixture of six individual lytic phages that provides broad-spectrum protection against pathogenic strains of the Salmonella genus, including Salmonella typhimurium, Salmonella Heidelberg, Salmonella Newport, Salmonella Kentucky, and Salmonella infantis.

[0109] FOP™ is a unique and exclusive mixture of 15 individual lytic phages that provides broad-spectrum protection against pathogenic strains of Salmonella enterica, Escherichia coli and Listeria monocytogenes.

[0110] The list of microorganisms used is given in Table 1 below. [Table 1]

[0111] The list of reagents used is shown in Table 2 below. [Table 2]

[0112] (Simulated gastric fluid (SGF)) Simulated gastric fluid is prepared (for 100 mL) according to the studies of Ma et al. (2008), Colom et al. (2015) and Vinner et al. (2018). 0.2 g NaCl 0.4g pepsin (500U / mg) ·Distilled water QS 100mL Adjust pH with HCl or NaOH to achieve pH lines: 2.5, 3.0, 3.5, 4.0

[0113] Enzyme activity (U / mL) was reproduced according to the study of Adouard et al. (2019). Once prepared, preheat the liquid to 37 °C.

[0114] For co-dried samples, pour 50 mL of SGF into a 180 mL jar and add 0.5 g of sample, which corresponds to a 1 in 100 dilution.

[0115] For phage in solution (control), pour 9.9 mL of SGF into a 60 mL jar and add 100 µL of phage solution into it. This corresponds to a 1 in 100 dilution. Dilute the phage solution to obtain the phage concentration closest to that of the co-dried sample.

[0116] The jars are then incubated at 37° C. with agitation (100 rpm) to mimic passage through the stomach. At time zero (immediately after adding the sample) and at 15, 30, 60, 120 minutes, phage counts by spot counting according to the method described below are performed, remembering to agitate the jars before withdrawing aliquots for dilution.

[0117] Yeast counts are also performed in gastric juice at pH 2.5 at the beginning (T0) and end (T120) of the experiment according to the method described.

[0118] At the end of the experiment the pH is measured again.

[0119] (Simulated intestinal fluid (SIF)) Simulated intestinal fluid is prepared (for 100 mL) according to the studies of Ma et al. (2008), Colom et al. (2015) and Vinner et al. (2018). 0.68g KH2PO4 1g porcine bile 2.16g pancreatin ·Distilled water QS 100mL pH adjusted to 6.8 with NaOH

[0120] Enzyme activity was reproduced according to the work of Adouard et al. (2019). Once prepared, preheat the liquid to 37 °C.

[0121] For the co-dried samples, 50 mL of SIF was poured into a 180 mL jar and 0.5 g of sample was added thereto, which corresponds to a 1 in 100 dilution.

[0122] For phages in solution (control), 9.9 mL of SIF is poured into a 60 mL jar and 100 μL of phage solution is added thereto, which corresponds to a 1 / 100 dilution. The phage solution is diluted to obtain a concentration of phage as close as possible to that of the co-dried sample. The jar is then incubated at 37 °C with stirring (100 rpm) to mimic intestinal passage. At time zero (immediately after addition of the sample) and at 30, 60, 120 min (Minekus et al. 2014), phage counts by spot according to the method described in the following paragraphs are performed, without forgetting to stir the jar before withdrawing an aliquot for dilution.

[0123] Yeast counts are also performed at the beginning (T0) and end (T120) of the experiment according to the methods described.

[0124] At the end of the experiment, the pH is measured again.

[0125] (Co-dried phage and yeast counts) Counting of phages co-dried with yeast is performed after a step of rehydrating the powder in a Stomacher blender, according to a method adapted from an internal procedure (counting of bacteriophage per PFU).

[0126] (Rehydration of co-dried samples) Weigh out 1 g of the co-dried phage-yeast sample using a precision balance and place it in a Stomacher bag; · 9 mL of 37°C sterile distilled water is added to the bag, which is then placed in a Stomacher blender at medium speed for 3 minutes.

[0127] (In-depth phage counting technique) TSA+YE (tryptocasein soy agar (TSA) + yeast extract (YE)) gelatin is dissolved in 6g / L of agar + cycloheximide (0.5%) and kept in a supercooled state; Prepare a 90 mm Petri dish containing approximately 15 mL of TSA+YE+cycloheximide (0.5%) medium and let it dry under the hood for 30 min. Inoculate 20 mL of TSB+YE (tryptocasein soy broth (TSB) + yeast extract (YE)) broth with a colony derived from a fresh culture; Incubate with agitation until a culture is obtained at the beginning of the exponential growth phase (e.g. for E. coli, Salmonella and Listeria, cultures are stopped at a DO of 0.2-0.7). · Read the DO value. · Prepare 1 / 10 dilutions decimals of the rehydrated co-dried samples in Eppendorf™ tubes containing 900 μL of SM solution. To a 13 mL tube containing 100 µL of host bacteria solution (DO between 0.2-0.7), add 100 µL of the desired phage dilution and leave in contact for 10 min. Add 3mL of TSA+YE medium, 6g / L agar+cycloheximide and pour into a pre-filled, dried gelatin dish. Leave to set. Incubate for 24 hours at the growth temperature of the host bacteria.

[0128] A technique for counting phages by spotting (Preparation of agar dishes) · Dissolve gelatin with 6g / L agar, then add 0.5% cycloheximide and keep it in a supercooled state. Inoculate 20 mL of TSB+YE broth with a colony derived from a fresh culture. Incubate with agitation until a culture is obtained at the beginning of the exponential growth phase (e.g. for E. coli, Salmonella and Listeria, stop the culture at a DO of 0.2-0.7), prepare a square Petri dish with 25 mL of TSA+YE+cycloheximide (0.5%) medium and dry it under the hood for 30 min, · Read the DO value. To a 13 mL tube containing 300 µL of host-bacteria solution (DO between 0.2-0.7), add 9 mL of TSA+YE medium, 6 g / L agar+cycloheximide and pour into a pre-poured and dried gelatin dish. Leave it until it hardens.

[0129] (Preparation of 1 / 10 dilution of phage solution) Fill columns of wells of a 96-well microplate with 180 μL of SM buffer, starting with column 3 and skipping every other well, to allow for spot seeding. To the wells of the first column, 100 μL of the solution resulting from the rehydration of the sample are added, which now constitutes a first dilution of 1 / 10. Using a multichannel pipette, collect 20 μL in the first column and transfer it to the wells of the next dilution column, continuing until the final dilution.

[0130] (Deposition on the plate) From one microplate line (96 wells), 10 μL should be collected with an appropriate multichannel pipette.

[0131] Next, a square Petri dish previously seeded with the host bacteria is divided into four columns and 10 μL spots of the phage / yeast solution are deposited, allowed to dry and incubated for 24 h at the growth temperature of the host bacteria.

[0132] Yeast counts are performed in parallel.

[0133] After rehydration of the sample, 100 μL is withdrawn and a 1 / 10 dilution is made in an Eppendorf™ tube filled with 900 μL of sterile distilled water.

[0134] Collect 100 μL of the desired dilution and place it on a YM gelatin dish and smear.

[0135] The dishes are then incubated at 25° C. for 3 hours.

[0136] Regardless of the technique, the results are expressed as follows:

[0137] Theoretical titer PFU / gDM (before drying): This titer is calculated from the phage assay titer of the phage suspension and from the actual composition of the preparation achieved in the laboratory taking into account all added components (e.g. yeast, fractions, protectants, additives). To avoid bias, this titer is referred to as the dry matter of the mixture (before drying).

[0138] Assayed Potency PFU / gDM (after drying): This is the actual potency as assayed from the dried end product and referred to as the actual extract of the dried end product.

[0139] Phage loss (Log10 PFU / g) is the difference between: Log10 (theoretical titer before drying PFU / g DM) - Log10 (assay titer after drying PFU / g DM) and is expressed as Log10 PFU / g.

[0140] (Gastrointestinal resistance test) Simplified in vitro gastrointestinal resistance tests were performed on the co-dried samples. The goal of these tests is to analyze the viability of phages and yeasts by counting over time at different time points in simulated gastrointestinal fluid. These tests repeat the counting method described above, but without the rehydration step with a Stomacher blender, which is now performed directly in the simulated fluid.

[0141] 0.5g of sample was placed in 50mL of liquid. The pH line was adjusted to 2.5-4.0 for gastric testing.

[0142] (Potentiel d'alcalinisation) In order to define which excipients are responsible for increasing intragastric pH, co-dried samples were generated by lyophilizing SalmoFresh™ solution with each phage excipient individually in equal proportions (99% DM excipient vs. 1% DM phage). Samples were generated in the laboratory.

[0143] 0.5 g of each sample was contacted with 50 mL of simulated gastric fluid at pH 2.5. After homogenization, the pH of each preparation was measured with a pH meter after homogenization.

[0144] (UV resistance test) UV resistance tests were performed on rehydrated co-dried samples using a Stomacher blender (1 g in 9 mL of sterile distilled water) for 3 min and on phages in solution in a UV-BS-03+UV-MA chamber with a UV-C bulb. The measured irradiance was 12 mW / cm. 2 For each sample format, three identical aliquots were made for three different exposure times (5, 15, and 30 min). These tests are adapted according to the tests carried out by the team of Ramirez et al. (2018). The viability of the phages and yeast was measured by counting methods.

[0145] The counts at TO are the same for the co-dried sample and its rehydrated form, which are performed according to the spotting method described above, and the counts of phage in solution are performed according to internal procedures.

[0146] For the co-dried samples, weigh out approximately 1.5 g and spread it in an open Petri dish in the center of the chamber. After exposure to UV, weigh out 1 g on a precision balance, then rehydrate in a Stomacher blender for 3 min and count the phages according to the method of spotting described above.

[0147] For the rehydrated co-dried sample, place 1 mL into an open Petri dish in the center of the chamber. After exposure to UV, withdraw 100 µL and count the phages following the spotting method above.

[0148] For phages in solution, place 1 mL into an open Petri dish in the center of the chamber. The phage concentration in the solution is as close as possible to the phage concentration in the co-dried sample by dilution. After exposure to UV, withdraw 100 µL and count the phages according to internal procedures.

[0149] Yeast counts are performed at T0 and T30 according to the described method.

[0150] (Stability test) The stability of phage-yeast co-dried products stored in packets or pills under vacuum was studied over a 3-month period under three temperature and humidity conditions: 25℃ / 60%RH 30℃ / 65%RH 40℃ / 75%RH

[0151] Phage counting by in-depth counting technique was performed at different time points (days 0, 15, 30, 60, and 90). w (Water activity) was measured at 25°C on days T0, 30 and 90 using an AquaLab Series 4TEV dew point hygrometer.

[0152] Water activity is an important parameter related to the quality of dried products. w The value is a measure of the potential for microbial growth and toxin production. Water activity values ​​vary between 0 (product dried to a non-reactive state because all water is bound to the product) and 1 (pure water with no solutes).

[0153] In fact, a w indicates the proportion of free water in the product, i.e. the proportion of water available for the growth of microorganisms, for example. w The higher the water activity, the more water is available for the growth of these microorganisms. Water activity values ​​above 0.6 can promote the growth of microorganisms. EXAMPLES

[0154] Example 1: General method for obtaining a dried yeast-bacteriophage mixture by drying in a fluidized bed The steps of the method are as follows: Control the pH of the yeast cream or yeast derivative, optionally adjusting the pH to 7.0 with 10% sodium hydroxide Filter the yeast cream on a plate filter under pressure to obtain compressed yeast (PY) Mixing the PY with a phage suspension and a drying or protecting agent (also called a drying excipient); - Granulate and mold by extrusion Dry the granules in a fluidized air bed dryer. Control the final moisture content of dried granules and package them under vacuum or inert atmosphere

[0155] Thus, 16.5 g of SalmoFresh™ phage suspension (0.6% dry matter), 2 g of trehalose dihydrate, 2 g of maltodextrin and 0.065 g of L-leucine are added to 200 g of compressed yeast (30% dry extract). After mixing for 1 minute in a Matfer AlphaMix 5L mixer beater with a movable dashboard for mixing, 60 g of starch (potato starch) is added and the mixture is mixed for 1 minute. The mixed mass, which contains about 40% dry extract, is then extruded on a DRC i10 extruder equipped with an extrusion grid with 1 mm openings. The resulting filaments are collected in a 5L beaker and manually broken by vigorous shaking. Drying is carried out in a Fluid Bed Dryer Tornado M501 (Sherwood) equipped with a multi-chamber support. 2 x 80 g of wet granules are placed into two glass drying chambers (60 mm diameter and 400 mm length) equipped with a 250 mesh stainless steel sieve (fluidized air inlet) at the base. A drying program is started and includes two steps, each carried out at a temperature of 40-60 °C. The fluidized and heated air is dehydrated by an air dehumidifier (Munters ComDry M190Y).

[0156] Drying is completed when the granules have a dry extract of at least 94%. The packaging of the granules is done under vacuum to ensure the best stability of the product over time.

[0157] Example 2: General method for obtaining a dried yeast-bacteriophage mixture by spray drying The steps of the method are as follows: Preparing the mixture by adding yeast or yeast derivatives and, optionally, drying the components in the phage suspension Mix vigorously until all ingredients are completely dissolved or dispersed. Control the pH of the mixture, possibly adjusting it to 7.0 with 10% sodium hydroxide Dry the mixture in a spray tower · Control the final moisture content and package the fine powder under vacuum or inert atmosphere

[0158] Therefore, a suspension of yeast shells is partially reconstituted by dispersing 75.3 g of Safmannan™ yeast shells in 336 g of demineralized water (Preparation 1). The pH of the yeast shell suspension could optionally be adjusted to 7.0 with 10% sodium hydroxide. Preparation 1 is cooled to a temperature between 2 and 10°C.

[0159] Next, 42 g of trehalose dihydrate and 2.7 g of L-leucine are dissolved in 112 g of warm demineralized water (Preparation 2). After cooling Preparation 2, 39 g of SalmoFresh™ phage suspension (2.5% dry extract) is added and mixed under vigorous stirring. This mixture is then added to Preparation 1 under vigorous stirring to obtain a mixture to be dried containing phage, yeast or yeast derivatives and various supports or components. This mixture is kept stirred at low temperature throughout the process.

[0160] Drying is carried out in a Mini Spray Dryer B-290 (Buchi) tower equipped with a compressed air two-fluid nozzle, a peristaltic pump, a cyclone to separate the dried particles from the moist air, and a polyester outlet filter. The equipment is complemented by an air dehumidifier (Munters ComDry M190Y) that treats the air at the inlet to the spray tower.

[0161] The operating parameters (air temperature and flow rate, feeding rate of the drying solution, etc.) are adjusted to dry the phages under moderate conditions by exposing them to moderate temperatures, thus controlling the outlet temperature not to exceed 60°C.

[0162] Drying is completed when the moisture content of the powder is less than 6%. The spray dried powder is packaged under vacuum.

[0163] Example 3: General method for obtaining a dried yeast-bacteriophage mixture by freeze-drying The steps of the method are as follows: -Preparing the mixture by adding yeast or yeast derivatives and possibly other components to the phage suspension Mix vigorously until all ingredients are completely dissolved or dispersed. Control the pH of the mixture, possibly adjusting it to 7.0 with 10% sodium hydroxide - Rapidly cool and freeze to at least -20°C Dry the frozen mixture in a freeze dryer under reduced pressure Grinding the freeze-dried material and controlling the final moisture content · Packaging micronized dry products under vacuum or inert atmosphere.

[0164] Thus, the pH of the freshly collected yeast cream (21.3% dry extract) is adjusted to 7.0 with 10% sodium hydroxide. A protection solution containing supports or excipients is prepared by dissolving 18.9 g of trehalose dihydrate, 18.9 g of maltodextrin and 0.63 g of L-leucine in 51.1 g of warm demineralized water. 7.9 g of a suspension of T5 phage (2.3% dry extract) is added to this protection solution after it has cooled. The mixture is kept under stirring and cooled. This mixture is added, while continuing to stir, to 113 g of the aforementioned yeast cream and cooled. It contains about 27.4% dry extract.

[0165] The liquid yeast / phage / protectant mixture is dispensed into glass trays or vials such that the layer height within the container does not exceed 15 mm. All containers are cooled to a temperature ≦-20° C. and quickly frozen in a freezer.

[0166] After several hours of freezing, the containers are placed into a laboratory freeze-dryer (Lyovapor L-200 Buchi) with pre-cooled freeze-drying shelves and chamber.

[0167] As discussed above, primary drying is followed by secondary drying, with the freeze-drying process being stopped at the end of 80-96 hours. The freeze-dried material is pulverized gently using a mortar mill and optionally passed through a 500 μm mesh sieve. The final moisture content must be controlled to be <3%. Packaging under vacuum is done quickly to avoid changes in the product over time.

[0168] The mixtures used in the tests described below were dried according to one of the three methods described in Examples 1-3.

[0169] In the context of the stability test, the counts were performed using the phage in-depth counting technique. In the context of the other tests, the counts were performed using the spot counting technique and a starting sample that constituted a 1 / 100 dilution. The detection limit of this method is therefore 4 Log (hereafter referred to as ND).

[0170] When confronted with acidic conditions, phages appear to become suddenly inactive below a certain threshold located around pH 3.5 (Ma et al. 2008, Davis et al. 1985). Therefore, four pH value lines (2.5, 3.0, 3.5, 4.0) were tested to straddle this threshold and to analyze the full spectrum that can be found in the fasting stomach or after absorption of a digestive bolus (gastric resistance test in gastric juice).

[0171] Regardless of the drying mode used, the T5 bacteriophage or the SalmoFresh™ bacteriophage cocktail retained activity that was fully satisfactory for industrial use. Similar results were obtained with the T4 and T7 bacteriophages as well as the FOP cocktail.

[0172] Example 4: Gastric resistance test in gastric juice with mixtures obtained by co-drying S. cerevisiae CNCM I-3856 yeast or yeast shells with T5 bacteriophage or SalmoFresh™ bacteriophage cocktail by spraying [Table 3]

[0173] Phage alone in solution are inactive at pH 4, but phages co-dried with yeast can be counted from pH 3 onwards.

[0174] At pH 2.5, none of the forms of phage tested were able to maintain activity.

[0175] At pH 3, T5 phage co-dried with yeast CNCM I-3856 remained active for at least 30 min before falling below the detection threshold.

[0176] At pH 3.5, after 2 hours, the loss of T5 phage co-dried with yeast CNCM I-3856 is seen as a higher Log PFU / g.

[0177] At pH 4, T5 phage co-dried with yeast CNCM I-3856 is in a more stable environment. Note that the concentration of PFU / g of the sample can increase between T0 and T30. This is most likely due to the fact that powders (especially spray-dried powders) can form aggregates that do not always dissolve very quickly.

[0178] Yeasts from samples in contact with gastric juice at pH 2.5 were counted at the beginning (T0) and end (T120) of the experiment. The results are similar for all tests. After 2 hours in an acidic environment, the yeast concentration decreases very slightly. [Table 4]

[0179] Example 5: Gastric resistance test in gastric juice using a mixture obtained by co-drying yeast with SalmoFresh™ bacteriophage cocktail by spraying [Table 5]

[0180] Comparing the gastric resistance of the phage solutions alone (T5 and SalmoFresh™), better survival was observed for the phages present in the SalmoFresh™ solution. It is also worth noting that the SalmoFresh™ solution contains a cocktail of phages, and the sensitivity of each phage to pH may differ.

[0181] Note that spray co-drying with yeast allows the phage to remain active for 30 min even at pH 2.5 before moving below the detection threshold.

[0182] At pH 3.0, 3.5 and 4.0 the samples retain activity over the 2 hour period of the experiment.

[0183] Similarly, yeast from samples in contact with gastric juice at pH 2.5 were counted at the beginning (T0) and end (T120) of the experiment. The results are similar for all tests. After 2 hours in an acidic environment, the yeast concentration decreases very slightly. [Table 6]

[0184] Example 6: Gastric resistance testing in gastric juice using mixtures obtained by co-drying yeast shells with T5 bacteriophage or SalmoFresh™ bacteriophage cocktail [Table 7]

[0185] These samples were prepared by co-drying with spray drying. Phage viability appears to be the same for pH 3.0, 3.5 and 4.0, even though the acidity is less neutralized by the samples compared to other spray drying studies using yeast CNCM I-3856.

[0186] At pH 3, T5 phage was observed to survive for 60 minutes, while phage from the SalmoFresh™ solution survived throughout the 120 minutes of the experiment. A possible hypothesis is that the powder obtained from these tests is even less soluble than the spray-dried powder obtained with live yeast, so that diffusion of the phage occurs later, which may be responsible for its protection. This hypothesis could also explain why more phage are counted after 30 minutes of exposure than in the counts performed at T0 minutes.

[0187] Example 7: Resistance testing in intestinal fluids using mixtures obtained by co-drying yeast with T5 bacteriophage or SalmoFresh™ bacteriophage cocktail [Table 8]

[0188] The intestinal resistance test is based on the same principle as the gastric test, except that only one pH is tested (pH 6.8), showing that intestinal fluids do not affect the viability of the phage or yeast. [Table 9]

[0189] (Example 8: Alkalization Potential Test) To determine which component from the formulation has this alkalizing effect that helps protect the phage in acidic media, five samples were made, all of which consisted of 1% SalmoFresh™ phage in DM (dry matter) / TDM (total dry matter) and 99% of the single excipient used. 0.5 g of these samples were contacted with 50 mL of gastric juice at pH 2.5. After dissolving the powder, the pH was measured.

[0190] The dry samples with yeast and L-leucine caused significant pH variations. The measured pH was 3.59, and the sample containing yeast was even able to exceed the inactivation threshold of 3.5. It can be observed that it is the yeast that constitutes the most effective dry support and is therefore present in significant amounts in the various formulations, unlike L-leucine. [Table 10]

[0191] (Example 9: UV resistance test) [Table 11]

[0192] Phages in solution and co-dried with yeast by spray drying were exposed to UV-C for 30 min and updated at 0, 5, 15, 30 min to count the phages. Counts were performed using the same samples by spotting as in the test for gastric resistance. The detection limit of this technique is 3 Log, since the first dilution available was a 1 / 10 dilution (derived from rehydration by Stomacher).

[0193] It can be observed that the phages in solution (T5 and SalmoFresh™ phages) are very sensitive to UV-C. Phages from SalmoFresh™ solution are no longer active after 5 minutes of exposure, and T5 phages only last for 5 minutes. Conversely, phages co-dried with yeast are only slightly affected by UV-C.

[0194] By counting the yeast before and after a 30 min exposure it was possible to show that for all the configurations tested, UV-C exposure did not affect their viability.

[0195] Example 10: Stability study over time A 3-month stability study was conducted on samples co-dried by spray drying at 25°C / 60% RH, 30°C / 65% RH and 40°C / 75% RH. w (Water activity) and phage loss were measured at the beginning (T0 day), middle (T30 day) and end (T90 day) of the experiment. [Table 12] [Table 13]

[0196] a w A very low value of indicates that the percentage of water released in the resulting product is very low, low enough to prevent microbial growth and thus ensure the stability of the product over time.

Claims

1. 1. A method for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said mixture being in the form of solid entities, each solid entity consisting of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and optionally at least one dry excipient, characterized in that the method is carried out by mixing the at least one yeast and / or yeast derivative and the at least one bacteriophage in suspension and drying the mixture.

2. 1. A method for producing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, comprising: - providing in suspension at least one yeast and / or yeast derivative, preferably in cream form, and at least one bacteriophage; - mixing said at least one yeast and / or yeast derivative with said at least one bacteriophage to form a mixture; - carrying out a drying step of said mixture to form a dry mixture; recovering the dry mixture; A method comprising:

3. 3. The method according to claim 1 or 2, characterized in that the drying step is carried out by freeze-drying or spray-drying or on a fluidized bed.

4. 3. The method according to claim 1 or 2, characterized in that the drying step is carried out in the presence of a drying excipient, preferably maltodextrin.

5. 3. The method according to claim 1 or 2, characterized in that the yeast is derived from a strain selected from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably from a strain selected from the Saccharomyces cerevisiae strain deposited on October 17, 2007 under number CNCM I-3856, the Saccharomyces cerevisiae strain deposited on March 22, 2018 under number CNCM I-5298, and the Saccharomyces boulardii strain deposited on August 21, 2007 under number CNCM I-3799.

6. The method described in claim 1 or 2, characterized in that one or more of the bacteriophages are selected from those having antibacterial activity against bacterial strains selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or Salmonella strains, and lactic acid bacteria.

7. 3. The method according to claim 1 or 2, characterized in that the yeast derivative is yeast shell.

8. A dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, characterized in that the dry mixture is in the form of a solid entity, each solid entity consisting of at least one yeast and / or at least one yeast derivative, at least one bacteriophage and, optionally, at least one dry excipient.

9. 9. The dry mix of claim 8, wherein the solid entities have the form of flakes, grains, vermicelli or granules.

10. 10. The dry mixture according to claim 8 or 9, characterized in that the yeast is derived from a strain selected from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably from a strain selected from the Saccharomyces cerevisiae strain deposited on October 17, 2007 under number CNCM I-3856, the Saccharomyces cerevisiae strain deposited on March 22, 2018 under number CNCM I-5298, and the Saccharomyces boulardii strain deposited on August 21, 2007 under number CNCM I-3799.

11. The dry mixture described in claim 8 or 9, characterized in that one or more of the bacteriophages are selected from those having antibacterial activity against bacterial strains selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or Salmonella strains, and lactic acid bacteria.

12. 10. A dry mixture according to claim 8 or 9, characterized in that the yeast derivative is yeast husk.

13. 10. The dry mixture according to claim 8 or 9 for use as a medicament.

14. 10. A dry mixture according to claim 8 or 9 for use in treating the acidity of gastric juices.

15. 9. A dry mix according to claim 8 or obtained from the method according to claim 1 for use in a composition for the treatment of acidity of gastric juices.

16. 10. Use of the dry mix according to claim 8 or the dry mix obtained from the method according to claim 1 in a composition for the stimulation, protection, biocontrol and / or nutrition of plants.

17. 10. Use of the dry mix according to claim 8 or the dry mix obtained from the method according to claim 1 in a food composition or a food supplement.

18. 10. Use of the dry mix according to claim 8 or the dry mix obtained from the method according to claim 1 in beer brewing and / or wine brewing.

19. 10. Use of the dry mix according to claim 8 or the dry mix obtained from the method according to claim 1 in bread making.

20. 10. Use of the dry mix according to claim 8 or the dry mix obtained from the method according to claim 1 in the production of bioethanol.