Probiotic compositions for the treatment of oral diseases

Abstract

Probiotic compositions comprising Bacillus subtilis for use in the prevention or treatment of an oral or dental disease caused by bacterial infections, such as periodontal disease, in mammals such as small pets, for example, dogs and cats. Also provided are methods of prevention and treatment of oral or dental diseases caused by bacterial infections.

Description

PROBIOTIC COMPOSITIONSFOR THE TREATMENT OF ORAL DISEASESFIELD OF THE DISCLOSURE

[0001] The present disclosure relates to probiotic compositions containing Bacillus subtilis for use in the prevention or treatment of an oral or dental disease caused by pathogens, such as periodontal disease, in mammals such as small pets, for example, dogs and cats. Also provided are methods of prevention and treatment of oral or dental diseases caused by pathogens.BACKGROUND OF THE DISCLOSURE

[0002] Periodontal disease is the most frequent problem diagnosed in small animal veterinary medicine. However, most cases do not present external signs of the disease, leading to more severe complications if not treated. Therefore, dental care at home for pets should be considered a key element in both the prevention and periodontal therapy of small animals such as cats and dogs. Among many of the passive dental care strategies at home is the supplementation of the animal diet with bioactive ingredients that can reduce or prevent periodontal disease in its initial or advanced state.SUMMARY OF THE DISCLOSURE

[0003] Additional features and advantages of the present disclosure may be described further below. This summary section is meant merely to illustrate certain features of the disclosure and is not meant to limit the scope of the disclosure in any way. The failure to discuss a specific feature or embodiment of the disclosure, or the inclusion of one or more features in this summary section, should not be construed to limit the claims.

[0004] In one aspect, the present disclosure provides methods for preventing or treating an oral disease in a subject by administering to the subject in need thereof a composition comprising Bacillus subtilis.

[0005] In one embodiment, the present invention is a method of preventing or treating an oral or dental disease in a subject in need thereof comprising administering to the subject a probiotic composition comprising Bacillus subtilis, a supernatant, a cell-free supernatant and / or a heat-killed cell-free supernatant thereof.

[0006] In another embodiment, the present invention is a probiotic composition comprising Bacillus subtilis, a supernatant, a cell-free supernatant and / or a heat-killed cell-free supernatant thereof for use in preventing or treating an oral or dental disease in a subject in need thereof.

[0007] In a further embodiment, the present invention is a probiotic composition comprising Bacillus subtilis, a supernatant, a cell-free supernatant and / or a heat-killed cell-free supernatant thereof for use in the manufacture of a medicament for preventing or treating an oral or dental disease in a subject in need thereof.

[0008] In embodiments, Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015).

[0009] In preferred embodiments, the Bacillus subtilis used in the methods and probiotic compositions of the present invention is a single strain of Bacillus subtilis, BS-MB40 strain (PTA- 122264 filed before ATCC on 24 June 2015), a supernatant, a cell-free supernatant and / or a heat- killed cell-free supernatant thereof.

[0010] In preferred embodiments, the subject is a mammal, preferably a pet, even more preferably a dog, a cat, or another small pet.

[0011] In further embodiments, the oral disease being treated is a disease caused by one or more pathogen selected among the group comprising Porphyromonas gingi valis. Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0012] In particularly preferred embodiments, the oral or dental disease being treated or prevented by the methods and probiotic compositions of the present invention is caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0013] In other preferred embodiments, the oral or dental disease being prevented or treated by the methods and probiotic compositions of the present invention is selected from the group comprising gingivitis, (inflammation of the gums due to plaque), early, moderate or advanced periodontitis (progressive destruction of periodontal tissues, ligament and bone), furcation disease (bone loss between roots of multirooted teeth), endodontic-periodontal lesions or infections (combined pulp and periodontal infections), stomatitis (inflammation of the mouth lining), plaque formation, tartar formation, poor oral health, poor gum health, gum inflammation, dental abscess, caries lesions, or tooth decay.

[0014] In some embodiments, the probiotic composition and methods of the invention inhibit the growth of one or more of pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0015] In some aspects, the composition inhibits growth of one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens. In embodiments, the oral disease is an inflammatory disease, gingivitis, periodontitis, dental abscess, caries lesions, or an endodontic infection.

[0016] In some embodiments, the probiotic compositions and methods of the present invention inhibit the formation of a microbial biofilm on a surface. Said surface may be inert surface or a biological tissue such as oral mucosa, teeth or gingives. In some aspects, the probiotic compositions and methods of the present invention inhibit the formation of biofilm growth caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens.

[0017] In some embodiments of the present invention, the probiotic compositions comprise Bacillus subtilis spores.

[0018] In preferred embodiments, the concentration of spores is, for example, from IxlO8to IxlO12CFU / g, or is at least about IxlO11CFU / g.

[0019] In one embodiment, the probiotic compositions and methods of the present invention may comprise one or more additional probiotics, in the same or separate composition, to be administered together, separately, simultaneously, or sequentially, selected from the group comprising Streptococcus oralis, Streptococcus uber is, Streptococcus rattus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus salivarius, Bacillus coagulans, and Bifidobaceterium (e.g., Bifidobaceterium breve, Bifidobaceterium animalis, Bifidobaceterium longum). In particular preferred embodiments said additional probiotic is selected from the group comprising Lactobacillus fermentum CECT5716, Lactobacillus salivarius CECT 5713 and / 'or Bacillus coagulans GBI-30, 6086.

[0020] In further embodiments of the present invention, the probiotic composition is in the form of a dry powder. The dry powder may be lyophilized, freeze dried, spray dried, or air-dried.In some embodiments, the dry powder may further comprise a cryoprotectant. In preferred embodiments, the dry powder further comprises maltodextrin.

[0021] In some preferred embodiments, the probiotic compositions of the present invention further comprise maltodextrin.

[0022] In further embodiments, Bacillus subtilis may be microencapsulated.

[0023] According to further preferred embodiments, the moisture content of the dry powder may be 10% or less as measured by Ohaus MB45 method.

[0024] In further preferred embodiments, the probiotic composition may additionally contain sodium chloride, calcium carbonate, and / or sodium bicarbonate.

[0025] According to preferred embodiments of the present invention, the probiotic composition comprising Bacillus subtilis for use in preventing or treating an oral or dental disease is a nutritional composition, such as, for example, a dental chew, a dental stick, a complete meal or a meal supplement formulated for administration to a pet.

[0026] In particularly preferred embodiments, the pet is a dog, a cat or another small pet. In one embodiment, the small pet is selected from the group comprising a rabbit, a hamster, a guinea pig, a ferret, or another small pet.

[0027] The invention further relates to a composition comprising Bacillus subtilis and / or a supernatant thereof, for use in preventing or treating an oral or dental disease in a subject in need thereof. Preferably, said subject is a mammal. In a preferred embodiment said mammal is a pet selected among a dog, a cat, or another small pet selected from the group comprising a rabbit, a hamster, a guinea pig, a ferret, or another small pet.

[0028] In a preferred embodiment of the present invention said Bacillus subtilis for use in preventing or treating an oral or dental disease in pets is Bacillus subtilis BS-MB40 (PTA-122264filed before ATCC on 24 June 2015) and / or a supernatant thereof. In a preferred embodiment theBacillus subtilis, or the supernatant thereof are in microencapsulated form.

[0029] The invention further relates to the spores of Bacillus subtilis strain BS-MB40 (PTA- 122264 filed before ATCC on 24 June 2015) for use in preventing or treating an oral or dental disease in pets.

[0030] The invention further relates to the use of Bacillus subtilis and / or a supernatant thereof for the prevention of biofilm formation on a surface. Said surface may be an inert surface or a biological tissue, such as such as oral mucosa, teeth or gingives. In particularly preferred embodiments the biofilm formation is caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The application contains the following figures provided only for illustrative purpose and not by way of limitation.

[0032] FIG. 1 shows the workflow for the preparation of cell-free supernatants (CFS) and heat-killed cell-free supernatants (HK-CFS) of Bacillus subtilis and testing their effects on pathogen growth inhibition.

[0033] FIG. 2, panels A-D shows the results of the Pathogen Inhibition Assay with cell- free supernatant as described in Example 4; MB40 (A) is CFS from a 24 hours culture of MB40; MB40 (B) is CFS from a 48 hours culture of MB40.

[0034] FIG. 3, panels A-D shows the results of the Pathogen Inhibition Assay with heat killed cell-free supernatant as described in Example 4; MB40 (A) is CFS from a 24 hours culture of MB40; MB40 (B) is CFS from a 48 hours culture of MB40.

[0035] FIG. 4, panels A-D show the results of the anti -biofilm assay of the bacteria Bacillus subtilis MEMO against different oral dog pathogenic bacteria as described in Example 5.

[0036] FIG. 5, panels A-D show the results of the anti-biofilm assay of the freshly prepared bacteria Bacillus subtilis MEMO against different oral dog pathogenic bacteria as described in Example 5.

[0037] FIG. 6, panels A-D show the dose ranging effect of CFS of Bacillus subtilis MB40 after 48 hours incubation on the biofilm formation of different oral dog pathogenic bacteria as described in Example 5.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] Before describing in more detail the present probiotic compositions, methods, and methodologies, it is to be understood that the disclosure is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope will be limited only in the appended claims.

[0039] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "a saccharide fatty acid ester" includes one or more saccharide fatty acid esters of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may be usedin the practice or testing of the disclosure, as it will be understood that modifications and variations are encompassed within the spirit and scope of the disclosure. Accordingly, the invention is limited only by the following claims.

[0041] Any range will be understood to encompass and be a disclosure of each discrete point and subrange within the range. Stated differently, the ranges in the present disclosure are equivalent to a subset of the unwieldy and lengthy description of every possible combination of these discrete values, presented in an easily understood shorthand format (i.e., a range).

[0042] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by a person of ordinary skill in the art and will vary in some extent depending on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" and "approximately" will mean plus or minus <10% of particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term. "Comprising" and "consisting essentially of have their customary meaning in the art.

[0043] As used herein, optional (e.g., the use of the term “may” include) and alternative elements (e.g., example items in a list) described herein may be explicitly excluded from the compositions and methods of the disclosure.

[0044] As used herein, the term “probiotic compositions” of the present invention includes compositions comprising Bacillus sublilis. a single strain of Bacillus sublilis. a combination of strains of Bacillus sublilis., Bacillus subtilis BS-MB40 strain (PTA- 122264 filed before ATCC on 24 June 2015), spores, supernatants, cell-free supernatants and / or heat-killed cell-free supernatants thereof. The bacterial strain acronyms “BS-MB40” and “MB40” will be used interchangeably in the context of the present disclosure.

[0045] As used herein, the term “supernatant” includes culture supernatant, cell-free supernatant or heat-killed cell-free supernatant.Probiotic compositions

[0046] In one aspect, the present disclosure provides a probiotic composition comprising Bacillus subtilis. In some aspects, Bacillus subtilis is MB40 strain, such as MEMO (PTA-122264) strain.

[0047] In some aspects, the probiotic composition of the invention is for use in preventing or treating an oral or dental disease.

[0048] The present invention provides a probiotic composition comprising Bacillus subtilis for use in preventing or treating an oral or dental disease in a subject in need thereof. In another embodiment, the present invention provides a probiotic composition comprising Bacillus subtilis and / or a supernatant thereof for use in preventing or treating an oral or dental disease in a subject in need thereof. In another embodiment, the present invention provides a probiotic composition comprising Bacillus subtilis a supernatant, a cell-free supernatant and / or a heat killed cell-free supernatant thereof for use in preventing or treating an oral or dental disease in a subject in need thereof. In another embodiment, the present invention provides a probiotic composition comprising a supernatant of Bacillus subtilis for use in preventing or treating an oral or dental disease in a subject in need thereof. In a preferred embodiment, said Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015). Said supernatant, is preferably a cell- free supernatant, even more preferably heat-killed cell-free supernatant. In another embodiment, the present invention provides a probiotic composition comprising spores of Bacillus subtilis for use in preventing or treating an oral or dental disease in a subject in need thereof.

[0049] In some embodiment of the present invention the activity of the supernatant used in the probiotic compositions of present invention may be due to the release of bioactive molecules during, for example, the heat treatment, and these may have some specificity against certain oral pathogens.

[0050] In a preferred embodiment of the present invention said Bacillus subtilis derives from a pure culture. In a preferred embodiment of the present invention said Bacillus subtilis BS- MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) is a purified strain. In a preferred embodiment of the present invention said Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) was derived using methods which ensure purity and reproducibility of results.

[0051] The oral or dental disease to be prevented or treated according to the methods or probiotic composition of the present invention may be selected from the group comprising gingivitis, (inflammation of the gums due to plaque), early, moderate or advanced periodontitis (progressive destruction of periodontal tissues, ligament and bone), furcation disease (bone loss between roots of multirooted teeth), endodontic-periodontal lesions or infections (combined pulp and periodontal infections), stomatitis (inflammation of the mouth lining), plaque formation, tartar formation, oral health, gum health, gum inflammation, dental abscess, caries lesions, or tooth decay. In particular preferred embodiments of the present invention said oral or dental disease is caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens. The disease may be, for example, an inflammatory disease, gingivitis, periodontitis, dental abscess, caries lesions, or an endodontic infection.

[0052] In a further embodiment, the probiotic compositions of the present invention inhibits the growth of one or more pathogen causing said disease, namely Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens.

[0053] According to an alternative embodiment of the invention, said probiotic composition may additionally comprises one or more probiotic selected from the group comprising Streptococcus oralis, Streptococcus uberis, Streptococcus rattus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus salivarius, Bacillus coagulans, and Bifidobaceterium spp. (e.g., Bifidobaceterium breve, Bifidobaceterium animalis, Bifidobaceterium longum) to be used in combination. Preferably, said additional probiotic may be selected among the group comprising: Lactobacillus fermentum CECT 5716, Lactobacillus salivarius CECT 5713 and / or Bacillus coagulans GBI-30, 6086. In some embodiments, said additional probiotic may be in the same or separate solution, suspension, emulsion, tablet, granule, powder or capsule. In some embodiments, said additional probiotic may be administered sequentially or simultaneously.

[0054] In some preferred embodiments, the probiotic composition of the invention may be lyophilized, freeze dried, spray dried, air-dried or frozen. The probiotic composition may further comprise a cryoprotectant. The cryoprotectant may be selected among the group comprising maltodextrin, polyvinylpyrrolidone, inulin, sugars (such as trehalose, sucrose, lactose, glucose and others), sugar alcohols (such as mannitol, sorbitol and others), the polymer hydroxyethyl starch, or mixtures thereof.

[0055] In some aspects, the probiotic composition of the present invention may be microencapsulated. In a further preferred embodiment, the probiotic (intended as the strain as such,the supernatant or the spores thereof) contained in the probiotic composition of the invention may be in encapsulated or in microencapsulated form. Encapsulation of the probiotic composition for use of the invention may have the advantage of allowing a controlled release, for example upon contact with saliva, or a continuous release over an extended period of time. Moreover, encapsulation of the probiotic compositions of the present invention may be protected from degradation improving the shelf life.

[0056] In some further preferred embodiments, the probiotic composition of the invention additionally contains maltodextrin. In other preferred embodiments the probiotic composition of the invention may additionally contain sodium chloride, calcium carbonate, and / or sodium bicarbonate.

[0057] In preferred embodiments, the probiotic composition of the present invention may be a dry powder. The moisture content of the probiotic composition may be 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Preferably, the moisture content of the probiotic composition may be about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, or about 5%. In other preferred embodiments, the moisture content of the probiotic composition may be, from 5 to 20%, from 5 to 19%, from 5 to 18%, from 5 to 17%, from 5 to 16%, from 5 to 15%, from 5 to 14%, from 5 to 13%, from 5 to 12%, from 5 to 11%, from 5 to 10%, from 5 to 9%, from 5 to 8%, from 5 to 7%, from 5 to 6%. In further preferred embodiments, the moisture content of the probiotic composition may be from 6 to 20%, from 7 to 20%, from 8 to 20%, from 9 to 20%, from10 to 20%, from 11 to 20%, from 12 to 20%, from 13 to 20%, from 14 to 20%, from 15 to 20%,from 16 to 20%, from 17 to 20%, from 18 to 20%, or from 19 to 20%. Even more preferably, the moisture content of the probiotic composition may be from about 8% to about 12%, from 9% to about 11%, or from about 9% to about 10%.

[0058] The moisture content in the probiotic compositions of the present invention may be measured, for example, by Ohaus MB45 method, or another method known in the art.

[0059] In specific embodiments of the invention the probiotic composition may contain Bacillus subtilis spores. According to preferred embodiments, the spore concentration may be about IxlO6CFU / g, about IxlO7CFU / g, about IxlO8CFU / g, about IxlO9CFU / g, about IxlO10CFU / g, about IxlO11CFU / g, about IxlO12CFU / g, about IxlO13CFU / g, about IxlO14CFU / g, or about IxlO15CFU / g. Preferably, the spore concentration may be at least IxlO6CFU / g, at least IxlO8CFU / g, at least IxlO9CFU / g, at least IxlO10CFU / g, at least IxlO11CFU / g, at least IxlO12CFU / g, at least IxlO13CFU / g, at least IxlO14CFU / g, at least IxlO15CFU / g. Even more preferably, the spore concentration may be from IxlO8to IxlO15CFU / g, from IxlO8to IxlO12CFU / g, from IxlO8to IxlO11CFU / g, from IxlO8to IxlO10CFU / g, from IxlO8to IxlO9CFU / g, from IxlO9to IxlO15CFU / g, from IxlO10to IxlO15CFU / g, from IxlO11to IxlO15CFU / g, from IxlO12to IxlO15CFU / g, from IxlO13to IxlO15CFU / g, or from IxlO14to IxlO15CFU / g. In the most preferred embodiments, the spore concentration may be from about IxlO8to about IxlO12CFU / g, from about IxlO9to about IxlO11CFU / g, or from about IxlO10to about IxlO11CFU / g.

[0060] According to further embodiments, the probiotic composition of the present invention contains Bacillus subtilis by total weight of the composition of from about 5% to 50%, from about 6% to 50%, from about 7% to 50%, from about 8% to 50%, from about 10% to 50%, from about 15% to 50%, from about 20% to 50%, from about 25% to 50%, from about 30% to50%, from about 40% to 50%, from about 45% to 50%, from about 5% to 20%, from about 5% to15%, from about 5% to 10%, from about 10% to 70%, from about 40% to 60%, from about 40% to 50%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 9%, at least 8%, at least 7%, at least 6%, at least 5%, at least 4%, at least 2%, or at least 1%. Preferably, the probiotic composition contains Bacillus subtilis by total weight of the composition of from about 50% to 95%, from about 60% to 95%, from about 70% to 95%, from about 75% to 95%, from about 80% to 95%, from about 85% to 95%, from about 90% to 95%, from about 50% to 90%, from about 60% to 90%, from about 70% to 90%, from about 75% to 90%, from about 80% to 90%, from about 85% to 90%, from about 5% to 75%, from about 10% to 70%, from about 40% to 60%, or from about 40% to 50%.

[0061] In some preferred embodiments of the present invention, the amount of viable spores in the probiotic composition relative to the total amount of spores may be about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In further preferred embodiments, the amount of viable spores in the probiotic composition relative to the total amount of spores is 10% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0062] In some aspects, the total viable spore count in the probiotic compositions of the present invention is at least IxlO2, at least IxlO3CFU / g, at least IxlO4CFU / g, at least IxlO5CFU / g, at least IxlO6CFU / g, at least IxlO7CFU / g, at least IxlO8CFU / g, at least IxlO9CFU / g, at least IxlO10CFU / g, at least IxlO11CFU / g, at least IxlO12CFU / g, at least IxlO13CFU / g, at least IxlO4CFU / g, or at least IxlO15CFU / g.

[0063] According to a preferred embodiment of the probiotic composition of the present invention, the spores are prepared by fermentation of a pure culture of Bacillus subtilis. Following fermentation, acid may be added to the bacterial culture to decrease the pH to 4.5. The stabilized culture may be concentrated, for example, by centrifugation, followed by addition of maltodextrin so that the total solids content is up to 10% by weight of the total preparation, and then spray dried. The resulting product may be blended with additional maltodextrin (or other food-grade diluents, such as, for example, sodium chloride, calcium carbonate, and sodium bicarbonate), resulting in the final spore preparation.

[0064] In further embodiments, the total solids content in the spore preparation before spray drying, freeze drying or lyophilization is, for example, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 40%, or about 50%. Preferably, the total solids content is, for example, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 11%, up to 12%, up to 13%, up to 14%, up to 15%, up to 16%, up to 17%, up to 18%, up to 19%, up to 20%, up to 25%, up to 30%, up to 40%, or up to 50%.

[0065] According to preferred embodiments, the pH of the spore preparation is, for example, from about 2.0 to 9.5, about 8.0, about 7.0, about 6.5, about 6.0, about 5.5, about 5.0, about 4.5, about 4.0, about 3.5, about 3.0, 9.0 or less, 8.0 or less, 7.5 or less, 7.0 or less, 6.5 or less, 6.0 or less, 5.5 or less, 5.0 or less, at least 4.5, at least 4.0, less at least 3.5, or 3.0 or less. Preferably, the pH of the spore preparation is from about 3.0 to about 8.0, from about 3.5 to about 8.0, from about 4.0 to about 8.0, from about 4.5 to about 8.0, from about 5.0 to about 8.0, from about 3.0 to about 7.0, from about 3.5 to about 7.0, from about 4.0 to about 7.0, from about 4.5 to about 7.0, from about 5.0 to about 7.0, from about 5.5 to about 7.0, from about 6.0 to about 7.0, from about3.0 to about 5.0, from about 3.5 to about 5.0, from about 4.0 to about 5.0, or from about 4.5 to about 5.0.

[0066] In some embodiment, in the probiotic compositions and methods of the present invention the probiotic composition is in the form of a solution, suspension, emulsion, tablet, granule, powder or capsule.

[0067] In some aspects, the compositions is a dry powder having the properties as specified in paragraphs above.

[0068] In some embodiment, daily dosage of administration of Bacillus subtilis in the probiotic compositions of the present invention ranges between 500 million CFU / day and 10 billion CFU / day, between 1 billion CFU / day and 9 billion CFU / day, between 2 billion CFU / day and 8 billion CFU / day, 3 billion CFU / day and 7 billion CFU / day, 4 billion CFU / day and 6 billion CFU / day. In one embodiment the daily dosage of administration of Bacillus subtilis in the probiotic composition of the invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 billion CFU / day. In one embodiment the daily dosage of administration of Bacillus subtilis in the probiotic composition of the invention is 4, 5, or 6 billion CFU / day. In a preferred embodiment the daily dosage of administration of Bacillus subtilis in the probiotic composition of the invention is 5 billion CFU / day.

[0069] In some embodiments, the probiotic compositions and methods of the present invention contains a supernatant of Bacillus subtilis,' in preferred embodiment said supernatant is a cell-free supernatant (CFS); in a even more preferred embodiment the CFS is heat-killed CFS (HK CFS).

[0070] According to a preferred embodiment of the probiotic compositions and methods of the present invention the CFS or HK CFS is obtained by culturing Bacillus, subtilis for 24 hours,48 hours, 60 hours, 72 hours, or 96 hours.

[0071] In a preferred embodiment of the invention said Bacillus Subtilis may be cultured in TSA-II or TSB media at 37°C under constant agitation (for example, at 250 rpm) under aerobic conditions for 24, 48, 60, 72 or 96 hours. Cell debris may be removed by centrifugation, optionally sterilized (for example, 20 minutes at 121°C) to prepare HK-CFS, pH adjusted to 7.0 and filtered using a 0.2 pm filter.

[0072] In some aspect, the present disclosure provides a nutritional composition. In some aspects the nutritional composition contains dry powder containing Bacillus subtilis and having the properties as specified in paragraphs above.

[0073] The present disclosure further provides a nutritional probiotic composition. In some aspects the nutritional composition comprises a dry powder comprising Bacillus subtilis and / or a supernatant thereof as a probiotic. In some aspect, the nutritional composition has the properties as specified in the paragraphs above. In particularly preferred aspects, the nutritional composition of the invention contains a supernatant. Said supernatant is preferably a cell-free supernatant (CFS); even more preferably the CFS is heat-killed CFS (HK CFS).

[0074] In some aspects the CFS or HK CFS is obtained by culturing Bacillus subtilis for 24 hours, 48 hours, 60 hours, 72 hours, or 96 hours.

[0075] Bacillus subtilis may be cultured, for example, in TSA-II or TSB media at 37°C under constant agitation (for example, at 250 rpm) under aerobic conditions for 24, 48, 60, 72 or 96 hours. Cell debris may be removed by centrifugation, optionally sterilized (for example, 20 minutes at 121°C) to prepare HK-CFS, pH adjusted to 7.0 and filtered using a 0.2 pm filter.

[0076] According to particular embodiments of the present invention the probiotic nutritional compositions of the present disclosure may be formulated as a chew, a dental stick, a complete meal, or a meal supplement for a pet. As above mentioned, the pet may be a dog, a cat, or another small pet, such as a rabbit, a hamster, a guinea pig or a ferret.

[0077] The nutritional composition of the present invention may contain further nutritional ingredients, such as meet, fat, grain, protein, vitamins, minerals, amino acids, nucleic acids, and other nutritional supplements suitable for administration in veterinary field.

[0078] The nutritional composition may contain further ingredients that are used in pet foods, such as flavor agents, dyes, fillers, binding agents, stabilizers, emulsifiers, sweeteners, colorants, buffers, salts, coatings, condiments, preservatives, and the like.

[0079] The probiotic composition of the present invention may be administered multiple times a day, at every meal, once a day or as needed.

[0080] The invention further relates to the use of the probiotic composition for inhibiting the biofilm formation on a surface. Said surface may be an inert surface or a biological tissue, such as oral mucosa, teeth or gingives. In particularly preferred embodiments the oral biofilm formation is caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens.

[0081] In another embodiment, the probiotic compositions of present invention balance the bacterial composition of oral biofilms derived from saliva in favour of beneficial bacteria.Methods

[0082] In one embodiment, the present invention relates to a method of preventing or treating oral or dental diseases in a subject in need thereof by administering to the subject the probiotic compositions according to the present invention. In a preferred embodiment, in themethods of the present invention said probiotic composition comprises Bacillus subtilis a supernatant, a cell-free supernatant and / or a heat killed cell-free supernatant thereof. In a further preferred embodiment, in the methods of the present invention said probiotic composition comprises spores of Bacillus subtilis. In a preferred embodiment, in the methods of the present invention said probiotic composition comprises Bacillus subtilis, preferably Bacillus subtilis BS- MB40 strain (PTA-122264 filed before ATCC on 24 June 2015).

[0083] Said oral or dental disease may be selected among the group comprising gingivitis, (inflammation of the gums due to plaque), early, moderate or advanced periodontitis (progressive destruction of periodontal tissues, ligament and bone), furcation disease (bone loss between roots of multirooted teeth), endodontic-periodontal lesions or infections (combined pulp and periodontal infections), stomatitis (inflammation of the mouth lining), plaque formation, tartar formation, poor oral health, poor gum health, gum inflammation, dental abscess, caries lesions, or tooth decay. In particular preferred embodiments of the present invention said oral or dental disease is caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens. In a particularly preferred embodiment said disease may be an inflammatory disease, gingivitis, periodontitis, dental abscess, cariotic lesions, or an endodontic infection, and others.

[0084] In some embodiments, the probiotic compositions of the present invention employed in the methods of the invention inhibit the growth of one or more of the pathogen causing said disease, such as Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens. In a preferred embodiment, the Bacillus subtilis for use in the methods according to the present invention prevents, inhibits or reduces the growth of one or more pathogen selected among Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia, orPrevotella nigrescens. Preferably the Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) and / or the spores or the supernatant thereof.

[0085] According to an alternative embodiment of the methods of the present invention, said probiotic compositions of the present invention may be simultaneously or sequentially administered in combination with one or more additional probiotic selected from the group comprising Streptococcus oralis, Streptococcus uber is, Streptococcus rattus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus salivarius, Bacillus coagulans, and Bifidobaceterium spp. (e.g., Bifidobaceterium breve, Bifidobaceterium animalis, Bifidobaceterium longum). Preferably, said additional probiotic may be selected from the group comprising: Lactobacillus fermentum CECT5716, Lactobacillus salivarius CECT 5713 and / or Bacillus coagulans GBI-30, 6086.

[0086] In some aspects, the subject in needs thereof may be a mammal. Preferably said mammal is a pet. More preferably, said pet is a small pet, such as a dog, a cat, a rabbit, a hamster, a guinea pig or a ferret. The compositions may be administered multiple times a day, at every meal, once a day or as needed.Strain

[0087] The present invention further relates to Bacillus subtilis and / or a supernatant thereof, for use in preventing or treating an oral or dental disease in a subject in need thereof. In a preferred embodiment, said Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) and / or a supernatant thereof. Said supernatant, is preferably a cell-free supernatant (CFS), even more preferably heat-killed cell-free supernatant (HK-CFS).

[0088] In a preferred embodiment of the present invention said Bacillus subtilis derives from a pure culture.

[0089] In a further preferred embodiment of the present invention said Bacillus subtilis is in encapsulated or microencapsulated form.

[0090] A further object of the present invention are Bacillus subtilis spores for use in preventing or treating an oral or dental disease in a subject in need thereof. In a preferred embodiment, said Bacillus subtilis spores derive from BS-MB40 strain (PTA- 122264 filed before ATCC on 24 June 2015). In a preferred embodiment said spores derive from a pure culture of Bacillus subtilis.

[0091] In a preferred embodiment, the subject in needs thereof is a mammal. Said mammal is a pet, preferably a small pet. More preferably, said small pet is selected from a dog, a cat, a rabbit, a hamster, a guinea pig or a ferret.

[0092] The invention further relates to the use of Bacillus subtilis and / or a supernatant thereof and / or its spores for the prevention of biofilm formation on a surface. Said surface may be an inert surface, a biological tissue, such as the oral mucosa, teeth or gingives. In particularly preferred embodiments the oral biofilm formation is caused by one or more pathogen selected among the group comprising Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia or Prevotella nigrescens.

[0093] In some embodiments the present invention is:

[0094] 1. A method for preventing or treating an oral disease in a subject by administering to the subject in need thereof a composition comprising Bacillus subtilis.

[0095] 2. The method of statement 1, wherein Bacillus subtilis is BS-MB40 strain(PTA- 122264).

[0096] 3. The method of statement 1, wherein the subject is a mammal.

[0097] 4. The method of statement 3, wherein the mammal is a dog.

[0098] 5. The method of any one of statements 1-4, wherein the oral disease is a disease caused by one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0099] 6. The method of any one of statements 1-5, wherein the oral disease is an inflammatory disease, gingivitis, periodontitis, dental abscess, caries lesions, or an endodontic infection.

[0100] 7. The method of any one of statements 1-6, wherein the composition inhibits growth of one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0101] 8. The method of any one of statements 1-7, wherein the composition is a dry powder.

[0102] 9. The method of statement 8, wherein the dry powder is lyophilized, freeze dried, spray dried, air-dried or frozen.

[0103] 10. The method of any one of statements 8-9, wherein the dry powder further comprises a cryoprotectant.

[0104] 11. The method of any one of statements 8-10, wherein the dry powder further comprises maltodextrin.

[0105] 12. The method of any one of statements 8-11, wherein Bacillus subtilis comprises Bacillus subtilis spores.

[0106] 13. The method of statement 12, wherein the concentration of spores is from1x108 to 1x1012 CFU / g.

[0107] 14. The method of statement 12, wherein the concentration of spores is at least about 1x1011 CFU / g.

[0108] 15. The method of any one of statement s 8-14, wherein Bacillus subtilis is mi croencap sul ated .

[0109] 16. The method of any one of statements 8-15, wherein the moisture content is10% or less as measured by Ohaus MB45 method.

[0110] 17. The method of any one of statements 8-16, wherein the composition further comprises sodium chloride, calcium carbonate, and / or sodium bicarbonate.[OH l] 18. The method of any one of statements 8-17, wherein the composition comprising Bacillus subtilis is a nutritional composition.

[0112] 19. The method of statement 18, wherein the nutritional composition is a dental chew, a dental stick, a complete meal or a meal supplement formulated for administration to a pet.

[0113] 20. The method of statement 19, wherein the pet is a dog.

[0114] 21. A composition comprising Bacillus subtilis BS-MB40 strain (PTA-122264).

[0115] 22. The composition of statement 21, wherein the composition is a dry powder.

[0116] 23. The composition of statement 22, wherein the dry powder is lyophilized, freeze dried, spray dried, air-dried or frozen.

[0117] 24. The composition of any one of statements 22-23, wherein the dry powder further comprises a cryoprotectant.

[0118] 25. The composition of any one of statements 22-24, wherein the dry powder further comprises maltodextrin.

[0119] 26. The composition of any one of statements 21-25, wherein Bacillus subtilis comprises Bacillus subtilis spores.

[0120] 27. The composition of statement 26, wherein the concentration of spores is from 1x108 to 1x1012 CFU / g.

[0121] 28. The composition of statement 26, wherein the concentration of spores is at least about 1x1011 CFU / g.

[0122] 29. The composition of any one of statements 21-28, wherein Bacillus subtilis is microencapsulated.

[0123] 30. The composition of any one of statements 21-29, wherein the moisture content is 10% or less as measured by Ohaus MB45 method.

[0124] 31. The composition of any one of statements 21-30 further containing sodium chloride, calcium carbonate, and / or sodium bicarbonate.

[0125] 32. The composition of any one of statements 21-31, wherein the composition is a nutritional composition.

[0126] 33. The composition of statement 32, wherein the nutritional composition is a dental chew, dental stick, a complete meal, or a meal supplement formulated for administration to a pet.

[0127] 34. The composition of statement 33, wherein the pet is a dog.

[0128] 35. The composition of any one of statements 21-34 for use for preventing or treating an oral disease in a subject.

[0129] 36. The composition of statement 35, wherein the oral disease is a disease caused by one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0130] 37. The composition of any one of statements 35-36, wherein the oral disease is an inflammatory disease, gingivitis, periodontitis, dental abscess, caries lesions, or an endodontic infection.

[0131] 38. The composition of any one of statements 35-37, wherein the composition inhibits growth of one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

[0132] 39. The composition of any one of statements 35-38, wherein the subject is a mammal.

[0133] 40. The composition of statement 39, wherein the mammal is a dog.EXAMPLES

[0134] The embodiments of the present disclosure are described in further detail by means of the following Examples that should not be considered to be limiting for the scope of the present invention.Example 1Dry powder of Bacillus subtilisTable 1. Dry powder of Bacillus subtilis content and characteristicsExample 2Bacillus subtilis strain BS-MB40 (PTA- 122264) spore preparation

[0135] Spores are prepared by fermentation of a pure culture. Following fermentation, acid is added to the bacterial culture to decrease the pH to 4.5. The stabilized culture is concentrated by centrifugation, followed by addition of maltodextrin so that the total solids is up to 10%, and then spray dried. The resulting product is blended with additional maltodextrin (or other food-grade diluents, such as sodium chloride, calcium carbonate, and sodium bicarbonate), resulting in the final spore preparation.

[0136] The final spore preparation contains: total viable spore count (not less than 1011CFU / g); moisture (< 10%); heavy metals, including lead (< 0.5 mg / kg); yeast and mold (< 300 CFU / g); coliforms (< 30 CFU / g); Escherichia coli (negative / 25 g); Salmonella serovars (negative / 25 g); Staphylococcus aureus (not detected); and Listeria spp. (negative / 25 g). The results of stability testing indicate tha Bacillus subtilis BS-MB40 PTA-122264 spore preparation is stable for up to 30 months at room temperature (21±2 °C).Example 3Preparation of cell-free supernatants (CFS) and heat-killed cell-free supernatants (HK-CFS) of Bacillus subtilis

[0137] Bacillus subtilis MB40 was tested in pathogen inhibition analysis using cell-free supernatants (CFS) or heat-killed cell-free supernatants (HK-CFS).

[0138] The CFS and HK-CFS were prepared by adding one cryovial in 50 mL of sterile TSB and incubating in aerobic condition at 37°C under constant agitation at 250 rpm for 24 and 48 hours.

[0139] Cell debris was removed by centrifugation, optionally sterilized (20 minutes at 121°C) to prepare HK-CFS, pH adjusted to 7 and finally filtered using a 0,2 pm filter.

[0140] CFS samples were stored at 4°C and pre-reduced for 24 hours in an anaerobic chamber prior their use.Example 4Pathogen Inhibition assays - Broth method

[0141] Pathogenic bacteria preparation

[0142] The four pathogenic bacteria listed in Table 2 were purchased from American Type Culture Collection (ATCC). The bacteria were put in culture 48 hours prior to experimentation and incubated in an anaerobic chamber in ATCC media 2722 under (5% H2, 10% CO2, 85% N2).

[0143] Pathogen inhibition assay

[0144] Bacillus subtilis MB40 was tested in pathogen inhibition analysis by using cell-free supernatants (CFS) or heat-killed cell-free supernatants (HK-CFS) from the Bacillus subtilis culture prepared according to Example 3.

[0145] A BioScreen system was used with the provided custom-designed plate(HoneyComb plate).

[0146] Samples were dissolved, suspended and / or diluted in IxPBS and tested at 10% (v / v; CFS and HK-CFS).

[0147] Each well was filled with culture medium (ATCC medium 2722), pathogenic bacteria (30 uL of an overnight culture) and samples (10-40 uL; 2 replicates) to obtain a final volume of 300 pL.

[0148] Microplates were incubated in an anaerobic chamber at 37°C (see Table 2 for details) for 24 and 48 h and OD 600 nm was measured after incubation to evaluate bacterial growth.

[0149] The overall flow of the process is schematically depicted in FIG. 1.Table 2. Pathogens incubation conditions (broth media).XATCC broth medium 2722 pre-reduced for 24 h in A atmosphere.A: Anaerobic chamber with a gas mixture comprising 5% of hydrogen, 10% of carbon dioxide and 85% of nitrogen.

[0150] The results with CFS are shown in FIG. 2.

[0151] After 24 hours incubation of Porphyromonas gingivalis with 48 hours CFS, 94.3% inhibition was observed.

[0152] After 48 hours incubation of Porphyromonas gingivalis with 48 hours CFS, 99.86% inhibition was observed.

[0153] After 24 hours incubation of Porphyromonas gulae with 48 hours CFS, 96.03% inhibition was observed.

[0154] After 48 hours incubation of Porphyromonas gulae with 48 hours CFS, 92.96% inhibition was observed.

[0155] After 24 hours incubation of Prevotella intermedia with 48 hours CFS, 93.96% inhibition was observed.

[0156] After 48 hours incubation of Prevotella intermedia with 48 hours CFS, 89.77% inhibition was observed.

[0157] After 24 hours incubation of Prevotella nigrescens with 48 hours CFS, 82.46% inhibition was observed.

[0158] After 48 hours incubation time of Prevotella nigrescens with 48 hours cell-free supernatant (CFS) 93% inhibition was observed.

[0159] The results with HK-CFS are shown in FIG. 3.

[0160] After 24 hours incubation of Porphyromonas gingivalis with 48 hours HK-CFS, 100% inhibition was observed.

[0161] After 48 hours incubation of Porphyromonas gingivalis with 48 hours HK-CFS, 100% inhibition was observed.

[0162] After 24 hours incubation of Porphyromonas gulae with 48 hours HK-CFS, 96.57% inhibition was observed.

[0163] After 48 hours incubation of Porphyromonas gulae with 48 hours HK-CFS, 99.9% inhibition was observed.

[0164] After 24 hours incubation of Prevotella intermedia with 48 hours HK-CFS, 95.18% inhibition was observed.

[0165] After 48 hours incubation of Prevotella intermedia with 48 hours HK-CFS, 93.67% inhibition was observed.

[0166] After 24 hours incubation of Prevotella nigrescens with 48 hours HK-CFS, 94.55% inhibition was observed.

[0167] After 48 hours incubation of Prevotella nigrescens with 48 hours HK-CFS, 96.05% inhibition was observed.

[0168] As can be seen in the Figures, strong inhibitory properties were observed against all pathogenic bacteria when using 48 hours supernatant from Bacillus subtilis.

[0169] As can be seen in FIG. 3, inhibitory properties and in most cases strong inhibitory properties were observed against all pathogenic bacteria when using 24 hours heat killed cell-free supernatant from Bacillus subtilis.

[0170] After 24 hours incubation of Porphyromonas gingivalis with 24 hours HK-CFS, 94.31% inhibition was observed.

[0171] After 48 hours incubation of Porphyromonas gingivalis with 24 hours HK-CFS, 31.22% inhibition was observed.

[0172] After 24 hours incubation of Porphyromonas gulae with 24 hours HK-CFS, 0.52% inhibition was observed.

[0173] After 48 hours incubation of Porphyromonas gulae with 24 hours HK-CFS, 14.69% inhibition was observed.

[0174] After 24 hours incubation of Prevotella intermedia with 24 hours HK-CFS, 92.19% inhibition was observed.

[0175] After 48 hours incubation of Prevotella intermedia with 24 hours HK-CFS, 90.21% inhibition was observed.

[0176] After 24 hours incubation of Prevotella nigrescens with 24 hours HK-CFS, 86.05% inhibition was observed.

[0177] After 48 hours incubation of Prevotella nigrescens with 24 hr HK-CFS, 37.89% inhibition was observed.Example 5Anti-biofilm activities of Bacillus subtilis MB40 against various pathogenic bacteria

[0178] Objective

[0179] To verify the anti-biofilm capacity of the bacteria Bacillus subtilis MB40 against different pathogenic bacteria.

[0180] Materials and equipmentConsumable and chemical products

[0181] Procedures

[0182] Culture media preparation

[0183] At first a hemin solution stock was prepared with 0.5g of Hemin, 1.74g of K2HPO4 and 100 mL of distilled water. All the above ingredients were boiled in a large flask. Thereafter the ATCC Medium 2722 was prepared as follow: 30 g of Tryptic Soy Broth, 5 g of Yeats Extract, 0.5 g of L-cysteine hydrochloride, 1 mL of hemin stock, 0.2 mL of Vitamin K-l and lOOOmL of distilled water was added. Then the medium was autoclaved at 121 °C. Each bottle was wrapped in aluminium foil as the hemin and vitamin K are light sensitive.

[0184] Preparation of the cell-free supernatant (CFS):

[0185] Preparation of Bacillus subtilis MB40 supernatants was realized by preparing 4 erlenmeyer of 100 mL each containing 50 mL of sterile tryptone soya broth medium (TSB). Each erlenmeyer was inoculated with a cryovial of the MB40 strain and incubated aerobically at 37°C under constant agitation (225 rpm) for 24 hours and 48 hours. After the different incubation periods, one erlenmeyer incubated for 24 hours and another for 48 hours were put in an autoclave to kill the bacteria (121°C for 30 minutes). After sterilization, the contents of each erlenmeyer were transferred into a 50 mL conical tube and left at room temperature so that the liquid cooled to room temperature. Then, each culture, heat-killed or not, was centrifuged for 40 minutes at 4,150 rpm. The supernatant was then harvested, and the pH measured. In each case, a portion of the supernatant was kept at its own pH and another portion was adjusted to a pH of 7. Subsequently,each sample (CFS and HK-CFS for heat-killed cell-free supernatants) was filtered using a filtration unit with a 0.2pM filter. Each sample was kept at 4°C until use.

[0186] Static biofilm formation assay to verify the anti -biofilm activity of MB40 strain

[0187] To verify if the Bacillus subtilis MB40 strain has an anti-biofilm effect a static biofilm formation assay was performed. Briefly, an overnight culture was diluted with fresh ATCC 2722 medium to obtain an OD 600nm of 0.5. The cells were aliquoted into the wells of a 96-well microtiter plate (260 pL per well), 20 pL of either the CFS-MB40 (Heat-killed or CFS) or TSB was added and incubated anaerobically at 37°C for 24 h. The supernatant of the culture was aspirated, and then the well was washed twice with 300 pL phosphate-buffered saline (150 mM NaCl, 3 mM KC1, 10 mM ISfeHPCU, and 1.5 mM KH2PO4, pH 7.4). The biofilms were stained by incubation of each well with 100 pL of 0.1% crystal violet (CV) for five minutes. The plate was then washed twice with 200 pL distilled water and destained with 95% ethanol (200 pL per well) for five minutes. The solubilized CV was transferred to a new microtiter plate, and the OD540nm was measured. Biofilm formation was qualitatively determined to be proportional to the absorbance of the CV.

[0188] Results

[0189] Inhibition of biofilm formation

[0190] Tests were performed to validate whether the CFS of MB40 strain could reduce or inhibit bacterial biofilm formation. Different pathogenic strains were used to verify this effect. The strains Porphyromonas gulae ATCC 51700, Prevotella intermedia ATCC 25611, Prevotella nigrescens ATCC 33563 and Porphyromonas gingivalis ATCC 33277 were used, as they are strains known to cause periodontal disease, where biofilm formation seems to act as a virulence factor of adherence of the strains and have also been used previously in the same project. Testswere therefore carried out on the same eight samples used previously (antimicrobial effect), but using two different batches. Two independent cultures, performed on different days, were used to validate the observed effects on biofilm formation. Results shown in FIG.s 4, panels A-D and 5, panels A-D represent the OD 540 nm results obtained after exposing the four pathogenic strains mentioned above to the different MB40 samples and stained with crystal violet, as mentioned in the protocol section. All tests were performed in triplicate, i.e., on three different experiments and each condition was tested a minimum of five times in each test. Statistical calculations were performed using GraphPad Prism 10.5.0 and using an Ordinary One-Way ANOVA by performing a multiple comparison against the results obtained for each of the pathogenic strains cultured alone in the different tests. An asterisk indicates a significantly significant value, i.e., a p-value < 0.05.

[0191] Thus, the results for the two batches of MB40 show that the strain shows an effect on biofilm formation of the four strains used. However, the effect appears to be more pronounced for the strains Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611. The results also demonstrate that the antibiofilm effect is more pronounced for samples that have been previously incubated for 48 hours, i.e., a 2-day incubation of the MEMO strain before the recovery of the supernatants. This effect also appears to persist even after the same supernatants have been sterilized at a temperature of 121°C for 30 minutes, indicating a potential presence of a heat-resistant molecule with the ability to interfere with the biofilm formation of certain pathogenic bacteria.

[0192] Further analysis to see the effect of these samples were performed by checking the minimum dose having an effect on biofilm formation. To do this, the following dilution range was used for the MEMO samples incubated for 48 hrs: 0.001, 0.01, 0.1, 1, 10, 25 and 50% v / v, where 10% was the reference value initially tested. The results, in FIG.s 6 A-D, appear to show an effecton biofilm formation for almost all the strains used, only from a concentration of 10% v / v. The effect is even more marked when the concentrations of 25 and 50% are used for the strainsPorphyromonas gulae ATCC 51700 and Prevotella nigre scens ATCC 33563. The capacity does not appear to improve when these same concentrations are used with the other 2 pathogenic strains. The effect appears to be almost the same from 10 to 50% v / v.

[0193] These preliminary analyses of the Bacillus subtilis MB40 strain show that: supernatants of the MB40 strain cultured after 24 and 48 hours, with or without neutralized pH and killed by sterilization appear to have an effect on biofilm formation of various pathogenic strains involved in oral diseases in animals. the effect on biofilm formation is significantly enhanced when the strain is cultured for 48 hours. This effect also appears to be present, but to a lesser extent, when the strain is incubated for 24 hours (not statistically significant). the effect is also maintained for samples cultured for 48 hours, even after they have been heated to 121°C for 30 minutes (sterilization), suggesting the presence of a heat-resistant molecule. the effect on biofilm formation is significant when the concentration is at least 10% of the final volume.

[0194] The disclosure has been described in detail with reference to the above embodiments. However, the disclosure should not be construed as being limited thereto. It should further be apparent to those skilled in the art that various changes in form and detail of the disclosure as shown and described above may be made. It is intended that such changes be included within the spirit and scope of the claims appended hereto.

Claims

CLAIMS1. A probiotic composition comprising Bacillus subtilis and / or a supernatant thereof for use in the prevention or treatment of an oral or dental disease in a subject in need thereof.

2. The probiotic composition for use according to claim 1, wherein said Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015).

3. The probiotic composition for use according to any one of claims 1 -2, wherein said subject is a mammal.

4. The probiotic composition for use according to claim 3, wherein said mammal is a dog, a cat or a small pet.

5. The probiotic composition for use according to any one of claims 1-4, wherein the oral or dental disease is selected from the group comprising gingivitis, early, moderate or advanced periodontitis, furcation disease, endodontic-periodontal lesions or infections, stomatitis, plaque formation, tartar formation, poor oral health, poor gum health, gum inflammation, dental abscess, caries lesions or tooth decay.

6. The probiotic composition for use according to claim 5, wherein said oral or dental disease is caused by one or more pathogen selected from Porphyromonas gingivalis, Porphyromonas gulae. Prevotella intermedia and Prevotella nigrescens.

7. The probiotic composition for use according to any one of claims 1-6, wherein the composition inhibits the growth of one or more pathogen selected from Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

8. The probiotic composition for use according to any one of clams 1-7, wherein the probiotic composition comprises Bacillus subtilis spores.

379. The probiotic composition for use according to claim 8, wherein the concentration of spores is from IxlO8to IxlO12CFU / g.

10. The probiotic composition for use according to claim 9, wherein the concentration of spores is at least about IxlO11CFU / g.

11. The probiotic composition for use according to any one of claims 1-10, wherein said probiotic composition is a dry powder.

12. The probiotic composition for use according to claim 11, wherein said dry powder is lyophilized, freeze dried, spray dried, air-dried or frozen.

13. The probiotic composition for use according to any one of claims 11-12, wherein the dry powder further comprises a cryoprotectant.

14. The probiotic composition for use according to any one of claims 11-13, wherein the dry powder further comprises maltodextrin.

15. The probiotic composition for use according to of any one of clams 13-14, wherein the probiotic composition is encapsulated or microencapsulated.

16. The probiotic composition for use according to any one of 11-15, wherein the moisture content is 10% or less as measured by Ohaus MB45 method.

17. The probiotic composition for use according to any one of 11-16, wherein the probiotic composition further comprises sodium chloride, calcium carbonate, and / or sodium bicarbonate.

18. The probiotic composition for use according to any one of claims 11-17, 5 which is a nutritional composition.3819. The probiotic composition for use according to claim 18, wherein said nutritional composition is a dental chew, a dental stick, a complete meal or a meal supplement formulated for administration to a pet.

20. The probiotic composition for use according to claim 19, wherein the pet is a dog, a cat or a small pet.

21. The probiotic composition for use according to any one of the preceding claims further comprising one or more probiotic selected from the group comprising Streptococcus oralis, Streptococcus uberis, Streptococcus rattus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus salivarius, Bacillus coagulans, and Bifidobaceterium spp.

22. The probiotic composition for use according to claim 21, wherein the additional probiotic is selected from the group comprising Lactobacillus fermentum CECT5716, PP Lactobacillus salivarius CECT 5713 and Bacillus coagulans GBI-30, 6086.

23. The probiotic composition for use according to anyone of the preceding claims, wherein the daily dosage of Bacillus subtilis ranges between 500 million CFU / day and 10 billion CFU / day, preferably between 100 billion CFU / day and 9 billion CFU / day, more preferably between 2 billion CFU / day and 8 billion CFU / day, or even more preferably between 3 billion CFU / day and 7 billion CFU / day.

24. The probiotic composition for use according to anyone of the preceding claims, wherein the Bacillus subtilis supernatant is a cell-free supernatant or a heat killed cell-free supernatant.

25. The probiotic composition for use according to anyone of the preceding claims, wherein the Bacillus subtilis is derived from a pure culture.

26. A probiotic composition comprising Bacillus subtilis and / or a supernatant thereof for use in the prevention or treatment inhibition of biofilm formation on a surface caused by one or more pathogen.

27. The probiotic composition for use according to claim 26, wherein said Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) and / or a supernatant thereof.

28. The probiotic composition for use according to claim 26 or 27, wherein said pathogen is selected from the group c,ompdsm§,Porphyromonasgingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

29. The probiotic composition for use according to any one of claims 26-28, wherein said surface is an inert surface or a biological tissue selected among oral mucosa, teeth or gingives.

30. The use of a composition comprising Bacillus subtilis BS-MB40 strain (PTA- 122264 filed before ATCC on 24 June 2015) and / or a supernatant thereof for the inhibition of the biofilm formation caused by one or more pathogen on a surface.

31. Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) for use in the prevention or treatment of an oral or dental disease in a pet.

32. A supernatant of Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) for use in the prevention or treatment of an oral or dental disease in a pet.

33. A supernatant of Bacillus subtilis BS-MB40 for use according to claim 32, which is a cell-free supernatant or a heat killed cell-free supernatant.

34. The spores of Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) for use in the prevention or treatment of an oral or dental disease in a pet.

35. A Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) a supernatant, and / or the spores thereof for use according to any one of claims 31-34, wherein the Bacillus subtilis derives from a pure culture.

36. A Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) a supernatant, and / or the spores thereof for use according to any one of claims 31-35, wherein the Bacillus subtilis derives from a pure culture in microencapsulated form.

37. A Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) a supernatant, and / or the spores thereof for use according to any one of claims 31-36, wherein the pet is a dog, a cat or a small pet.

38. A Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) a supernatant, and / or the spores thereof for use according to any one of claims 31-37, wherein said oral or dental disease is selected from the group comprising gingivitis, early, moderate or advanced periodontitis, furcation disease, endodontic-periodontal lesions or infections, stomatitis, plaque formation, tartar formation, oral health, gum health, gum inflammation, dental abscess, caries lesions, or tooth decay.

39. A Bacillus subtilis BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015) a supernatant, and / or the spores thereof for use according to any one of claims 31-38, wherein said oral or dental disease is caused by one or more pathogen selected from a disease caused by one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

40. A method for preventing or treating an oral or dental disease in a subject in need thereof by administering to the subject a probiotic composition comprising Bacillus subtilis and / or a supernatant thereof.

41. The method according to claim 40, wherein Bacillus subtilis is BS-MB40 strain (PTA-122264 filed before ATCC on 24 June 2015).

42. The method according to any one of claim 40-41, wherein the subject is a mammal.

43. The method according to claim 42, wherein the mammal is a pet selected between a dog, a cat or a small pet.

44. The method according to any one of claims 40-43, wherein said oral or dental disease is a disease selected from the group comprising gingivitis, early, moderate or advanced periodontitis, furcation disease, endodontic-periodontal lesions or infections, stomatitis, plaque formation, tartar formation, oral health, gum health, gum inflammation, dental abscess, caries lesions, or tooth decay.

45. The method according to claim 44, wherein said oral or dental disease is caused by one or more pathogen selected from a disease caused by one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

46. The method according to any one of claims 40-45, wherein the probiotic composition inhibits the growth of one or more of Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia and Prevotella nigrescens.

47. The method according to any one of claims 40-46, wherein the probiotic composition comprises Bacillus subtilis spores.4248. The method according to claim 47, wherein the concentration of spores is fromIxlO8to IxlO12CFU / g.

49. The method according to claim 48, wherein the concentration of spores is at least about IxlO11CFU / g.

50. The method according to any one of claims 40-49, wherein the probiotic composition is a dry powder.

51. The method according to claim 50, wherein the dry powder is lyophilized, freeze dried, spray dried, air-dried or frozen.

52. The method according to any one of claims 50-51, wherein the dry powder further comprises a cryoprotectant.

53. The method according to any one of claims 50-52, wherein the dry powder further comprises maltodextrin.

54. The method according to any one of claims 50-53, wherein the probiotic composition is encapsulated or microencapsulated.

55. The method according to any one of claims 50-54, wherein the moisture content of the dry powder is 10% or less as measured by Ohaus MB45 method.

56. The method according to any one of claims 40-55, wherein the probiotic composition further comprises sodium chloride, calcium carbonate, and / or sodium bicarbonate.

57. The method according to any one of claims 40-56, wherein the probiotic composition further comprises one or more probiotic selected from the group comprising Streptococcus oralis, Streptococcus uberis, Streptococcus rattus, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus,43Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus salivarius, Bacillus coagulans, and Bifidobaceterium spp.

58. The method according to claim 57, wherein the additional probiotic is selected from the group comprising Lactobacillus fermentum CECT5716, Lactobacillus salivarius CECT 5713 and Bacillus coagulans GBI-30, 6086.

59. The method according to any one of claims 40-58, wherein the daily dosage of Bacillus subtilis ranges between 500 million CFU / day and 10 billion CFU / day, preferably between 100 billion CFU / day and 9 billion CFU / day, more preferably between 2 billion CFU / day and 8 billion CFU / day, or even more preferably between 3 billion CFU / day and 7 billion CFU / day.

60. The method according to any one of claims 40-59, wherein said Bacillus subtilis supernatant is a cell-free supernatant or a heat killed cell-free supernatant.

61. The method according to any one of claims 40-60, wherein the probiotic composition is a nutritional composition in the form of a dental chew, a dental stick, a complete meal or a meal supplement formulated for administration to a pet.

62. The method according to any one of claims 40-61 , wherein Bacillus subtilis derives from a pure culture.44

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