A mixture of at least one bacteriophage and at least one yeast, and their drying process

Abstract

This application relates to a process for manufacturing a dry mixture of at least one yeast and / or a yeast derivative and at least one bacteriophage, said process being characterized in that it is carried out by mixing at least one yeast and / or a yeast derivative and at least one bacteriophage in suspension and drying this mixture. It also relates to a dry mixture of at least one yeast and / or a yeast derivative and at least one bacteriophage, characterized in that it is in the form of solid entities, each solid entity being composed of at least one yeast and / or at least one yeast derivative and at least one bacteriophage and optionally at least one drying excipient, and its uses.

Description

Title of the invention: Mixture of at least one bacteriophage and at least one yeast and their drying process Technical field

[0001] The present invention relates to a method for manufacturing a dry mixture of at least one bacteriophage and at least one yeast and / or yeast derivative, a mixture comprising solid entities each of which is composed of at least one bacteriophage and at least one yeast and / or yeast derivative and different uses of such a mixture. Prior art

[0002] Both humans and animals or plants can act as hosts for bacteria giving rise to bacterial infections. The use of bacteriophages has been adopted to treat diseases or digestive disorders of bacterial origin. For example, document US20190255122 describes a method for treating or preventing gastrointestinal inflammation or pain in a human being comprising the oral administration of a composition comprising one or more bacteriophages chosen from bacteriophages of the Siphoviridae family or the Myoviridae family. In particular, the bacteriophage(s) are chosen from UïQl-Myoviridae, EL5-Siphoviridae, T4-D-Myoviridae and EL12-Myoviridae. The bacteriophages would therefore play a role as prebiotics making it possible to protect the gastrointestinal microflora.

[0003] Moye et al. (A Bacteriophage Cocktail Eliminates Salmonella typhimurium from the Human Colony 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) discusses bacteriophage cocktails administered to human patients to eliminate Salmonella strains from the intestine without disrupting the indigenous microbiota. This cocktail also prevents the risk of Salmonella invasion of the intestinal epithelium. The bacteriophage cocktail studied in this article was prepared from three phage preparations commercially available from In-tralytix, Inc.: ListShield™, EcoShield PX™, and SalmoFresh™.

[0004] Dissanayake et al. (Bacteriophages Reduce Pathogenic Escherichia coli Counts in Mice Without Distorting Gut Microbiota. Front Microbiol. 2019 Sep 10; 10:1984) used the same bacteriophage cocktail against the Escherichia coli O157 / H7 strain. The researchers concluded that such a cocktail has a similar antibiotic effect to ampicillin without showing as detrimental an effect on the gut microbiota as the latter.

[0005] Furthermore, it is known that yeasts have a beneficial role in human nutrition and health, as well as in animal nutrition and health or plant nutrition and health. For example, certain strains of Saccharomyces cerevisiae are considered probiotic yeasts that promote intestinal health. As seen above, bacteriophages can be used to prevent or treat a bacterial infection in humans, animals or plants. It is therefore appropriate to combine at least one bacteriophage and at least one yeast to prevent or treat bacterial infections while allowing the yeasts to develop and play their nutritional, protective and stimulating role in human, animal or plant health.

[0006] An attractive innovation concept consists of combining probiotic yeast and phages in the same product to combine their respective effects during application (antibacterial effect of the phages and protection offered by the yeasts). For example, document US20180161382 relates to compositions comprising at least one type of bacteriophage as a prebiotic agent and at least one probiotic agent that can be chosen from Saccharomyces boulardii or Saccharomyces cerevisiae. The bacteriophage has a role as a promoter in the development of beneficial bacteria by reducing the populations of harmful bacteria and by releasing nutrients into its environment intended to be used by the beneficial bacteria in the digestive system in an individual. The prebiotic and probiotic agents cited are added separately within the composition. According to this document, each bacteriophage has a specificity for an undesirable bacteria.Therefore, as such, bacteriophages do not directly affect any other organisms in the digestive tract or any probiotics. Thus, specific unwanted bacteria would be lysed and their cellular material is available as nutrients for the probiotic or endogenous organism. Furthermore, by weakening the population of the specific unwanted bacteria, the probiotic organisms can successfully compete and establish colonization producing an environment that is suitable for them but inhospitable to the unwanted organism.

[0007] The presentation of microorganisms in dry form promotes handling, long-term storage stability and the possibility of being used in capsules or in other forms of presentation and dosages adapted for specific applications (animal feed, foodstuffs, etc.).

[0008] Currently, different methods can be used for drying living microorganisms but not all of them allow a satisfactory final viability rate to be obtained. Among the existing methods, there are, for example, freeze-drying, spray drying and drying in a fluidized air bed.

[0009] The applicant has extensive knowledge of the processes for obtaining yeasts in dry form: frozen intermediate humidity yeast, dry yeast active (ADY for active dry yeast), active instant yeast (IDY for instant dry yeast).

[0010] An example of a method for drying living microorganisms is given in document FR 2708621 in which bacteria are co-dried with yeasts. Bacteria have complex walls that are not very sensitive to degradation by yeast proteases, which allows this co-drying.

[0011] Bacteriophages are viruses that infect bacteria. They have walls made up solely of proteins, walls that are thinner and more fragile than those of bacteria. Thus, when it comes to drying a suspension of bacteriophages (a family or a mixture of phages), in most cases it is necessary to use supports to promote survival and / or obtaining a final dry product with the required properties (shape, particle size, dry extract, porosity, solubilization or instantaneous properties, compressibility, etc.). It is in fact impossible to dry as is because the dry matter content of the suspension (from a culture of lysed bacteria or a saline solution suitable for preserving phages) is generally very low (< 5%). Drying under these conditions would not be economically advantageous and, above all, too harmful for the phages.

[0012] Thus the drying support is closely linked to the notion of formulation which amounts to implementing one or more ingredients or supports or excipients with the microorganism so that drying is possible and easy.

[0013] Furthermore, and provided that bacteriophages are available in powder form, the mixture of yeast powders and bacteriophages in a finished industrial product would require overcoming certain difficulties such as ensuring the homogeneity of the mixed powder or filling capsules or other presentation and dosage forms with a limited volume. To avoid the problems associated with a powder form, it is possible to imagine a mixture of phages with yeasts in the form of yeast cream or pressed yeast. However, yeast cream and pressed yeast are acidic (pH 5.8) and could be associated with a protease activity which could modify both the phage titer and the lytic activity of the phages on the bacterial target.

[0014] Finally, phages are biological entities requiring protection against stresses generally encountered during storage and depending on their use (application on an inert or living support, in a protected atmosphere or in the open air, ingestion by a human or an animal, etc.).

[0015] Thus, the prevention of bacterial infections using bacteriophages can be complicated by the harsh conditions found in the animal or human stomach (pH ~ 2-3), as well as by exposure to bile and digestive enzymes in the gastrointestinal tract, which render the phages inactive. Similarly, the transient persistence of phages in different plant environments remains a concern. major challenge in biological control using phages against plant pathogens. Indeed, UV irradiation from sunlight can inactivate the phage during storage and hinder its potential application as a biological control agent.

[0016] It is also known that foods and beverages fermented with fresh yeast can also be subject to contamination by bacteria. As seen above, bacteriophages can then be used to combat these bacteria. Among these foods and beverages, bakery products or fermented beverages such as wine or beer can be mentioned.

[0017] There is therefore a need for mixtures of yeasts and bacteriophages having resistance to pH variations and UV rays while ensuring the activity of the yeasts or yeast derivatives and the lytic activity of the bacteriophages.

[0018] A phage is a virus that infects a bacterium. According to the invention, the terms “phage” and “bacteriophage” are interchangeable. Summary of the invention

[0019] The invention improves the situation.

[0020] According to a first aspect, the present invention relates to a method for manufacturing a dry mixture of at least one yeast and / or one yeast derivative and at least one bacteriophage, said method being characterized in that it is carried out by mixing at least one yeast and / or one yeast derivative and at least one bacteriophage in suspension and drying this mixture.

[0021] According to a second aspect, the present invention relates to a dry mixture of at least one yeast and / or one yeast derivative and at least one bacteriophage, characterized in that it is in the form of solid entities, each solid entity being composed of at least one yeast and / or at least one yeast derivative and at least one bacteriophage and optionally at least one drying excipient.

[0022] Preferably, said mixture is obtained by the method according to the first aspect.

[0023] According to a third aspect, the present invention relates to a use of the dry mixture according to the second aspect or the dry mixture obtained from the method according to the first aspect as a medicament in a composition for decreasing the acidity of gastric juice.

[0024] According to a fourth aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect in a composition for the stimulation, protection, biocontrol and / or nutrition of plants.

[0025] According to a fifth aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect in a food composition or in a food supplement.

[0026] According to a sixth aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect in brewing and / or in oenology.

[0027] According to a seventh aspect, the present invention relates to a use of the dry mix according to the second aspect or of the dry mix obtained from the process according to the first aspect in breadmaking. Statement of the invention

[0028] Thus, according to a first aspect, the present invention relates to a method for manufacturing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said method being characterized in that it is carried out by mixing at least one yeast and / or yeast derivative or yeast derivative and at least one bacteriophage and drying this mixture.

[0029] Such a method makes it possible to produce stable formulations containing phages ensuring their optimal protection and their delivery to the sites of action, such as the gastrointestinal tract or the phyllosphere of plants (aerial parts). Dry forms are preferred due to their ease of handling and their long-term storage stability, for example at ambient temperatures, thus avoiding the need for a cold chain for storage.

[0030] Yeasts or yeast derivatives, used as probiotics in animal, human and plant health have been characterized for their tolerance and resistance to several stress factors associated with both manufacturing procedures (downstream processes, e.g. drying, storage) and stressful conditions of the intestinal tract (gastric pH, bile and digestive enzymes).

[0031] The combination of yeast and / or yeast derivative and phage constitutes an alternative to protect the phages by the yeast or its derivatives and transport them to the target site. Thus, the yeasts (or its derivatives) have a dual role: on the one hand they play the role of probiotics and, on the other hand, they serve as a support during the drying step, as shown by the examples below. This solution makes it possible to maximize the beneficial effects of each of the components of the combination.

[0032] Furthermore, and in particular in the case of use for plant protection, the mixture obtained by the process of co-drying phage with yeast and / or yeast derivatives makes it possible to improve the protection of the phages against environmental conditions and UV effects (in vitro conditions), while retaining the activity and beneficial properties of the yeast.

[0033] Such a mixture makes it possible to reduce the costs associated with the use of the compositions comprising both yeasts or yeast derivatives and bacteriophages by optimizing stock management and supply. It is therefore ready to use, reduces the risk of losses and does not generate additional costs related to mixing dry yeasts or yeast derivatives with bacteriophages, while avoiding the risk of handling errors in the management of microorganisms, such as dosage errors. Indeed, these errors can occur in particular during the preparation of yeast powder mixtures when the user must add the ferments in specific mass proportions. The use of this mixture facilitates the preparation of compositions involving the use of yeasts or yeast derivatives.

[0034] Another notable advantage of this method compared to a conventional dry / dry mixture between at least one yeast and one bacteriophage lies in the homogeneity of the mixture obtained. Thus, the risk of loss of homogeneity is greatly minimized. This is due to the fact that each solid entity making up the dry mixture obtained using the method according to the invention simultaneously comprises yeasts or yeast derivatives and bacteriophages. This dry mixture also makes it possible to limit the handling of powdered products and therefore to limit health risks. In other words, a single dry mixture is used.

[0035] Thus, this new approach of mixing yeasts with bacteriophages before drying them allows for improved performance, elimination of the risks of handling errors linked to the use of several powders or micro-organisms on the same site, practicality and simplification, cost reduction, and very significant limitation of losses following changes in orders and market fluctuations.

[0036] The method according to the invention can be implemented using active yeasts and / or yeast derivatives.

[0037] The term “active yeast,” which is synonymous with “live yeast” or “fresh yeast,” refers to a population of yeast cells that are metabolically active. When so-called “fresh” yeasts are used, activity refers to their viability.

[0038] A yeast derivative according to the invention is defined as a fraction obtained during the degradation of yeast by physical or chemical action, for example by plasmolysis, hydrolysis or autolysis of the yeast. Yeast-derived products are all products capable of being obtained from whole or fractionated yeast cells, by physical or chemical action. They include in particular yeast extracts obtained by autolysis or autolysates, yeast cell walls, man-noproteins, inactivated yeasts. They are most often in the form of a more or less fine powder after grinding or in suspension in a rehydration medium, in the form of yeast cream or pressed yeast. In a completely Advantageously, the yeast derivative is yeast hull or yeast extract. Even more advantageously, the yeast derivative is yeast hull.

[0039] Yeast hulls can be obtained or prepared according to techniques known to those skilled in the art, in particular by enzymatic lysis or by mechanical lysis (separation, concentration, etc.).

[0040] In one embodiment, the hulls are produced by enzymatic lysis (autolysis by their own proteolytic enzymes or heterolysis) of yeast cells followed by separation of the soluble and insoluble parts, for example by physical means, such as centrifugation, and recovery of the insoluble part. The insoluble part is thus typically recovered by removal of the soluble part by centrifugation. The insoluble part corresponds to the yeast hulls. The soluble part resulting from this process, which is light in color and has low turbidity, is called yeast extract.

[0041] Preferably, the process for manufacturing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage is characterized in that it comprises:

[0042] - providing at least one yeast and / or yeast derivative, preferably in the form of cream, and at least one bacteriophage in suspension;

[0043] - mixing said yeast and / or yeast derivative with said at least one bacterium riophage so as to form a mixture;

[0044] - carrying out a step of drying the mixture so as to form a dry mixture;

[0045] - recover the dry mixture.

[0046] Preferably, the bacteriophages are suspended 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.

[0047] According to certain embodiments, the drying step is carried out by freeze-drying or atomization or on a fluidized air bed.

[0048] Preferably, the level of dry matter of yeast and / or yeast derivative in the mixture before the step of drying at least one yeast or yeast derivative and at least one bacteriophage is between 20 and 60% relative to the total quantity of dry matter in the mixture to be dried.

[0049] Preferably, the drying step may be preceded by a dehydration step to increase the dry matter content. This dehydration step is then followed by a drying step proper to obtain the final dry mixture to be recovered. In other words, the drying step would be carried out in two stages.

[0050] Preferably, the drying step may be followed by a step in which the dry mixture is further divided, for example, by grinding it.

[0051] Drying by lyophilization makes it possible to obtain a lyophilizate which can be ground under form of flakes or fine powder whereas spray drying produces a finely divided dry product.

[0052] The principle of spray drying is to dehydrate liquid droplets in a stream of hot gas (air for example) which circulates in a drying tower. The liquid to be dried (solution or suspension or mixture, with a dry extract adapted so as not to be too viscous) is nebulized in the form of fine droplets using an atomization device (nozzle or turbine) which is generally placed at the top of the tower. This is a drying by entrainment, the droplets are transformed almost instantly into solid particles which are separated from the air at the end of drying to obtain a fine powder or a micro-granulated powder, depending on the configuration of the dryer (single, double or multiple effect).

[0053] If the air inlet temperatures are generally high, for example between 100 and 300°C, preferably between 120 and 250°C, the temperature of the outlet air and especially the temperature of the product inside the tower is several tens of degrees lower because the particles, which are surrounded by a film of water, cool during the change of state from liquid water to vapor. A person skilled in the art will be able to adapt these temperatures according to his needs.

[0054] Nevertheless, even if the product temperature is lower, this technique can be destructive for the dehydration of living products such as microorganisms. It is however possible to partially preserve viability by implementing operating conditions (in particular the addition of a support or drying additives in the formulation of the initial mixture and choice of temperature scale) that are adapted and more gentle. Furthermore, the residence time of the particles in the drying tower can also impact viability; it will therefore be necessary to minimize it, but taking care to aim for a final humidity of the powder that is compatible with the desired shelf life of the product (preservation).

[0055] Spray drying of the phage suspension alone as obtained (bacterial lysate) is not possible because the dry extract is too low and the operation would not be economically advantageous but could also be harmful to the micro-organisms (use of high air temperatures). In order to increase the dry extract, according to the invention, yeasts and / or yeast derivatives are used, and possibly secondary ingredients or excipients having a particular effect (protective for example).

[0056] Particular attention will be paid to the dry extract targeted by the preparation to be dried: the liquid must not exceed a certain viscosity in order to be able to be pumped and transformed into fine droplets by the atomization device.

[0057] The preparation of the mixture is carried out until the ingredients or supports are completely dissolved or dispersed and it will be dried in an atomization tower as quickly as possible. possible to avoid any degradation of the product or microbial proliferation. Preferably the mixture will be kept at a low temperature throughout the drying period. A person skilled in the art will know how to choose the necessary temperature.

[0058] The drying parameters are adapted according to the configuration of the tower and the atomization device but also the characteristics of the mixture (viscosity, dry extract), the aim being to obtain a fine powder with a maximum final humidity of 10%, preferably a maximum of 8%, more preferably 6%.

[0059] In addition to yeasts and / or yeast derivatives, additional drying supports may be mentioned: such as maltodextrin, native starch, trehalose, L-leucine.

[0060] Generally speaking, freeze drying is a drying technique that allows the vacuum drying of liquid or semi-pasty products that have been previously frozen. This technique is often used for fragile products that cannot withstand direct drying, and thus ensures the stability of perishable products, stops the metabolism of biological products, and produces powdery products that are easily rehydratable. Thus, a freeze-dried product has a very high affinity for the solvent it contains (generally water).

[0061] From a practical point of view, this operation involves 3 important phases and we therefore speak of a freeze-drying cycle.

[0062] The first phase is a freezing operation of the product which allows the matrix to solidify and, above all, to crystallize the water it contains in the form of ice. For this, it is necessary to lower the temperature of the product sufficiently below its total solidification temperature. A person skilled in the art will know how to choose the necessary temperature.

[0063] The second phase is a primary drying or sublimation step. During this phase it is necessary to lower the pressure in the freeze-drying chamber and therefore to create a high vacuum. Thus the pressure must be lower than the vapor pressure of the ice, at the temperature considered. Furthermore, the temperature of the product must remain lower than the starting melting temperature.

[0064] The third phase is a secondary drying step which allows dehydration to be completed by eliminating the last traces of water by desorption. It is characterized by the lowest possible pressure in the chamber and a high product temperature which will remain below its denaturation temperature.

[0065] Thus with this last phase it will be possible to obtain dry products with very low residual humidity (for example < 1%).

[0066] Finally, the additional operations of the freeze-drying cycle are:

[0067] - the preparation of the product to be lyophilized which generally consists of combining it with a mixture of excipients or carriers with protective and cryoprotective effect;

[0068] - the protection of the final freeze-dried product which is often unstable and can resume ra quickly the solvent it contained (hygroscopicity if aqueous product) due to its highly porous structure. In this case, it is a question of isolating the lyophilisate from the external environment by packaging it appropriately.

[0069] - the phage suspension is formulated with adjuvants or excipients which will have a role as a support and / or cryoprotective agent. It is necessary to increase the dry matter (for example around 25-30%) to concentrate the phage suspension and thus reduce the amount of water to be eliminated. According to the invention, this increase in dry matter is made possible by mixing with yeasts and / or yeast derivatives. Those skilled in the art will know how to choose a “bacteriophage / yeast and / or yeast derivative” ratio in order to obtain the best viability of the phages after drying.

[0070] - the preparation of the mixture is carried out until the ex are completely dissolved or dispersed containers or yeast and / or yeast derivative.

[0071] In addition to yeasts and yeast derivatives, other drying supports can be cited: such as maltodextrin, native starch, trehalose, L-leucine.

[0072] During this step the mixture will be completely cooled (for example < 8°C) and distributed in trays or bottles respecting a certain layer height (for example 15 mm), before undergoing the freezing phase at at least -20°C in a freezer or deep freezer.

[0073] After checking the solidification of the mixture, which must be complete (water completely crystallized), the containers or bottles are placed on the shelves of the freeze dryer, previously cooled by starting the cold trap (for example -55°C).

[0074] The person skilled in the art will know how to choose how to implement the drying steps and adapt the temperatures accordingly.

[0075] At the end of the freeze-drying cycle, the freeze-dried product generally has the appearance of a porous meringue. Depending on the case, this meringue is reduced to a fine powder by gentle grinding and this operation will preferably be carried out in a humidity-controlled enclosure (to prevent it from absorbing water), before rapid packaging under vacuum or in an inert atmosphere.

[0076] Preferably, the method according to the invention further comprises a step of extruding the dehydrated mixture so as to form an extruded mixture, the drying step being carried out in a fluidized bed on the extruded mixture so as to form a dry mixture.

[0077] Fluidized bed drying makes it possible to obtain a dry granular product or in the form of vermicelli.

[0078] Generally speaking, the principle of fluidized bed drying is to dehydrate wet solid particles in a stream of hot air. These wet solid particles are obtained in this case after a granulation step which allows to obtain a shape and density of solid suitable for fluidization in air. The particles (also called granules) are thus suspended in the hot air without touching each other and it is their entire surface of contact with the air which can be dried uniformly. The dry granules are characterized by a residual humidity of less than 10%, preferably 8%, more preferably 5%, which ensures good stability over time of the products (preservation).

[0079] The advantage of this technique is that it can be used for gentle drying at low temperatures, making it a method of choice for drying living microorganisms or fragile biological products. The size of the particles to be dried, after the granulation step, is important, and the smaller it is, the faster the product will dry. The product's exposure time to heat will also be reduced, which will promote a better viability rate after drying.

[0080] From an application point of view, this technique is commonly used for drying baker's yeast and in this case, instant dry yeast is obtained in the form of porous granules which are capable of rehydrating very quickly in water or in a powdery mixture (flour) to which water is added.

[0081] Granulation, also called extrusion, is the step preceding fluidized bed drying and can only be carried out on pasty or semi-pasty products having a dry extract compatible with this operation of passing through a die. For example, the filtration of a yeast cream gives pressed yeast which has this property.

[0082] In fact, the mass to be dried is shaped in a granulator (also called an extruder) which produces continuous fine filaments, which are then fragmented into short vermicelli to obtain the granules. If the mass to be extruded is a little too wet, the filaments tend to stick together after extrusion and it will no longer be possible to dry them in individual form: at the end of drying, large aggregates will be obtained which will retain a certain humidity.

[0083] A person skilled in the art will know how to adapt the humidity of the mass to be extruded according to his needs.

[0084] For example, one way to counter this difficulty is the addition of a drying excipient. Preferably, the drying excipient is chosen from maltodextrin, trehalose, native starch or L-leucine.

[0085] Preferably, the drying step is carried out in the presence of a drying excipient, preferably maltodextrin.

[0086] The yeast may be derived from a strain chosen from the species Saccharomyces ce-revisiae and Saccharomyces boulardii, preferably the yeast is derived from a strain chosen from the strain Saccharomyces cerevisiae deposited on October 17, 2007 under number CNCM 1-3856, the strain Saccharomyces cerevisiae deposited on March 22, 2018 under number CNCM 1-5298, the Saccharomyces boulardii strain deposited on August 21, 2007 under number CNCM 1-3799, the Saccharomyces cerevisiae strain deposited on August 31, 2016 under number CNCM 1-5129, the Saccharomyces cerevisiae strain deposited on August 31, 2016 under number CNCM 1-5130 or the Saccharomyces cerevisiae strain deposited on February 9, 2011 under number CNCM 1-4444.

[0087] Preferably, the yeast is derived from a strain chosen from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably the yeast is derived from a strain chosen from the strain Saccharomyces cerevisiae deposited on October 17, 2007 under the number CNCM 1-3856, the strain Saccharomyces cerevisiae deposited on March 22, 2018 under the number CNCM 1-5298 and the strain of Saccharomyces boulardii deposited on August 21, 2007 under the number CNCM 1-3799.

[0088] Preferably, the bacteriophage(s) are chosen from those having antibacterial activity against strains of bacteria chosen from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium per-fringens or strains of the genus Salmonella. By antibacterial activity is meant a lytic activity on the part of the bacteriophage on the bacterium following infection of the bacterium by the latter.

[0089] Known bacteriophages that can be used are chosen from T4 marketed by DSMZ under the reference DSM 4505 and belonging to the Myoviridae family, T5 marketed by DSMZ under the reference DSM 16353 and belonging to the Siphoviridae family or T7 marketed by DSMZ under the reference DSM 4623 and belonging to the Podoviridae family, or a mixture of these or from the SalmoFresh™ or FOP™ phage mixtures marketed by Intralytix Inc. The bacteriophages T4, T5 and T7 have antibacterial activity against VE scherichia coli. The cocktail of 6 bacteriophages belonging to the Myoviridae family marketed under the reference SalmoFresh™ has antibacterial activity against pathogenic strains of the genus Salmonella, for example Salmonella enterica or Salmonella typhimurium, Salmonella Heidelberg, Salmonella Newport, Salmonella Kentucky, Salmonella infantis.FOP™ is a unique and proprietary blend of fifteen individual lytic phages that provide broad protection against pathogenic strains of Salmonella enterica, Escherichia coli and Listeria monocytogenes.

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

[0091] Preferably, the mixture is obtained according to a method described according to the first aspect.

[0092] Preferably, the yeast derivatives are yeast hulls.

[0093] Preferably, the dry mixture is divided into powder form which comprises solid entities and, possibly, a drying excipient.

[0094] Preferably, the solid entities are in the form of flakes, grains, vermicelli or granules.

[0095] Preferably, the drying excipient is chosen from maltodextrin, trehalose, native starch or L-leucine.

[0096] Preferably, the yeasts and bacteriophages are chosen from those described above.

[0097] According to a third aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect as a medicament in a composition intended to reduce the acidity of gastric juice.

[0098] According to a fourth aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect in a composition for the stimulation, protection, biocontrol and / or nutrition of plants.

[0099] More particularly, this is for the treatment or protection of plants against diseases produced or caused by pathogenic agents, in particular fungal, bacterial or viral, for the induction or stimulation in a plant of natural defenses against pathogenic agents.

[0100] According to a fifth aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect in a food composition or in a food supplement.

[0101] According to a sixth aspect, the present invention relates to a use of the dry mixture according to the second aspect or of the dry mixture obtained from the process according to the first aspect in brewing and / or in oenology.

[0102] According to a seventh aspect, the present invention relates to a use of the dry mix according to the second aspect or of the dry mix obtained from the process according to the first aspect in breadmaking. Materials and methods

[0103] Bacteriophage T4 or Phage T4 belonging to the Myoviridae family which have a long contractile tail

[0104] Bacteriophage T5 or Phage T5 belonging to the Siphoviridae family which have a long non-contractile tail

[0105] Bacteriophage T7 or Phage T7 belonging to the Podoviridae family which have a small non-contractile tail

[0106] These 3 phages are lytic phages that infect Escherichia coli.

[0107] SalmoFresh™ is a unique and proprietary blend of six individual lytic phages which offer broad protection against pathogenic strains of the genus Salmonella, for example Salmonella enterica or Salmonella typhimurium, Salmonella Heidelberg, Salmonella Newport, Salmonella Kentucky, Salmonella infantis.

[0108] FOP™ is a unique and proprietary blend of fifteen individual lytic phages that provide broad protection against pathogenic strains of Salmonella enterica, Escherichia coli and Listeria monocytogenes.

[0109] The list of microorganisms used is given in Table 1 below.

[0110] [Tables 1] Names Type Reference Phage T4 Bacteriophage DSMZ 4505 Phage T5 Bacteriophage DSMZ 16353 Phage T7 Bacteriophage DSMZ 4623 SalmoFresh™ Bacteriophages Mixture of 6 phages produced by Intralytix targeting pathogenic strains of the genus Salmonella FOP™ Bacteriophages Mixture of 15 phages produced by Intralytix targeting Salmonella enterica, Escherichia coli and Listeria monocytogenes Salmonella enterica Bacteria SN388 Escherichia coli Bacteria Escherichia coli DSMZ 613 (Migula 1895) Castellani andChalmers 1919 Escherichia coli Bacteria Escherichia coli 342-5 Listeria monocytogenes Bacteria LM 114 000689 Saccharomyces cerevisiae Yeast CNCM 1-4444 (filed on February 9, 2011) Saccharomyces cerevisiae Yeast CNCM 1-3856 (filed on October 17, 2007) Saccharomyces boulardii Yeast CNCM-L3799 filed on August 21, 2007

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

[0112] [T ableaux2] Name Reference CAS No. Sodium Chloride Merck 31434 7647-14-5 Magnesium Sulfate Heptahydrate Merck M2773 ​​10034-99-8 Cycloheximide 1% 66-81-9 Potassium Phosphate Monobasic Merck P0662-500G-M 7778-77-0 Tris-HCl IM (pH7.5) Merck T2319 Gelatin 2% Merck G1393 9000-70-8 Hydrochloric Acid 37% Sigma 320331-500ml Sodium Hydroxide Merck 30620-1KG-M Yeast Extract Procel 351 pw Trypto Casein Soy Broth bioMérieux 51019 Trypto Casein Soy Agar bioMérieux 51044 Agar BD 214530 Difco YM Agar BD 271210 Porcine Bile Extract Sigma B88631-100G 8008-63-7 Pancreatin from Porcine Pancreas 4X USP Sigma P1750-100G 8049-47-6 Pepsin 500U / mg Sigma 77160-100G

[0113] Simulated Gastric Fluid (SGF)

[0114] Simulated gastric fluid is prepared according to the work of Ma et al. (2008), Colom et al. (2015) and Vinner et al. (2018) (per 100ml):

[0115] - 0.2g NaCl

[0116] - 0.4g pepsin (500 U / mg)

[0117] - Distilled water QSP 100 ml

[0118] - pH adjusted with HCl or NaOH pH range achieved: 2.5, 3.0, 3.5, 4.0

[0119] The enzymatic activities (U / ml) were reproduced according to the work of Adouard et al.2019. Once prepared, the liquid is preheated to 37°C.

[0120] For co-drying samples 50ml of SGF is poured into a 180ml pot and 0.5g of the sample is added, which corresponds to a 1E-02 dilution.

[0121] For the phages in solution (controls) 9.9 ml of SGF are poured into a 60 ml pot in which 100 pl of phage solution are added, which corresponds to a 1E-02 dilution, the phage solution is diluted to obtain a concentration of phages closest to that of the co-dried sample.

[0122] The pots are then incubated at 37°C with shaking (100 rpm) to mimic the passage through the stomach. At times 0 (just after adding the sample), 15, 30, 60, 120 min, spot phage counts are carried out using the method described below, remembering to shake the pot before taking the aliquot for dilution.

[0123] Yeast counts are also carried out in the gastric fluid at pH 2.5 at the start (T0) and at the end (T 120) of the experiment according to the method described.

[0124] The pH is remeasured at the end of the experiment.

[0125] Simulated Intestinal Fluid (SIF)

[0126] Simulated intestinal fluid is prepared according to the work of Ma et al.2008, Colom et al.2015 and Vinner et al.2018 (for 100ml):

[0127] - 0.68g KH2PO4

[0128] - 1g of porcine bile

[0129] - 2.16g of pancreatin

[0130] - Distilled water QSP 100 ml

[0131] - pH adjusted to 6.8 with NaOH

[0132] The enzymatic activities were reproduced according to the work of Adouard et al. 2019. Once prepared, the liquid is preheated to 37°C.

[0133] For co-drying samples 50ml of SIF is poured into a 180ml pot into which 0.5g of the sample is added, which corresponds to a 1E-02 dilution.

[0134] For the phages in solution (controls) 9.9 ml of SIF are poured into a 60 ml pot in which 100 pl of phage solution are added, which corresponds to a 1E-02 dilution, the phage solution is diluted to obtain a phage concentration as close as possible to the concentration of the co-dried sample. The pots are then incubated at 37°C with shaking (100 rpm) to mimic the passage through the intestine. At times 0 (just after adding the sample), 30, 60, 120 min (Minekus et al. 2014) phage spot counts are carried out according to the method described below in paragraph 141, without forgetting to shake the pot before taking the aliquot for dilution.

[0135] Yeast counts are also carried out at the start (T0) and end (T120) of the experiment according to the method described.

[0136] The pH is remeasured at the end of the experiment.

[0137] Enumeration of co-dried phages and yeasts

[0138] The counts of the phages co-dried with the yeast are carried out after a step of rehydration of the powder in the Stomacher homogenizer and are carried out according to a method adapted from an internal procedure: Counting of bacteriophages per PFU.

[0139] Rehydration of co-drying samples

[0140] - weigh 1g of phage-yeast co-drying sample using a balance precision and place it in a stomacher bag;

[0141] - add 9ml of sterile distilled water at 37°C into the bag then pass it for 3 min in the Stomacher homogenizer at medium speed.

[0142] In-depth phage counting technique

[0143] - melt the TSA+YE agar (Trypto casein Soy Agar (TSA) + Extract of yeast (YE) at 6g / L of agar + cycloheximide (0.5%) and keep it supercooled;

[0144] - prepare 90 mm Petri dishes with approximately 15 ml of TSA+YE + cyclo medium heximide (0.5%), dry them under a hood for 30 minutes;

[0145] - from a colony from a fresh culture, inoculate 20ml of TSB+YE broth (Trypto casein Soy broth (TSB) + Yeast extract (YE);

[0146] - incubate with shaking until a culture is obtained at the beginning of the exponential phase growth (example: stop the culture at an OD between 0.2 and 0.7 for E. coli, Salmonella and Listeria);

[0147] - raise the value of DO.

[0148] Prepare decimal dilutions of the rehydrated co-drying sample in Eppendorf tubes containing 900pL of SM solution

[0149] In a 13ml tube containing 100pl of host bacteria solution (OD between 0.2 and 0.7), add 100pl of the desired phage dilution and leave in contact for 10 min

[0150] Add 3ml of TSA+YE medium 6g / l of agar + cycloheximide and pour onto a previously poured and dried agar plate. Allow to solidify.

[0151] Incubate at the growth temperature of the host bacteria for 24 hours.

[0152] Phage spot counting technique

[0153] Preparation of the agar plates

[0154] - melt the agar to 6g / L of agar then add 0.5% of cycloheximide, maintain it supercooled.

[0155] - from a colony from a fresh culture, inoculate 20ml of TSB+YE broth. Incubate with shaking until a culture is at the beginning of the exponential growth phase (e.g., stop the culture at an OD between 0.2 and 0.7 for E. coli, Salmonella, and Listeria). Prepare square Petri dishes with 25 ml of TSA+YE + cycloheximide (0.5%) medium and let them dry under a hood for 30 min.

[0156] - raise the value of DO.

[0157] - in a 13ml tube containing 300pl of host bacteria solution (DO between 0.2 and 0.7), add 9ml of TSA+YE medium 6g / l of agar + cycloheximide and pour onto a previously poured and dried agar plate;

[0158] - let solidify.

[0159] Preparation of decimal dilutions of the phage solution

[0160] - fill 96-well microplate well columns with 180 µl of buffer SM starting at column 3 and skipping every other column to allow spot seeding.

[0161] - add 100pl of solution from the re to the wells of the first column hydration of the sample which here constitutes the first dilution to 10 (1E-01).

[0162] - using a multichannel pipette, take 20pl from the first column and transfer to the wells of the following dilution column and so on until the last dilution.

[0163] Deposit on box

[0164] It is necessary to collect the Opl on a microplate line (96 wells) with a suitable multichannel pipette.

[0165] Then, divide the square Petri dish previously seeded with the host bacteria into 4 columns and deposit the spots of the Op1 of the phage / yeast dilutions, let dry and incubate at the growth temperature of the host bacteria for 24 hours.

[0166] The yeast count is carried out in parallel.

[0167] Once the sample is rehydrated, 100pl are taken to make decimal dilutions in Eppendorf tubes filled with 900pl of sterile distilled water.

[0168] 100pl of the dilution of interest are taken, placed on a YM agar plate and spread.

[0169] The plates are then incubated for 3 days at 25°C.

[0170] Regardless of the technique, the results are expressed as follows:

[0171] Theoretical PFU / g MS ​​titer (before drying) - this titer is calculated from the phage titer of the phage suspension and the actual composition of the formulation produced in the laboratory, which takes into account all the added ingredients (yeast, fraction, protectors, additives, etc.). To avoid bias, this titer is reduced to the dry matter of the mixture (before drying).

[0172] Assayed PFU / g MS ​​titer (after drying) - this is the actual titer as assayed on the dry finished product and reduced to the actual extract of the dry finished product.

[0173] Phage losses (LoglO PFU / g) are equal to the difference between:

[0174] LoglO (theoretical PFU / g MS ​​titer before drying) - LoglO (dosed PFU / g MS ​​titer after drying) and expressed in LoglO PFU / g.

[0175] Gastro resistance tests

[0176] Simplified in vitro gastrointestinal resistance tests were carried out on the co-drying samples. These tests aim to analyze the survival of phages and yeasts by counting at different points over time in simulated gastrointestinal fluids. These tests repeat the counting method described above without the rehydration phase with the Stomacher homogenizer, which here is done directly in the simulated liquid.

[0177] 0.5g of sample is placed in 50ml of liquid. A pH range was carried out for the gastric test ranging from 2.5 to 4.0.

[0178] Alkalization potential

[0179] In order to define which excipient is responsible for the rise in gastric pH, co-drying samples were produced by lyophilization of the SalmoFresh™ solution with each excipient individually in the same proportions (99% DM excipient versus 1% DM phage). The samples were made in the laboratory.

[0180] 0.5g of each sample was brought into contact with 50ml of gastric fluid simulated at pH 2.5. After homogenization, the pH of each preparation was measured with a pH meter after homogenization.

[0181] UV resistance tests

[0182] UV resistance tests were carried out on the co-drying samples rehydrated for 3 min with a Stomacher homogenizer (1g in 9ml of sterile distilled water) and on the phages in solution in the BS-03 +UV-MA UV chamber with UV-C bulbs. The measured irradiation is 12mW / cm2. For each sample form, three identical aliquots are made to be exposed for 3 different times (5, 15, 30 min). These tests are adapted from the tests carried out by the team of Ramirez et al. 2018. The survival of phages and yeasts is measured by the counting method.

[0183] The count at T0 is identical for the co-drying sample and for its rehydrated form, it is done according to the spot method described above, the count of the phage in solution is done according to an internal procedure.

[0184] For co-drying samples, approximately 1.5g are weighed and spread in an open Petri dish in the center of the chamber. After UV exposure, 1g is weighed on a precision balance and then rehydrated for 3 min in a Stomacher homogenizer so that the phages can be counted according to the spot method described above.

[0185] For rehydrated co-drying samples, 1 ml is placed in an open Petri dish in the center of the chamber. After UV exposure, 100 pl are taken to count the phages according to the spot method described above.

[0186] For phages in solution, 1 ml is placed in an open Petri dish in the center of the chamber. The phage concentration of the solution is as close as possible to the phage concentration in the co-drying sample by dilutions. After exposure to UV, 100 pl are taken to count the phages according to an internal procedure.

[0187] The yeast count is carried out at T0 and T30 according to the method described.

[0188] Stability tests

[0189] The stability of phage-yeast co-drying products stored in vacuum bags or pill boxes was studied over 3 months under 3 temperature conditions and humidity:

[0190] - 25°C / 60%RH

[0191] - 30°C / 65%RH

[0192] - 40°C / 75%RH

[0193] Phage counts using the depth counting technique are carried out at different points in time: 0, 15, 30, 60, 90 days. The aw (water activity) is measured at 25°C at times T0, 30 and 90 days using an AquaLab Series 4TEV dew point hygrometer.

[0194] Water activity is an important parameter related to the quality of dry products. The aw values ​​are an indicator of the possibility of growth of microorganisms and production of toxins. The value of water activity varies between 0 (dry product to the point that all the water is bound to the product, and therefore without reactive quality) and 1 (pure water and without solute).

[0195] Indeed, the aw indicates the proportion of free water in a product, i.e. available for example for the growth of microorganisms. The higher the aw, the more water there is available for the development of these microorganisms. Water activity values ​​greater than 0.6 could promote the growth of microorganisms. Examples

[0196] Example 1: general process for obtaining a dry yeast-bacteriophage mixture by drying in a fluidized air bed

[0197] The steps of the process:

[0198] - control of the pH of the yeast cream or yeast derivatives and possibly pH adjustment to 7.0 with 10% sodium hydroxide;

[0199] - filtration of yeast cream on a pressure plate filter: obtaining pressed yeast (LP)

[0200] - mixing the LP with the phage suspension and the desiccating or pro agents detectors (also called drying excipients)

[0201] - granulation and shaping by extrusion

[0202] - drying of granules in a fluidized air bed dryer

[0203] - control of the final humidity of the dry granules and vacuum packaging or under inert atmosphere

[0204] Thus, 16.5 g of a 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 pressed yeast (30% dry matter). After mixing for 1 min in an Alphamix Matfer 5 L mixer equipped with a paddle as a mixing blade, 60 g of starch (potato starch) are added and the mixture is mixed for 1 min. The mixed mass containing approximately 40% dry matter is then extruded on a DRC ilO extruder equipped with an extrusion grid with 1 mm holes. The filaments obtained are collected in a 5L beaker and fragmented manually by strong shaking. Drying is carried out on a Fluid Bed Dryer Tomado M501 (Sherwood) equipped with a multi-chamber support. 2 x 80 g of wet granules are placed in 2 glass drying chambers (diameter 60 mm and length 400 mm) equipped at their base with a 250 mesh stainless steel sieve (fluidization air inlet). The drying program is launched and includes two stages, each carried out at a temperature between 40 and 60 ° C. The fluidization and heating air is dehydrated by an air dehumidifier (Munters ComDry M190Y).

[0205] Drying is complete when the dry extract of the granules reaches at least 94%. The granules are vacuum-packed to ensure the best stability of the product over time.

[0206] Example 2: general process for obtaining a dry yeast-bacteriophage mixture by spray drying

[0207] The steps of the process:

[0208] - preparation of the mixture by adding yeast or yeast derivatives and, if necessary actually, drying ingredients in the phage suspension

[0209] - vigorous mixing until all ingredients are completely dissolved or dispersed ingredients

[0210] - control of the pH of the mixture and possibly adjustment to 7.0 with sodium hydroxide 10%

[0211] - drying the mixture in an atomization tower

[0212] - final humidity control and packaging under vacuum or atmosphere inert fine powder

[0213] Thus, a suspension of yeast cell walls is partially reconstituted by dispersing 75.3 g of dry Safmannan® cell walls in 336 g of demineralized water (preparation 1). The pH of the yeast cell wall suspension is optionally adjusted to 7.0 with 10% sodium hydroxide. Preparation 1 is cooled to a temperature between 2 and 10 °C.

[0214] Then, 42 g of trehalose dihydrate and 2.7 g of L-leucine are dissolved hot in 112 g of demineralized water (preparation 2). After cooling preparation 2, 39 g of a SalmoFresh™ phage suspension (2.5% dry extract) are added and mixed with vigorous stirring. Then this mixture is added to preparation 1 with vigorous stirring to obtain the mixture to be dried comprising the phages, yeasts or yeast derivatives and the various supports or ingredients. This mixture is kept stirring at low temperature throughout the process.

[0215] Drying is carried out on a Mini Spray Dryer B-290 tower (Büchi) equipped with a compressed air bi-fluid nozzle, a peristaltic pump, a cyclone for separating dry particles from humid air and a polyester outlet filter. The equipment is completed by an air dehumidifier (Munters ComDry M190Y) which treats the air entering the atomization tower.

[0216] The operating parameters (temperature and air flow, feed rate of the solution to be dried, etc.) are adjusted to dry the phages under gentle conditions by exposing them to moderate temperatures, so the outlet temperature is controlled so as not to exceed 60°C.

[0217] Drying is complete when the humidity of the powder is less than 6%. The atomized powder is vacuum-packed.

[0218] Example 3: general process for obtaining a dry yeast-bacteriophage mixture by drying by lyophilization

[0219] The steps of the process:

[0220] - preparation of the mixture by adding yeasts or yeast derivatives and, optionally, other ingredients in the phage suspension

[0221] - vigorous mixing until all ingredients are completely dissolved or dispersed ingredients

[0222] - control of the pH of the mixture and possibly adjustment to 7.0 with sodium hydroxide 10%

[0223] - rapid cooling and freezing to at least -20°C

[0224] - drying the frozen mixture in a freeze dryer under reduced pressure

[0225] - grinding of the lyophilisate and control of the final humidity

[0226] - packaging under vacuum or in an inert atmosphere of the finely dried product split.

[0227] Thus, the pH of a freshly harvested yeast cream (dry extract of 21.3%) is adjusted to 7.0 with 10% sodium hydroxide. A protective solution comprising the supports or excipients is prepared: 18.9 g of trehalose dihydrate, 18.9 g of maltodextrin and 0.63 g of L-leucine are dissolved hot in 51.1 g of demineralized water. 7.9 g of a suspension of T5 phages (2.3% dry extract) are added to this protective solution, after it has cooled. The mixture is kept stirring and cooled. This mixture is added to 113 g of the previous yeast cream, still stirring and cooled. It contains approximately 27.4% dry extract.

[0228] The liquid yeast / phage / protectant mixture is distributed into glass trays or vials in such a way that the layer height in the container does not exceed 15 mm. All containers are cooled and frozen quickly in the freezer at a temperature < -20°C.

[0229] After freezing for a few hours, the containers are placed in a laboratory freeze dryer (Lyovapor L-200 Büchi) whose shelves and freeze-drying chamber will have been previously cooled.

[0230] As mentioned previously, primary desiccation followed by desiccation secondary are started and the freeze-drying process is stopped after 80 to 96 hours. The freeze-dried products are finely ground into powder using a mortar and pestle mill and possibly passed through a 500 µm mesh sieve. The final humidity is controlled and must be < 3%. Vacuum packaging is carried out quickly to prevent the product from deteriorating over time.

[0231] The mixtures used in the tests described below were dried according to one of the three methods described in Examples 1-3.

[0232] In the stability tests, the counts were carried out using the deep phage technique. In the other tests, the counts were carried out using the spot technique and with a starting sample which constitutes the dilution lE-02, the detection limit of this method is therefore 4 Log (hereinafter referred to as ND).

[0233] When confronted with acidic conditions, phages appear to become abruptly inactivated below a certain threshold which would be around pH 3.5 (Ma et al. 2008, Davis et al. 1985). A range of four pHs (2.5, 3.0, 3.5, 4.0) was therefore tested to frame this threshold and analyze the entire spectrum that can be found in the stomach when fasting or after absorption of a food bolus (gastro-resistance test in gastric liquid).

[0234] Regardless of the drying method used, T5 bacteriophages or the SalmoFresh™ bacteriophage cocktail retain activity that is entirely satisfactory for industrial use. Similar results were obtained with T4 and T7 bacteriophages and the FOP cocktail.

[0235] Example 4. Gastro resistance test in gastric liquid with a mixture resulting from co-drying by atomization of a yeast S. cerevisiae CNCM1-3856 or yeast cell walls with the bacteriophage T5 or with the bacteriophage cocktail SalmoFresh™ PH 0 30 min 120 min Time and mixture tested 2.5 ND ND ND Log PFU / G yeast CNCM 1-3856 + bacteriophage T5 3 6.6 5.58 <4 3.5 6.73 6.72 6.09 4 6.55 6.67 6.55 2.5 ND ND ND Log PFU / G bacteriophage T5 alone 3 ND ND ND 3.5 ND ND ND 4 6.28 6.15 4.88

[0236] Phage alone in solution is inactivated under pH4 while it is possible to count co-dried phages with yeast from pH 3.

[0237] At pH 2.5, none of the phage forms tested remained active.

[0238] At pH 3, T5 phages co-dried with CNCM 1-3856 yeast remain active for at least 30 min before falling below the detection threshold.

[0239] At pH 3.5, after 2 hours, a loss of T5 phages co-dried with the CNCM 1-3856 yeast is observed in higher Log PFU / g.

[0240] At pH 4, T5 phages co-dried with CNCM 1-3856 yeast are in a more stable environment. Note that the PFU / g concentration of the samples may increase between times T0 and T30. This is most certainly due to the fact that the powder (especially the atomized powder) can form agglomerates which do not always dissolve very quickly.

[0241] The yeasts in the samples placed in contact with gastric fluid at pH 2.5 were counted at the beginning (T0) and then at the end (T120) of the experiment. The results are similar in all the tests; after 2 hours in an acidic environment, the yeast concentration is very slightly reduced. time 0 120 min Log CFU / g 9.01 8.91 Example 5. Gastro resistance test in gastric liquid with a mixture resulting from co-spray drying of yeast with the SalmoFresh™ bacteriophage cocktail PH 0 30 min 120 min Time and mixture tested 2.5 6.19 5.11 <4 Log PFU / G yeast CNCM 1-3856 + SalmoFresh™ bacteriophage cocktail 3 7.72 7.59 5.70 3.5 7.95 8.08 7.63 4 8.03 8.03 7.92 2.5 ND ND ND Log PFU / G SalmoFresh™ bacteriophage cocktail alone 3 ND ND ND 3.5 5.90 5.31 ND 4 7.53 6.80 6.19

[0242] Comparing the gastric resistance of the phage solutions alone (T5 and SalmoFresh™) with each other, better survival is observed for the phages present in the SalmoFresh™ solution. It should also be noted that the SalmoFresh™ solution contains a cocktail of phages and that the pH sensitivity of each of the phages composing it may be different.

[0243] It should be noted that co-drying with yeasts by atomization allows the phages to remain active for 30 min even at pH 2.5 before falling below the detection threshold.

[0244] At pH 3.0, 3.5 and 4.0, the samples remain active during the 2 hours of the experiment.

[0245] Similarly, the yeasts of the samples brought into contact with the gastric liquid at pH 2.5 were counted at the beginning (T0) and then at the end (T120) of the experiment. The results are similar in all tests, after 2 hours in an acidic environment, the yeast concentration is very slightly reduced. time 0 120 min Log CFU / g 9.75 9.46 Example 6. Gastro resistance test in gastric liquid with a mixture resulting from the co-drying of yeast cell walls either with bacteriophage T5 or with the bacteriophage cocktail SalmoFresh™. PH 0 30 min 60 min 120 min Time and mixture tested 2.5 ND ND ND ND Log PFU / G yeast cell walls + bacteriophage T5 3 7.7' 7.60 6.57 ND 3.5 8.02 8.33 8.19 7.81 4 8.23 ​​8.43 8.34 8.19 2.5 ND ND ND ND Log PFU / G yeast cell walls + bacteriophage cocktail SalmoFresh™ 3 7.09 8.34 7.79 6.78 3.5 8.67 8.29 7.53 7.25 4 9.09 8.83 8.40 7.41

[0246] These samples were made by co-spray drying. Phage survival appears to be identical for pH 3.0, 3.5, and 4.0 despite the acidity being more weakly neutralized by the samples compared to other spray-dried tests using CNCM 1-3856 yeast.

[0247] It was observed at pH3 that T5 phages survived for 60 min and phages in the SalmoFresh™ solution survived for 120 min of the experiment. The probable hypothesis is that since the powder obtained with these tests is even less soluble than the atomized powder obtained with live yeasts, the diffusion of the phages would occur later, which could contribute to their protection. This hypothesis could also explain why more phages are counted after 30 min of exposure compared to the counts carried out at T0 min.

[0248] Example 7. Resistance test in intestinal liquid with a mixture resulting from the co-drying of yeasts either with bacteriophage T5 or with the bacteriophage cocktail SalmoFresh™ Drying Method 0 30 min 60 min 120 min Time and Mixture Tested Control 6.87 6.95 6.85 6.90 Bacteriophage T5 Fluidized Bed 6.18 5.78 6.11 6.20 Log PFU / G Yeast L13 + Bacteriophage T5 Spray Drying 6.92 7.24 7.29 7.37 Log PFU / G Yeast CNCM 1-3856 + Bacteriophage T5 Freeze Drying 7.58 7.66 7.68 7.75 Control 8.04 8.04 8.10 7.92 Bacteriophage Cocktail Only Fluidized Bed 6.56 6.39 6.36 6.48 Log PFU / G Yeast CNCM 1-3856 + Bacteriophage Cocktail SalmoFresh™ Spray Drying 8.34 8.57 8.68 8.70 Freeze drying 8.01 8.05 8.15 8.19

[0249] Intestinal resistance tests are based on the same principle as gastric tests, except that only one pH is tested (pH 6.8). This shows that there is no impact on the survival of phages or yeasts in the intestinal fluid. time 0 120 min Tested mixture Log CFU / g 8.79 8.70 CNCM I-3856 yeast + T5 bacteriophage Log CFU / g 9.40 9.57 L47 yeast + SalmoFresh™ bacteriophage cocktail Example 8. Potential alkalization test

[0250] To determine which ingredients of the formulations had this alkalizing effect which allows to protect the phages in an acidic environment, five samples were made all composed of 1% of SalmoFreshTM phages in DM (dry matter) / MST (total dry matter) and 99% of a single excipient used. 0.5g of these

[0251] samples were contacted with 50ml of gastric fluid at pH 2.5. After dissolving the powders, the pH was measured. The samples dried with yeast and L-leucine caused significant pH variations. With a pH measured at 3.59, the sample with yeast even exceeded the inactivation threshold of 3.5. It can be observed that yeast is the most effective drying medium and is therefore present in significant quantities in the different formulations, unlike L-leucine. Drying Dried phages Formula pH after addition in 50ml of SGF at pH 2.5 Freeze-drying SalmoFresh™ L-leucine 3.40 Trehalose 2.50 maltodextrin 2.48 starch 2.49 yeast 3.59 Example 9. UV resistance test

[0252] 0 5 min 15 min 30 min Time and mixture tested 6.22 5.06 ND ND Log PFU / G bacteriophage T5 alone (control) 6.54 6.51 5.90 5.55 Log PFU / G yeast CNCM 1-3856 + bac-teriophage age T5 8.03 8.03 7.92 8.75 ND ND ND Log PFU / G cocktai 1 bacteriophage SalmoF resh™s eul 6.27 5.88 5.90 5.70 Log PFU / G yeast CNCM 1-3856 + cocktail 1 bacteriophage SalmoF resh™ time 0 30 min Tested mixture Log CFU / g 9.41 9.29 yeast CNCM I-3856 + bacteriophage T5 Log CFU / g 9.50 9.57 yeast CNCM I-3856 + bacteriophage cocktail SalmoFresh™

[0253] Phages in solution and co-spray-dried phages with yeast were exposed to UV-C for 30 min with points at 0, 5, 15, 30 minutes to count the phages. The counts are carried out by spot as for the gastroresistance tests and with the same samples. The first available dilution being the 1E-01 dilution (coming from rehydration in the Stomacher) the detection limit of the technique is 3 Log.

[0254] It can be observed that the phages in solution (T5 and SalmoFresh™ phages) are very sensitive to UV-C. The phages in the SalmoFresh™ solution are no longer active after 5 minutes of exposure and the T5 phages only resist for 5 minutes. Conversely, the phages co-dried with yeast are only very slightly affected by UV-C.

[0255] Counting the yeasts before and after 30 min of exposure demonstrated that for all the configurations tested, exposure to UV-C had no impact on their survival. Example 10. Stability test over time

[0256] 3-month stability tests were performed at 25°C / 60%RH, 30°C / 65%RH and 40°C / 75%RH on the co-spray-dried samples. The aw (water activity) and phage loss were measured at the beginning (T0 days), middle (T30 days) and end (T90 days) of the experiment. Time and conditions 0 30 d 90 d Test mixture 25°C / 60%RH 8.35 7.24 6.63 Log PFU / G S. boulardii + bacteriophage T5 30°C / 65%RH 8.35 7.12 6.23 40°C / 75%RH 8.35 6.51 6.23 25°C / 60%RH 8.83 8.54 8.37 Log PFU / G S. boulardii + bacteriophage cocktail SalmoFresh™ 30°C / 65%RH 8.83 8.52 8.19 40°C / 75%RH 8.83 8.17 6.93 0 30 d 90 d time 25°C / 60%RH 0.05 0.07 0.09 awS. boulardii + bacteriophage T5 30°C / 65%RH 0.05 0.06 0.08 40°C / 75%RH 0.05 0.07 0.18 25°C / 60%RH ND 0.07 0.09 aw S. boulardii + SalmoFresh™ bacteriophage cocktail 30°C / 65%RH ND 0.06 0.08 40°C / 75%RH ND 0.08 0.19

[0257] The very low values ​​of aw indicate that the share of free water in the product obtained is very low, sufficiently to prevent the growth of microorganisms and therefore to ensure the stability of the product over time.

Claims

Claims

1. A method of manufacturing 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 and at least one bacteriophage and optionally at least one drying excipient, said method being characterized in that it is carried out by mixing at least one yeast and / or yeast derivative and at least one bacteriophage in suspension and drying this mixture.

2. A method of manufacturing a dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, said method being characterized in that it comprises: - providing at least one yeast and / or yeast derivative, preferably in cream form and at least one bacteriophage in suspension; - mixing said at least one yeast and / or yeast derivative with said at least one bacteriophage so as to form a mixture; - carrying out a step of drying the mixture so as to form a dry mixture; - recovering the dry mixture.

3. Method according to one of claims 1 or 2, characterized in that the drying step is carried out by freeze-drying or atomization or on a fluidized air bed.

4. Method according to one of claims 1 to 3, characterized in that the drying step is carried out in the presence of a drying excipient, preferably maltodextrin.

5. Method according to one of claims 1 to 4, characterized in that the yeast is derived from a strain chosen from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably the yeast is derived from a strain chosen from the strain Saccharomyces cerevisiae deposited on October 17, 2007 under number CNCM 1-3856, the strain Saccharomyces cerevisiae deposited on March 22, 2018 under number CNCM 1-5298 and the strain of Saccharomyces boulardii deposited on August 21, 2007 under number CNCM 1-3799.

6. Method according to one of claims 1 to 5, characterized in that the bacteriophage(s) are chosen from those having antibacterial activity against strains of bacteria chosen from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or strains of the genus Salmonella.

7. Method according to one of claims 1 to 5, characterized in that the yeast derivatives are yeast hulls.

8. Dry mixture of at least one yeast and / or yeast derivative and at least one bacteriophage, characterized in that it is in the form of solid entities, each solid entity consisting of at least one yeast and / or at least one yeast derivative and at least one bacteriophage and optionally at least one drying excipient.

9. Dry mixture according to claim 8, characterized in that the solid entities are in the form of flakes, grains, vermicelli or granules.

10. Dry mixture according to one of claims 8 or 9, characterized in that the yeast is derived from a strain chosen from the species Saccharomyces cerevisiae and Saccharomyces boulardii, preferably the yeast is derived from a strain chosen from the strain Saccharomyces cerevisiae deposited on October 17, 2007 under number CNCM 1-3856, the strain Saccharomyces cerevisiae deposited on March 22, 2018 under number CNCM 1-5298 and the strain of Saccharomyces boulardii deposited on August 21, 2007 under number CNCM 1-3799.

11. Dry mixture according to one of claims 8 to 10, characterized in that the bacteriophage(s) are chosen from those having antibacterial activity against strains of bacteria chosen from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or strains of the genus Salmonella.

12. Dry mixture according to one of claims 8 to 11, characterized in that the yeast derivatives are yeast hulls.

13. Dry mixture according to one of claims 8 to 12 for use as a medicament.

14. Dry mixture according to one of claims 8 to 12 for use in the treatment of gastric juice acidity.

15. Dry mixture according to one of claims 8 to 12 or dry mixture obtained from the process according to one of claims 1 to 7 for use in a composition for the treatment of gastric juice acidity.

16. Use of the dry mixture according to one of claims 8 to 12 or of the dry mixture obtained from the process according to one of claims 1 to 7 in a composition for the stimulation, protection, biocontrol and / or nutrition of plants.

17. Use of the dry mixture according to one of claims 8 to 12 or of the dry mixture obtained from the process according to one of claims 1 to 7 in a food composition or in a food supplement.

18. Use of the dry mixture according to one of claims 8 to 12 or of the dry mixture obtained from the process according to one of claims 1 to 7 in brewing and / or in oenology.

19. Use of the dry mix according to one of claims 8 to 12 or of the dry mix obtained from the process according to one of claims 1 to 7 in breadmaking.