WO2025149376A1 - Lactiplantibacillus plantarum strain and its oral health related uses - Google Patents

Abstract

The present invention relates to a strain of the species Lactiplantibacillus plantarum with deposit number CECT 9161, and/or a heat-treated product thereof, its culture supernatant, and/or a composition comprising them, their use for the treatment and/or prevention of periodontal disease and halitosis, and their non-therapeutical use for reducing the dental plaque in a subject, or for ameliorating the halitosis in a subject.

Description

LACTIPLANTIBACILLUS PLANTARUM STRMN AND ITS ORAL HEALTH RELATED USES

The present invention relates to a strain of the species Lactiplantibacillus plantarum, both in its viable and non-viable form, as well as a culture supernatant thereof, and a composition comprising any of them, for its therapeutic and non-therapeutic uses in periodontal disease, amelioration of dental plaque and reduction of halitosis in a subject. Therefore, the present invention could be included in the field of medicine and veterinary, in particular, in the field of oral health improvement, in the prevention and treatment of oral diseases.

BACKGROUND ART

The oral microbiota is stable, with a highly resilient population. A balanced oral microbiota leads to good oral health and consequently good overall systemic health. However, there are several factors, nutrition, environment or the introduction of some opportunistic pathogens that can alter the composition and quantity of microorganisms inhabiting the mouth inducing dysbiosis. Thus, frequent carbohydrate intake or decreased saliva production can lead to caries, and excessive plaque accumulation increases the risk of periodontal disease.

Periodontal disease is an inflammatory condition that damages the periodontium, the specialized tissues that surround and support the tooth. The periodontium is composed of the gum, cementum, periodontal ligament and alveolar bone. Periodontal disease is separated into two conditions: gingivitis and periodontitis. Gingivitis is a reversible condition in which the gum becomes red and inflamed. Periodontitis is irreversible, but often manageable, in which the tissues supporting the tooth become inflamed, ultimately resulting in a loss of attachment due to destruction of the periodontal ligament, the cementum and the alveolar bone. Periodontal disease is one of the most common diseases in a subject such as domestic dogs, being 20-30% in small breed dogs and 15% in cats. The majority of dogs in the population have gingivitis and the likelihood of developing periodontitis increases with age.

The etiology of periodontal diseases is multifactorial in origin and involves host, environmental and infectious factors associated with bacteria embedded in biofilm on teeth and gums, commonly known as dental plaque. As mentioned above, periodontal disease is caused by an alteration of the ecology of the oral microbiota, and pathogens with anaerobic, proinflammatory and proteinase capacity play an important role in the formation of oral biofilm. The activity of such pathogens is associated with tooth loss, an extremely painful chronic periodontal inflammation in its later stages, which could compromise the overall health of a subject such as pets. In addition, periodontal disease sometimes does not present visible symptoms until very late in the condition. Different research papers have described the incidence of pathogenic bacteria of the genera Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella or Porphyromonas, among others, in the occurrence of periodontal disease (Belibasakis, et aL, In Periodontology 2000. January 2023; Jia et aL, Frontiers in Cellular and Infection Microbiology, 262. July 2019; Mujica Troncoso et al., Revista Clfnica de Periodoncia, Implantologfa y Rehabilitation Oral. 3(3):1 18-122. December 2010).

A balanced mouth microbiota (also known as homeostasis) protects against the proliferation of pathogenic bacteria thus preventing oral diseases and has the ability to form dental plaque. Periodontal disease usually begins with excessive accumulation of dental plaque due to poor dental hygiene. The increase in biofilm creates an anaerobic environment where pathogenic bacteria can thrive. In response to the appearance of bacteria, the immune system reacts by raising the temperature and initiating an immune response involving antibodies, immune cells and proteins. Inadvertently, this immune response creates a protein-rich environment that proteolytic pathogens use to increase their numbers and create a neutral or even alkaline environment. In this aberrant environment, a strong selection pressure is created in the oral cavity, favoring periodontopathogenic bacteria and creating a loop that is difficult to break.

If gingival inflammation persists over time, osteoclast activation occurs, reducing bone mass in the gums, leading to tooth loss due to repeated cycles. Not only inflammation is associated with periodontitis, but also relevant is the appearance of metabolites related to dysbiosis, such as ammonia or sulfur-related volatile compounds associated with halitosis.

In conclusion, plaque accumulation and the resulting inflammatory response of the host can lead to a positive feedback loop that causes gingivitis and, in some cases, if the host is susceptible, periodontitis.

Despite brushing, plaque builds up and hardens into calculus, known as tartar, causing irritation and inflammation of the gum line and surrounding areas. This condition, known as gingivitis, represents the early stages of gum disease and thus the starting point of periodontal disease.

The use of dental treats (such as chew toys and food) has become very popular in recent years; it is actually the strategy used to reduce the amount of discomfort and to help with insufficient general oral hygiene and bad breath. However, the chances of real success of this strategy are limited, and the room for improvement in this regard remains to be explored.

Neutralization or inhibition of disease-causing factors, along with the identification and promotion of health-promoting species and functions, e.g., by prebiotics and probiotics, could improve the resilience of the microbiome and lead to new strategies to prevent disease. In this direction, prebiotics can drive beneficial changes in the oral microbiota and could increase resistance to dysbiosis and recovery of health. However, the use of probiotic and postbiotic products to prevent periodontal disease is still an under-researched field with an extreme lack of product development.

Therefore, there is a need to investigate the fundamental bases of the microbiology involved in the early stages of periodontal disease and/or halitosis in order to develop innovative products aimed at improving the oral health in humans and non-human animals.

DESCRIPTION OF THE INVENTION

The inventors of the present invention have identified a strain of the species Lactiplantibacillus plantarum (formerly called Lactobacillus plantarum), hereinafter “L. plantarum”, which has been selected among several strains screened in vitro, which represents a solution for these oral health problems, in particular which is useful for the treatment/prevention of periodontal disease, for the amelioration of dental plaque (oral biofilm inhibition) and for the treatment/prevention of halitosis. In addition, the postbiotic form obtained by heat treatment of this L. plantarum strain was tested in vitro (see Example 2, Example 3, Example 5, Example 7, Example 8, Example 9, Figure 2, Figure 3, Figure 5, Figure 6, Figure 7), ex vivo (Example 10 and Example 11 , Figure 9, Figure 10, Figure 15, Figure 16, Figure 17) and in vivo in a canine oral microbiome study (Example 12, Example 13 Example 14 and Example 15, Figure 1 1 , Figure 12, Figure, 13, Figure 14, Figure 19, Figure 20, Figure 21 ).

Surprisingly, both the probiotic form (strain in form of viable cell) and the postbiotic form (strain in form of non-viable cell), which may be obtained from the heat-treatment of the live form of the strain, are able to inhibit the growth of oral pathogens in the subjects' mouth. In addition, the ability of the probiotic form (the live form of the strain) as well as of the postbiotic form (the non-viable form of the strain) to inhibit biofilm formation on teeth is better than the ability of other commercial strains belonging to the same species also tested by the inventors for the purposes of comparison (see Examples below). The live form of the strain also showed the ability to degrade molecules known to be responsible for halitosis using them as a substrate for cell growth (Example 4, Figure 4)

Particularly, the inventors have evaluated the L. plantarum CECT 9161 strain of the invention (also named “CECT 9161” or“BPL7”), the postbiotic obtained by heat treating of said strain (also named “HT-CECT 9161 or “HT-BPL7”) and the supernatant obtained from an anaerobic fermentation of the strain (also named “CECT 9161 supernatant” or “BPL7 supernatant”) as candidates for use in oral health improvement of a subject. In addition, they have carried out comparative assays of the previous mentioned products with other strains belonging to the same species: L. plantarum 1 (from a private collection) and L. plantarum CECT 7481 (a commercial product, also named Benchmark 3 in the examples). The results showed the following:

(i) CECT 9161 probiotic (live form of the strain) was able to grow degrading putrescine and/or cadaverine, biogenic amines related with halitosis, and using any of them as substrate (Example 4, Figure 4).

(ii) HT-CECT 9161 inhibited Neisseria zoodegmatis and Neisseria weaveri (colonising strains) growth up to 99.9% with a dose of 1 E9 CFU/mL. HT- CECT 9161 inhibited up to 99% Neisseria weaveri with a lower dose of 1 E8 CFU/mL, up to 30% with a dose of 1 E7 CFU/mL and up to 36% with a dose of 1 E6 CFU/mL. L plantarum 1 was showing a low capability of inhibiting the pathogen growth and the biofilm formation if compared with other strains such as HT-BPL7 among others (Example 5 and Example 6, Figure 5 and Figure 6). Further, HT-CECT 9161 inhibited pathogen biofilm formation. Comparison of biofilm formation capability was assayed for HT-CECT 9161 and benchmarked L. plantarum CECT 7481 postbiotic strain against three pathogens, and the biofilm inhibition results were significantly improved with HT-CECT 9161 (Example 7, Figure 7).

The anti-inflammatory properties of the heat-treated live cells of strains, HT-CECT 9161 , L. plantarum 1 and CECT 7481 , in an oral cell line were evaluated. HT-CECT 9161 was one of the strains with the highest efficacy (29±3% IL-8 inhibition and 72±6% CXCL-10 inhibition vs control) and was able to significantly inhibit cytokines. Both L. plantarum strains, namely L. plantarum 1 and L. plantarum CECT 7481 , in their non- viable cells form tested inhibited the secretion of cytokines (respectively, 5±8% and 25±3% IL-8 inhibition and 56±10% and 74±2% CXCL-10 inhibition vs control), but HT- CECT 9161 enhanced IL-6 secretion (71 ±9% IL-6 secretion vs control (TNF-a)) whereas both, L. plantarum 1 and CECT 7481 in their respective forms of non-viable cells, demonstrated lower IL-6 values (respectively, 46±22% and 55±11 % IL-6 secretion vs control (TNF-a)) (Example 8).

The inventors evaluated ex vivo the postbiotic inhibitory effect on the biofilm formation with dog and cat saliva. The capability of HT-CECT 9161 of inhibiting the biofilm formation, and also of lowering the presence of microbial cells in the biofilm collected from the HT-CECT 9161 cells, was corroborated, indicating that HT-CECT 9161 decreases plaque formation and decreases the risk of periodontal disease and halitosis development (Example 10 and Example 1 1 , Figure 9, Figure 15).

(iii) CECT 9161 supernatant significantly reduced the growth of the dental plaque- associated pathogens Neisseria zoodegmatis and Neisseria weaver/' in a liquid culture in comparison with the other strains tested, including a commercial product (Example 2, Figure 2). CECT 9161 supernatant inhibited the biofilm formation by all the tested Neisseria strains by more than 90% compared to the control or with the other strains tested, including a commercial product. Thus, the inhibition of biofilm produced by CECT 9161 supernatant is a promising activity that plays a key-roll in avoiding biofilm development and therefore contributing to maintenance of a good oral health (Example 3, Figure 3).

On the other hand, an acerola powder inhibited the oral pathogens Neisseria zoodegmatis and Neisseria weaveri (with 12.5 mg/mL it is possible to see inhibition effect, and with 50 mg/mL around 75% of inhibition is found). Also, an acerola powder showed a unexpected and surprising effect in combination with CECT 9161 supernatant: the acerola powder itself at 25 mg/mL inhibited pathogen growth around 85%, and the CECT 9161 supernatant inhibited between 75% and 50% in this assay. The combination of both (acerola powder and CECT 9161 ) increased inhibition, between 90-94%, resulting in 90-94% inhibition of N. zoodegmatis oral bacteria, demonstrating a surprising effect of this combination (Example 9, Figure 8, Table 3).

Thus, the present invention refers to an isolated strain of Lactiplantibacillus plantarum that has been deposited at the Spanish Type Culture Collection under the deposit or registration number CECT 9161 , hereinafter “the strain of the invention” or “CECT 9161” or “BPL7”.

In the context of the present invention, the strain of the invention may be in the form of viable (live) cells or non-viable (inactivated or dead) cells. In a preferred embodiment, the strain of the invention is in the form of non-viable cells (e.i. postbiotic). More preferably, the non-viable cells or the postbiotic has been obtained by means of a heattreatment. The strain of the invention showed strong anti-inflammatory properties in a buccal epithelial model by inhibiting the secretion of cytokines IL-8 and CXCL-10, involved in neutrophil and T-cell recruitment respectively, as well as enhanced IL-6 secretion in TNF-a-stimulated TR146 cells. Furthermore, the heat-treated strain of the invention performed better than L plantarum 1 (used in the Examples below for comparison) in all assays where the strains were tested and better than the reference L. plantarum CECT 7481 with respect to IL-6 secretion.

The strain of the invention was further able to inhibit the growth of multi-species biofilms in an oral biofilm simulation. Furthermore, the results obtained highlighted the heat- treated strain of the invention as the best performing strain of the five strains tested (even better than those also belonging to L. plantarum). The ability of the heat-treated strain of the invention to enhance the presence of specific bacteria known to be beneficial to oral health in the microbiome analysis was also demonstrated.

Taken together, these results allow to conclude that both (i) the live (viable) strain (probiotic), (ii) the non-viable, preferably the heat-treated, strain (postbiotic) and (iii) the supernatant of the strain of the invention showed exceptional ability to improve overall oral health in subjects.

Based on this, a number of inventive aspects has been developed and will be described below.

Thus, in a first aspect, the present invention refers to an isolated strain of the species Lactiplantibacillus plantarum deposited in the Spanish Type Culture Collection under the deposit number CECT 9161. This strain was isolated from the mouth swap of a healthy person. CECT 9161 strain is a gram-positive facultative anaerobe, rod-shaped, non-spore-forming bacteria. It is capable of resisting long term to lysozyme as well as strong acid environment and high concentration of bile. This ability suggests that strain of the invention is capable of surviving the gut environment. The deposit of the strain of the invention was made under the Budapest Treaty on July 4, 2016 in the Spanish Type Culture Collection as International Depositary Authority (located at Building 3 CUE, Parque Cientffico de la Universidad de Valencia, C/ Catedratico Agustin Escardino, 9, 46980 Paterna (Valencia) SPAIN) by Biopolis, S.L. as depositant. The terms "strain of the invention", "microorganism of the invention", "L. plantarum CECT 9161 ", “ Lactiplantibacillus plantarum CECT 9161 ”, “ Lactiplantibacillus plantarum strain CECT 9161 ”, and “BPL7” (BPL0007) are used interchangeably herein to refer to Lactiplantibacillus plantarum strain CECT 9161.

Former members of the genus Lactobacillus are found in the gastrointestinal tract, urinary and genital systems of humans and other animals. It is one of the most studied groups in the food industry and many of its species have become microorganisms of great importance due to their probiotic properties and common basic beneficial health effects, but certain benefits, however, may depend on the species and even of the strain. Because of their purported health-promoting properties, these bacteria have been incorporated into many functional foods as active ingredients. Lactobacilli group promotes gut maturation and integrity, and it is generally antagonistic to pathogens and contribute to the modulation of intestinal immunity.

Former Lactobacillus genus comprises gram-positive, facultative or microaerophilic, bacilliform, non-spore-producing bacteria. Former members of the genus Lactobacillus are the main group of lactic acid bacteria, their production of lactic acid makes their environment acidic, which inhibits the growth of pathogenic bacteria. Many species are involved in the decomposition of plant and animal matter. Due to their technological properties, some species of former members of the genus Lactobacillus are used industrially for the production of yogurt, cheese and other fermented foods. The strain of the invention is described as follows: Kingdom: Bacteria; Phylum: Firmicutes; Class: Bacilli; Order: Lactobacillales; Family: Lactobacillaceae; Genus: Lactiplantibacillus. The present invention also encompasses strains derived from the strain L. plantarum CECT 9161 , which may be mutants and genetically modified organisms that present variations in their genome compared to the genome of the strain described in the invention, but where such variations do not affect the ability of the strain to prevent, remit and/or improve periodontal disease, halitosis or inhibition of oral biofilm formation. Strains derived from L plantarum CECT 9161 may occur naturally or intentionally by mutagenesis methods known in the state of the art such as, for example, but not limited to, the growth of the original strain in the presence of mutagenic or stress-causing agents or by genetic engineering aimed at obtaining the desired mutation. An assay to check whether a microorganism has the ability to prevent, remit and/or improve periodontal disease, halitosis or inhibition of oral biofilm formation in a subject is described in the examples accompanying this description.

Furthermore, the present invention also extends to cellular components, metabolites and / or molecules secreted by L. plantarum CECT 9161 as well as compositions that comprise said cellular components, metabolites and/or secreted molecules, and uses thereof for the treatment and/or prevention of periodontal disease, halitosis or for the inhibition of oral biofilm formation. The “cellular components” may include components of the cell wall (such as, for example, peptidoglycan), nucleic acids, membrane components, or others such as proteins, lipids and carbohydrates, and combinations thereof (such as lipoproteins, glycolipids or glycoproteins). The "metabolites" include any molecule produced or modified by the bacterium as a result of its metabolic activity, during its growth, its use in technological processes or during the storage of the product (composition of the invention). Examples of these metabolites include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals, and nucleic acids. "Secreted molecules" include any molecule secreted or released by the bacteria during its growth, its use in technological processes (e.g. food or drug processing) or during product storage. The molecules secreted by the strain of the invention are, for example, bacteriocins or plantaricins.

Examples of these molecules include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, minerals, salts, and nucleic acids.

In a preferred embodiment of the strain of the invention said strain is in the form of a non-viable cells or viable cells. As illustrated in the Examples section, viable and non- viable cells of the strain of the invention, as well as the culture supernatant of any of them, have shown to have oral health beneficial effects in in vitro and ex vivo models. Thus, in a more preferred embodiment of the strain of the invention, said non-viable cells have been inactivated by heat (i.e. heat-treated or obtained by heat-treatment).

Another aspect of the invention refers to a supernatant of a culture of the strain of the invention, hereinafter “the supernatant of the invention”. Preferably, said supernatant is obtained after 16-48 hours anaerobic fermentation at 30-37^QC (it can be, or not, neutralized) of the viable or non-viable form of the strain of the invention, and then the liquid medium resulting from the fermentation is filtered by 0.22 pm filter to obtain a free cell supernatant that is neutralized until pH=7.

The supernatant referred to in the present invention comprises, preferably, the remaining of the culture medium in which the strain of the invention has grown, including but without limitation, glucose, yeast extract, some salts, and metabolites produced by the strain during the fermentation process, including but without limitation, lactic acid and the bacteriocins plantaricins.

Another aspect of the invention refers to a composition, hereinafter “the composition of the invention”, comprising the strain of the invention or the supernatant of the invention, preferably wherein the strain of the invention is in the form of viable or non-viable cells, more preferably, in which the non-viable cells have been heat-treated.

When the composition of the invention comprises the strain of the invention in the form of non-viable cells and/or the supernatant of the invention, said composition is a postbiotic composition. When the composition of the invention comprises the strain of the invention in the form of viable cells said composition is a probiotic composition.

As used herein, the term "non-viable", "inanimate", "inactive" or "inactivated" refers to any microorganism or cell, in the context of the present invention the strain L. plantarum deposited at the Spanish Type Culture Collection under the registration number CECT 9161 , that is metabolically or physiologically inactive, i.e. does not retain its metabolic activity or its ability to elongate or proliferate even after nutrient administration.

With reference to the foregoing, in the present invention "postbiotic" refers to inactivated microorganisms and/or their microbial cell structures or fragments, preferably included in a composition, which confer a health benefit to the host administered with it. With respect thereto, in the present invention "postbiotic composition" is understood as a preparation comprising intact inanimate microbial cells and/or microbial cell structures or fragments, such as cell wall, membranes, exopolysaccharides, pili, etc. and/or metabolites or end products such as organic acids, peptides, enzymes, vitamins, bacteriocins, etc. The microorganism of the postbiotic composition may be inactivated by any method known in the state of the art.

Some techniques known in the prior art to turn live microorganisms into non-viable cells are, without limitation, thermal/heat processing, freezing, or radiation, such as ultraviolet radiation. Thermal processing is used in many cases to inactivate microorganisms, as there is a long history of thermal processing in the food industry. A common procedure for inactivating microorganisms is heat treatment. Heat is lethal to microorganisms, but each species has its own tolerance to heat. During a thermal destruction process, such as pasteurization, tyndallization and autoclaving, the rate of destruction is logarithmic, as it is their growth rate. Thus, bacteria subjected to heat die at a rate proportional to the number of organisms present. The process depends on both the exposure temperature and the time required at this temperature to achieve the desired destruction rate. Preferably, the heat-treatment is carried out, in the present invention, by boiling the cells of the strain of the invention for 1 hour (+/- 10 minutes) at 100^QC (+/- 30^QC) [preferably, for industrial processes, the cells are pasteurized and dried by spray-drying instead of boiling them], and then, the boiled cells are concentrated and lyophilized. These conditions have been tested by the inventors so that the L. plantarum CECT 9161 is inactivated but retains the therapeutic properties indicated in this invention. In a preferred embodiment, the present invention encompasses a postbiotic composition comprising L plantarum CECT 9161 which has been heat-treated.

Lactiplantibacillus plantarum is a bacterium commonly used as a probiotic, mostly found in sauerkraut, kimchi, yogurt, kefir and other fermented products, which can help to regulate the intestinal transit of the subject.

In the present invention "probiotic" or "probiotic composition" means a live microorganism or a composition comprising at least one live microorganism which, when ingested, interacts with the metabolism of the individual and produces a beneficial effect on them.

The strain of the invention and the supernatant of the invention are characterized by their ability to treat and/or prevent periodontal disease or halitosis and to reduce dental plaque and oral biofilm formation.

As shown in the examples, the composition of the invention may also comprise an acerola powder which provides a surprising and unexpected effect together with the strain, or the supernatant of the invention, against oral pathogens. Thus, a preferred embodiment of the composition the invention refers to the composition of the invention further comprising an acerola powder. The term "acerola” refers to a fruit that contains approximately 50-100 times the amount of vitamin C than an orange or lemon. It can be found in beverages, supplements, baked goods, personal care, prebiotic fortification). Acerola powder standardized to 17% vitamin C (dry basis), analyzed by High Performance Liquid Chromatography, was produced after preselection of the acerola juice, direct spray drying process employing only tapioca maltodextrin as excipient, and adjusting atomization conditions to obtain a fine powder.

The composition of the invention may be formulated for pharmaceutical or veterinary administration, i.e., forming part of pharmaceutical or veterinary products that will be administered to a subject (for example, orally, topically, etc), and/or for food administration, i.e., forming part of food that is consumed in a subject's diet, and/or it can be administered as a nutritional or dietary complement or supplement. Thus, in a preferred embodiment of the composition of the invention, the composition is a pharmaceutical, a veterinary or a nutritional composition.

The "pharmaceutical or veterinary composition” comprises one or more active components or compounds that is made up of, at least, the strain or the supernatant of the invention, at any concentration, and which, in addition, may comprise one or more components or compounds having some biological and/or pharmacological activity which, after administration to a subject, may increase, reinforce and/or boost the activity of the strain or the supernatant included in the composition of the invention. As understood by the expert skilled in the art, the additional components or compounds must be compatible with the strain and supernatant of the composition of the invention.

In another preferred embodiment, the composition of the invention is formulated in liquid, semisolid or solid form, preferably in solid form. In another preferred embodiment, the composition of the invention is in a form selected from the list consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles, coated tablets, dispersible strips, dispersible films, oral solution, suspension, steam solution, droplet, emulsion, syrup, dental stick, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

In the present invention, the dental stick, the dental chew, the soft chews, the sachet stick pack, the dry kibble, the fast melts for water bowl, and the toothpaste may both comprise the composition of the invention inside or being coated with it.

The composition of the invention may also be a nutritional composition. The "food composition” or "nutritional composition" refers to a food or nutritional or dietary supplement that beneficially affects one or more bodily functions, thus improving the state of health and well-being of the individual who consumes it. In this invention, said food composition is intended to prevent, remit and/or improve periodontal disease, halitosis or inhibition of oral biofilm formation. The food composition referred to in this invention includes, but is not limited to, a food, a functional food, a feed (for non-human animal feeding), a probiotic, a postbiotic or a nutritional complement or supplement. In cases where the composition of the invention is formulated as a nutritional composition, said composition may be a food or be incorporated into a food or foodstuff intended for animals, including humans. Thus, in a more preferred embodiment, the food composition is selected from a foodstuff (which can be, but is not limited to, a food for specific nutritional purposes or a medicinal food) and a nutritional supplement.

The terms “complement”, "supplement” and "additive", are synonymous with any of the following terms: "dietary supplement", "nutritional supplement", "food supplement", “diet supplement” or "dietary additive", and refer to products or preparations intended to supplement the normal diet, consisting of concentrated sources of nutrients or other substances having a beneficial nutritional or physiological effect on the individual. The food supplement can be found in a simple or combined form and marketed in dosage form, i.e., in capsules, tablets, pills and other similar forms, sticks, sachets of powder, ampoules of liquid and dropper bottles and other similar forms of solids, liquids and powders to be taken in a unit amount.

Examples of foods that may comprise the composition of the invention include, but are not limited to, feed, dairy products, plant products, meat products, snacks, dental sticks, chocolates, beverages, dehydrated powdered foods, food gels, baby foods, cereals, fried foods, industrial pastry and cookies. Examples of dairy products include, but are not limited to, products derived from fermented milk (e.g., yogurt or cheese) or unfermented milk (e.g., ice cream, butter, margarine or whey). The plant-based product is, for example, a cereal in any form, fermented (e.g. a fermented soy-based product or fermented oat-based product) or unfermented, or a snack. Beverages may be unfermented milk or smoothies in liquid form or in powder for reconstitution with water. In a particular embodiment, the feed or food product comprising the composition of the invention is selected from the group consisting of: fruit or vegetable juices, ice-cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, pastry products, dairy products, plant-based products, meat products, fish products, beverages and confectionery, gum-based products (for example, fruit gums, with or without added sugars).

In a more preferred embodiment, the nutritional composition is a food, a feed composition or nutritional supplement.

In a preferred embodiment, the composition of the invention is in the form of a stick, since in that form the post- or probiotic composition of the invention may stay longer in the subject’s oral cavity and exert a better effect. In another preferred embodiment, the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, meat products, meat beverages. In another preferred embodiment, the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, meat products, meat beverages, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

In another preferred embodiment, the composition of the invention is formulated for oral administration.

In another preferred embodiment, the composition of the invention is administered to a subject through diet.

The dosage form of the composition of the invention shall be adapted to the route of administration used. Therefore, the composition may be formulated as a solution, suspension, emulsion, syrup, stick or any other suitable dosage form. Taking into account that the preferred route of administration is oral, the composition of the invention is preferably presented in solid, semi-solid or liquid form, more preferably solid, for oral administration. Examples of solid formulations include tablets, capsules, powders, granules or granulated products, particles or coated tablets, suppositories, tablets, sticks, pills, gels, dispersible films, microspheres, dental chews, soft chews, sachets, dry kibbles, fast melts, or toothpaste. More preferably, the composition of the invention is presented in stick form.

Alternatively, sustained-release forms can be used to deliver the composition of the invention, including, for example, its encapsulation in liposomes, micro-bubbles, micro- particles or microcapsules, and similar. Appropriate sustained-release forms, as well as materials and methods for their preparation, are widely known in the state-of-the- art. Thus, the orally administered form of the composition of the invention could be a sustained-release form that additionally comprises a coating or matrix. The sustained- release coating or matrix includes, but is not limited to, water-insoluble or modified, natural, semi-synthetic or synthetic polymers, proteins, waxes, fats, fatty alcohols, fatty acids, semi-synthetic or synthetic natural plasticizers, or a combination of two or more of the above. Enteric coatings can be applied using conventional processes known to experts in the art.

Another aspect of the invention relates to the composition of the invention for use as a medicament. The term "medicament", as used in this specification, refers to any substance used for the prevention, relief, treatment, reduction or cure of diseases or clinical conditions a subject.

Another aspect of the invention refers to the strain, the supernatant or the composition of the invention for use in the prevention and/or treatment of periodontal disease in a subject. Alternatively, this aspect refers to the use of the strain, the supernatant or the composition of the invention for the manufacture of a medicament for the prevention and/or treatment of periodontal disease in a subject. Analogously, the present invention relates to a method for the prevention and/or treatment of periodontal disease in a subject, comprising administering the strain, the supernatant or the composition of the invention to said subject.

Another aspect of the invention refers to the strain, the supernatant or the composition of the invention for use in the treatment and/or prevention of halitosis in a subject. Alternatively, this aspect refers to the use of the strain, the supernatant or the composition of the invention for the manufacture of a medicament for the treatment and/or prevention of halitosis in a subject. Analogously, the present invention relates to a method for the prevention and/or treatment of halitosis in a subject, comprising administering the strain, the supernatant or the composition of the invention to said subject.

As used herein, the term "to treat" or "treatment" comprises inhibiting the disease or pathological condition, i.e., stopping its development; relieving the disease or pathological condition, i.e., causing regression of the disease or pathological condition; and/or stabilizing the disease or pathological condition in a subject.

As used herein, the term "prevention" means the avoidance of occurrence of the disease or pathological condition in a subject, particularly when the subject has predisposition for the pathological condition, but has not yet been diagnosed.

The strain, the supernatant or the composition of the invention can prevent or treat periodontal disease and/or halitosis because they have the ability to inhibit the growth of oral pathogens in the mouth of the subject.

In a preferred embodiment of the medical uses of the invention, the periodontal disease and/or the halitosis is caused by/ correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria, or bacterial genera with potential pathogenicity, are Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella Porphyromonas, Veillonella, or Bacteroides. These pathogenic bacteria contribute to biofilm build-up together with genus Neisseria, preferably, with the species Neisseria zoodegmatis and/or Neisseria weaver/'.

In another aspect the invention refers to a non-therapeutic use of the strain, the supernatant or the composition of the invention for reducing the dental plaque in a subject or the ameliorating the oral biofilm formation giving rise to said dental plaque. In another aspect the invention refers to a non-therapeutic use of the strain, the supernatant or the composition of the invention for ameliorating the halitosis in a subject.

In another aspect the invention refers to a non-therapeutic use of the strain, the supernatant or the composition of the invention for ameliorating the effects of periodontal disease in a subject.

In a preferred embodiment of the non-therapeutic uses of the invention, the dental plaque or the halitosis is caused by/ correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria are or bacterial genera with potential pathogenicity, are Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella Porphyromonas, Veillonella, or Bacteroides. These pathogenic bacteria contribute to biofilm build-up together with genus Neisseria, preferably with the species Neisseria zoodegmatis and/or Neisseria weaver/'.

In another preferred embodiment of the non-therapeutic uses of the invention, the subject is a non-human animal, preferably a domestic animal or a pet, more preferably dogs or cats, even more preferably dogs.

As used herein, the term "subject" refers to all animals, more preferably mammals, including humans. For example, the subject may be a domestic or tamed animal. Subjects may be cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc., and can be domestic animals or pets.

Therefore, in a preferred embodiment of the strain, the supernatant or the composition for the therapeutic use according to the present invention, the subject is a non-human animal, preferably a domestic animal or a pet, more preferably dogs or cats. In another preferred embodiment of the non-therapeutic used of strain, the supernatant or the composition of the invention, the subject is a non-human animal, preferably a domestic animal or a pet, more preferably dogs or cats.

In another aspect, the present invention relates to acerola, or composition comprising it, for use in the inhibition of oral pathogen bacteria growth in a subject, or for use in the treatment and/or prevention of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

In another aspect, the present invention relates to the non-therapeutic use of acerola, or composition comprising it, for reducing the dental plaque in a subject, for ameliorating the halitosis in a subject, or for ameliorating effects of periodontal disease in a subject.

As use herein, the term “acerola” refers to the fruit coming from the Barbados cherry tree which common name is Malpighia emarginata D.C., and its taxonomic classification is class Magnoliopsida, order Malpighiales, family Malpighiaceae, genus Malpighia, and species M. emarginata D.C. The acerola fruit is a drupe and when ripe, has a thin epicarp, a mesocarp (pulp) that represents 70%-80% of total fruit weight, and a tri-lobed endocarp enveloping an average of one seed with 3-5 mm in diameter, an oval shape and two cotyledons. After fruit establishment, development happens over an average 22 day-period leading to a ripe edible pulp which is fleshy, soft, juicy with an acidic flavour and extremely rich in vitamin C.

In a preferred embodiment, the acerola is in powder form.

All the definitions and preferred embodiments disclosed in previous paragraphs for the composition of the invention are applicable to the present inventive aspect relating to acerola. In another preferred embodiment, the composition comprises a carrier and excipient. Imbalances in the oral microbiome may be associated with a variety of oral diseases such as, but not limited to, dental caries, gingivitis, periodontitis, oral candidiasis, halitosis, bad breath, dental plaque and dental calculus.

Thus, in another aspect, the present invention provides an in vitro method for predicting the risk of suffering from an oral disease selected from halitosis, gingivitis, periodontitis, dental plaque or dental calculus in a subject, comprising (hereinafter “method of the invention”): a) determining the presence of the microbiome comprising at least one or more of the following species: Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella and species of Peptostreptococcaceae, from an isolated biological sample from a subject, and b) comparing the microbiome obtained in step a) with a microbiome obtained from a control group, wherein if the subject presents the microbiome obtained in step a), then the subject shows high probability of suffering from halitosis or gingivitis or periodontitis or dental plaque or dental calculus.

In the present invention, the term “predicting the risk” refers to a measure of statistical probability that in the future a subject will suffer from or develop a disease, based on the presence of a characteristic or factor (or several) that increases the likelihood of adverse consequences. In the present invention the disease to be identified is an oral disease. Specifically, the diseases to be identified are halitosis and/or gingivitis and/or periodontitis and/or dental plaque and/or dental calculus and the clinical parameters is the presence/absence of the microbiome comprising at least one, two, three, four, five, six, seven, eight, nine or ten of the following species: of the microorganisms Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella and species of Peptostreptococcaceae obtained from an oral biological sample isolated from a subject.

As use herein, “dental calculus” or “tartar” is hardened dental plaque that can form on your teeth, both above and below your gum line. Dental calculus mostly contains dead bacteria that have mineralized, mixed with a small amount of mineralized proteins from your saliva.

To establish the risk in the method of the invention, first step a) is carried out, which consists in determine the presence of the microbiome comprising at least one, two, three, four, five, six, seven, eight, nine or ten of the following species: Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella and species of Peptostreptococcaceae, from an isolated biological sample from a subject.

In a preferred embodiment of the method of the invention, first step a) is carried out, which consists of determining the presence of the microbiome consisting of Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella and species of Peptostreptococcaceae, from an isolated biological sample from a subject. The term “microbiome” refers to the community of microorganisms (such as fungi, bacteria and viruses) that exists in a particular environment. In humans, the term is often used to describe the microorganisms that live in or on a particular part of the body, such as the oral, skin or gastrointestinal tract. These groups of microorganisms are dynamic and change in response to a host of environmental factors, such as exercise, diet, medication and other exposures. In the context of the present invention, the type of microbiome used to predict the risk of suffering from an oral disease is the oral microbiome, which is defined as the collective genome of microorganisms residing in the oral cavity.

Oral microbiome health is characterized by a balanced community of microorganisms, where microbial abundances are well-regulated. Disruptions in this balance can lead to dysbiosis, an imbalance that contributes to oral health issues such as periodontitis and cavities. An example of a dysbiotic oral community is one where certain pathogenic bacteria, such as Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum and species of Prevotella and Peptostreptococcaceae species, are overrepresented. This shift often leads to increased alpha diversity and a metabolic profile that favors inflammation, characterized by elevated reactive oxygen species (ROS) production and a reduction in anti-inflammatory metabolites like catechol.

The microorganisms included in the microbiome of the invention are Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella and species of Peptostreptococcaceae. The detection of a dysbiotic (unbalance) environment in an isolated biological sample can be perform using methods familiar to those skilled in the field, including but not limited to omics and statistical approaches. For instance, massive next-generation sequencing can be employed, followed by assessments of alpha diversity (e.g., Simpson Index, Shannon Index, Richness Index) and beta diversity (e.g., Bray-Curtis dissimilarity, Jaccard index). Additionally, statistical methods such as PERMANOVA (Permutational Multivariate Analysis of Variance) and differential abundance analysis can be used to evaluate variations in community composition and identify significant differences in microbial populations.

Thus, in a preferred embodiment of the method of the invention, step a) of presence determination is carried out by a measuring massive next-generation sequencing of the samples, and taxonomic annotation using advance tools like MetaPhlAn, Kraken, PhyloPhlAn, MG-RAST, Kaiju or similar ones.

In the present invention, “sample” means a part or small quantity of a thing which is considered representative of the whole and which is taken or separated therefrom for the purpose of study, analysis or experimentation. In particular, in the present invention the term sample encompasses samples of biological origin, which are isolated from a subject, including without limitation, faecal, saliva, gingival secretion (supragingival and subgingival) and oral fluid.

Thus, in another preferred embodiment of the method of the invention, the biological sample is a liquid sample; preferably an oral liquid sample. The person skilled in the art knows the techniques/methods required for the isolation of a sample of a liquid from the oral cavity of a subject.

In another preferred embodiment of the method of the invention, the isolated biological sample is selected from a list consisting of a sample of saliva, supragingival secretion subgingival secretion and oral fluid; more preferably the sample is gingival secretion, still more preferably, supragingival secretion.

In the present invention the term “saliva” refers to a watery liquid secreted into the mouth by glands, providing lubrication for chewing and swallowing, and aiding digestion.

In the present invention, the term “gingival secretion” or “gingival crevicular fluid” or “sulcular fluid” refers to a physiologic fluid secreted in the gingival crevice that is classified as inflammatory exudate during disease or serum transudate during health. GCF secreted in minute amount in healthy state, which is increased in response to inflammation. When bacterial dental plaque is located above the gum it is called “supragingival”, while if it is located below the gum it is called “subgingival”.

In the present invention, the term “oral fluid” is a mixture of saliva and "oral mucosal transudate". Saliva is produced by the salivary glands. Oral mucosal transudate enters the mouth by crossing the buccal mucosa from the capillaries. Oral fluids contain both pathogens and antibodies. In other words, oral fluid is the liquid found in the oral (mouth) cavity, consisting of saliva from the salivary glands, cells and tissues of the gum and cheek, cellular debris, microorganisms and food residues. Strictly speaking, “oral fluid” is the mixed saliva from the glands and other constituents present in the mouth. “Saliva” is the fluid collected from a specific salivary gland and is free from other materials.

Following step a), to establish the risk of developing an oral disease in a subject, the method of the invention comprises a step b) in which the microbiome obtained in step a) is compared with a microbiome obtained from a control group. In the present invention, the term “control group” refers, in the context of the risk prediction method, to the group of subjects who do not suffer from an oral disease.

Thus, if the subject presents the microbiome obtained in step a), then the subject shows high probability of suffering from halitosis or gingivitis or periodontitis or dental plaque or dental calculus.

The term “high probability of suffering from halitosis or gingivitis or periodontitis or dental plaque or dental calculus” indicates that the subject is expected (that is, predicted to have, or is at high risk of developing) to develop, halitosis or gingivitis or periodontitis or dental plaque or dental calculus. The term "high probability" refers to a higher likelihood or a higher risk than the average risk for a population that does not have a microbiome comprising at least one or more of the following species: Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella and species of Peptostreptococcaceae.

In a preferred embodiment of the method of the invention, the subject is a non-human animal, preferably a domestic animal or a pet, more preferably dogs or cats, even more preferably dogs.

As used herein, the term "subject" refers to all animals, more preferably mammals, including humans. For example, the subject may be a domestic or tamed animal. Subjects may be cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc., and can be domestic animals or pets.

On the other hand, the inventors have observed that the presence of Porphyromonadaceae bacterium H1 in the oral microbiome is related to a good oral health, so that high abundance of this microorganism with respect to the whole microorganisms comprising the oral microbiome results in a low amount of the pathogen bacteria responsible for the dental plaque.

Thus, in another aspect, the present invention relates to a Porphyromonadaceae bacterium H1 , or a composition comprising it, for use as a medicament (hereinafter, “second use of the invention”).

In a particular embodiment of the second use of the invention, the Porphyromonadaceae bacterium H1 , or a composition comprising it, is for use in the prevention and/or treatment of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

As use herein, the term “Porphyromonadaceae bacterium H1” is understood as the bacteria with the taxonomy ID: 1658779 (NCBI). The full lineage of this bacteria is cellular organisms; Bacteria; Pseudomonadati; FCB group; Bacteroidota/Chlorobiota group; Bacteroidota; Bacteroidia; Bacteroidales; Porphyromonadaceae; and unclassified Porphyromonadaceae, according to the NCBI (Schoch CL, et al. NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford).2020:Baaa062. PubMed: 32761142, PMC7408187). Bacteria of the Porphyromonadaceae family have coccobacilli shapes, and are obligately anaerobic, non-spore forming, and non-motile.

In a particular embodiment of the second use of the invention, the Porphyromonadaceae bacterium H1 is in the form of a non-viable cells or viable cells. In a more particular embodiment of the second use of the invention, said non-viable cells have been heat-treated.

In another particular embodiment of the second use of the invention, the composition further comprises acerola power. In another particular embodiment of the second use of the invention, the composition is a pharmaceutical, a veterinary or a nutritional composition.

In another particular embodiment of the second use of the invention, the composition is formulated in liquid or in solid form.

In another particular embodiment of the second use of the invention, the composition is in a form selected from the list consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles, coated tablets, dispersible strips, dispersible films, oral solution, suspension, steam solution, droplet, emulsion, syrup, dental stick, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

In another particular embodiment of the second use of the invention, the nutritional composition is a food, a feed composition or nutritional supplement.

In another particular embodiment of the second use of the invention, the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, plant-based products, plant-based beverages, meat products, meat beverages, fish products, fish beverages, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

In another particular embodiment of the second use of the invention, the periodontal disease and/or the halitosis correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria is selected from Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella, Porphyromonas, Veillonella, Bacteroides and/or combinations thereof. In another particular embodiment of the second use of the invention, the subject is a non-human animal, preferably a domestic animal or a pet.

In another aspect, the present invention also relates to a non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, for reducing the dental plaque in a subject, for ameliorating the halitosis in a subject or for ameliorating effects of periodontal disease in a subject (hereinafter, “second non-therapeutic use of the invention”).

In a particular embodiment of the second non-therapeutic use of the invention, the Porphyromonadaceae bacterium H1 is in the form of a non-viable cells or viable cells. In a more particular embodiment of the second non-therapeutic use of the invention, the said non-viable cells have been heat-treated.

In another particular embodiment of the second non-therapeutic use of the invention, the composition further comprises acerola power.

In another particular embodiment of the second non-therapeutic use of the invention, the composition is a pharmaceutical, a veterinary or a nutritional composition.

In another particular embodiment of the second non-therapeutic use of the invention, the composition is formulated in liquid or in solid form.

In another particular embodiment of the second non-therapeutic use of the invention, the composition is in a form selected from the list consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles, coated tablets, dispersible strips, dispersible films, oral solution, suspension, steam solution, droplet, emulsion, syrup, dental stick, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

In another particular embodiment of the second non-therapeutic use of the invention, the nutritional composition is a food, a feed composition or nutritional supplement.

In another particular embodiment of the second non-therapeutic use of the invention, the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, plant-based products, plant-based beverages, meat products, meat beverages, fish products, fish beverages, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

In another particular embodiment of the second non-therapeutic use of the invention, the periodontal disease and/or the halitosis correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria is selected from Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella, Porphyromonas, Veillonella, Bacteroides and/or combinations thereof.

In another particular embodiment of the second non-therapeutic use of the invention, the subject is a non-human animal, preferably a domestic animal or a pet.

All the explanations, definitions and particular embodiments disclosed for the first therapeutic use and non-therapeutic use of the invention are applicable to the second therapeutic use and non-therapeutic use of the invention.

Likewise, in another aspects, the present invention also relates to: the use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, in the manufacture of a medicament, in particular, in the manufacture of a medicament (or composition) for the prevention and/or treatment of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

- the non-therapetucic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, for the prevention and/or treatment of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

- A method for the prevention and/or treatment of periodontal disease in a subject, or for the prevention and/or treatment of halitosis in a subject comprising administering a Porphyromonadaceae bacterium H1 , or a composition comprising it, to a subject in need thereof, particularly, the

Porphyromonadaceae bacterium H1 , or a composition comprising it, is administered in an effective amount.

- A non-therapeutic method for reducing the dental plaque in a subject, for ameliorating the halitosis in a subject or for ameliorating effects of periodontal disease in a subject comprising administering a Porphyromonadaceae bacterium H1 , or a composition comprising it, to a subject in need thereof, particularly, the Porphyromonadaceae bacterium H1 , or a composition comprising it, is administered in an effective amount.

All the explanations, definitions and particular embodiments disclosed for the first therapeutic use and non-therapeutic use of the invention are applicable to the above- mentioned aspects. DESCRIPTION OF THE FIGURES

Figure 1. Dog oral pathogens biofilm formation inhibition of four Neisseria strains (Neisseria zoodegmatis and Neisseria weaver/' species) by the addition of CECT 9161 strain pure culture free-cells supernatant.

Figure 2. Dog oral pathogens growth inhibition in Neisseria zoodegmatis (left) and N eisseria weaveri (right) liquid cultures tests with CECT 9161 strain and Benchmark 1 and Benchmark 2. These benchmarks are commercially available.

Figure 3. Dog oral pathogens biofilm formation inhibition of two Neisseria strains (Neisseria zoodegmatis (upper) and Neisseria weaveri (bottom)) by the addition of CECT 9161 strain, Benchmark 1 and Benchmark 2 culture free-cells supernatant.

Figure 4. A) Microbial growth (CFU/mL) CECT 9161 in i) MRS medium, ii) MRS medium without glucose, iii) MRS medium without glucose + putrescine, iv) MRS medium without glucose + cadaverine. B) HPLC results of the content of putrescine and cadaverine during the growth of CECT 9161 , after 24h and 48 h of incubation at 37^QC.

Figure 5. Pathogens Neisseria zoodegmatis and Neisseria weaver/ growth inhibition is presented in the follow graphs for four different doses. The CFU/mL were calculated per triplicate and the error bar represents the standard deviation between the measurements.

Figure 6. Pathogen growth inhibition for four different doses of HT-CECT 9161 and the control. CFU/mL was calculated per triplicate and the error bar represents the standard deviation between the measurements. Figure 7. Pathogen growth inhibition for two different doses of HT-CECT 9161 , CECT 9161 and heat-treated L. plantarum CECT 7481 (Benchmark 3) and the control. Percentage of biofilm inhibition is shown.

Figure 8. A) Pathogen growth inhibition by acerola powder, measured as OD at 595 nm; B) Pathogen (N. zoodegmatis) inhibition growth by acerola, CECT 9161 supernatant and combined effect.

Figure 9. Cell index xCELLigence values after BHI control subtraction. All differences between the control (C) and the postbiotic curves (CECT 9161 , L. plantarum 1, L. reuteri 1 , and L. reuteri 7) were significant (p<0.05), except timepoint 54 minutes for L. reuteri 1 , and L. reuteri 7 and the timepoints 54 minutes to 1 h 54 min for L. plantarum 1. The BHI values were subtracted of the original xCELLigence values.

Figure 10. Results of Ex vivo microbiome assay

A) Boxplots that represent the alpha diversity measures of the community profiles at species level. From left to right the alpha diversity indices that are shown are Richness, Simpson and Shannon.

B) Table with the result of the PERMANOVA statistical test used with beta-diversity results. The columns show the degree of freedom, the sum of squares, the R2, the statistic F and the p-value of each group tested. Both group and Subject show significant differences (*:p-val<0.05, **:p-val<0.01 , ***:p-val<0.001 ).

C) Barplot of log2FoldChange of Escherichia and Rothia genus between Groups. Positive values mean a higher presence of that bacteria in the group compared to control, and negative values a lower presence. Significant differences are drawn based on their p-value (#:p-val<0.1 , *:p-val<0.05, **:p-val<0.01 , ***:p-val<0.001 ). D) Barplot of log2FoldChange of Bifidobacterium breve and Rothia nasimurium species between Groups. Positive values mean a higher presence of that bacteria in the group compared to control, and negative values a lower presence. Significant differences are drawn based on their p-value (#:p-val<0.1 , *:p-val<0.05, **:p-val<0.01 , ***:p- val<0.001).

Figure 11 . Boxplots of the mean time to consume by treatment (the right plot excludes dogs identified as outliers).

Figure 12. Box plots of the percentage diet consumed throughout the trial per dog and by treatment.

Figure 13. Boxplots of the change in plaque from baseline at day 29 and 57 by treatment type.

Figure 14. Predicted change in plaque by treatment group.

Figure 15. The results of the curves of the ex-vivo trial were statistically analysed. See with triangles statistical of Benchmark vs. C+ and in squares statistical of HT-CECT 9161 vs. C+ The T-test of C+ vs condition in each recorded time; n.s.= Not significant; * = p-value <0.05; **= p-value <0.01 ; **** p-value <0.0001 .

Figure 16. The results of the ex-vivo trial were analyzed taking in account different parameters: the area under the curve (AUC), the maximum cell index rate value of the curve (Maximum) and the cell index rate value at the end of the experiment (Final Point). ANOVA + Dunnett test vs C+; n.s.= Not significant; * = p-value <0.05; ** = p- value <0.01 ; **** = p-value <0.0001 . Figure 17. The specific clinical information of each of the 12 feline donors (all sterilized) were examined to determine whether HT-CECT 9161 can be effective independently of their gender (male or female) or clinical conditions (periodontal diagnosis and halitosis symptoms or healthy symptoms). The differences were not statistically significant (two-way ANOVA). The use of human food in cat’s diet very significantly (two-way ANOVA; p value = 0.0083) increased the biofilm formation and this result was statistically significantly ameliorated by the presence of HT-CECT 9161 (two-way ANOVA; p value = 0.0140).

Figure 18. Boxplots of richness, Shannon and Simpson indexes of each group. Wilcoxon test was applied between groups. (*): p-value < 0.05.

Figure 19. Heatmap is split in two for easy reading. The left side of every heatmap displays correlation coefficients of the linear model between clinical factors and species abundances, while the right side represents the 'log Fold Change' (log2FC) of group comparisons at the species level. In the right side of the heatmaps, the significance takes into consideration that the taxon must be present in minimum 50% of the samples of at least one of the two compared groups. The barplot shows the mean normalized abundance of each taxon. Clades without a valid name were not represented. (*): adj p-value <0.05.

Figure 20. Boxplots of richness, Shannon and Simpson indexes on each group at gene level. Wilcoxon test was applied between groups. (*): p-value < 0.05.

Figure 21. Schematic of module M00529 Denitrification (left) and violin/boxplots showing the stat from DESeq2 on each set of genes (right). Stat is calculated by the log2FC divided by its standard error, and it is used to rank the genes for the GSEA. EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors that reveal the capacity of the strain of the invention to inhibit the oral biofilm formation.

I. Materials and Methods

IN VITRO ASSAYS

Bacterial strains

Probiotic strains were cultured in MRS (De Man, Rogosa and Sharpe) broth (BD, San Jose, CA). The probiotics were incubated under anaerobic conditions at 37°C for 24 h.

The isolated pathogens, Neisseria zoodegmatis and Neisseria weaver/ were cultured in BHI (Brain Heart infusion) broth (Oxoid Distributors US) and incubated under aerobic conditions and at 37°C for 24 h.

Pathogen isolation

Pathogens were isolated from 6 healthy dogs. Samples of gum, teeth and palate were collected from each dog. All samples were cultivated in MRS+ 0.5% cysteine, brain heat infusion (BHI), and blood agar media in anaerobiosis or aerobiosis condition. After 48h of incubation different colonies were isolated and identified by 16S rRNA gene sequencing.

Heat treatment process

A pure culture of probiotic was obtained by fermentation of the strain in the optimal conditions. Growth media was removed and probiotic cells were washed twice by centrifugation and subsequent resuspension of the cells in NaCI solution 0.9% (w/v). Heat treatment (HT) was produced by boiling the cells during 1 h at 100^QC. The boiled cells were concentrated and lyophilized without any cryoprotectant or carrier addition. The confirmation of heat killing efficacy was checked by flow cytometry. Live-dead staining with SYTO 9 and propidium iodide (PI) was run in the cytometer for check the cell completeness. Flow cytometer settings were obtained from Bunthof et al work (Bunthof and Abee, Applied and Environmental Microbiology, 68(6), 2934. July 2002). Samples were stained with final concentration of 0.1 nM of SYTO9 and 30 nM of PI. At the same time the potency of HT cells was determined by flow cytometer. The efficacy of HT process was also confirmed by cultured of the cells in solid MRS.

Evaluation of supernatant as a growth inhibition substance

Inhibition assays in liquid medium were performed with probiotic supernatant obtained from a 24h hours anaerobic fermentation at 37^QC. After fermentation, supernatant was filtered by 0.22 pm filter to obtain a cell free supernatant and neutralized (until pH=7). The supernatant was added to pathogen suspensions and BHI medium: 20 pL of pathogen suspension at OD: 0.26; 162 pL of cell free probiotic supernatant and 18 pL of BHI broth. Reaction mixes, in duplicates, were incubated under aerobic conditions for 48 h at 37^QC. Optical density was measured in multiskan spectrophotometer at 595 nm.

Evaluation of biofilm inhibition with probiotic supernatant

The analysis of supernatant was performed with cell free supernatants by filtration on 0.22 pm filter in multiwell plates. The assays were run by triplicates. A total of 100 pl cell free supernatant was added in each well and 100 pl of pathogen suspension at OD 0.26. The plate was incubated at 37^QC in static conditions for 24 h. After the incubation, supernatant was removed and the wells were softly washed tree times with NaCI solution at 0.9% (w/v). Once washed, the plate was incubated for 1 hour at 60^QC. Then the biofilm was stained with 150 pl violet crystal at 0.1% (w/v) during 15 minutes at room temperature. After the staining, the crystal violet was removed, and the wells were washed twice with 150 pl of Elix water. The plate was dried at 37^QC for 15 minutes. Finally, 150 pl of ethanol 96% (v/v) was added to each well and the plate was read at in multiskan spectrophotometer at 545 nm. The biofilm staining with crystal violet was executed as previously described. The different controls were also stained. Degradation of biogenic amines

A pure culture of the tested probiotic was obtained by fermentation of the selected strain in the optimal conditions. The strain was inoculated at final OD: 0.1 in 10 mL tubes with different culture media. The degradation of biogenic amines was assessed by addition of 5 g/L of cadaverine or 5 g/L of putrescine in MRS without glucose (MwG). MwG was used as a negative control and MRS was used as positive control. Each condition was run in duplicate. Plate counting was performed at time 0. Also, 2 mL of medium were stored for the following analysis. At 48 h of fermentation, the colony forming unit (CFU) quantification was also performed by plate counting, and 2 ml of this culture was centrifugated 6 min at 4000 rpm and the supernatant was stored at - 20^QC until analysis.

Before the High Performance Liquid Chromatography (HPLC) determination, the samples were derivatized with 2-undecanol method developed by Aflaki et al. (Aflaki et al., Analytical Methods, 6(5):1482-1487. February 2014). HPLC was configured with the following method:

• Equipment: Acquity ARC, Waters, Method, KinetC18_2_DAD_2

• Column: Kinetex C18, 2.6pm 100*4.6mm, Phenomenex, with pre-column Serie SNH1 9-235505

• Temperature: Room temperature

• Eluent: A: MiliQ Water, B: CH3CN

• Flow: 1 ml/min

• Gradient (Table 8 below):

Table 8: Gradient.

• Detector: PDA 2998 (A254 nm Sampling rate 20 points /sec, Resolution 1 .2nm) Waters

• Injection volume 5 pL. Degradation capacity of the molecules responsible for halitosis with alive probiotics

Nine different live form of the strains (8. longum 1, W.cibaria 7, L. casei 1, CECT 9161, L. rhamnosus 1, L. animalis 1, L. animalis 2, L paracasei 1 and L. reuteri 3) were tested to evaluate their capability of degradation of the molecules responsible for halitosis, such us putrescine and cadaverine. Inhibition capacity of HT-strains on microorganisms causing periodontal disease.

Ten different heat-treated strains (Table 1 ) were tested to evaluate their capability of inhibiting growth and biofilm formation of the oral cavity pathogens isolated Neisseria zoodegmatis and Neisseria weaver/'.

Table 1 . Selected strains for the screening of suitable probiotics and postbiotics against periodontitis in dogs.

The different probiotic strains were treated 1 h at 100^QC in saline solution for inactivating the bacterial cells for testing their effect as postbiotics. The heat-treated cells were lyophilized to have a stabilized stock of material.

The capacity of inhibiting the growth of the oral cavity pathogenic strains in presence of the HT-postbiotic strains was assayed following Chen et al, 2020 (Chen et al., Lett Appl Microbiol, 70(4):310-317. April 2020). The assay was performed in duplicate and with two positive controls where only the pathogen strain was inoculated.

The formulated HT strains were then resuspended in BHI medium and dosed (at 1 E9, 1 E8, 1 E7 and 1 E6 CFU/mL, adjusted measuring by flow cytometry). The pathogenic strains were dosed at 1 E6 CFU/mL (adjusted measuring by flow cytometry corresponding to OD595=0.025 in the case of Neisseria weaver/' and OD595=0.05 in the case of Neisseria zoodegmatis) and inoculated in different tubes containing BHI medium with the HT-postbiotic strains disclosed in Table 1 dosed at 1 E9, 1 E8, 1 E7, 1 E6 CFU/mL. The culture tubes were incubated in optimal conditions for the pathogen for 24h. Drop cell plate counting was performed for each tube.

Inhibitory capacity of acerola powder and synergistic effect with CECT 9161 supernatant.

Neisseria strains were incubated in BHI with the addition of different concentration of the selected acerola powder. They were incubated for 48 hours at 37^QC in aerobic conditions. Optical density was measured at the end of the assay in a multiskan spectrophotometer.

For the synergistic assessment, the acerola powder was employed at 25 mg/mL concentration. The pathogens were cultured in the presence of 162 pL of filtrated supernatant of CECT 9161 and 36 pL of BHI medium, plus the acerola at final concentration of 25 mg/mL.

Growth inhibition with heat treated postbiotics

To evaluate the antimicrobial activity of the postbiotics obtained by heat treatment (HT), the strains were cultured in MRS media at 37°C for 24 h and then inactivated by HT process. HT postbiotic and pathogen strains were cocultured as previously described (Chen et al., Lett Appl Microbiol, 70(4):310-317. April 2020). Each probiotic was assayed in 4 different doses: 1 E9, 1 E8, 1 E7 and 1 E6 cells/mL in fresh BHI medium. Every dose was inoculated with 1 E7 CFU/mL of the isolated Neisseria zoodegmatis or Neisseria weaver/'. Also, a condition with the pathogen alone was done as a control. All the conditions were incubated 24 h at 37^QC and 330 rpm. To determinate the inhibition effect of the postbiotic, drop recount was performed by triplicate in BHI medium plate and cultured 24 h at 37^QC.

Biofilm inhibition with heat treated postbiotics

The biofilm evaluation was performed by a modification of the protocol developed by Pye et al. (Pye et al., Vet Dermatol, 24(4);446-9, e98-9. August 2013). The postbiotics were resuspended in BHI medium at final doses of 1 E9, 1 E8, 1 E7 and 1 E6 cell/mL. A total of 100 pL of each dose was plated by triplicate in a multiwell plate of 96 wells. A solution of pathogen, incubated for 24 h at 37^QC in static aerobic conditions, was inoculated in each well adding 100 pL of solution until obtain a final concentration of OD: 0.13. Only pathogen cells were inoculated in BHI medium as a positive control. Also, postbiotic doses were mixed with BHI to control the background signal. Multiplate was cultivated for 24h at 37^QC in static conditions. The biofilm staining with crystal violet was executed as previously described. Also, the different controls was stained.

EX VIVO ASSAYS

Dogs assays

Biofilm inhibition of canine microcosmos

Ex vivo assay was run using the Real Time Cell Analysis (RTCA) xCELLigence model (Agilent, USA). 50 pL of dog saliva (n=12) were added to 100 pL of BHI medium supplied by vitamin K1 and hemin. Also 100 pL of BHI with or without postbiotic was add. The postbiotics were treated in a way that the final concentration in the well was 1 E9 cell/mL. The condition without postbiotic was used as control. Also, postbiotics without saliva inoculum were evaluated as an analytical control. All tested conditions were plated by duplicate in wells of the specific plates of xCELLigence. The assay was running during 12 h. At the end point pH was measured in all conditions.

DNA quantification

After supernatant removal, the biofilm from the wells was resuspended in 100 pL PBS. DNA was isolated using the MagNA Pure LC 2.0 Instrument using the MagNA Pure LC DNA Isolation Kit III for Bacteria and Fungi (both Roche Diagnostics) and quantified by Qubit 3 Fluorometer (Thermo Scientific) as previously described (Rosier eta/., Scientific Reports, 10(1 ), 12895. July 2020).

Study of the anti-inflammatory capability of HT-CECT 9161 on buccal mucosa cells

TR146 cell line derived from a squamous carcinoma of the buccal mucosa was obtained from Applied Biological Materials Inc, Canada. Cells were grown in Ham's F12 medium supplemented with 10% FBS, 2 mM L-glutamine and Penicilin- Streptomicin (100 U/mL- 100 pg/mL) at 37°C in a humidified atmosphere with 5% CO2. Reagents were purchased from ThermoFisher Scientific. Under this culture conditions, TR146 cells form a confluent stratified non-keratinized squamous cell layer that resembles normal buccal mucosa.

TR146 were seeded in 96 well-plates (4E4 cells/well, 200 pL) and cultured for 7 days to form cell monolayers. The day of the experiment, cells were washed once with PBS and then, mixtures containing TNF-a (10 ng/mL) and the selected heat-treated strains (Doses: 10E8 cells/mL and 10E9 cells/mL) were prepared in culture medium without antibiotics. They were immediately added to each well and plates were incubated for 3h at 37^QC in the CO2 incubator. After inflammation induction, cell supernatants were harvested and stored at -20^QC until cytokine quantitation. To rule out, cytokine inhibition release due to viability cell loss, cell viability was determined using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytokine analysis in cell culture supernatants was performed using Luminex 200™ System with a 3- cytokine /chemokine panel which included IL-6, IL-8 and CXCL10 from Thermo Fisher Scientific according to the manufacturer’s instructions. For each heat-treated, percentage of inhibition (%) for each cytokine was calculated. Results are expressed as Average ± standard deviation (SD) of at least two independent experiments with three technical replicates. One-way ANOVA followed by a Dunnett’s post hoc test was used for the statistical data analysis using GraphPad Prism 9.0. A p-value of < 0.05 was considered statistically significant.

The following heat-treated strains were considered for this study: HT-CECT 9161 , L. plantarum 1 and L. plantarum CECT 7481 as benchmark were included.

Metagenome analysis

Sequencing library (16S)

In the first amplification step primers were designed containing 16S rRNA gene universal primers. In the second and last assay a universal linker sequence together with indexes and sequencing primers by Nextera XT Index kit (ILLUMINA) were included with PCR. 16S based libraries were quantified by fluorimetry using Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermofisher).

Libraries were pooled prior to sequencing on the MiSeq platform (Illumina), 300 cycles paired reads configuration. The size and quantity of the pool were assessed on the Bioanalyzer 2100 (Agilent) and with the Library Quantification Kit for Illumina (Kapa Biosciences), respectively. PhiX Control library (v.3) (Illumina) was combined with the amplicon library (expected at 20%).

Data preprocessing (16S)

Raw sequences, forward (R1 ) and reverse (R2), were merged using PEAR v.0.9. and Cutadapt v.3.7 plugin was used to filter specific V3-V4 16S region adapters. Low- quality reads were filtered using BBMap's Reformat script v.36.92 and were truncated where they started to lose quality (Q20). Filtered reads were imported into QIIME2 platform and DADA2 algorithm was applied using 'dada2 denoise-single' function and ASVs ('Amplicon Sequence Variants') were generated internally in the same function, while quimera sequences were also removed. Taxonomy of the resulting ASVs was annotated using blastn v2.2.29+) against 16S specific database from the NCBI (v. May 2022). Assigned taxonomies with an identity percentage lower than 97% were reassigned using Naive-Bayes algorithm against SILVA v.138 SSU Ref NR 99 database.

Sequencing library (Metagenomics)

Nextera XT Library kit (Illumina) was used following the manufacturer’s instructions. DNA was simultaneously fragmented and tagged with dual index sequencing adapters that enable accurate assignment of reads per sample. Library quality control was ensured by profiling and length distribution analysis using HSD5000 kit in the TapeStation 4200 equipment (Agilent). NovaSeq 6000 sequencing platform in a 150 paired end reads configuration generated *.bcl files as primary sequencing output (NovaSeq Control Software (NCS) v.1.6). Bcl2fastq v.2.20 program was used to translate the sequencing reads from bcl (Base Calling) to FASTQ format. This step also removed sequencing adapters.

Data preprocessing (Metagenomics)

The Clumpify tool from the BBToolssuite was used to remove optical duplicates. Reads with less Phred quality score than Q20 and less length than 50 nucleotides were filtered out using the program BBMap v38.36 (Bushnell B, 2014 Human genome presence was filtered using NGLess v1 .0.0-Linux64 using the built-in Homo Sapiens hg19 genome as reference. Those reads with alignments of more than 45 bases and 97% similarity to the human genome were discarded. The remaining sequences were called ’High Quality sequences’ and were meant to be the final sequences.

Metaphlan (v.4) was used to assign the taxonomy. The reads of each sample were aligned against single-copy genetic markers present in almost all bacteria. From these alignments, the estimated number of reads contributed to a given clade for each identified taxa were obtained computationally.

MEGAHIT genome assembler (v.3.13.0) was used to perform the assembly of the 'High-Quality Sequences'. Six assemblies were conducted at k-mer sizes of 21 , 33, 55, 77, 99 and 127. Metagenome quality was assessed for all assemblies using QUAST v.5.0.2, and the assembly with K-mer size 127 was selected. Contigs larger than 500 bp were used to predict genes using Prodigal (v2.6.3). The gene quantification was calculated with the SALMON software (v. 1.5.1 ). KEGG functional annotation was performed using the web server GhostKoala.

Statistical analysis The relative taxonomic abundances of the samples were displayed with collapsed histograms plotted by 'ggplot2' (v.3.4.0) library in R. Data was normalized using the rarefaction technique from 'phyloseq' R package in order to perform alpha diversity analysis. Shannon, Simpson and Richness indexes were calculated using 'vegan' (v.2.5-6) R package. Violinplots were created with 'ggpubr' (v.0.4.0) library in R,and Wilcoxon test was performed through 'stats' (v.3.6.0) R package) to find significant differences between groups.

To illustrate taxonomic dissimilarities based on ASVs, Principal Coordinates Analysis (PCoA) across the samples was carried out using the Bray-Curtis distance matrix calculated with the 'phyloseq' (v.1.28.0) R package and represented with 'ggplot' (v.3.4.0) R package. The effects of the factors on taxonomy date were evaluated with a permutational multivariate analysis of variance (PERMANOVA) with 'vegan' (v.2.5-6) R package using the Bray-Curtis dissimilarity matrix that was previously calculated considering the relative abundances of functional categories in all samples.

For biomarkers identification, the inventors normalized the feature table using calcNormFactors function, with the trimmed mean of M-values (TMM) option. After normalization, the inventors used the limma (v.3.42.2) function voom to convert normalized counts to Iog2-counts-per-million and assign precision weights to each observation based on the mean-variance trend. The inventors used the functions Im Fit, eBayes, and topTable in the limma R package to fit weighted linear regression models, performed tests based on an empirical Bayes moderated t-statistic and obtained BH FDR-corrected p-values. A taxon was considered differentially abundant if the corrected p-value < 0.05 and if it was present on at least 50% of the samples of one of the compared groups. The linear model included the subject variable as a random effect. Heatmaps were constructed using 'ComplexHeatmap' R package (v.2.10.0) for visualization. A Gene Set Enrichment Analysis (GSEA) was additionally conducted in the metagenomic dataset, using fgsea package in R v1.16 on KEGG L3 pathways and KEGG modules. The t statistic of KOs/annotated genes from limma-voom differential abundance analysis were used to perform the GSEA.

Cats assays

Sampling of cats’ saliva

A sterile cotton swab was used for sampling the cat saliva, slightly rubbing the mouth. The swab was then inserted in a two-level tube and posterior centrifuged 10 min at 4000 rpm. Samples were maintained at room temperature until the experiment started.

Ex-vivo assay with feline saliva

Samples were collected from healthy cats 3 hours before starting the experiment. The early-stage biofilm formation was explored using the xCELLigence RTCA (Agilent) adapted for oral microbiome model. Each well was filled with 100 pl of BHI medium, enriched with 5pg/ml of hemin and 1 pg/ml of Vitamin K. Additionally, 50 pl of saliva was added to each well. A final concentration of 1.00+E9 cells/ml of HT-CECT 9161 was added to the assay wells. The xCELLigence model was then incubated at 38^QC under aerobic conditions for 20 hours. The cell index was recorded every 10 minutes. The final supernatant product was collected and filtered for subsequent pH measurement.

CLINICAL TRIALS

In vivo Efficacy Study

A randomized placebo controlled trial was conducted with the objective of comparing the efficacy of a high and low dose intervention compared to a placebo on plaque in 60 colony dogs. Each was randomly assigned to one of three interventions that were added directly to their diet, with 20 dogs per study arm. The interventions were:

- Placebo;

- Low dose: 0.5 billion Cells of HT-CECT 9161 daily dose;

- High dose: 2.5 billion Cells of HT- CECT 9161 daily dose

Dogs underwent mouth cleaning on Day 0 and were then administered either HT-CECT 9161 according to the dosage above, or placebo, once daily as a top dressing on kibble. Plaque was measured at day 29 and 57.

A linear model was fitted to the formation of plaque. A fixed time effect was included in the model to account for whether a change occurs between baseline and day 29 or day 57. A mixed-effects linear model was used and included a random effect for dogs in the model, as the change scores between baseline and days 29 and 57 for individual dogs are likely to be correlated with each other.

Data were recorded on two additional variables:

- Time to consume diet (g)

- Average diet consumed (%)

The time required to consume the diet for each of the 60 dogs was recorded on one day of the study. It is important to explore whether time to consume is associated with outcome measures, as it is possible that the longer it takes a dog to consume its diet, the longer the intervention will take to take effect. Since the intervention is mixed into the diet, it is possible that those dogs that do not consume as much of their diet will consume less of the intervention.

The weight of the diet offered (g) and the diet consumed (g) was recorded for each dog on each day of the study and from this the percentage of diet consumed each day was calculated. This was to ensure that each dog ate a sufficient proportion of the diet offered to ensure that the treatment had a chance to take effect. The intervention consisted of a small amount (mg) mixed with the main diet. Since the amount of treatment consumed cannot be measured, the amount of diet consumed in general is used as an indicator.

Modelling started with the full model which included the following fixed effects (as well as the random effect for the dog): a) Treatment effect (placebo, low or high dose) b) Timing (day 27 or day 59) c) Interaction between treatment and timing d) Time to consume e) Average diet consumed

The interaction between treatment and timing examines whether the treatments had a greater or lesser effect at the two follow-up time points. A stepwise backtracking process was performed in which each non-significant term was removed from the model (choosing the least significant term based on the F test). In reporting model results, the model was treated with fixed effects a) to c) as the minimum model and these terms were added back to the final model whether they were statistically significant or not. For all reported models, model fit was verified by using residual plots.

Consumption study

A consumption study was conducted in 20 dogs comparing the time required to consume the diet between the low- and high-dose interventions. For this purpose, the time required to consume the diet for each of the 20 dogs (in minutes and seconds) was recorded for 6 consecutive days. Each diet was mixed with either a low or high dose intervention. Ten dogs were randomly assigned the low dose on the first day and the remaining dogs were assigned the high dose intervention. Dogs consistently received the same dose for the first three days of the study and then switched to the other dose for the last three days of the study.

The mean time to consume for each intervention (low and high dose) was calculated for each dog over the three days it was offered to compare the distributions of mean time to consume by intervention.

To test more formally whether time to consume was associated with intervention (low or high dose), a linear mixed-effects model was fitted to time to consume with intervention as a fixed effect. A random effect for dogs was included in the model, since it is likely that the values of time to consume on each of the recorded days are correlated with each other.

Supragingival plaque microbiome study

Study design and Supragingival Plaque Sample Collection

Sixty healthy dogs were selected for the study, receiving oral treatment (20 dogs for each treatment: placebo, postbiotic high dose or postbiotic low dose) for 57 days. Plaque samples were collected only after 57 days of administration.

Supragingival plaque samples were collected on Day 57 using collection kits from DNA Genotek (Ottawa, ON, Canada). Samples were stored according to manufacturer’s instructions at room temperature until analysis.

DNA Isolation and Metagenomic Next Generation Sequencing of Supragingival Plaque Samples

Microbiome DNA extraction of supragingival plaque samples included enzymatic lysis using 100 mg/ml lysozyme; 1 mg/ml lysostaphin and 25 KU/ml mutanolysin (Sigma- Aldrich, St. Louis, MO, USA) for 30 minutes at 37^QC and bead beating; FastPrep-24- 5G, one round of 60 seconds at 6.0 followed by processing of samples using the QIAsymphony PowerFecal Pro DNA Kit (Qiagen, Barcelona, Spain) robotic magnetic bead-based kit. DNA concentration was assayed using the Qubit dsDNA System (Thermo Fisher Scientific, Waltham, MA, USA) and samples were normalized accordingly to generate high-quality functional libraries. Library construction, and preprocessing metagenomic analysis for supragingival plaque samples are described below.

Preprocessing Metagenomic Analysis.

The Clumpify tool from the BBToolssuite was used to remove optical duplicates. Reads with less Phred quality score than Q20 and less length than 50 nucleotides were filtered out using the program BBMap v38.36. Human and dog genome presence was filtered using NGLess (vl .0.0) using the built-in Homo Sapiens ‘hg19’ genome as reference first and ‘GCF_000002285_CanFam3.1_genomic’ genome as reference second. Those reads with alignments with more than 45 bases and 97% similarity to the reference genome were discarded. The remaining sequences were called ’High Quality sequences’ and were meant to be the final sequences.

Metaphlan (v.4) was used to assign the taxonomy. The reads of each sample were aligned against the ‘CHOCOPhlAn’ database that contains single-copy genetic markers present in almost all bacteria, specifically the January 2021 version (‘mpa_vJan21_CHQCQPhlAnSGB_202103’) for the preclinical phase and the October 2022 version (‘mpa_vOct22_CHOCOPhlAnSGB_202212’) for the clinical phase. From these alignments the estimated number of reads contributed to a given clade for each identified taxon were obtained computationally.

MEGAHIT genome assembler (v.1 .2.9) was used to perform the assembly of the 'High- Quality Sequences'. Six assemblies were conducted at k-mer sizes of 21 , 33, 55, 77, 99 and 127 and the automatically selected ‘final. contigs. fa’ file was used, which corresponded to the 127 k-mer size assembly for both preclinical and clinical phase cases. Contigs larger than 500 bp were used to predict genes using Prodigal (v2.6.3). KEGG functional annotation was performed on the predicted genes using the web server GhostKoala. The gene quantification was calculated with SALMON software (v. 1 .5.1 ). Reads from genes from heme biosynthesis module were aligned using BLASTn) against nt database (version July 2022) to study the taxonomy of those genes.

Metagenomic Next Generation Sequencing of Supragingival Plaque Samples.

Libraries were prepared with the Nextera XT Library kit (Illumina San Diego, CA, USA) following the manufacturer’s instructions. Library quality control was ensured by profiling and length distribution analysis using HSD5000 kit in the TapeStation 4200 equipment (Agilent, Santa Clara, CA, USA). The libraries were sequenced on NovaSeq 6000 platform in a 150 paired end reads attaining a minimum of 20 million reads per sample. The configuration generated *.bcl files as primary sequencing output (NovaSeq Control Software (NCS) v.1.6). Bcl2fastq v.2.20 program was used to translate the sequencing reads from bcl (Base Calling) to FASTQ format. This step also removed sequencing adapters (data deposited in NCBI SRA under BioProject PRJNA1 150971 ).

Statistical Analysis of the Supragingival Plaque Microbiome

The relative taxonomic abundances of the samples were displayed with collapsed histograms plotted by 'ggplot2' (v.3.4.0) library in R version 4.2.3. Taxa and genes data were normalized using the rarefaction technique from 'phyloseq' (v.1 .34) R package to perform alpha diversity analysis. Shannon, Simpson, and Richness indexes were calculated using 'vegan' (v.2.5-7) R package. Violin plots were created with 'ggpubr' (v.0.4.0) library in R and Wilcoxon signed-rank test was performed through 'stats' (v.3.6.0) R package to determine significance between groups. To illustrate taxonomic dissimilarities based on Ampicon Sequence Variants (ASVs), Principal Coordinates Analysis (PCoA) across the samples was carried out using the Bray-Curtis distance matrix calculated with the 'phyloseq' (v.1 .034) R package and represented with 'ggplot' (v.3.4.0) R package. The effects of the factors on taxonomy date were evaluated with PERMANOVA with 'vegan' (v.2.5-7) R package using the Bray-Curtis dissimilarity matrix that was previously calculated considering the relative abundances of functional categories in all samples.

Identification of Taxonomic and Functional Features in the Microbiome of Supragingival Plaque

Differential taxa and gene abundance analysis was performed using the DESeq2 R package v.1.30.1. The normalization was based on the ‘Relative Log Expression’ method. ‘EstimateSizeFactors’ function was used to calculate the scaling factors using the median ratio between taxa and genes abundances and the geometric mean. The ‘poscounts’ method was used to address the taxa and genes that had multiple zeros in the samples. A taxon was considered differentially abundant with a Benjamini- Hochberg ("BH") multiple testing correction adjusted p<0.05 and if it was present in at least 50% of the samples of one of the compared groups. To determine significance of the functional profiles between groups a Gene Set Enrichment Analysis (GSEA) was additionally conducted in the metagenomic dataset using fgsea (v1 .16) R package on KEGG modules. The ‘stat’ statistic of genes from DESeq2 differential abundance analysis was used to rank the genes to perform the GSEA. Heatmaps were constructed using ComplexHeatmap R package v.2.1 1 .1 .

II. RESULTS

Example 1. Strain classification, characterization and safety

The novel strain Lactiplantibacillus plantarum CECT 9161 was isolated from the mouth swap of a healthy person. CECT 9161 strain is a gram-positive facultative anaerobe, rod-shaped, non-spore-forming bacteria. It was showed capable of resisting long term to lysozyme as well as strong acid environment and high concentration of bile. This ability suggests that strain CECT 9161 is capable of surviving the gut environment.

The strain of the invention was demonstrated to be a safe strain, belonging to a species included in the Qualified presumption of safety (QPS) list of European Food Safety Authority (EFSA) with no transferable antibiotic resistances according to the EFSA requirements. Moreover, the whole genome analysis showed that in the genome of the strain of the invention the genes needed for the biogenic amine production are not present.

Example 2. Study of the effect of supernatant in pathogen growth effect.

The effect of neutralized supernatant was tested in some oral pathogens isolated from the dog mouth, N. zoodegmatis and N. weaver/'. These pathogens are related with bad oral health due to their proteolytic capacity, which induce a neutral/basic ambient in the mouth inducing a dysbiosis. The strain CECT 9161 reduced significantly the growth of both the tested pathogens in a liquid culture (Figure 1 ).

The effect of CECT 9161 strain was also compared with two commercial products present in the market: Benchmark 1 and Benchmark 2. The results showed that CECT 9161 strain inhibited the growth of the pathogens better than the proposed commercial benchmarks (Figure 2).

The components of benchmark 1 are: Bifidobacterium lactis, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, Lactobacillus case/', Lactobacillus plantarum, Bifidobacterium breve, Streptococcus thermophilus, Saccharomyces cerevisiae (boulardii), Bifidobacterium animalis, Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus sporogenes, Lactobacillus fermentum, Lactobacillus reuteri and Lactobacillus salivarius. The components of benchmark 2 are: Streptococcus uberis KJ2®, Streptococcus oralis KJ3®, Streptococcus rattus JH 145®.

Benchmark 1 , benchmark 2 and benchmark 3 are commercially available.

Example 3. Study of biofilm inhibition by supernatant.

The dental plaque is one of the most influent factors in the oral health. The plaque allows the formation of anaerobic conditions that can induce the microbiota modulation as a dysbiotic microbiota with high content of anaerobic bacteria which usually are pathogenic or related with periodontitis. Since the plaque is mostly formed by biofilm, the capability of an oral care product to inhibit the formation of biofilm is a fundamental parameter to be evaluated.

The free-cells supernatant of a CECT 9161 strain pure culture was used for inhibiting the biofilm formation of four Neisseria strains. As reported in Figure 1 , CECT 9161 strain free-cells supernatant inhibits the biofilm formation by all the tested Neisseria strains by more than 90% compared to the control (single-strain culture without the addition of supernatant).

The effect of free-cells supernatant obtained culturing Benchmark 1 and Benchmark 2 commercial benchmarks, was tested on the growth of biofilms of N. zoodegmatis and N. weaver/' isolated from dog mouth sample and compared to CECT 9161 strain supernatant results (Figure 3). Supernatant of CECT 9161 significantly outperformed Benchmark 2 and showed better results than Benchmark 1 .

The inhibition of biofilm produced by CECT 9161 strain is a promising activity that can have a critical impact on the creation of plaque and play a key-roll in the maintenance of a good oral health in dogs. Example 4. Study of the degradation capacity of the molecules responsible for halitosis with CECT 9161 alive strain

The presence of biogenic amines such as putrescine and cadaverine is related with the odor of dogs oral cavity. Thus, the degradation of these molecules by the probiotic strain may be related to the reduction of the odors of the oral cavity. A specific assay allowed to confirm that CECT 9161 alive strain was able to grow degrading putrescine and/or cadaverine and using them as substrate (Figure 4.A).

CECT 9161 strain demonstrated to be able to grow degrading both cadaverine (33.82% less after 48h) and putrescine (26.43% less after 48h), that is it uses these molecules as substrate for its growth (Figure 4.B). This reduction could be especially important since it is known that two of the molecules play an important role in halitosis.

Example 5. Study of the inhibition capacity of HT-probiotics on microorganisms contributing to periodontal disease

The results obtained are presented in Figure 5 for each HT-probiotic, doses showing growth percentage with respect to the control.

The results clearly indicate that both pathogens tested are sensitive to the presence of some of the HT-probiotic strains and specially to the strains of the Lactobacillus genus. The growth of Neisseria weaver/' and Neisseria zoodegmatis was inhibited up to 99% with a dose of 1 E9 CFU/mL of HT- Lactobacillus strains.

Neisseria zoodegmatis growth was inhibited up to 99% by all the tested HT- Lactobacillus, HZ. cibaria 7, and up to 50% by B. animalis 1. HT-L paracasei 1 also inhibited the N zoodegmatis growth up to 23% with a dose of 1 E8 CFU/mL. However, the most interesting results are those related to the HT-CECT 9161 strain, that inhibited both pathogen strains growth up to 99.9% with a dose of 1 E9 CFU/mL. HT-CECT 9161 inhibited up to 99% Neisseria weaveri with a lower dose of 1 E8 CFU/mL, up to 30% with a dose of 1 E7 CFU/mL and up to 36% with a dose of 1 E6 CFU/mL

Example 6. Study of the capacity to inhibit pathogen growth by HT- CECT 9161

The assay [protocol adapted from the article by Chen, et al. (Chen, et al., Letters in Applied Microbiology, 70(4), 310-317. April 2020) for testing the inhibition of the growth of the oral cavity pathogenic strains in presence of the HT-CECT 9161 was performed in duplicate and with two positive controls where only the pathogen the strain was inoculated.

The formulated HT strains were then resuspended in BHI medium and dosed (at 1 E9, 1 E8, 1 E7 and 1 E6 CFU/mL adjusted measuring by flow cytometry). The pathogenic strains were dosed using at 1 E7 CFU/mL (adjusted measuring by flow cytometry corresponding to OD595=0.025 in the case of Neisseria weaveri and OD595=0.05 in the case of Neisseria zoodegmatis) and inoculated in different tubes containing BHI medium with the HT-probiotic strain dosed at 1 E9, 1 E8, 1 E7, 1 E6 CFU/mL. The tubes were incubated in optimal conditions for the pathogen for 24h. Drop cell plate count was performed for each tube. The results obtained are presented in Figure 6.

The results indicate that both pathogens are sensitive to the presence of the HT-CECT 9161 , that was inhibiting both pathogen strains growth up to 99.9% with a dose of 1 E9 CFU/mL. HT-CECT 9161 was inhibiting up to 99% Neisseria weaver/ with a lower dose of 1 E8 CFU/mL, up to 30% with a dose of 1 E7 CFU/mL and up to 36% with a dose of 1 E6 CFU/mL. Example 7. Study of the capacity to inhibit pathogen biofilm formation by HT- CECT 9161

The capability of the HT- CECT 9161 to inhibit pathogens biofilm formation was tested using a semiquantitative method based on a 96-multiwell plate.

Each pathogen was inoculated (final OD595=0. 13) in 96-multiwell plates (final volume=0.2L each well) in a suspension (1 :1 ) of fresh medium and HT-probiotic lyophile resuspended in fresh medium (per triplicate). The positive control (consisting in fresh medium inoculated with the pathogen), and the negative control (only fresh media), were also incubated per triplicate in the same plate. As analytic control fresh medium with HT-probiotic doses (from 1 E9 to 1 E6) was added by triplicate in the same plate. After 24h of incubation at 37^QC without shaking, the liquid was removed from the wells without touching the bottom and each single well was washed three times with sterile saline solution. The empty 96-multiwell plate was incubated for 60 min at 60^QC. The wells were refilled with 150 pL of 0.1 % crystal violet commercial 1 % (w/v) diluted (1 :10) and the plate was incubated at room temperature for 15 minutes. After washingout the crystal violet, the plate was dried at 37^QC for 15-30 min. The crystal violet was then solubilized using 150 pL (each well) of 96% v/v ethanol and the OD at 545nm was read. Table 2 presents the capability of the HT-strains to inhibit the pathogen biofilm formation.

Table 2. Pathogen biofilm inhibition results. The effect of the specific dose of HT- CECT 9161 (from 1 E9 to 1 E6 CFU/mL) on Neisseria zoodegmatis and Neisseria weaveri biofilm formation was classified as severe (+++; less than 20% of biofilm compared to the control), moderate (++; between 20 and 50% of biofilm compared to the control), minimum (+; more than 50% of biofilm compared to the control) or none effect (-; more than 70% of biofilm compared to the control).

The biofilm formation assay (Table 2) confirms the results of the previous inhibition test: both pathogen strains Neisseria weaver/' and Neisseria zoodegmatis were sensitive to the presence of a range of the HT-CECT 9161 concentration (1 E7-1 E9 cells/mL).

Comparison of biofilm formation capability was assayed for HT-CECT 9161 and benchmark strain HT-CECT 7481 , against three pathogens (Figure 7).

HT-CECT 9161 outperforms the benchmark strain against the three the pathogens tested.

Example 8. Study of the anti-inflammatory capability of CECT 9161 on buccal mucosa cells

The anti-inflammatory properties of the heat-treated strains in an oral environment were evaluated using the human TR146 cell line. Analysis of the following cytokines was carried out: IL-8, which is directly involved in neutrophil infiltration; CXCL-10, which promotes the recruitment of CD8+ and CD4+ T-cells and IL-6, known to modulate the differentiation of osteoblasts and osteoclasts. Thus, inflammatory cytokine production (IL-8, IL-6 and CXCL-10) was evaluated by incubating TR146 cell monolayers with TNFa in the presence or not of the HT strains at two different doses (1 E8 cells/mL and 1 E9 cells/mL).

Results showed that heat treated probiotic strains displayed different degree of inhibition for IL-8 and CXCL-10 cytokines at 1 E9 cells/mL. In function of the potency of the effect, the strains were categorized. In general terms, HT-CECT 9161 was one of the strains with the highest efficacy (29±3% IL-8 inhibition and 72±6% CXCL-10 inhibition vs control (TNF-a)) was able to inhibit significantly both cytokines at the evaluated dose (1 E9 cells/mL). Both the HT-L. plantarum strain tested (HT-L. plantarum CECT 7481 and HT-L. plantarum 1 ) inhibiting the secretion of IL-8 cytokine 56±10% and 74±2% CXCL-10 inhibition vs control (TNF-a)).

The heat-treated L. plantarum 1 was obtaining lower value and the benchmark product, hea-treated L. plantarum CECT 7481 , was reaching similar values (respectively 5±8% and 25±3% IL-8 inhibition and 56±10% and 74±2% CXCL-10 inhibition vs control (TNF- a)). These reported results were statistically significant (p-value < 0.05 TNF-a vs HT- strain, one-way ANOVA, Dunnett’s multiple comparisons test).

Regarding IL-6, all probiotic strains failed to inhibit its secretion in TR146 cells upon TNF-a challenge. HT-CECT 9161 may even enhance IL-6 secretion in TR146 cells stimulated with TNF-a for 3h by heat-treated probiotic strains at 1 E9 cells/mL dose (71 ±9% IL-6 secretion vs control (TNF-a)). Both the HT-L. plantarum 1 and the benchmark product, HT-L. plantarum CECT 7481 , were reaching lower values (respectively 46±22% and 55±1 1 % IL-6 secretion vs control (TNF-a)).

HT-CECT 9161 strain displayed excellent anti-inflammatory properties in a buccal epithelial model as this strain was able to inhibit the secretion of IL-8 and CXCL-10 cytokines, involved in neutrophil and T-cell recruitment respectively, as well as enhance IL-6 secretion in TR146 cells stimulated with TNF-a.

The obtained results indicated that HT-CECT 9161 was performing similarly to the benchmark strain HT-L. plantarum CECT 7481 and better than HT-L. plantarum 1 inhibiting the secretion of IL-8 and CXCL-10 cytokines, but better than both the HT-L. plantarum strains tested (heat-treated L. plantarum CECT 7481 and heat-treated L. plantarum 1 ) enhancing IL-6 secretion. Example 9. Inhibitory capacity of acerola powder and synergistic effect with CECT 9161 supernatant against oral pathogens.

Acerola powder is effective against the oral pathogens tested: with 12. 5 mg/mL it is possible to see inhibition effect, and with 50 mg/mL around 75% of inhibition is found, as can be observed in Figure 8.A.

For the combined effect assessment, the acerola powder was employed at 25 mg/mL concentration. The pathogen was cultured in the presence of 162 pL of filtrated supernatant of CECT 9161 and 36 pL of BHI medium, plus the acerola at final concentration of 25 mg/mL (Table 3).

Table 3. Percentages of OD reached by the pathogen (isolated N. zoodegmatis) growing in the presence of probiotic supernatant, acerola, and combination of both.

As can be observed in Figure 8.B, the acerola itself at 25 mg/mL inhibited pathogen growth around 85%, and the probiotic supernatant inhibited between 75% and 50% in this assay. The combination of both increased inhibition around 90%, demonstrating a surprising effect of this combination (Table 3).

Example 10. Ex vivo with microbiome assay in dogs

The evaluation of postbiotic inhibitory effect on the biofilm formation on the teeth surface (preventive effect) was simulated by using a Real Time Cell Analysis (RTCA) xCELLigence system. The RTCA xCELLigence is multi-well impedance based system adapted for the study of single-species and multi-species biofilm growth in real time. In this study the ex vivo dog saliva was collected and used as inoculum to form multiplespecies biofilms (protocol adapted from Mira et al. (Mira et al., Journal of Oral Microbiology, 11(1). May 2019). The saliva samples (dog (D, n=12)) were collected and inoculated with BHI in wells of a xCELLigence plate (12h of incubation). Different conditions were tested in duplicate: control (C), 1 E9 cells/mL HT- CECT 9161 1 E9 cells/mL HT-L plantarum 1 , 1 E9 cells/mL HT-L reuteri 1 ,1 E9 cells/mL HT- L. reuteri 2 postbiotic 1 E9 cells/mL. Duplicates of different controls were added without saliva. Biofilm growth was measured in real time by impedance (Cell Index values). Duplicates of different controls were added without saliva.

After 12h of incubation with canine saliva, the pH dropped in all conditions because of sugar fermentation by canine bacteria. No relevant variation was recorded for the pH in the wells treated with HT-CECT 9161 (protocol adapted from Rosier et al. (Rosier et al., Scientific eports, 10(1), 12895. July 2020) indicating that the presence of HT- CECT 9161 was not inducing the drop of pH in this fermentation.

All differences between the control (C+) and the postbiotic curves (CECT 9161 , L. reuteri 1 , L. reuteri 2, L. plantarum 1 ) were significant (p<0.05), except timepoint 54 minutes for L. reuteri 1 and L. reuteri 2 and the timepoints 54 minutes to 1 h 54 min for L. plantarum 1 (Figure 9). This indicates that the postbiotics decreased cell growth and/or attachment of the canine oral communities.

The capability of HT-CECT 9161 of inhibiting the biofilm formation, but also to lowering the presence of microbial cells in the biofilm collected from the HT-CECT 9161 treated wells, was corroborated by the quantification of DNA extracted from the biofilms formed on the bottom of each well. The value obtained for HT-CECT 9161 wells compared with the control condition, indicated that less DNA was obtained from the biofilms of the HT- CECT 9161 indicating a lower presence of cells in the biofilm assays (p<0.05 by Wilcoxon test, HT-CECT 9161 vs control). Summarizing HT-CECT 9161 was performing better than the compared strains in this assay under all the tested parameters. In fact, HT-CECT 9161 obtained a higher decreasing of biofilm growth (lower cell index in the xCELLigence system and less DNA detection in final biofilms) indicating that HT-CECT 9161 could decrease canine plaque formation in vivo and decrease the risk of periodontal disease and halitosis development.

The DNA obtained was used for the genomic analyses showing that while no significant differences were observed in alpha-diversity indexes (Figure 10.A), while Bray-Curtis dissimilarity between the HT-CECT 9161 treated group versus the control group show significant dissimilarities (R2=0.037; p-valor 0.036) (Figure 10.B). Rothia sp. and Bifidobacterium breve, previously described either as potentially beneficial or found in healthy dogs’ saliva, tended to be overrepresented in treatment group respect to control. On the other hand, Escherichia genus, which species has been described like potential harmful bacteria, was significantly (p-valor<0.05) less abundant in HT-CECT 9161 treated group versus the control group (Figure 10 C and D).

Example 11. Ex vivo assays with feline saliva

For confirming that HT-CECT 9161 is effective for cats oral health applications, a trial using the xCELLigence, a state-of-art ex vivo with microbiota model for the study of the oral biofilm formation, was performed using cat’s saliva. Twelve independent assays were performed using saliva form twelve different cats (Figure 15).

All the data obtained were compared and the results are presented in Figure 16.

The samples obtained from a cat were analysed in triplicate with and without (C+) the addition of HT-CECT 9161 in an ex vivo assay using the Real Time Cell Analysis (RTCA) xCELLigence system (Agilent). The data obtained (Figure 17) showed that the presence of HT-CECT 9161 was reducing the biofilm formation by >60% (n=12) after 18 hours.

The curves in data were analyzed are reported in Figure 17. The curves were analyzed taking in account different parameters: the area under the curve (AUC), the maximum cell index rate value of the curve (Maximum) and the cell index rate value at the end of the experiment (Final point).

The specific clinical information of each of the 12 feline donors (all sterilized) were examined to determine whether HT-CECT 9161 can be effective independently of the clinical conditions:

• 8 out of 12 cats are female.

• 2 out of 12 cats has periodontal diagnosis and halitosis symptoms.

• 3 out of 12 cats include human food in their diet.

The results presented in Figure 18 suggest that HT-CECT 9161 can be equally effective in male and female cats, although not statistically significant. Additionally, when cats are diagnosed with periodontal disease or exhibit halitosis symptoms, introducing human food into their diet increases biofilm production (p value = 0.0083). However, the use of HT-CECT 9161 significantly ameliorates this effect (p value = 0.0140).

Example 12. Consumption Study in dogs

Boxplots of the mean time to consume by treatment group are given in Figure 1 1 where the boxplots on the right exclude outliers. There are clearly a small number of dogs (four in total) that are defined as outliers (with values greater than 1.5 times the interquartile range) and take a longer time to consume their diet compared to the remaining dogs. The boxplots highlight that there is an overlap between the mean time to consume for each treatment; visually there does not seem to be a difference in mean time to consume between the low and high dose treatment.

When fitting the statistical model to daily consumption time, it was found that there was no evidence of a treatment effect in the model. There is no evidence that the type of treatment is associated with the time it takes a dog to consume its diet. It should be noted that the delivery of the invention as a top dressing on kibble in the present study is likely to be associated with a comparatively shorter contact time (c. 4 minutes in the present study) than delivery of the invention in a format such as a dental chew. It is expected that extending the contact time of the invention would correspondingly increase the magnitude of effect.

Example 13. Efficacy Study in dogs: Diet Consumed

Boxplots of the diet consumed (%) per dog and treatment group are provided in Figure 12. Typically, most dogs consume a high proportion of their diet on each day (close to 100%). Some dogs do have days when they consume less (highlighted by the points on the plots) and there are a few dogs whose median diet consumed is lower. Based on these results, it was agreed the following:

1 . One dog was removed from the analysis as the only dog that was deemed to have consumed an inadequate amount of the interventional product (the dog’s median diet consumed was 80% with a range of between 33% and 100%).

2. The mean diet consumed over all days was included in the model as a potential covariate as a proxy for amount of intervention consumed (Table 4). Table 4 Summary statistics of the diet consumed by dogs in the efficacy study.

Example 14. Efficacy Study in dogs: Change in Plaque from Baseline The change from baseline in plaque at day 29 and 57 by treatment group are illustrated in the boxplots in Figure 13 and the summary statistics are provided in Table 5. These boxplots illustrate that:

• All distributions are greater than 0 which means that all dogs saw an increase in plaque from baseline at both day 29 and day 57.

• There is some evidence of a difference between treatments with a lower increase in plaque for the low dose intervention at day 29 compared to placebo and high dose. Both the low dose and high dose interventions have a lower on average increase in plaque at day 57 compared to placebo and the high dose group demonstrated a significant reduction in plaque between day 29 and day 57.

Table 5. Summary statistics of the change in plaque from baseline by Treatment and Time Point.

The model results (coefficients) for the change in plaque from baseline by time point and treatment are shown in Table 6. Using the F test, there were no significant effects at the 5% significance level in the model, but there was an interaction between treatment and time at a 10% level of significance. The model coefficients illustrate that overall, the low dose has a lower value for change in plaque compared to the placebo (at the 10% level), but that there is no difference between the high dose and the placebo. There is evidence that the levels of plaque for the high dose are significantly lower at day 57 compared to day 29.

Table 6. Regression coefficients for change in plaque by treatment group and time point. The estimated changes in plaque from baseline for the treatment groups at each timepoint are plotted in Figure 14 (and the values are tabulated in Table 7).

Table 7. Predicted change in plaque by treatment group and time point.

Figure 14 illustrates that the confidence intervals for all treatment groups are greater than 0 at both day 29 and day 57 which means that there is evidence of an increase in plaque at these time points across all treatments. At day 29 the estimated increase in plaque for the low dose is smaller than for the high dose and placebo. There is a reduction in plaque between day 29 and day 57 for the high dose treatment which is not evident in the low dose or placebo groups.

Example 15. Effects on supragingival plaque microbiota diversity and composition in dogs

Microbiota composition

Between 28% and 50% of sequences of all the samples (60/60) were taxonomically classified. From those sequences that were classified, a total of 153 distinct species were bacteria and archaea. The supragingival plaque microbiome population was dominated by species of the Porphyromonas genus: Porphyromonas gulae (14.54 ±

There was a significant increase in Shannon alpha diversity metric in High Dose group when compared to Low Dose group (Wilcoxon test; Figure 18).

Differential abundance analysis

At species level, only a species identified as Porphyromonadaceae bacterium H1 had its abundance significantly increased in Low Dose group when compared to Placebo (log2FC = 0.721 ± 0.220, adj. p = 0.037) (Figure 19), and was inversely correlated with gingivitis, plaque, and calculus scores, indicating a positive association with oral health.

Correlation between clinical factors and supragingival plague species

The inventors observed significant positive associations of potentially pathogenic species with the mean values of gingivitis, plaque and tartar, but not with the halitosis mean score (Figure 19), including Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum and species of Prevotella and Peptostreptococcaceae, indicating a negative association with oral health. Abundance of other species including Gemella palaticanis, Actinomyces bowdenii, Canibacter oris, P. dagmatis ATCC 51570 and species of Neisseria, were negatively correlated with oral health scores, indicating a positive association with oral health. Abundance of Porphyromonadaceae bacterium H1 significantly increased (log2FC = 0.721 ± 0.220, adj. p = 0.037) in the Low Dose group when compared with the Placebo group and was inversely correlated with gingivitis, plaque, and calculus scores, indicating a positive association with oral health. Among all oral microorganisms, species of the genus Porphyromonas are capable of invading and damaging the epithelial layer as well as to induce inflammatory responses leading to attachment loss and periodontal destruction. Species P. gingivalis is considered the key pathogen in periodontal infections due to the production of different virulence factors, and P. gulae is a prevalent species in subgingival biofilm of dogs with periodontitis. Other periodontophatic species according to literature include Treponema parvum, Campylobacter rectus and Filifactor alocis.

Effects on supragingival plaque microbiota functionality

Gene annotation

The overall percentage of annotation for all the predicted genes was approximately 40%. Samples mapped 94-97% of their reads against the set of predicted genes. After filtering the genes by abundance and prevalence, 28% of genes remained and 30% of those genes had bacterial or archaeal annotation.

Richness values and Shannon and Simpson alpha diversity indexes were calculated on the genes annotated by KEGG database on each sample. According to Wilcoxon test, there was a higher number of genes (richness) in Placebo group compared to Low Dose, and a higher Simpson diversity index in Placebo group than in Low Dose. For Shannon index, the same comparison was almost significant with p-value=0.052 (Figure 20).

Gen set enrichment analysis on KEGG module

A gene set enrichment analysis (GSEA) was conducted comparing the differential module enrichment between groups, based on the abundances of the genes annotated with the KEGG database. Only the bacteria and archaea related KEGG modules were studied. Some of the more remarkable results of the GSEA are:

• Abundance of genes involved in hydrogen sulphide (H2S; Normalized Enrichment Score [NES] = 1 .769, adj. p = 0.002) and ammonia production (NES = 1.548, adj. p = 0.015) increased in the High Dose group compared with the Placebo group.

• Abundance of genes involved in tryptophan biosynthesis (NES = 1 .520, adj. p = 0.001 ) and catechol production (NES = 1 .840, adj. p = 0.017) was increased in the High Dose group compared with the Placebo group. Catechol has been found to inhibit the development of oral pathogens.

• For Low dose and High dose groups, the abundance of genes involved in reactive oxygen species (ROS) catalases (NES = 1 .790, adj. p = 0.003; NES = 1 .768, adj. p = 0.003, respectively) were increased compared with the Placebo group.

• Abundance of genes involved in heme biosynthesis was also increased for Low soe and High dose groups compared with the Placebo group (NES = 1 .306, adj. p = 0.007; NES = 1 .224, adj. p = 0.024, respectively), and a BLASTn alignment of the genes against NCBI nt database (version July 2022) indicated that, of those genes that had a match, most were taxonomically classified as Porphyromonas cangingivalis (33.98 ± 9.58%) and Porphyromonas gingivalis (17.38 ± 6.65%).

• The abundance of genes involved in biofilm formation were increased in the High group compared with both the Low dose and Placebo groups (NES = 1.590, adj. p = 0.001 and NES = 1.539, adj. p = 0.002, respectively). Biofilm formation can lead to caries or periodontal diseases when the competitiveness of certain bacteria is altered and dysbiosis occurs.

• Abundance of genes involved in the biofilm formation were increased in High Dose when compared to Placebo and to Low Dose. • Detailed analysis of modules with significant gene enrichment results in between-group comparisons revealed that module M00529 (denitrification) was significantly enriched in the HIGH group compared with the CON group. However, this module is incomplete, as no mapped reads were found against subunit C of the nor gene (Figure 21 ).

Genes that had a matching sequence in the nt database were primarily taxonomically classified as Porphyromonas. It has previously been reported that species of Porphyromonas capable of synthesizing their own heme are associated with good canine oral health (O'Flynn, C., et aL, Genome Biol Evol, 2015. 7(12): p. 3397-413). O'Flynn, C., et aL, postulated that Porphyromonas species possessing heme biosynthesis pathways could occupy the same ecological niche as pathogenic variants of Porphyromonas, with the autonomous capability for heme production imparting metabolic flexibility and a competitive advantage to outcompete periodontal pathogens.

Claims

1 . An isolated strain of Lactiplantibacillus plantarum deposited at the Spanish Type Culture Collection under the registration number CECT 9161 .

2. The strain according to claim 1 , wherein said strain is in the form of a non-viable cells or viable cells.

3. The strain according to claim 2, wherein said non-viable cells have been heat- treated.

4. A supernatant of a culture of the strain according to any one of claims 1 to 3.

5. A composition comprising the strain according to any one of claims 1 to 3 or the supernatant according to claim 4.

6. The composition according to claim 5, further comprising an acerola powder and/or Porphyromonadaceae bacterium H1 .

7. The composition according to claims 5 or 6, wherein the composition is a pharmaceutical, a veterinary or a nutritional composition.

8. The composition according to claim 5 to 7, wherein the composition is formulated in liquid or in solid form.

9. The composition according to any one of claims 5 to 8, wherein the composition is in a form selected from the list consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles, coated tablets, dispersible strips, dispersible films, oral solution, suspension, steam solution, droplet, emulsion, syrup, dental stick, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

10. The composition according to claim 7 or 8, wherein the nutritional composition is a food, a feed composition or nutritional supplement.

11. The composition according to claim 10, wherein the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, plant-based products, plant-based beverages, meat products, meat beverages, fish products, fish beverages, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

12. The strain according to any one of claims 1 to 3, the supernatant according to claim 4 or the composition according to any one of claims 5 to 1 1 , for use as a medicament, preferably, for use in the prevention and/or treatment of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

13. The strain, the supernatant or the composition for use according to claim 12, wherein the periodontal disease and/or the halitosis correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria is selected from Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella, Porphyromonas, Veillonella, Bacteroides and/or combinations thereof.

14. The strain, the supernatant or the composition for use according to claim 12 or 13, wherein the subject is a non-human animal, preferably a domestic animal or a pet.

15. A non-therapeutic use of the strain according to any one of claims 1 to 3, the supernatant according to claim 4 or the composition according to any one of claims 5 to 1 1 for reducing the dental plaque in a subject, for ameliorating the halitosis in a subject or for ameliorating effects of periodontal disease in a subject.

16. A non-therapeutic use of the strain according to claim 15, wherein the dental plaque or the halitosis correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria is selected from Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella Porphyromonas, Veillonella, Bacteroides and/ or combinations thereof.

17. The non-therapeutic use according to claim 15 or 16, wherein the subject is a non-human animal, preferably a domestic animal or a pet.

18. Acerola for use in the inhibition of oral pathogen bacteria growth in a subject, or for use in the treatment and/or prevention of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

19. Non-therapeutic use of acerola for reducing the dental plaque in a subject, for ameliorating the halitosis in a subject, or for ameliorating effects of periodontal disease in a subject.

20. An in vitro method for predicting the risk of suffering from an oral disease selected from halitosis, gingivitis, periodontitis, dental plaque or dental calculus in a subject, comprising: a) determining the presence of the microbiome comprising at least one or more of the following species: Porphyromonas macacae, Corynebactum cam's, Peptoniphilus mikwangi, Porphyromonas crevoricanis, Filofactor alocis, Treponema parvum, Fretibacterium fastidiosum, species of Prevotella, and species of Peptostreptococcaceae, from an isolated biological sample from a subject, and b) comparing the microbiome obtained in step a) with a microbiome obtained from a control group, wherein if the subject presents the microbiome obtained in step a), then the subject shows high probability of suffering from halitosis or gingivitis or periodontitis or dental plaque or dental calculus.

21. A method according to claim 20, wherein the biological sample is an oral sample, preferably, gingival secretion sample, more preferably, supragingival secretion sample.

22. A method according to claim 20 or 21 , wherein the subject is a non-human animal, preferably a domestic animal or a pet.

23. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use as a medicament.

24. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to claim 23, in the prevention and/or treatment of periodontal disease in a subject, or for use in the prevention and/or treatment of halitosis in a subject.

25. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to claim 24, wherein said bacterium is in the form of a non-viable cells or viable cells.

26 A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to claim 25, wherein said non-viable cells have been heat-treated.

27. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 23 to 26, wherein the composition further comprises acerola power.

28. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 23 to 27, wherein the composition is a pharmaceutical, a veterinary or a nutritional composition.

29. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 23 to 28, wherein the composition is formulated in liquid or in solid form.

30. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 23 to 29, wherein the composition is in a form selected from the list consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles, coated tablets, dispersible strips, dispersible films, oral solution, suspension, steam solution, droplet, emulsion, syrup, dental stick, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

31 . A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 28 to 30, wherein the nutritional composition is a food, a feed composition or nutritional supplement.

32. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 28 to 31 , wherein the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, plant-based products, plant-based beverages, meat products, meat beverages, fish products, fish beverages, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

33. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 24 to 32, wherein the periodontal disease and/or the halitosis correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria is selected from Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella, Porphyromonas, Veillonella, Bacteroides and/or combinations thereof.

34. A Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 24 to 33, wherein the subject is a non-human animal, preferably a domestic animal or a pet.

35. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, for reducing the dental plaque in a subject, for ameliorating the halitosis in a subject or for ameliorating effects of periodontal disease in a subject.

36. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to claim 35, wherein said bacterium is in the form of a non-viable cells or viable cells.

37 A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to claim 36, wherein said non-viable cells have been heat-treated.

38. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 35 to 37, wherein the composition further comprises acerola power.

39. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 35 to 38, wherein the composition is a pharmaceutical, a veterinary or a nutritional composition.

40. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 35 to 39, wherein the composition is formulated in liquid or in solid form.

41. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 35 to 40, wherein the composition is in a form selected from the list consisting of tablets, lozenges, sweets, soft sweets, gummies, candies, lollipops, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles, coated tablets, dispersible strips, dispersible films, oral solution, suspension, steam solution, droplet, emulsion, syrup, dental stick, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

42. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 39 to 41 , wherein the nutritional composition is a food, a feed composition or nutritional supplement.

43. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 39 to 42, wherein the nutritional composition is selected from the list consisting of fruit juices, vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, powdered milk, cereals, baked goods, milk-based products, plant-based products, plantbased beverages, meat products, meat beverages, fish products, fish beverages, dental chew, soft chews, sachet stick pack, dry kibble, fast melts for water bowl, and toothpaste.

44. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, according to any one of claims 35 to 43, wherein the periodontal disease and/or the halitosis correlates with a biofilm comprising pathogenic bacteria, preferably, the pathogenic bacteria is selected from Aggregatibacter, Streptococcus, Fusobacterium, Tannerella, Treponema, Campylobacter, Prevotella, Porphyromonas, Veillonella, Bacteroides and/or combinations thereof.

45. A non-therapeutic use of a Porphyromonadaceae bacterium H1 , or a composition comprising it, for use according to any one of claims 24 to 32, wherein the subject is a non-human animal, preferably a domestic animal or a pet.