Use of bacillus inaquosorum alone or in combination with a yeast product to reduce or prevent the formation of dental biofilm

Abstract

The present invention relates to the field of oral / dental hygiene and health. The invention relates to the use of the strain Bacillus inaquosorum, cells derived therefrom or a composition comprising same, for reducing and / or preventing the formation of dental biofilm, in particular by reducing or inhibiting the growth of bacteria of the species Peptostreptococcus canis.

Description

[0001] Use of Bacillus inaquosorum alone or in combination with a yeast product in the field of oral hygiene and health

[0002] technical field

[0003] The present invention relates to the field of oral hygiene and health. The invention relates to the use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, to reduce and / or prevent the formation of dental biofilm, in particular by reducing or inhibiting the growth of bacteria of the species Peptostreptococcus canis.

[0004] Technical background

[0005] In mammals, the oral cavity is a complex ecosystem, both open to the outside and the inside, inhabited by numerous microorganisms.

[0006] In companion animals, particularly dogs and cats, the oral cavities are also complex ecosystems inhabited by numerous microorganisms (see, for example, the article: Holcombe et al., PlosOne, 2014, 9, 12, e113744). Most of these microorganisms are commensal and necessary to maintain the balance of this ecosystem. However, a disruption of this balance can occur and lead to the development of infectious diseases of the oral cavity.

[0007] The main infectious diseases of the oral cavity are periodontal diseases. Indeed, dental caries are very rare in dogs and cats. Among the bacterial species most frequently implicated in periodontal diseases in pets are bacteria of the species Porphyromonas cangingivalis, Porphyromonas gulae, and Porphyromonas spp.

[0008] To prevent or treat infectious diseases of the oral cavity, it is helpful to maintain or restore the balance between non-pathogenic and pathogenic microorganism populations within the oral microbiota. This allows for the control of dental biofilm formation, development, and composition. Many pathogenic bacteria can be present in the canine or feline oral microbiota and can stimulate the formation, development, and composition of dental biofilm, potentially contributing to the development of infectious diseases of the oral cavity.

[0009] Bacteria of the species Peptostreptococcus canis express virulence factors that allow them to colonize the subgingival space. Colonization of the oral microbiota by Peptostreptococcus canis bacteria and their growth within the dental biofilm contribute to the onset and development of infectious diseases of the oral cavity in pets. Indeed, this anaerobic bacterium is considered a late colonizer that adheres to the dental biofilm, thus allowing progression to periodontal disease. Periodontal diseases are among the most common inflammatory diseases in small pets, with a prevalence reaching 80%. Furthermore, these periodontal diseases can have major systemic consequences.

[0010] Therefore, there is a need to provide a product that reduces and / or prevents the formation of dental biofilm, particularly in pets. There is also a need to provide a product that reduces and / or prevents the formation of dental biofilm caused by the growth of Peptostreptococcus canis bacteria, and thus reduces or inhibits the growth of these bacteria.

[0011] Summary of the invention

[0012] The invention relates to the non-therapeutic use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, to reduce and / or prevent the formation of dental biofilm.

[0013] The present invention also relates to the Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use to reduce and / or prevent the formation of dental biofilm.

[0014] In some embodiments, the Bacillus inaquosorum strain is the strain deposited on September 12, 2024 with the Agricultural Research Culture Collection under the Treaty of Budapest and having received the registration number NRRL B-68429.

[0015] In some embodiments, the formation of dental biofilm is reduced or prevented by reducing or inhibiting the growth of bacteria of the species Peptostreptococcus canis.

[0016] In some embodiments, the Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, is administered to mammals; preferentially to dogs and / or cats; very preferentially to dogs.

[0017] In some embodiments, the Bacillus inaquosorum strain, and / or cells derived from it, are in dry form.

[0018] In some embodiments, the Bacillus inaquosorum strain, and / or cells derived from it, is administered in combination with a yeast product. In some embodiments, the yeast product is a yeast product comprising at least 20% p-glucans, by weight per total weight of the yeast product.

[0019] In embodiments, the yeast product is a yeast extract having a pH between 6.0 and 7.0; and comprising, by weight per total weight of the yeast product, 35 to 65% protein (nitrogen x 6.25), 10 to 30% ash and 3% or less sodium chloride.

[0020] In some embodiments, the composition of the Bacillus inaquosorum strain, and / or cells derived from it, is in dry form.

[0021] In some embodiments (non-therapeutic use), the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, is administered to a healthy subject.

[0022] In some embodiments, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, is administered to a subject orally.

[0023] In some embodiments (therapeutic use), the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, is administered to a subject with a disease of the oral cavity, or at risk of having one.

[0024] In embodiments, the composition comprising the Bacillus inaquosorum strain, and / or cells derived from it, is selected from a food supplement, a parapharmaceutical composition or a pharmaceutical composition.

[0025] The inventors have shown, surprisingly, that the Bacillus inaquosorum strain, and / or cells derived from it, optionally in combination with yeast products, can reduce and / or prevent the formation of dental biofilm, and in particular reduce or inhibit the growth of bacteria of the species Peptostreptococcus canis, which is a periodontogenic microorganism, the growth of which contributes to the appearance and development of infectious diseases of the oral cavity of pets.The Bacillus inaquosorum strain, and / or cells derived from it, optionally in combination with yeast products, or the composition comprising it(s), is particularly interesting in that it can be used in healthy subjects (non-therapeutic use) or in subjects with a disease of the oral cavity, or at risk of having one (therapeutic use), these two populations of subjects being distinct.

[0026] A more detailed description of the invention is given below. Detailed description

[0027] The invention is now described in more detail and in a non-limiting manner in the following description.

[0028] Definitions

[0029] By “Saccharomyces cerevisiae yeast strain”, we mean a relatively homogeneous population of Saccharomyces cerevisiae yeast cells obtained by culturing (or multiplying) the starting strain.

[0030] The term "oral microbiota" refers to all the microorganisms, particularly bacteria, present in the oral cavity. The terms "oral microbiota," "buccal microbiota," "human oral microbiota," "dog and cat oral microbiota," "oral cavity microbiota," "oral flora," and "oral flora" can be used interchangeably. The microorganisms present in the oral microbiota can be non-pathogenic or pathogenic. The balance between non-pathogenic and pathogenic microorganism populations therefore determines, from a clinical and biological perspective, whether the oral microbiota is healthy or not.Des microorganismes présent peuvent être, par exemple, les baccteria appartant au genre Actinomyces, Actinomycetemcomitans, Aggregatibacter, Alloprevotella, Alloscardovia, Anaeroglobus, Atopobium, Bacteroides Bifidobacterium, Capnocytophaga, Catonella, Corynebacterium, Dialister, Eggerthia, Eubacteria, Fusobacterium, Haemophilus, Lachnoanaerobaculum, Leptotrichia, Mogibacterium, Moryella, Neisseriaceae, Olsenella, Oribacterium, Parvimonas, Peptoniphilus, Peptostreptococcus, Propionibacterium, Porphyromonas, Prevotella, Rikenellaceae, Ruminococcaceae, Selenomonas, Shuttleworthia, Slackia, Solobacterium, Stomatobaculum, Streptococcus, Tannerella, Treponema ou Veillonella. Pathogenic microorganisms are for example: Tannerella forsythia (T. forsythia), Porphyromonas gingivalis (P. gingivalis), Treponema denticola (T. denticola), Porphyromonas cangingivalis (P. cangingivalis), Porphyromonas gulae (P. gulae), Porphyromonas endodontalis {P. endodontalis), Prevotella intermedia {P.intermedia), Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). Non-pathogenic microorganisms include, for example: Streptococcus oralis (S. oralis), and Streptococcus mitis (S. mitis).

[0031] The term "biofilm" refers to the film formed on the surface of teeth and oral mucosa, such as the mucous membranes of the tongue, gums, palate, and cheeks, by the oral microbiota. The terms "biofilm," "dental biofilm," "dental plaque," "polymicrobial biofilm," and "polymicrobial film" can be used interchangeably.

[0032] By "periodontium" we mean all the supporting tissues of the tooth, the gums, the bone tissue, the cementum and the periodontal ligament.

[0033] An "infectious disease of the oral cavity" is a medical condition located in the oral cavity (mouth) that results from infection by a pathogenic microorganism. Infectious diseases of the oral cavity include, but are not limited to, dental caries and periodontal disease.

[0034] By "dental caries" or "cavity", we mean an infectious disease of the tooth, which causes damage to the enamel, dentin and / or cementum (pulp).

[0035] By "periodontal health" we mean the absence of inflammation or the presence of a low level of inflammation in an intact or reduced but stable periodontium.

[0036] Periodontal disease refers to inflammation of the tissues supporting the teeth, namely the bone and gums. These infections are caused by the accumulation of pathogenic bacteria and their toxins on the gum line surrounding the teeth. It initially manifests as gingivitis and then, if left untreated, progresses to periodontitis. The terms "periodontal disease" and "periodontal disease" can be used interchangeably.

[0037] Gingivitis refers to inflammation of the gums, stage 1 of periodontal disease. The terms gingivitis, gum disease, and gum inflammation can be used interchangeably.

[0038] By "periodontitis" we mean the inflammation of the deep tissues of the periodontium, stage 2 of periodontal disease.

[0039] By "postbiotic" we mean a preparation of inanimate microorganisms and / or their components that confer a health benefit to the host.

[0040] The term "treatment" means a method intended to: (1) delay or prevent the onset of a disease or clinical condition; (2) slow or stop the progression, worsening, or deterioration of disease symptoms; (3) improve disease symptoms; and / or (4) cure the disease. A treatment may be administered before the onset of the disease, for prophylactic action (referred to as "prevention"), or it may be administered after the disease has begun, for therapeutic action. In the context of the invention, the term "treatment" generically refers to the reduction and / or prevention of dental biofilm formation, particularly through the growth of periodontogenic and / or cariogenic bacteria.

[0041] By "physiologically acceptable excipient" is meant any medium or additive which does not interfere with the effectiveness of the biological activity of the active ingredient and which is not excessively toxic to the subject at the concentrations at which it is administered.

[0042] The term “pharmaceutical active ingredient” means any compound or substance whose administration has a therapeutic effect or whose administration has a beneficial effect on the health or general condition of a subject to whom it is administered.

[0043] Unless otherwise stated, all percentages for the quantities indicated are mass percentages. For p-glucan percentages, these are expressed as an equivalent mass of glucose. For mannan percentages, these are expressed as an equivalent mass of mannose.

[0044] The Bacillus inaquosorum strain, the cells derived from it, and their composition

[0045] The present invention relates to the use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, to reduce and / or prevent the formation of dental biofilm, in particular by reducing or inhibiting the growth of bacteria of the species Peptostreptococcus canis.

[0046] The Bacillus inaquosorum strain is a subspecies of the bacterium Bacillus subtilis (also known as Bacillus subtilis subsp. inaquosorum). The Bacillus inaquosorum strain was documented in the article by AP Rooney et al., "Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov," Int J Syst Evol Microbiol., 2009 Oct, 59, 2429-2436.

[0047] The Bacillus inaquosorum strain was deposited on September 12, 2024 with the International Depository Authority Agricultural Research Culture Collection (1815 N. University Street, Peoria, IL, 61604, USA) on behalf of Envera LIC LLC (220 Garfield Ave. West Chester, PA, 19380 USA) under the Treaty of Budapest and was given the registration number NRRL B-68429.

[0048] The cells can be obtained by culturing the Bacillus inaquosorum strain in a culture medium, according to processes well known to those skilled in the art, for example according to those described in the book "Biotechnology, 5th edition, R. Scriban, Edition Tec. & Doc., 1999, ISBN: 2-7430-0309-X".

[0049] Typically, a cell production process by strain culture implemented within the scope of the invention comprises the following steps:

[0050] - inoculation of a culture medium with an inoculum of the strain;

[0051] - culture under aerobic conditions, to obtain cell multiplication; and

[0052] - Separation of the biomass from its culture medium to obtain cells. The cells thus obtained are predominantly in their vegetative form. The "vegetative form" of a bacterium refers to the form of a bacterium under favorable conditions. The expression "predominantly in its vegetative form" means that at least 70% of the cells are in their vegetative form, preferably at least 80%, and more preferably at least 90%. An example of favorable conditions is a non-limiting culture medium at a temperature and pH conducive to bacterial growth. A non-limiting culture medium contains all the nutrients necessary for cell growth.

[0053] The cell production process may also include an intermediate step, between the aerobic culture stage and the biomass separation stage, of placing the cells under unfavorable conditions. The cells thus obtained are then predominantly in spore form.

[0054] The "spore-forming" form of a bacterium refers to the form of a bacterium in unfavorable conditions. The expression "predominantly in spore form" means that at least 70% of the cells are in spore form, preferably at least 80%, and even more preferably at least 90%. The spore-forming form is a resistant form that allows cells to withstand a hostile environment such as a lack of nutrients (i.e., a nutrient-limiting environment), water stress, significant variations in pH or temperature, or passage through the digestive tract.

[0055] Unfavorable cell conditions can be achieved, for example, by: not renewing the bacterial culture medium, stopping feeding into the culture medium, using a limiting culture medium, changing the temperature and / or pH, and controlling aeration and / or agitation to maintain a chlorophyll pressure above 20% in the culture medium and a CO2 pressure below 1.5% in the outgoing gases or their combination. It is also possible to obtain predominantly spore-forming cells by adding glutamic acid to the culture medium in an amount of approximately 0.75 g / L.

[0056] In a preferred embodiment, the cells are predominantly in spore form.

[0057] The cell production process may also include a subsequent step of drying the cells to obtain them in a dry form. Drying methods include, for example, freeze-drying, fluidized bed drying, or spray drying.

[0058] In one particular embodiment, the cells are in dry form. The expression "cells in dry form" advantageously means that the biomass obtained at the end of the cell production process advantageously comprises more than 90% dry matter, preferably more than 95% dry matter. The Bacillus inaquosorum strain, and / or cells derived from it, may be formulated in any form suitable for administration to mammals; preferably to dogs and / or cats; most preferably to dogs.

[0059] The present invention can be implemented by providing a composition comprising the Bacillus inaquosorum strain, and / or cells derived from it.

[0060] The composition may be in dry form. It may be formulated as a capsule (encapsulated composition), sachet, or powder. It may also be coated with a protective layer by spraying or coating. The composition may also be coated onto a substrate, such as kibble, biscuits, or treats. The composition may also be formulated as an oral gel.

[0061] Yeast products

[0062] The Bacillus inaquosorum strain, and / or cells derived from it, can be used in combination with at least one yeast product.

[0063] Yeast, as a living microorganism, comprises cytoplasm surrounded by a cytoplasmic membrane (also called the inner membrane or cell membrane). The cytoplasm contains intracellular compartments, including the nucleus, mitochondria, and Golgi apparatus. The cytoplasmic membrane is surrounded by a cell wall or cortex (the terms "yeast cell walls" and "yeast cortex" are used interchangeably and refer to the insoluble portion of yeast cells, i.e., the cell wall and the plasma membrane of yeast) composed primarily of p-glucans and mannans. The area between the cell membrane and the cell wall forms the periplasm (also called the periplasmic space).

[0064] Yeast products are obtained from the yeast Saccharomyces cerevisiae, and more specifically from a strain of Saccharomyces cerevisiae. Saccharomyces cerevisiae is also known as brewer's yeast or baker's yeast. Many strains of Saccharomyces cerevisiae are known to the art. They are widely used in the food industry for their role in the production of various foods, including breads and fermented beverages. A yeast strain can be obtained from a clone, a clone being a population of yeast cells derived from a single yeast cell. Culturing a strain of Saccharomyces cerevisiae can be carried out using any suitable method. Yeast culture methods are known in the prior art, and those skilled in the art know how to optimize the culture conditions for each strain according to its specific characteristics.Thus, a Saccharomyces cerevisiae yeast can be obtained by multiplying a strain in an appropriate culture medium, for example, as described in the reference work "Yeast Technology", 2. ème edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31982-8.

[0065] In some embodiments, the yeast product is an extract of p-glucan-rich cell walls of Saccharomyces cerevisiae yeast, preferentially rich in p-1,3 / p-1,6-glucans (p-1,3 / p-1,6-glucans).

[0066] By "rich in p-glucans" is meant a product comprising at least 20% p-glucans by weight of the total weight of the product. The yeast product may comprise from 20 to less than 30%, alternatively from 30 to less than 40%, alternatively from 40 to less than 50%, alternatively from 50 to less than 60%, alternatively from 60 to less than 70%, alternatively from 70 to less than 80%, alternatively from 80 to less than 90%, alternatively at least 90%, of p-glucans, by weight of the total weight of the yeast product.

[0067] The p-glucans originating from the yeast cell wall are called "(3-cell wall glucans). These p-glucans are essentially glucose polymers in which the glucose units of the main chain are linked by p-1,3 bonds and whose branches are linked by p-1,6 bonds. p-glucans are insoluble and have low viscosity.

[0068] Mannans from yeast cell walls are called "cell wall mannans." These mannans are polysaccharides composed mainly of mannose, more precisely of copolymers of neutral or acidic sugars (with 5 or 6 carbon atoms), linked together by glycosidic bonds and associated with proteins. In Saccharomyces cerevisiae cell wall mannans, mannose is present as a skeleton of 50 or more mannose residues linked by α-(1,6) chains, branched by short chains of mannose linked by α-(1,2) and α-(1,3) chains.

[0069] The yeast product may comprise, by weight per total weight of the yeast product:

[0070] - at least 50%, preferably from 50 to 90%, preferably from 50 to 80%, preferably from 50 to 70%, preferably from 50 to 60%, of p-glucans;

[0071] - 5% or less, preferably between 1 and 5%, of mannans;

[0072] - 10% or less protein; and

[0073] - 10% or less of glycogen.

[0074] This yeast product may comprise at least 94%, preferably at least 96%, of dry matter, by weight per total weight of the yeast product. β-glucans and mannans may be present in a (weight / weight) ratio of 12 to 40, preferably 15 to 30, and most preferably 18 to 22.

[0075] For example, a product of this type is disclosed in application WO 2023194609 A1 published on October 12, 2023.

[0076] The yeast product may comprise, by weight per total weight of the yeast product:

[0077] - at least 20%, preferably between 20 and 30%, of p-glucans;

[0078] - at least 20%, preferably 20 to 30%, of mannans;

[0079] - 25% or less, preferably 10 to 25%, of protein; and

[0080] - 10% or less of glycogen.

[0081] This yeast product may comprise at least 94%, preferably at least 96%, of dry matter, by weight per total weight of the yeast product.

[0082] For example, a product of this type is disclosed in international application WO 2020152229 A1 published on July 30, 2020.

[0083] The yeast product can be obtained from any process that yields a product rich in p-glucans.

[0084] Yeast product refers to the insoluble fraction of yeast. This insoluble fraction can be obtained using conventional methods. The method can be biochemical and / or mechanical. For example, mechanical methods can be implemented using glass beads, a high-pressure homogenizer, ultrasound, or microwaves. Biochemical methods, for instance, can be implemented through autolysis, thermal plasmolysis, enzymatic hydrolysis, osmotic shock, or repeated freeze-thaw cycles. Specifically, the process may include the following steps: autolysis or enzymatic hydrolysis of the whole yeast; separation of the insoluble fraction and removal of the soluble fraction; and optionally, drying of the soluble fraction.The soluble fraction is conventionally called "yeast extract" and comprises the majority of amino acids, including glutamic acid, peptides, and minerals. The insoluble fraction comprises yeast cell walls, polymers, polysaccharides, nucleotides, and thermocoagulated proteins. Conventional extraction methods are disclosed in the reference work "Yeast Technology," 2. ème edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31982-8.

[0085] In some embodiments, the yeast product is a yeast extract having a pH between 6.0 and 7.0; and comprising, by weight per total weight of the yeast product, 35 to 65% protein (nitrogen x 6.25), 10 to 30% ash, and 3% or less sodium chloride. The yeast product may also be a yeast extract having a pH between 6.1 and 6.9; and may comprise, by weight per total weight of the yeast product, 40 to 61% protein (nitrogen x 6.25), 13 to 27% ash, and 2% or less sodium chloride.

[0086] The yeast product may be a yeast extract having a pH between 6.2 and 6.8; and may comprise, by weight per total weight of the yeast product, 45 to 57% protein (nitrogen x 6.25), 16 to 24% ash, 1% or less sodium chloride.

[0087] The protein content was measured using the Kjedhl method (6.25 times the total nitrogen). The sodium chloride content was measured using the sodium chloride method. The pH was measured at room temperature (18°C) in a solution containing 8.33% dry matter.

[0088] The yeast product can be in powder form.

[0089] The yeast product may comprise at least 94%, preferably at least 96%, by weight of dry matter per total weight of yeast product. Alternatively, the yeast product may comprise 100% or less, preferably 99% or less, by weight of dry matter per total weight of yeast product. The dry matter content may be measured, for example, by a drying method carried out for 5 hours at 105 °C.

[0090] The yeast product may be soluble. By "soluble" we mean a yeast product which can be homogeneously dissolved in water at a concentration of 50 g / L, under stirring and at a temperature of 50°C, the solution comprising less than 500 mg of solid residues i.e. undissolved.

[0091] The yeast product can be obtained by implementing a preparation process comprising the following steps:

[0092] - obtaining a yeast cream by fermentation followed by concentration by centrifugation;

[0093] - washing of the yeast cream obtained by adding water and centrifugal concentration;

[0094] - heating the washed yeast cream to a temperature between 60 and 95°C, preferably between 75 and 90°C;

[0095] - incubation under stirring of the yeast cream heated to a temperature of 55 to 90°C, preferably between 55 and 75°C, for a duration of 1 to 5 hours;

[0096] - separation of yeast extract and yeast cell walls by centrifugation;

[0097] - concentration of the yeast extract by evaporation and / or by reverse osmosis; and - optionally drying of the concentrated yeast extract.

[0098] The yeast product is available under the trade name Springer® 4101 / 0-PW-L from the company Biospringer by Lesaffre (Lesaffre group).

[0099] Springer® 4101 / 0-PW-L yeast product is in powder form, has a pH between 6.3 and 6.7 (8.33% solution) and comprises at least 94% by weight dry matter, less than 1% by weight sodium chloride, 7.5 to 9.0% by weight total nitrogen, 46.9 to 56.3% by weight protein (nitrogen x 6.25), and 18 to 22% by weight ash (excluding sodium chloride) by total weight of yeast product.

[0100] Uses

[0101] The present invention relates to the use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, as a probiotic in the field of oral hygiene and health. Indeed, the Bacillus inaquosorum strain, and / or cells derived from it, selectively inhibits the growth of host microorganisms, thus conferring a health benefit.

[0102] The Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, is used to reduce or inhibit the growth of the bacterium of the species Peptostreptococcus canis, in the oral microbiota.

[0103] The action of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, on reducing or inhibiting the growth of the Peptostreptococcus canis bacterium helps maintain or restore a healthy oral microbiota and thus prevents or treats infectious diseases of the oral cavity, particularly periodontal diseases. Reducing or inhibiting the growth of Peptostreptococcus canis bacterium also helps reduce and / or prevent the formation of dental biofilm.

[0104] The use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, including its therapeutic or non-therapeutic use, depends on the subject to whom it is administered.

[0105] If the individual is in good health, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered to maintain the individual's well-being, particularly to maintain satisfactory oral hygiene. This is a non-therapeutic use of Bacillus inaquosorum, or the composition containing it. Bacillus inaquosorum, or the composition containing it, may be formulated as a food supplement, a parapharmaceutical composition, or any other appropriate non-therapeutic form. If the individual has an infectious disease of the oral cavity, or is at risk of developing one, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered to prevent, limit, or treat the disease. This is a therapeutic use of Bacillus inaquosorum, or the composition containing it.The Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, may be formulated as a pharmaceutical composition or any other appropriate therapeutic form.

[0106] The method according to the invention can be used to treat a first episode of infectious disease of the oral cavity, in particular gingivitis or periodontitis, or a recurrence.

[0107] The Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered alone. Alternatively, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered with a yeast product as defined above. Alternatively, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered with another therapy, for example, an antibiotic or an antiseptic. Alternatively, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered with another prebiotic, probiotic, and / or postbiotic product.Alternatively, the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, may be administered in combination with a mechanical or surgical procedure, including scaling, root planing, curettage, filling, restoration or devitalization.

[0108] Topics

[0109] The Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing them, may be administered to a subject. The subject may already have an infectious disease of the oral cavity, or is at risk of developing one. When the subject has such a disease, the term "patient" may be used interchangeably.

[0110] The Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, may be administered to an animal, preferably to a pet, very preferably to a mammal, more preferably to a dog or a cat.

[0111] Effective quantity

[0112] The use of Peptostreptococcus canis to reduce or inhibit the growth of the bacterium (and the corresponding method of treatment and / or prevention) involves administering to an animal an effective amount of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition containing both. The effective amount, which may be administered in one or more doses, may be determined by the veterinarian, the breeder, or any other appropriate person. The exact amount to be administered may vary from one animal to another, depending on the species, age, weight, general condition of the animal, the presence or absence of an infectious disease of the oral cavity, and if so, its nature, severity, and / or extent. The effective amount may also vary depending on the desired therapeutic effect (reduction and / or prevention of dental biofilm formation).

[0113] Form of administration

[0114] The Bacillus inaquosorum strain, and / or cells derived from it, can be administered as such or in the form of a preparation or composition.

[0115] The Bacillus inaquosorum strain, and / or cells derived from it, can be administered as a dietary supplement to reduce or inhibit the growth of Peptostreptococcus canis bacteria. The dietary supplement may be in the form of powder, sachets, capsules (encapsulated form), biscuits, treats, gummies, paste, sticks, strips, granules, kibble, or may be incorporated directly into food during its manufacture, particularly pet food such as kibble.

[0116] The Bacillus inaquosorum strain, and / or cells derived from it, can be administered in the form of a parapharmaceutical composition to reduce or inhibit the growth of the bacterium Peptostreptococcus canis. The parapharmaceutical composition may be in the form of sachets, powders, capsules, softgels, dental pastes, gummies, or gels.

[0117] The Bacillus inaquosorum strain, and / or cells derived from it, can be administered as a pharmaceutical composition to reduce or inhibit the growth of the bacterium Peptostreptococcus canis. The pharmaceutical composition may be available by prescription or over the counter. It may be administered using any combination of dosage and route of administration effective in achieving the desired therapeutic or prophylactic effect. The effective amount to be administered may vary from one individual to another, depending on the species, age, weight, general condition of the individual, the presence or absence of an infectious disease of the oral cavity, and, if applicable, its nature, severity, and / or extent, etc.

[0118] The pharmaceutical composition may be intended for topical or oral administration. The pharmaceutical composition may include a physiologically acceptable excipient. A physiologically acceptable excipient may be one suitable for administration to mammals, particularly dogs and cats.

[0119] The pharmaceutical composition may include at least one additional active pharmaceutical ingredient having soothing, anti-irritant, analgesic, pain-relieving, anti-inflammatory, healing, antibiotic, antipyretic or antifungal activity.

[0120] The pharmaceutical composition may include at least one additive, for example an additive chosen from the group consisting of preservatives, sweeteners, flavorings, thickening agents, colorings, humectants, disintegrating agents, absorption accelerators, lubricating agents, and mixtures thereof.

[0121] The pharmaceutical composition may be in any form suitable for administration to a subject, preferably a mammal, most preferably a dog or a cat.

[0122] The pharmaceutical composition may be in the form of tablets, pills, dragees, capsules, pearls, syrups, emulsions, ointments, pastes, gels, powders, sachets or injectable solutions.

[0123] The Bacillus inaquosorum strain, and / or cells derived therefrom, optionally in combination with a yeast product, or the composition comprising it(s), may be administered by incorporation into a dental medical device comprising the Bacillus inaquosorum strain, and / or cells derived therefrom, as defined above, to reduce or inhibit the growth of bacteria of the species Peptostreptococcus canis.

[0124] Example

[0125] The following example illustrates the invention without limiting it.

[0126] Example 1

[0127] Product tested

[0128] Product 1: Suspension of Bacillus inaquosorum bacteria filed on September 12, 2024 with Agricultural Research Culture Collection and having the registration number NRRL B-68429.

[0129] Study model

[0130] The spot-on-the-lawn test was implemented to evaluate the inhibitory activity of product 1 against Peptostreptococcus canis bacteria using bacterial mats with 3 different concentrations: 10 6 CFU / ml, 10 7 CFU / ml and 10 8 CFU / ml.

[0131] Results concerning the growth of bacteria of the species Peptostreptococcus canis

[0132] The results concerning the growth of bacteria of the species Peptostreptococcus canis in the presence of product 1 are reported in Table 1.

[0133] Table 1

[0134] The results reported in Table 1 demonstrate that product 1 achieves inhibition of the growth of bacteria of the species Peptostreptococcus canis at the tested concentrations.

[0135] Example 2

[0136] Product tested

[0137] Product 1: Suspension of Bacillus inaquosorum bacteria filed on September 12, 2024 with Agricultural Research Culture Collection and having the registration number NRRL B-68429.

[0138] Study model

[0139] In vitro model of a polymicrobial biofilm comprising five different bacterial species mimicking a canine dental biofilm, namely Neisseria zoodegmatis CCUG 52598T, Corynebacterium canis CCUG 58627T, Porphyromonas cangingivalis DSMZ VPB 4874, Peptostreptococcus canis CCUG 57081 and an isolate of Enterococcus faecalis.

[0140] Analysis

[0141] The tests were performed in triplicate. A dental plaque biofilm was artificially created from five microorganisms described in the literature as being involved in periodontal disease: early plaque colonizers such as Neisseria zoodegmatis and Corynebacterium canis, other anaerobic bacteria such as Peptostreptococcus canis and Porphyromonas cangingivalis, and Enterococcus faecalis, which is involved in the development of periodontal disease in dogs. A bacterial suspension of each strain was deposited into the wells of a 96-well microplate placed on a Calgary device. A lid with pegs, previously incubated for two hours in artificial canine saliva, was then added to the microplate.After 48 hours of incubation at 37°C under microaerophilic conditions, biofilm formed on the surface of the PEGs. Following incubation, the PEGs were washed three times in 0.9% NaCl and transferred to a new microplate supplemented with product 1 at a concentration of 10% of each product. The plate was then incubated for 24 hours at 37°C under microaerophilic conditions, after which the percentage of biofilm formation inhibition was determined by Crystal Violet staining (by determining optical density).

[0142] Results regarding biofilm inhibition

[0143] The results (average of the three trials) concerning the inhibition of biofilm formation in the presence of product 1 are reported in Table 2.

[0144] Table 2

[0145] The results reported in Table 2 demonstrate that product 1 enables inhibition of biofilm formation.

[0146] Example 3

[0147] Products tested

[0148] Product 2: Bacillus inaquosorum suspension filed on September 12, 2024 with Agricultural Research Culture Collection and having the registration number NRRL B-68429.

[0149] Product 3: product comprising 20 to 30% p-glucans, 20 to 30% mannans, 10 to 25% protein and 10% or less glycogen, by weight by total weight of product (Safmannan® product, Phileo by Lesaffre, Lesaffre group) Product 4: product comprising 50 to 60% p-glucans, 1 and 5% mannans, 10% or less protein and 10% or less glycogen, by weight by total weight of product (Safglucan® product, Phileo by Lesaffre, Lesaffre group). Product 5: product having a pH between 6.3 and 6.7 (8.33% solution) and comprising at least 94% by weight of dry matter, less than 1% by weight of sodium chloride, 7.5 to 9.0% by weight of total nitrogen, 46.9 to 56.3% by weight of protein (nitrogen x 6.25), and 18 to 22% by weight of ash (excluding sodium chloride) by total weight of product (Springer® 4101 / 0-PW-L, Biospringer by Lesaffre, Lesaffre Group).

[0150] Study model

[0151] In vitro model of a polymicrobial biofilm according to the protocol of example 2.

[0152] Analysis

[0153] Analysis according to the protocol of example 2.

[0154] Results concerning biofilm inhibition The results concerning the inhibition of biofilm formation in the presence of the product combinations are reported in Table 3.

[0155] Table 3

[0156] The results reported in Table 3 demonstrate that the product combinations make it possible to achieve inhibition of biofilm formation.

Claims

DEMANDS 1. Non-therapeutic use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, to reduce and / or prevent the formation of dental biofilm.

2. Non-therapeutic use of the Bacillus inaquosorum strain, and / or cells derived from it, or a composition comprising them, whereby the Bacillus inaquosorum strain is the strain deposited on September 12, 2024 with the Agricultural Research Culture Collection under the Treaty of Budapest and having received the registration number NRRL B-68429.

3. Non-therapeutic use according to claim 1, wherein the formation of dental biofilm is reduced or prevented by reducing or inhibiting the growth of Peptostreptococcus canis.

4. Non-therapeutic use according to any one of the preceding claims, wherein the Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, is administered to mammals; preferably to dogs and / or cats; very preferably to dogs.

5. Non-therapeutic use according to any one of the preceding claims, wherein the Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, is administered in combination with a yeast product.

6. Non-therapeutic use according to claim 5, wherein the yeast product is a yeast product comprising at least 20% p-glucans, by weight per total weight of the yeast product.

7. Non-therapeutic use according to claim 5, wherein the yeast product is a yeast extract having a pH between 6.0 and 7.0; and comprising, by weight by total weight of the yeast product, 35 to 65% protein (nitrogen x 6.25), 10 to 30% ash and 3% or less sodium chloride.

8. Non-therapeutic use according to any one of the preceding claims, whereby the Bacillus inaquosorum strain, and / or the cells derived from it, or a composition comprising them, is administered to a healthy subject.

9. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use to reduce and / or prevent the formation of dental biofilm.

10. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to claim 9, wherein the Bacillus inaquosorum strain is the strain filed on September 12, 2024 with Agricultural Research Culture Collection under the Treaty of Budapest and having received the registration number NRRL B-68429.

11. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to any one of claims 9 and 10, wherein the formation of dental biofilm is reduced or prevented by reducing or inhibiting the growth of Peptostreptococcus canis.

12. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to any one of claims 9 to 11, whereby Bacillus inaquosorum, or the composition comprising it, is administered to mammals; preferably to dogs and / or cats; very preferably to dogs.

13. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to any one of claims 9 to 12, wherein Bacillus inaquosorum is administered in combination with a yeast product.

14. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to claim 13, wherein the yeast product is a yeast product comprising at least 20% p-glucans, by weight per total weight of the yeast product.

15. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to the claim 13, according to which the yeast product is a yeast extract having a pH between 6.0 and 7.0; and comprising, by weight per total weight of the yeast product, 35 to 65% protein (nitrogen x 6.25), 10 to 30% ash, 3% or less sodium chloride.

16. The Bacillus inaquosorum strain, and / or cells derived therefrom, or a composition comprising them, for therapeutic use according to any one of claims 9 to 15, whereby Bacillus inaquosorum, or the composition comprising it, is administered to a subject having a disease of the oral cavity, or at risk of having one.

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