Self-film-forming composition for oral care

Abstract

The name of the invention is a self-film-forming composition for oral care. A self-film-forming composition in powder form, reconstituted formulations and kits for oral use are provided that enable sufficient colonization of probiotics in the oral cavity and sufficient residence time to achieve beneficial effects against pathogens associated with oral conditions. Thus, the provided items are suitable for use in the prevention and / or treatment of conditions associated with alterations in oral microbiota, in particular peri-implantitis. The self-film-forming composition in powder form comprises at least one gelling agent and / or at least one bioadhesive and at least one strain of lactic acid bacteria.

Description

[0001] This application is a divisional application. The application date of the original application is July 11, 2016, the application number is 2016800417801 (PCT / EP2016 / 066456), and the name of the invention is “Self-forming film composition for oral care”. Technical Field

[0002] The present invention relates to the fields of medicine and microbiology, and more particularly to compositions for dental care and treatment. In particular, the present invention includes compositions for treating and / or preventing peri-implantitis and other oral conditions associated with microbial dysbiosis. Background Art

[0003] Inflammatory lesions that occur in the tissues surrounding implants are collectively referred to as peri-implant disease and include two disease entities: mucositis and peri-implantitis. Peri-implantitis and peri-implant mucositis have similar signs, but crestal bone loss is absent in peri-implant mucositis. Mucositis is defined as a reversible modification of the soft tissues around implants without bone loss. Peri-implantitis affects the deep soft tissues and bone around the implants inversely, and it is defined as an inflammatory process that affects the tissues close to the osseointegrated implant in a manner that causes loss of supporting bone loss as well as inflammation of the mucosa. The prevalence of peri-implantitis depends on the clinical threshold used to define the prevalence, with recent results varying between 6.6% and 47% [Albertini, M. et al. 2014].

[0004] Peri-implantitis is one of the main causes of implant failure. Plaque accumulation and bacterial imbalance have been proposed as one of the main causes, as there is a positive correlation between plaque accumulation and alveolar crestal bone loss. In peri-implantitis, bacteria, stress, or a combination of both can stimulate bone loss around the implant. Once bone loss increases and deep pockets form, an anaerobic environment is established that will enhance the dominance of anaerobic bacteria. These then become the main cause of continued bone loss. It has been shown that the microbial composition of the biofilm around peri-implantitis is different from that present in healthy implants. In general, diseased implants present a reduced proportion of beneficial bacterial species and an increased proportion of putative pathogens compared to healthy implants. Peri-implantitis sites often present a higher proportion of some of the identified periodontal pathogens from the orange complex, such as Fusobacterium nucleatum or Prevotella intermedia. Other fastidious pathogens from the red complex are present in these sites, including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia [Da Silva, ESC et al. 2013]. Although there is great controversy, we believe that other average levels of periodontal pathogens such as Aggregatibacter actinomycetemcomitans are often associated with peri-implantitis [Persson, GR et al. 2013].

[0005] Because the development of biofilm on dental implant surfaces plays an important role in the development of peri-implantitis, treatment is mainly based on reducing bacterial colonization on the implant surface or mechanically eliminating the bacterial microbiota. Surgical and non-surgical techniques have been developed to achieve this.

[0006] Surgical procedures are based on resection techniques to reduce pocket depth and stabilize soft tissue morphology and regenerative techniques based on guided orthopedic surgery using bone placement. Generally, these procedures are costly, painful or uncomfortable for the patient and are therefore limited as treatment options when bone loss is advanced or continues despite initial preventive treatment. [Ata-Ali, J. et al. 2011].

[0007] Therefore, non-surgical techniques are preferred as a first-line treatment option. Generally, these techniques involve the elimination of plaque through chemical plaque control in the form of a 0.12% chlorhexidine rinse (e.g., 12 hours for 15 days) and / or antibiotics. However, the side effects of chlorhexidine have long been known. Chlorhexidine irritates and damages the oral mucosa, causing discoloration and staining of teeth and other oral surfaces, increasing tartar formation and altering taste perception. Oral irritation and localized hypersensitivity-type symptoms have also been reported. Mucosal side effects, including stomatitis, gingivitis, and ulcers, have also been reported. Some of these effects are due to changes in the oral microbiome caused by the antibacterial effects of chlorhexidine [ L. et al. 1971].

[0008] Antibiotic therapy can cause a variety of side effects and drug interactions. For example, broad-spectrum systemic antibiotics occasionally have the problem of altering the commensal microbiota, which can lead to antibiotic-associated diseases, including Clostridium difficile-associated colitis, diarrhea, vaginosis, vaginitis, etc. Antibiotic use also favors the development of bacterial resistance, which can compromise treatment and even make further treatment more cumbersome [Slots, J. et al. 1990].

[0009] Recent research has turned to probiotics, "live microorganisms that when administered in adequate amounts confer a health benefit on the host." The impact of probiotics on oral health is relatively new, and much research is ongoing.

[0010] A known oral probiotic on the market is Lactobacillus reuteri (L. reuteri) Prodentis. Recent studies have shown that oral treatment with tablets containing the probiotic strain L. reuteri induced a significant reduction in proinflammatory cytokine responses and improved clinical parameters in a large proportion of patients with chronic periodontitis [Szkaradkiewicz, AK et al. 2014]. Another study aimed to evaluate the role of probiotic lozenges containing L. reuteri as an adjunct to scaling and root planing (SRP) in patients with chronic periodontitis. A reduction in Porphyromonas gingivalis was observed in the probiotic-treated SRP group, but no changes were seen in the other pathogens studied [Teughels, W. et al. 2013]. However, the effect of L. reuteri on the oral microbiota remains questionable, as in a parallel study, daily intake of probiotic lozenges containing L. reuteri (ATCC55730 and ATCC PTA5289) during experimental gingivitis did not appear to significantly affect plaque accumulation, inflammatory status, or the microbial composition of biofilms [Flallstroem, H. et al. 2013].

[0011] Recently published Flichy-Fernandez, AJ et al. 2015 described the effect of Lactobacillus reuteri Prodentis probiotic tablets containing strains Lactobacillus reuteri ATCC PTA5289 and Lactobacillus reuteri DSM 17938 on peri-implant mucositis. The study enrolled twelve patients with peri-implant mucositis who took the probiotic tablets for 30 days. Compared to placebo, patients showed a decrease in gingival index, which was mainly due to a decrease in inflammatory cytokines. The document does not address the effect of Lactobacillus reuteri on peri-implantitis. Compared to mucositis, peri-implantitis is characterized by damage to bone structure, which is mainly due to the effect of pathogens. Because the document does not describe the effect of probiotic tablets on peri-implantitis or on related pathogens, it remains a question whether probiotics are suitable for the treatment of peri-implantitis.

[0012] Another example is a recent study describing the effects of oral administration of lozenges containing L. rhamnosus GG and B. animalis subsp. lactis BB-12 on the amount of dental plaque, gingival inflammation and oral microbiota in healthy young adults. The probiotic lozenges reduced dental plaque and gingival index, but no probiotic-induced changes were found in the composition of salivary microbiota in either group. It was concluded that the probiotic lozenges improved the periodontal status in terms of gingival inflammation, but did not affect the oral microbiota [Toiviainen, A. et al. 2015].

[0013] Other probiotics such as Lactobacillus brevis CD2 have been investigated for their effects in periodontitis, but have not been demonstrated for peri-implantitis and mucositis [Maekawa, T. et al. 2014].

[0014] JP20100053062 discloses an oral composition containing lactic acid bacteria, which is suitable for preventing and / or treating oral diseases, including periodontal disease and dental caries. Specifically, it has been shown that the bacterial strain belonging to the species of Leuconostoc mesenteroides has good coaggregation and adhesion ability, and is therefore able to attach to the mucosa and inhibit pathogen biofilm. The oral composition can include bacterial strains and excipients, adhesives, lubricants and solubilizers, and is prepared according to a conventional method.

[0015] A recent review of this field [Yanine, N. et al. 2013] concluded that the effectiveness of probiotics for the prevention and treatment of periodontal disease remains questionable. Regarding the primary outcome probing pocket depth, there was no clinical beneficial effect of probiotics. Regarding the secondary outcomes, probiotics showed a slight benefit on plaque index and gingival inflammation. In summary, from the prior art it can be seen that probiotics can help improve inflammation in periodontal and gingival processes, but it remains a question whether probiotics have a role in the dysbiosis that occurs in these processes, which is the main cause of bone loss in peri-implantitis.

[0016] Another problem related to the efficacy of current therapies is that there are significant limitations to the techniques and methods used to deliver probiotics in the oral cavity, including incorporation into food, tablets, chewing gum, gel or toothpaste. These limitations include negative effects on the stability of the probiotics and their ability to elicit beneficial effects due to their short residence time in the oral cavity. This is a challenge when probiotics must be delivered to inaccessible cavities in the mouth, such as around dental implants, which compromises the antagonistic activity of the probiotics against oral pathogens for the prevention or treatment of peri-implantitis.

[0017] Furthermore, implant areas adjacent to bone often have wound beds that aid implant adhesion and osseointegration and remain contaminated despite antimicrobial therapy, with subsequent further bone loss and peri-implant pocket formation. Efforts to manage pathogenic colonization of these areas have met with limited success. Because these areas are inaccessible and smaller than most brush heads and other devices, dental care has proven to be of little use. Systemic prophylaxis is of limited benefit because bacteria tend to rapidly aggregate into protected clusters, increasing the likelihood of the development of antibiotic-resistant bacteria.

[0018] A delivery system capable of delivering probiotics to specific desired oral sites is needed to effectively modulate the oral microbiota. The delivery system should be able to slowly release live, metabolically active probiotics into the oral cavity, allowing the probiotics to fully colonize the desired sites and remain there long enough to achieve beneficial effects. Summary of the invention

[0019] One problem to be solved by the present invention may be seen as relating to the provision of a delivery system capable of delivering probiotics to a desired specific oral site, which is required to effectively modulate the oral microbiota involved in pathological conditions at said site.

[0020] The solution is based on providing a composition in powder form, a reconstitution formula and a kit for oral use which achieves sufficient colonization of the probiotics at the desired site and sufficient residence time to provide a beneficial effect against pathogens associated with oral conditions.

[0021] Therefore, a first aspect of the present invention relates to a self-film forming composition in powder form that forms a film on its own under stirring in the presence of a liquid medium, the composition comprising: (i) at least one gelling agent in powder form, (ii) at least one bioadhesive agent in powder form, and (iii) at least one lactic acid bacteria strain in powder form, wherein (i), (ii) and (iii) are in a single container or in separate containers, and wherein the self-film forming composition is administered topically.

[0022] Thus, the present invention provides a self-filming composition in powder form, wherein the self-filming is formed under stirring in the presence of a liquid medium. The term "film-forming" means capable of forming a film after application to a solid surface; i.e. leaving a soft, cohesive, continuous covering layer on the surface. The term "self-filming" means that no other components or conditions are required to form a film in addition to the indicated components or conditions. "Under stirring" should be understood broadly in this specification, including manual stirring or with the aid of an electric mixer; continuous or discontinuous stirring, and for a suitable period of time, preferably between 5 seconds and 60 minutes, more preferably 10 seconds to 30 minutes or 30 seconds to 10 minutes.

[0023] By increasing the viscosity of the composition with a gelling agent and by increasing the adhesion of the composition with a bioadhesive, a film is formed on the surface. The term "gelling agent" refers to a substance that increases the viscosity of a liquid and forms a gel in this article. The gelling agent can be dissolved in a liquid phase to form a colloidal mixture, and the colloidal mixture forms an internal structure with weak adhesion. It can also be referred to as a thickener, stabilizer or emulsifier in the art. The term "bioadhesive" refers to a naturally occurring polymer that acts as an adhesive, that is, it is combined together and resists separation after being applied to the surface of the material. Preferred gelling agents and bioadhesives are discussed in further detail below.

[0024] Without being limited to theory, it is believed that the solution proposed herein can deliver lactic acid bacteria (preferably with probiotic properties) to inaccessible cavities in the mouth, such as cavities and gaps around dental implants and the wound surface of the implant itself, which are often contaminated by pathogens. As for the second advantage, the solution proposed here realizes the full colonization of probiotics at the desired position and sufficient retention time to achieve the beneficial effects of pathogens related to oral conditions. More specifically, the gelling agent and bioadhesive included in the composition realize and enhance the growth of lactic acid bacteria at the desired position, thereby forming a beneficial biofilm that seals the problematic cavities (i.e., a physical barrier is formed for further pathogen contamination). It is worth noting that in the case of pathogens that are concerned with avoiding the undesirable growth of pathogens, the prebiotic effect of gelling agents and bioadhesives can be ignored. Therefore, while the development of beneficial probiotic biofilms is enhanced, the development of pathogenic biofilms is reduced. Thus, the formation of probiotic biofilm together with the antagonistic activity of lactic acid bacteria against oral pathogens prevents the vicious cycle resulting from the action of pathogenic bacteria, i.e. bone loss in the peri-implant area.

[0025] The second and third aspects of the present invention relate to a method for preparing a reconstituted formulation and a reconstituted formulation obtained therefrom, the method comprising mixing under stirring the self-film-forming composition in powder form of the first aspect with a liquid medium.

[0026] The fourth aspect of the present invention relates to a kit for oral use, comprising: (1) the self-film-forming composition in powder form of the first aspect or the reconstituted formula of the third aspect; and (2) a device for applying the self-film-forming composition in powder form or the reconstituted formula to the buccal cavity.

[0027] A fifth aspect of the present invention relates to a self-film forming composition or reconstituted formulation in powder form for use as a medicament.

[0028] The sixth aspect of the present invention relates to the self-film-forming composition in powder form of the first aspect or the reconstituted formulation of the third aspect for use in preventing and / or treating a condition selected from the group consisting of peri-implantitis, mucositis, periodontitis, gum disease, caries, oral candidiasis, herpes labialis and pustules.

[0029] A seventh aspect of the present invention relates to the non-pharmaceutical use of the self-film forming composition in powder form or reconstituted formulation for oral care.

[0030] An eighth aspect of the present invention relates to an isolated strain belonging to the genus Pediococcus for use in preventing and / or treating a condition selected from the group consisting of peri-implantitis and mucositis.

[0031] The ninth aspect of the present invention relates to an isolated strain belonging to the genus Pediococcus deposited in the Spanish Type Culture Collection, selected from the group consisting of the strain deposited under accession number CECT8903, strain CECT 8904, strain CECT 8905, strain CECT 8906.

[0032] The tenth aspect of the present invention relates to a self-film-forming composition in powder form that self-forms a film under stirring in the presence of a liquid medium, the composition comprising: (i) at least one agent in powder form selected from the group consisting of a gelling agent and a bioadhesive agent, and (ii) at least one Pediococcus strain in powder form, wherein (i) and (ii) are in a single container or in separate containers.

[0033] The eleventh and twelfth aspects of the present invention relate to a method for preparing a reconstituted formulation and a reconstituted formulation obtained therefrom, the method comprising mixing the self-film-forming composition in powder form of the tenth aspect with a liquid medium under stirring.

[0034] The thirteenth aspect of the present invention relates to a kit for oral use, comprising: (1) the self-film-forming composition in powder form of the tenth aspect or the reconstituted formula of the twelfth aspect; and (2) a device for applying the self-film-forming composition in powder form or the reconstituted formula to the buccal cavity.

[0035] The fourteenth aspect of the present invention relates to the self-film-forming composition in powder form of the tenth aspect or the reconstituted formulation of the twelfth aspect for use in preventing and / or treating a condition selected from the group consisting of peri-implantitis, mucositis, periodontitis, gum disease, caries, oral candidiasis, herpes labialis and pustules.

[0036] The embodiments and examples shown below are presented for the purpose of explaining the present invention clearly and completely enough to those skilled in the art, and should not be considered as limited to the essential aspects covered therein, which are presented in the foregoing parts of this specification. DETAILED DESCRIPTION

[0037] The present invention provides a self-film-forming composition in the form of a powder comprising lactic acid bacteria and a method for preparing the same, the method comprising mixing with a liquid medium.

[0038] In one aspect, the self-film forming composition comprises at least one gelling agent, at least one bioadhesive agent, and at least one lactic acid bacteria strain. In another aspect, the self-film forming composition comprises at least one gelling agent or at least one bioadhesive agent and at least one Pediococcus strain.

[0039] Self-film-forming composition in powder form

[0040] The lactic acid bacteria included in the self-filming composition in powder form of the present invention may be in the form of living cells or non-living cells. Strains in the form of living cells are generally used. However, it can also be extended to non-living cells, such as killed cultures or cell lysates (obtained by, for example, exposure to a changed pH, ultrasonic treatment, radiation, temperature, pressure or other means of killing or lysing bacteria) or compositions containing beneficial factors (such as bacteriocins and anti-inflammatory substances) previously produced by any strain.

[0041] In a particular embodiment, the at least one lactic acid bacteria strain included in the powder composition is in a form comprising living cells. This means that the composition consists essentially of living cells or consists partly of living cells.

[0042] In a particular embodiment, at least one agent in powder form selected from the group consisting of a gelling agent and a bioadhesive agent has no bactericidal effect on at least lactic acid bacteria. The term "bactericidal", also known as "bacteriocidal", means killing bacteria, i.e. reducing the number of viable cells in the composition. Therefore, the agent included in the composition is an agent that does not kill lactic acid bacteria. It is known to those skilled in the art that depending on the storage conditions and the use of protective agents, the storage conditions include temperature, atmosphere, protective agents such as cryoprotectants or other carriers, the number of viable cells in the product containing bacteria naturally decreases over time. In this specification, the expression "no bactericidal effect" means that when stored or incubated under the same conditions, the concentration of viable cells in the presence of a gelling agent and / or a bioadhesive agent is not reduced compared to the concentration of viable cells in the absence of these agents.

[0043] Preferably, the concentration of viable cells in the powder composition is reduced by no more than 10,000 times (i.e., 4 logs) in the presence of a gelling agent and / or a bioadhesive agent compared to the same composition without these agents. For example, this means that if the concentration of viable cells in the powder without a gelling agent and a bioadhesive agent is 1E+09 cfu / g, then the concentration of viable cells in the presence of these agents is no less than 1E+05 cfu / g.

[0044] More preferably, the concentration of viable cells in the powder composition in the presence of these agents is reduced by no more than 1000-fold (3 logs), 100-fold (2 logs), or 10-fold (1 log) compared to the same composition in the absence of these agents.

[0045] In a particular embodiment, in addition to having no bactericidal effect, the gelling agent and the bioadhesive agent also have no bacteriostatic effect on lactic acid bacteria. This means that the agents neither kill bacteria nor inhibit the growth of bacteria (they do not prevent bacteria from growing and dividing). Therefore, the agents included in the composition are agents that do not prevent the normal growth of bacteria.

[0046] In a more specific embodiment, the agents are agents that have a prebiotic effect. This means that they improve or enhance the growth of bacteria. This feature is shown in Examples 5 to 7 below.

[0047] In an even more specific embodiment, the prebiotic effect of the relevant agent is studied by incubating the relevant lactic acid bacteria in the presence of the agent. The relevant agent is supplemented to a final concentration of 0.5% (w / v) with artificial saliva containing 1 g / L 'Lab-lemco' powder (Oxoid, Basingstoke, UK), 2 g / L yeast extract (Oxoid), 5 g / L proteose peptone (Oxoid), 2.5 g / L porcine gastric mucin (Sigma Chemical Co., Poole, UK), 35 g / L sodium chloride (BDH Chemicals Ltd, Poole, UK), 0.2 g / L calcium chloride (BDH), 0.2 g / L potassium chloride (BDH) in distilled water. Artificial saliva without a gel component is also prepared to compare the effect of the relevant agent on bacterial growth in the absence of supplemented medium. After autoclaving, 1.25 mL of water (w / v) containing 40% urea was added per liter of artificial saliva medium. 200 microliters of the prepared different culture media were pipetted into a 96-well plate. This was followed by the addition of 20 microliters of a suspension of the relevant lactic acid bacteria standardized to 1E+07 CFU / mL in PBS. The same amount of PBS without bacterial inoculum was used as a negative control. The culture plates were incubated anaerobically at 37°C for 24 hours, and bacterial growth was monitored by measuring the optical density at 625 nm. The ability of the probiotic candidate to grow using the relevant reagent was calculated according to the following formula:

[0048] ΔGrowth = (ΔDO gp -ΔDO g0 )-(ΔDO sp -ΔDO s0 )

[0049] Where ΔDO gp is the difference between the optical density at 625 nm after 24 hours compared to 0 hours in wells supplemented with relevant reagents and inoculated with relevant lactic acid bacteria (LAB) candidates;

[0050] ΔDO g0 is the difference between the optical density at 625 nm at 24 h compared to 0 h in wells supplemented with the relevant reagent but containing PBS instead of LAB;

[0051] ΔDO sp It is the difference between the optical density at 625 nm at 24 hours and at 0 hours in the wells that were not supplemented with relevant reagents and inoculated with LAB;

[0052] ΔDOs0 is the difference in optical density at 625 nm at 24 hours compared to 0 hours in wells not supplemented with the relevant reagents and containing PBS instead of LAB.

[0053] Based on the detailed analysis described in Examples 5 to 7, a skilled person will generally be able to repeat the analysis to objectively determine whether a particular agent of interest has a prebiotic effect. It should be noted accordingly that the description and conditions of the analysis disclosed in Examples 5 to 7 do not limit the scope of the present invention. The analysis is an analysis suitable for testing the ability of an agent of interest to test its prebiotic effect.

[0054] In a specific embodiment, the agent is considered to have a prebiotic effect when the Δ growth determined as described above is higher than 0.005. In a more specific embodiment, the Δ growth is higher than 0.01. In an even more specific embodiment, the Δ growth is higher than 0.02.

[0055] As discussed above, the composition in powder form comprises at least one agent, which may be a gelling agent. In a particular embodiment, the at least one gelling agent is in an amount for providing viscosity to the composition and is selected from the group consisting of:

[0056] (a) starch, preferably rice starch, corn starch, potato starch or sodium starch glycolate;

[0057] (b) gums, preferably tragacanth, acacia, xanthan gum, ghatti gum, gellangum, karaya gum, konjac gum or galactomannan gums (especially guar gum, locust bean gum and taragum);

[0058] (c) seaweed polysaccharides, preferably alginic acid, sodium alginate, agar, dextran or carrageenan;

[0059] (d) a polysaccharide selected from the group consisting of pectin and maltodextrin;

[0060] (e) cellulose derivatives, preferably methylcellulose, carboxymethylcellulose, sodium or calcium carboxymethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, ethylmethylcellulose, hydroxyethylcellulose, cellulose acetate phthalate or microcrystalline cellulose;

[0061] (f) a polypeptide selected from the group consisting of gelatin, collagen and casein; and

[0062] (g) a polymer selected from the group consisting of: an acrylate-based polymer, a vinyl-based polymer (preferably polyvinylpyrrolidone, also known as polyvidone or povidone), a cationic polysaccharide (such as cationic acetyl-glucosamine, preferably chitosan, cationic cyclodextrin and cationic dextran) and a polyalkylene glycol (preferably polyethylene glycol).

[0063] At least one agent may also be a bioadhesive. Specifically, the bioadhesive is present in an amount for providing adhesion to the composition and is selected from the group consisting of:

[0064] (a) gums, preferably hakea gum, xanthan gum, acacia gum, konjac gum, gellan gum or galactomannan gum (especially guar gum);

[0065] (b) seaweed polysaccharides, preferably alginic acid, sodium alginate, agar, dextran or carrageenan;

[0066] (c) cellulose derivatives, preferably methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl hydroxyethyl cellulose, hydroxyethyl cellulose;

[0067] (d) a polysaccharide selected from the group consisting of pectin and maltodextrin; and

[0068] (e) A polymer selected from the group consisting of an acrylate-based polymer, a vinyl-based polymer (preferably polyvinyl pyrrolidone or povidone), a cationic polysaccharide (preferably chitosan) and a polyalkylene glycol (preferably polyethylene glycol).

[0069] As mentioned before, the gelling agent is in an amount for providing viscosity to the composition and the bioadhesive is in an amount for providing adhesion to the composition. The compounds used as gelling agents and bioadhesives are well known in the art, so the amount of the reagent will be determined by a person skilled in the art. In a specific embodiment, the gelling agent is a gum. The gum can be, for example, xanthan gum, gum arabic, guar gum, tragacanth gum, karaya gum, locust bean gum, carob gum, acacia gum, ghatti gum, gellan gum, karaya gum, konjac gum, khaki gum and tara gum. These gums can be generally classified as carbohydrate gums with a negative charge as a whole. More preferably, the gelling agent is guar gum, also known as guar gum (gum cyamopsis), guar flour (guar flour) and guarana (guaran) in the art. Guar gum is extracted from the endosperm of some plant seeds of the leguminous family. They are galactomannans, with D-mannose units (1-4) bonded to form straight chains and single D-galactose units (1-6) bonded to form branches. Guar gum can be modified or derivatized. Examples of guar gum derivatives are hydroxypropylated or carboxyhydroxypropylated derivatives, cationic derivatives (Ecopol) and products resulting from the depolymerization of guar gum.

[0070] In a particular embodiment, the bioadhesive is a cellulose derivative. The main derivative is a cellulose ether, i.e., an alkyl modification of cellulose, produced by replacing some of the hydrogen atoms of the hydroxyl groups of anhydrous glucose units with an alkyl group. Non-limiting examples of cellulose ethers are: methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), ethylhydroxyethylcellulose (EHEC), ethylcellulose, sodium carboxymethylcellulose (CMCNa), quaternary ammonium salts of hydroxyethylcellulose with trimethylammonium substituents (Polyquaternium 10) and dimethyldiallylammonium chloride copolymers (Polyquaternium A). In a more preferred embodiment, the bioadhesive is hydroxyethylcellulose (HEC).

[0071] Non-limiting examples of starches include pregelatinized starches (corn, wheat, tapioca), pregelatinized high amylose starches, pregelatinized hydrolyzed starches (maltodextrin, corn syrup solids), or chemically modified starches, such as pregelatinized substituted starches (e.g., octenyl succinate-modified starches).

[0072] In a particular embodiment, the self-film forming composition in powder form comprises at least one gelling agent and at least one bioadhesive agent. In a more particular embodiment, the gelling agent is a compound other than the bioadhesive agent.

[0073] In a particular embodiment, the gelling agent is selected from the group consisting of gums and algal polysaccharides and the bioadhesive agent is selected from the group consisting of cellulose derivatives and vinyl-based polymers.

[0074] More specifically, the gelling agent is guar gum or alginate and the bioadhesive is hydroxyethylcellulose (HEC) or polyvinylpyrrolidone.

[0075] Thus, in a particular embodiment, the gelling agent is selected from the group consisting of guar gum and sodium alginate, and the bioadhesive agent is HEC.

[0076] The self-film forming composition according to the invention is in the form of a powder. Specifically, that means that the water content of the composition is less than 10%, more preferably less than 5%.

[0077] The skilled artisan will typically select the amounts of gelling agent and bioadhesive in powder form to obtain a reconstituted formulation that provides an appropriate degree of viscosity and adhesion depending on the volume of the reconstituted formulation.

[0078] In a particular embodiment, the amount of gelling agent and bioadhesive in the self-film forming composition is the amount required for each agent to obtain a concentration of 0.05 to 20% (w / v) in the final reconstituted formula. More specifically, the gelling agent in powder form is in an amount for obtaining a concentration of 1 to 5% (w / v), more specifically 2%, 3% or 4% (w / v) in the final reconstituted formula, and the bioadhesive in powder form is in an amount for obtaining a concentration of 4 to 10% (w / v), more specifically 6% (w / v) in the final reconstituted formula.

[0079] In a specific embodiment of peri-implantitis applications, the self-filming composition comprises guar gum and HEC in amounts necessary to obtain a final reconstituted formula comprising 4% (w / v) guar gum and 6% (w / v) HEC. In a specific embodiment of dental applications, the self-filming composition comprises guar gum and HEC in amounts necessary to obtain a final reconstituted formula comprising 2% (w / v) guar gum and 6% (w / v) HEC.

[0080] In a particular embodiment, the composition in powder form comprises between 0.05 and 90% gelling agent (w / w), more specifically 1% to 60% (w / w). In a more specific embodiment, the amount of gelling agent is 50%, 20%, 15% and 10% (w / w). In another specific embodiment, the amount of bioadhesive in the composition in powder form is between 0.05 and 90% (w / w), and specifically between 30 and 85% (w / w). In a more specific embodiment, the amount of bioadhesive is 68%, 50% and 40% (w / w).

[0081] The technician will select the appropriate volume of liquid medium according to the surface to be treated and the condition. In a specific embodiment, the volume used for reconstructing the formulation is no more than 500 mL. In a specific embodiment, the volume used is no more than 200 mL, 150 mL, 100 mL, 50 mL or 20 mL. In a more specific embodiment, the volume used for reconstructing the formulation is 6 mL. In a more specific embodiment, the volume used for reconstructing the formulation is in the range of 2 to 3 mL.

[0082] A person skilled in the art will find non-limiting examples of suitable amounts of gelling agent and bioadhesive in powder form, the final reconstituted formulation and the volume of the liquid medium below (section Reconstitution formulation and methods of obtaining the same).

[0083] The gelling agent and bioadhesive agent and the amount used in the present invention are not just used as carriers, excipients, adhesives, lubricants, surfactants or solubilizers, wetting agents, but are selected to give the final reconstituted composition suitable viscosity and adhesive properties to improve the administration of lactic acid bacteria. Suitable combinations allow the administration of lactic acid bacteria without reducing its efficacy and stability, or even increasing its efficacy and stability, for a period of time that is convenient for clinical practice.

[0084] As discussed above, the self-filming composition in powder form of the present invention also comprises at least one lactic acid bacteria strain. In a particular embodiment, the lactic acid bacteria strain is capable of antagonizing at least one oral pathogen selected from the group consisting of bacteria of the following genera: Porphyromonas, Fusobacterium, Prevotella and Aggregatibacter. More specifically, it is capable of antagonizing at least one oral pathogen selected from the group consisting of: Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. For example, the ability of the lactic acid bacteria strain to antagonize oral pathogens is tested with the analysis of Examples 8 to 9.

[0085] In a particular embodiment, the lactic acid bacteria strain is selected from the group consisting of bacteria of the following genera: Pediococcus, Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enterococcus, Bacillus, Bacteroides, Propionibacterium and Leuconostoc. Specifically, the lactic acid bacteria is Lactobacillus (e.g., Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus rhamnosus or Lactobacillus plantarum), Bifidobacterium or Streptococcus. More specifically, the lactic acid bacteria strain belongs to Pediococcus. More specifically, the strain is Pediococcus acidilactici or Pediococcus pentosaceus. More specifically, the lactic acid bacteria strain is selected from the group consisting of strain CECT 8903, strain CECT 8904, strain CECT 8905 and strain CECT 8906.

[0086] As noted above, at least the gelling agent, at least the bioadhesive agent and at least the lactic acid bacteria strain forming the composition in powder form are in a single container or in a separate container. The method for obtaining the composition of the previous aspects comprises: obtaining the lactic acid bacteria strain by culturing the lactic acid bacteria strain in a suitable culture medium and processing it into a powder form (e.g., freeze drying it), and if the composition is provided in a single container, then the lactic acid bacteria strain is mixed with the gelling agent and the bioadhesive agent in powder form. The method for cultivating and post-processing lactic acid bacteria is described below. The composition can also be provided in three or two different containers, and the containers include the elements of the composition, and these elements can be mixed by the user before application.

[0087] The self-filming composition is particularly suitable for topical administration. As used herein, the term "topical" means application to a body surface, such as the skin or mucous membrane, external or internal. Non-limiting examples of topical routes of administration are epidermal administration (directly to the skin), application to the conjunctiva of the eye or to the ear, administration to oral surfaces (such as teeth and gums), or vaginal administration.

[0088] Refactoring recipes and how to obtain them

[0089] As discussed above, one aspect of the present invention relates to a method for preparing a reconstructed formulation, the method comprising mixing the self-film-forming composition in powder form of the aforementioned aspects with a liquid medium under stirring. A reconstructed formulation is obtained. Stirring can be performed by manual stirring or by means of an electric mixer; continuous or discontinuous stirring, and for a suitable period of time. The mixing period is preferably between 5 seconds and 60 minutes, more preferably 10 seconds to 30 minutes or 30 seconds to 10 minutes. In a more preferred embodiment, stirring is performed for 1, 2, 3, 4 or 5 minutes. The formulation is preferably reconstructed before use and the formulation is allowed to stand and gel before the formulation is administered. In a specific embodiment, after mixing, the composition is allowed to stand for at least 10 seconds. More specifically, the reconstructed formulation is allowed to stand for at least 30 seconds, more specifically at least 1 minute before application. In order to avoid the gel from becoming too thick before application, it is also appropriate to avoid a long period of time, which makes it difficult to distribute on the surface to be treated. Therefore, in a specific embodiment, the formulation is applied to the surface to be treated within a period of time not longer than 120 minutes once reconstructed. More specifically, the reconstituted formulation is administered over a period of time no longer than 60 minutes, more specifically no longer than 30 minutes, more specifically no longer than 10 minutes.

[0090] The liquid medium is preferably water which is acceptable for human or animal consumption. However, the liquid medium may also be, for example, any suspension in water, oil, glycerol or vaseline.

[0091] The reconstituted formulation may preferably be in the form of a gel having suitable viscosity and / or adhesion for the desired oral cavity; in the form of a varnish if, for example, the formulation is to be applied to tooth surfaces; or in the form of a spray.

[0092] The amount of each agent of the gelling agent and the bioadhesive agent in the final reconstituted formulation is 0.05 to 20% (w / v). Preferably, the reconstituted formulation comprises 1-5% (w / v) of the gelling agent, more preferably 2%, 3% or 4% (w / v of the total reconstituted formulation); and 1-10% (w / v) of the bioadhesive agent, more preferably 4-10% (w / v), more preferably 6% (w / v of the total reconstituted formulation).

[0093] In a particular embodiment for peri-implantitis applications, the reconstituted formulation comprises, inter alia, 4% w / v guar gum and 6% HEC. In a particular embodiment for dental applications, the reconstituted formulation comprises 2% w / v guar gum and 6% HEC.

[0094] A preferred composition in powder form comprises 20% (w / w) guar gum and 30% HEC, and the amount of lactic acid bacteria is 50%. If 0.5 g of the powder blend is reconstituted with 2.5 ml of water, the final concentration in the gel is 4% (w / v) guar gum and 6% HEC.

[0095] A preferred composition in powder form comprises 10% (w / w) guar gum and 30% HEC, and the amount of lactic acid bacteria is 60% (w / w). If 0.5 g of the powder blend is reconstituted with 2.5 ml of water, the final concentration in the gel is 2% guar gum (w / v) and 6% HEC.

[0096] In another embodiment, the composition in powder form comprises 15% (w / w) sodium alginate, 30% HEC and 1.5% calcium acetate, and the amount of lactic acid bacteria is 53.5% (w / w). If 0.5 g of the powder blend is reconstituted with 2.5 ml of water, the final concentration in the gel is 3% (w / v) alginate and 6% HEC.

[0097] In another embodiment, the composition in powder form comprises 10% (w / w) sodium alginate, 30% HEC and 1% calcium acetate, and the amount of lactic acid bacteria is 59% (w / w). If 0.5 g of the powder blend is reconstituted with 2.5 ml of water, the final concentration in the gel is 2% alginate and 6% HEC.

[0098] In another embodiment, the composition in powder form comprises 20% (w / w) guar gum and 10% polyvinyl pyrrolidone, and the amount of lactic acid bacteria is 70% (w / w). If 0.5 g of the powder blend is reconstituted with 2.5 ml of water, the final concentration in the gel is 4% guar gum (w / v) and 2% polyvinyl pyrrolidone.

[0099] In another embodiment, the composition in powder form comprises 50% (w / w) guar gum, and the amount of lactic acid bacteria is 50% (w / w).If 0.5 g of the powder blend is reconstituted with 6 ml of water, the final concentration of guar gum in the gel is 4% (w / v).

[0100] In another embodiment, the composition in powder form comprises 72% (w / w) HEC, and the amount of lactic acid bacteria is 28% (w / w).If 0.5 g of the powder blend is reconstituted with 6 ml of water, the final concentration of HEC in the gel is 6%.

[0101] Kits for oral use

[0102] A particular embodiment of the present invention is a product comprising a sealed container, wherein a powder composition as provided herein is enclosed in the sealed container, preferably in unit dose and under aseptic conditions. The container preferably has a capacity sufficient to allow the composition to be reconstituted in situ. In general, it is found that a capacity of about 1 ml to about 20 ml, preferably about 5 ml to about 10 ml, will be suitable.

[0103] Thus, in some cases, the article of manufacture is simply a container with the self-film forming composition in powder form, as the practitioner has at his disposal other elements (eg, liquid medium, syringe and needle, and applicator) to use and administer the self-film forming composition to a patient.

[0104] Another product is a reconstituted formulation which can be stored frozen for a short period of time prior to use.

[0105] However, in some cases, the article of manufacture is a kit comprising other elements for convenient use and application. Thus, another aspect of the invention relates to a kit for oral use comprising: (1) a self-forming film composition in powder form as defined above; or a reconstituted formulation; and (2) a device for applying the self-forming film composition in powder form or the reconstituted formulation to the buccal cavity.

[0106] The self-film forming composition in powder form can be applied directly to the oral cavity and reconstituted in situ, for example by saliva. However, a preferred embodiment is to reconstitute the composition immediately prior to use.

[0107] In a specific embodiment, the device for application to the buccal cavity is selected from the group consisting of a brush, a syringe containing an injection needle, a syringe containing a blunt tip needle, and an oral guard.

[0108] In a more specific embodiment, the application device is a syringe and a needle with a blunt tip, similar to a periodontal probe. With the aid of the syringe and the blunt tip needle, the reconstitution solution is applied into the affected area, ie into the peri-implant pocket.

[0109] The applicator is an oral guard, also known as a bite splint, mouth protector, mouth piece, gum shield, gum guard, night guard, occlusal splint or oral splint or occlusal surface. This is a removable dental appliance that fits over the upper or lower dental arch. The oral guard or oral splint is filled with a composition in powder form or a reconstitution formulation and fitted over the teeth.

[0110] In another embodiment, for ease of use, the kit further comprises a container having a liquid medium for reconstituting the self-film-forming composition in powder form.

[0111] In another embodiment, also for the convenience of use, the kit further comprises a device for putting a liquid into a container having a self-film-forming composition in powder form. The device is preferably a syringe equipped with a disposable punch needle, which is used to draw the liquid from the container and put it into a container having a self-film-forming composition in powder form.

[0112] The term "container" is used herein to denote any small receiver with a closure suitable for packaging a unit dose of reconstituted powder, preferably packaged under sterile conditions. It should be understood that this embodiment of the present invention encompasses equivalent forms of packaging, such as vials, ampoules, disposable syringes, syringe barrels or prefilled syringes. Optionally, the vial can be a multi-compartment vial comprising, for example, two compartments, one compartment containing reconstituted powder and one compartment containing a liquid sufficient to dissolve the powder. In such vials, the two compartments are interconnected by an opening, wherein a stopper can be used to prevent the powder from contacting the solvent liquid before the vial is ready for use. When in use, the stopper is loosened or punctured by any suitable means to contact the liquid with the powder, such as a device, such as applying pressure or pushing a needle through a piston of the stopper. Examples of the multi-compartment vial include a double compartment barrel and a double compartment vial for a syringe, or a delivery cap and a vial closed with this cap.

[0113] A specific embodiment of the present invention consists of the following steps: a certain amount of the self-film-forming composition of the present invention in powder form is introduced into a glass vial provided with a septum and an aluminum capsule. After adding water, preferably deionized water or distilled water, with a syringe and shaking by hand, a reconstructed gel is formed in a very short time. This gel does not have a very high viscosity to be applied by means of a syringe and has an adhesion suitable for dental implant applications. The reconstructed formulation is then administered in the periodontal pocket or surgical site, for example, by a syringe and needle, a brush or a pressure applicator. The dental specialist can manipulate the system to obtain the best fit with the treatment site and overcome the inherent placement difficulties of other systems. When administered, the film system remains in the desired position, making it possible to seal the desired implant interior space with the formulation of the present invention.

[0114] Medical Applications

[0115] As discussed above, another aspect of the present invention relates to a self-filming composition or reconstituted formulation in powder form for use as a medicament. Specifically, the present invention provides a self-filming composition or reconstituted formulation in powder form for use in preventing and / or treating conditions associated with alterations in the oral microbiota. More specifically, such conditions are selected from the group consisting of peri-implantitis, mucositis, periodontitis, gum disease, dental caries, oral candidiasis, herpes labialis, and pustules. In a specific embodiment, the condition is peri-implantitis.

[0116] These aspects may alternatively be expressed as the use of any one of the compositions of the invention for the manufacture of a medicament, veterinary drug, pharmaceutical, food, food supplement, medical food or oral care product for the prevention and / or treatment of conditions associated with alterations in the oral microbiota, preferably peri-implantitis, mucositis, periodontitis, gum disease, caries, oral candidiasis, herpes labialis and pustules. This may alternatively also be expressed as a method for the prevention and / or treatment of conditions associated with alterations in the oral microbiota, the method comprising administering to a subject in need thereof an effective amount of any one of the compositions of the invention.

[0117] Application in the treatment and prevention of peri-implantitis

[0118] Dentures generally consist of two components: The implant component (also called an endosseous implant or fixture) is a screw that is inserted into the osteotomy. This part sits below the gum line and is considered an artificial tooth root. The other component is the crown, bridge, or denture, which is secured to the implant component and, in the case of crown replacements, is the visible part of the tooth. The third component may include the abutment, which is a ceramic or titanium component that ensures a secure fit between the implant and the crown. The abutment portion is the portion that sits at and above the gum line. Bacteria from the mouth can penetrate the spaces and cavities between these structures. These bacteria can then make their way back into the adjacent tissues and can cause peri-implantitis.

[0119] As a specific embodiment of the present invention for preventing and treating peri-implantitis, a reconstructed gel is formed in a very short time after adding water to a container containing a self-film-forming composition in powder form by a syringe and shaking it by hand. The reconstructed gel is administered, for example, by a syringe and a blunt tip needle and placed in the gaps, microgaps and cavities of the implant. Specifically, because vertical bone loss has occurred and because the local inflammatory process has produced a peri-implant pocket, the dental implant including the wound surface is exposed to the oral fluid, so the above composition is applied to the pocket in such a way that the composition adheres to the wound surface of the implant and also adheres to the mucous surface of the pocket. Therefore, by bending the blunt tip needle, simulating a periodontal probe, the blunt tip is located near the bottom of the pocket and the product is injected until the solution reaches the upper edge of the gums, thereby advantageously applying the solution to the peri-implant pocket.

[0120] Advantageously, the above-described self-filming composition is associated with a device for administering said composition to the peri-implant area affected by an inflammatory process near a dental implant, in particular, the composition is associated with a dental kit comprising a flexible blunt needle.

[0121] Specifically, a particular embodiment of the present invention for treating peri-implantitis consists of: removing the crown; giving the patient local anesthesia; cleaning the area to be treated; mechanically removing subgingival plaque by scraping; applying an antibiotic, such as chlorhexidine; applying a saline solution; and applying the reconstructed formulation of the present invention to the surrounding environment and cavity of the implant and inside the implant. After the needle is withdrawn from the bag, a saline solution wash and air spray (for about 10 seconds) are applied to the treated area. The crown is then placed in place. The patient is advised not to brush his teeth within 6 hours after treatment.

[0122] In the case of preventive treatment, this consists in particular of: removing the crown; optionally cleaning the area to be treated and mechanically removing subgingival plaque by scraping; and applying the reconstructing formulation of the invention.

[0123] Other oral applications: gingivitis and caries

[0124] The present invention may also be used for dental care in healthy subjects to prevent possible disease and, in addition to peri-implantitis, to treat or prevent oral conditions associated with oral dysbiosis.

[0125] In a specific embodiment, the solution of the present invention is used to prevent and treat periodontitis and gingivitis. Gingivitis and inflammation of the periodontal tissues and the onset of periodontal disease are usually associated with a change in the microbial balance in the dental sulcus, i.e., around the caries cavity between the tooth surface and the gums. It leads to the formation of plaque (biofilm) and an increase in Gram-negative pathogenic species present in these deposits. The exotoxins and metabolites produced by these bacteria accumulate and cause inflammation around the gingival tissue, which in turn leads to swelling and bleeding. During the inflammatory process, the protection of the teeth by the junctional epithelium becomes weaker, allowing bacteria to penetrate the subgingival area. Most mammals will develop periodontal disease, especially horses, cats, rodents, livestock and dogs. For example, 80% of all dogs over the age of 3 have periodontal disease.

[0126] Periodontal treatment usually begins with mechanical removal of subgingival plaque by scraping or by ultrasound; by this mechanical removal, the bacteria in the biofilm are released and rinsed away. Antibiotics and other bactericidal compositions are currently used in this treatment process, which are designed to prevent recolonization by Gram-negative bacteria. However, the regular and large-scale use of compositions containing antibiotics has the disadvantage of the risk of developing antibiotic resistance and the side effects that these products are often caused. As in the case of peri-implantitis, the reconstructive formulation of the invention can be applied, for example, by means of a syringe, in particular a syringe with a blunt-tip needle.

[0127] In another specific embodiment, the composition of the invention is used to prevent and treat dental caries. In this case, the reconstituted formulation of the invention may be in the form of a varnish that is easily applied to the tooth surface by a dentist, dental hygienist or other health care professional with the aid of a brush. The varnish may be applied to the enamel, dentin or cementum of the teeth as an adjuvant to other treatment modalities. They are not permanent varnishes, but their adhesive consistency allows them to remain in contact with the tooth surface for up to several hours, allowing colonization of the probiotics and providing the beneficial effects of the composition of the invention.

[0128] The preferred application procedures are described below. Although professional prophylaxis is not necessary before applying the solution of the present invention, it is recommended to clean the teeth with a toothbrush and, if necessary, clean thick plaque or debris. The teeth should be slightly air-dried or wiped dry with cotton gauze. Isolate the teeth (e.g., with a cotton roll or absorbent material) to prevent re-contamination by saliva. Dispense a small amount of varnish (e.g., 0.5 ml). Only 0.3-0.6 ml is needed to treat the entire dentition. The varnish will adhere even if the teeth are wet. Then apply the varnish with a small brush or applicator. Instruct the patient to avoid brushing the teeth for the rest of the day. The normal oral hygiene routine can be started again the next day.

[0129] In applications such as periodontitis, gingivitis or caries, the composition of the invention may also be self-administered by the patient. The commercial product may be a dental kit as described above, comprising a brush for convenient application.

[0130] In other embodiments, the compositions and kits of the invention are used by a dental professional in acute treatment and the patient may then undergo maintenance treatment with an oral care product such as a chewing gum, toothpaste, mouthwash, mouth spray, lozenge or orodispersible tablet comprising the self-forming composition of the invention or lactic acid bacteria in powder form with common excipients.

[0131] Other uses and product forms

[0132] The present invention also provides a self-filming composition in powder form or a reconstituted formulation for oral care, i.e. for non-pharmaceutical use. Thus, it is well known to those skilled in the art that the composition of the present invention can be formulated in the form of mouthwashes, sprays, oral gels and toothpastes with the aid of conventional ingredients used in these oral forms.

[0133] As known to those skilled in the art, mouthwashes are aqueous or aqueous-alcoholic solutions for gargling, having well-known conventional formulations, and may include, in addition to water, polyols such as glycerol or glycols (e.g., propylene glycol, nonionic surfactants, etc.) and other additives for improving appearance, odor, and preservation.

[0134] Sprays are compositions equivalent to or similar to mouthwashes, but are dispensed from a spray bottle, allowing for convenient application of the dose required to moisten and protect the oral cavity without the need for subsequent rinsing.

[0135] Oral gels include polymers that allow direct, stable application to the oral cavity. With regard to these polymers, for the purposes of the present invention, it is preferred to use a combination of polymers generally known as polycarbophil and carbomer, because they can keep the gel structure stable for a very long time under extreme temperature conditions. The gel may also include a certain amount of natural, non-cariogenic sweeteners, such as sorbitol.

[0136] Toothpaste formulations are well known to those skilled in the art. In toothpaste compositions, nonionic (e.g., sugar-containing fatty acid esters) or amphoteric (e.g., cocoa-derived betaine) surfactants are preferably used, since anionic surfactants have an adverse effect on the delicate epithelial tissue of the gums. In the case of toothpaste, the use of sodium bicarbonate to neutralize oral acidity is also particularly preferred. In addition, toothpastes may contain thickeners, such as xanthan gum; abrasive silica fillers; and other supplements in addition to the agents commonly used in the toothpaste industry. Preferably, the lactic acid bacteria are introduced into the toothpaste after being encapsulated or protected in other forms.

[0137] Lactic acid bacteria strains

[0138] Other aspects of the invention relate to novel strains of the genus Pediococcus, in particular strains that are useful for the prevention and treatment of peri-implantitis and other oral conditions associated with oral dysbiosis. These novel strains are the result of extensive studies of different lactic acid bacteria strains isolated from healthy humans. Pediococcus pentosaceus PERI1, Pediococcus acidilactici PERI2, Pediococcus pentosaceus / lactici PERI3, Pediococcus pentosaceus / lactici PERI4 were deposited by depositor AB-Biotics, SA at the Spanish Type Culture Collection on June 16, 2015. de Cultivos Tipo, CECT, CUE Building 3, Parc Científic Universitat de València, Prof. Agustín Escardino, 9, 46980-Paterna, Valencia, Spain), located at the UAB Research Park (Parc deRecerca UAB), Campus UAB, s / n Edifici Eureka, 08193 Cerdanyola del Vallès (Barcelona, ​​Spain). After the International Authority of Deposit declared the strains viable, they received the accession numbers CECT 8903, CECT 8904, CECT 8905 and CECT 8906.

[0139] The strains were selected because of the following distinguishing characteristics / abilities:

[0140] The strain has a good ability to survive in the oral cavity because it can tolerate the presence of lysozyme and hydrogen peroxide well, which are two common antiseptics present in the oral cavity.

[0141] Ability to tolerate and grow well in the presence of gelling agents and / or bioadhesives. For example, growth of the selected strain in saliva is enhanced in the presence of gelling agents such as guar gum and / or bioadhesives such as hydroxyethylcellulose.

[0142] Ability to inhibit the growth of oral pathogens. For example, the strains are able to antagonize bacteria associated with peri-implantitis, including Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and / or Aggregatibacter actinomycetemcomitans.

[0143] The ability to aggregate, which is a requirement for the formation of biofilms, which can have a protective effect against colonization by other pathogens by forming a natural barrier.

[0144] Various lactic acid bacteria species have a long history of apparent safe use. The European Food Safety Authority has developed a system that awards "Qualified Presumption of Safety (QPS)" status to taxa that have demonstrated a long history of apparent safe use. The strains of the present invention belong to bacterial species with QPS status [Andreoletti, O. et al. 2008].

[0145] In summary, it is believed that no prior art describes strains of the genus Pediococcus, in particular strains of Pediococcus acidilactici and Pediococcus pentosaceus, having the above-mentioned characteristics and for the prevention and / or treatment of peri-implantitis. It is noteworthy that strains Pediococcus CECT 8903, Pediococcus CECT 8904, Pediococcus CECT 8905, Pediococcus CECT 8906 all meet all the previously mentioned characteristics and are therefore suitable for the treatment of peri-implantitis.

[0146] In a specific embodiment, the isolated strain has been fermented in an artificial culture medium and subjected to post-treatment after fermentation to obtain bacterial cells, and the resulting bacterial cells are in a liquid culture medium or in solid form. Specifically, the post-treatment is selected from the group consisting of: drying, freezing, freeze drying, fluidized bed drying, spray drying, and refrigeration in a liquid culture medium, and more specifically freeze drying.

[0147] The strain of the present invention is produced by culturing (or fermenting) bacteria in a suitable artificial culture medium and under suitable conditions. The expression "artificial culture medium" of microorganisms should be understood as a culture medium containing natural substances, and optionally containing synthetic chemicals, such as polymer polyvinyl alcohol, which can reproduce some functions of serum. Common suitable artificial culture medium is nutrient broth, which contains elements, including carbon sources (such as glucose), nitrogen sources (such as amino acids and proteins), water and salts required for bacterial growth. The growth medium can be in liquid form or often mixed with agar or other gelling agents to obtain a solid culture medium. The strain can be cultured alone to form a pure culture, or mixed cultured together with other microorganisms, or by culturing different types of bacteria alone, and then combining these bacteria in a desired ratio. After cultivation, and depending on the final formulation, the strain can be used in the form of purified bacteria, or bacterial culture or cell suspension can be used, used as is or after appropriate post-treatment. In this specification, the term "biomass" should be understood as a bacterial strain culture obtained after cultivation (or fermentation, fermentation is a term synonymous with cultivation).

[0148] The term "post-treatment" should be understood in the context of the present invention as any treatment of biomass for the purpose of obtaining storable bacterial cells. The purpose of post-treatment is to reduce the metabolic activity of cells in biomass, and thus slow down the rate of harmful cell reactions. Because of post-treatment, bacterial cells can be in solid or liquid form. When in solid form, the stored bacterial cells can be powders or granules. In any case, solid and liquid forms containing bacterial cells do not exist in nature, so they are not naturally produced because they are the result of artificial post-treatment methods. The post-treatment method may require the use of one or more of the so-called post-treatment reagents in a specific embodiment. In the context of the present invention, the expression "post-treatment reagent" refers to a compound for carrying out the post-treatment method described herein. Among the post-treatment reagents, non-limiting dehydrating agents, antibacterial agents, cryoprotective agents (cryoprotectants), inert fillers (also known as freeze-drying protectants), carrier materials (also known as core materials), etc. are included, used alone or in combination.

[0149] There are two basic ways to reduce the metabolic activity of bacterial cells, and therefore, there are two ways to perform post-treatment. The first is to reduce the rate of all chemical reactions, which can be done by cooling or freezing using refrigerators, mechanical freezers and liquid nitrogen freezers to reduce the temperature. Alternatively, the rate of all chemical reactions can be reduced by adding substances that inhibit the growth of bacterial cells, i.e., bacteriostatics, which are abbreviated as Bstatic.

[0150] A second method of post-treatment is to remove water from the biomass, which may include using a freeze dryer to sublime the water. Suitable techniques for removing water from the biomass are drying, freeze drying, spray drying, or fluidized bed drying. Post-treatment to produce a solid form may be drying, freezing, freeze drying, fluidized bed drying, or spray drying.

[0151] The post-treatment is preferably freeze drying, which includes removing water from the frozen bacterial suspension by sublimation under reduced pressure. This method consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most of the water, and secondary drying to remove bound water. Because of the objective and expected variability of the industrial process of manufacturing and isolating freeze-dried bacterial cultures, freeze-dried bacterial cultures usually contain a certain amount of inert fillers, also known as freeze-drying protectants. Its function is to standardize the content of live probiotics in the product. The following inert fillers in commercial freeze-dried cultures are used: sucrose, saccharose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin and other pharmaceutically acceptable non-hygroscopic fillers. Optionally, other stabilizers or cryoprotectants, such as ascorbic acid, are also used to form a viscous paste, which is subjected to freeze drying. In any case, the material obtained in this way can be ground into a suitable size, including powder.

[0152] It is obvious that by using the deposited strain as starting material, the skilled person will be able to generally obtain further mutants of the strain by conventional mutagenesis or re-isolation techniques, which mutants retain or enhance the relevant features and advantages described herein of the strain forming the composition of the present invention. In a specific embodiment, the mutants are obtained by using recombinant DNA technology. In another embodiment, the mutants are obtained by random mutagenesis. Therefore, another aspect of the present invention relates to a method for obtaining a mutant of at least one Pediococcus strain, wherein the strain is selected from the group consisting of: strain CECT 8903, strain CECT 8904, strain CECT 8905 and strain CECT 8906, wherein the method comprises using the deposited strain as starting material and applying mutagenesis, and wherein the mutants obtained at least further retain or enhance the ability of the deposited strain to antagonize at least one oral pathogen selected from the group consisting of bacteria of the following genera: Porphyromonas, Clostridium, Prevotella and Aggregatibacter.

[0153] Further aspects of the invention are pharmaceuticals, veterinary drugs, medical foods, foods, food supplements and oral care products comprising an effective amount of at least one of the strains as defined above and a suitable amount of an acceptable excipient.

[0154] The selection of excipients and the most appropriate formulation method in view of the particular purpose of the composition is within the capabilities of one of ordinary skill in the art of pharmaceutical technology.The strains of the present invention can be formulated as the sole active agent or in admixture with one or more other active agents.

[0155] The term "excipient" should be understood in its broad sense in this specification, including any natural or synthetic substance formulated with the active ingredients of pharmaceuticals, veterinary drugs, medicines, food supplements, medical foods and oral care products. Excipients are selected from the group comprising, but not limited to, fillers / diluents / bulking agents, binders, anti-adherents, disintegrants, coatings, anti-caking agents (e.g., magnesium stearate, colloidal silicon dioxide or talc), antioxidants, lubricants, sweeteners, flavoring agents, colorants, surfactants and other types of pharmaceutically and veterinarily acceptable excipients.

[0156] The effective amount of colony forming units (cfu) of the strain in the composition will be determined by a person skilled in the art and will depend on the final formulation. For example, when no other active agent is required for oral administration, the total amount of the strain of the invention present in a single dose composition is an amount that provides the following effective daily dose: 10 7 to 10 12 cfu, according to current regulations, preferably 10 9 to 10 11 cfu. The term "colony forming unit" ("cfu") is defined as the number of bacterial cells as revealed by microbial counting on agar plates. Food supplements usually contain probiotic strains in amounts ranging from 10 7 with 10 12 The range of cfu / g.

[0157] The term "drug" should be understood in its broad sense in this specification, including any composition comprising an active ingredient and a pharmaceutically acceptable excipient, in which case the active ingredient is a strain of the present invention preferably in the form of a composition. This term is not limited to drugs. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with tissues of subjects (e.g., humans) within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions or other problems or complications, and are matched with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with other formulation ingredients. Suitable carriers, excipients, etc. can be found in standard drug textbooks.

[0158] Medicine can adopt different forms or different names, and this depends on product approval pathway and also depends on country.For example, medicine is a kind of special medicine.Medical food is another kind of special medicine.Term " medical food " or " food for specific medical purpose " is used to refer to the food that special preparation is prepared and is intended to be used for the disease with special nutritional needs to carry out dietary management in some countries, and described special nutritional needs can not be satisfied only by normal diet.It is defined in regulations, such as the Food and Drug Administration's 1988 Orphan Drug Act Amendments of the U.S. Food and Drug Administration in 1988, and European Commission Directive (CommissionDirective) 1999 / 21 / EC.Medical food is different from the food supplement of wider category and the traditional food that carries health claim.Therefore, in a specific embodiment, bacterial strain of the present invention is formulated as medical food.

[0159] Probiotic compositions are often considered as food supplements, such as the probiotic compositions disclosed herein. Food supplements, also known as dietary supplements or nutritional supplements, are considered to be another special kind of medicine. This is a preparation or product intended to supplement the diet, made from compounds commonly used in food, which provides nutrients or beneficial ingredients that are not usually consumed or not consumed in sufficient quantities in a normal diet. Food supplements are mainly considered as foods, but they are sometimes also defined as drugs, natural health products or nutraceutical products. In the sense of the present invention, food supplements also include nutraceuticals. Food supplements are usually sold "over the counter", i.e., without a prescription. If a food supplement is in the form of pills, capsules, tablets or powders, it contains the same excipients as those used in drugs. However, food supplements can also be in the form of foods fortified with some nutrients (e.g., nutrition bars or yogurt). Therefore, in a specific embodiment, the strains of the present invention are formulated as food supplements. Food supplements can be given as is; can be mixed with a suitable drinkable liquid, such as water, yogurt, milk or juice; or can be mixed with solid or liquid foods. In this case, the food supplement may be in the form of tablets or lozenges, pills, capsules, granules, powders, suspensions, sachets, sweeteners, bars, syrups and the corresponding administration forms, usually in unit dosage form.

[0160] The strain of the present invention can also be included in various foods, such as dairy products (yogurt, cheese, fermented milk, milk powder, fermented products based on milk, ice cream, products based on fermented cereals, powders based on milk), bread, nutrition bars, spreads, biscuits and cereals, beverages, different types of oils or condiments. The term "food" is used in its broadest sense herein, including any type of product, in any presentation form, which can be ingested by animals, but does not include medicines and veterinary drugs. Other food examples are meat products, chocolate spreads, fillings and frostings, chocolate, candy, baked products, soy sauces and soups, juices and coffee whitening oils. Foods of particular interest are food supplements and infant formulas. Foods preferably include carrier materials, such as oatmeal, lactic acid fermented foods, resistant starch, dietary fiber, carbohydrates, proteins and glycosylated proteins. In a specific embodiment, the strain of the present invention is encapsulated or coated.

[0161] Composition of the present invention is intended for oral health application.Therefore, another aspect of the present invention provides a kind of oral care product, and described oral care product comprises composition as mentioned above and pharmaceutical excipient or cosmetic acceptable excipient or other edible ingredients.In this sense, composition is such oral product, it is not intentionally swallowed so that the systemic administration of specific therapeutic agent, but on the contrary keeps enough time in the oral cavity to contact substantially all tooth surfaces and / or oral tissues for the purpose of oral activity.Non-limiting examples of described product are toothpaste, dentifrice, tooth powder, local oral gel, mouthwash, denture product, oral spray, chewing gum, dental floss, dental cleaning tape, sandblasting powder, polishing paste, dental varnish, fissure sealant, filling material, oral paste or gel, candy, lozenge, oral dispersible tablet or oral tablet or can directly be sprinkled into the powder in the oral cavity.Oral care product can also comprise flavoring compound, for example menthol.

[0162] The embodiments described above are applicable to the fields of treatment, therapy and prevention, and also to veterinary medicine, especially dogs, cats, cows, horses, monkeys, sheep, goats. Specific animal products are products for chewing, biting and gnawing, sticks, animal treats, pet food, small balls or pet toys.

[0163] It will therefore be appreciated that the strains of the present invention are suitable for use in the management of oral dysbiosis regardless of the form of the composition, ie whether it is a drug, a medicament, a food, a food supplement, a medical food or an oral care product.

[0164] Another aspect of the present invention relates to a solid composition comprising a cryoprotectant; freeze-dried biomass comprising at least one lactic acid bacterium; and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is preferably selected from emulsions, gels, pastes, granules, powders and gums. Another aspect of the present invention provides oral care products, pharmaceutical compositions and edible products, dietary supplements and cosmetic compositions comprising an effective amount of the composition defined in the previous aspect. In a specific embodiment, the oral care product is chewing gum, toothpaste, oral spray, lozenge or orodispersible tablet. In a specific embodiment, the pharmaceutical composition, edible product or dietary supplement is a lozenge or orodispersible tablet.

[0165] Throughout the specification and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, components or steps. For those skilled in the art, additional objects, advantages and features of the present invention will become apparent after consulting the specification, or may be learned by practicing the present invention. In addition, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1 : Growth of probiotic strains in artificial saliva supplemented with guar gum (GG) compared to unsupplemented artificial saliva (G).

[0167] Figure 2 : Growth of probiotic strains in artificial saliva supplemented with hydroxyethylcellulose (HEC) compared to unsupplemented artificial saliva (G).

[0168] Figure 3 : Growth of probiotic strains in artificial saliva supplemented with sodium alginate (SA) compared to unsupplemented artificial saliva (G).

[0169] Figure 4 : Growth of probiotic strains in artificial saliva supplemented with methylcellulose (MC) compared to unsupplemented artificial saliva (G).

[0170] Figure 5 : Inhibitory activity of probiotic candidates against Bacillus actinomycetemcomitans.

[0171] Figure 6: Strain genotype analysis by random amplified polymorphic DNA (RAPD). For 1, PERI1; 2, PERI2; 3, PERI3; 4, PERI4, the graphs obtained after random amplification.

[0172] Examples

[0173] Example 1. Isolation of microorganisms

[0174] Lactic acid bacteria candidates were isolated from fresh feces and oral secretions (oral hyssops) of children aged 0-9 years. The samples were dissolved in PBS buffer (pH 7.4), divided and applied to MRS supplemented with various antibiotic combinations. The strains were cultured at 37 or 30 ℃ under microaerobic conditions (5% CO 2). The incubation time depends on the growth rate, but usually 24 hours to 3 days are carried out. Separate the strains individually with the same selection medium, and then, in order to obtain the first identification, carry out Gram staining (Gram staining). Once grown, the isolated strains were stored in PBS 0.1× containing 15% skim milk powder by freeze drying.

[0175] Example 2. Genus and species identification

[0176] Species identification was performed by sequencing the 16S rRNA gene. Briefly, the strains were sequenced using a 16S rRNA gene from Bio-Rad Laboratories (Barcelona, ​​Spain). The strain DNA was extracted with 100 μg of resin. The complete sequence of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal primers for eubacteria 27F and 1492R as previously described [Weisburg, WG et al. 1991; Muyzer, G. et al. 1998]. The integrity of the PCR product was checked in agarose gel using SYBR green dye (Invitrogen, Life Technologies, Madrid, Spain). The PCR product was sequenced using 27F, 357F, 907R and 1492 primers [Weisburg, WG et al., supra; Muyzer, G. et al., supra], v3.1 cycle sequencing kit and 3130XL genetic analyzer (Applied Biosystems, Life Technologies, Madrid, Spain, from Madrid, Spain). The obtained sequences were aligned and compared with those provided by the National Center for Biotechnology Information (NCBI) and the Ribosomal Database Project (RDP). Strains were identified based on the highest hit score.

[0177] The 16S rRNA sequence corresponds to the genus Pediococcus. When compared with the NCBI and RDP databases, the sequence of PERI1 corresponds to Pediococcus pentosaceus (100% identity); the sequence of PERI2 corresponds to Pediococcus acidilactici (100%); the sequence of PERI3 corresponds to either Pediococcus pentosaceus or Pediococcus acidilactici with 100% identity; and the sequence of PERI4 corresponds to either Pediococcus pentosaceus or Pediococcus acidilactici with 99% identity. They are deposited at the Spanish Type Culture Collection (CECT) with accession numbers CECT 8903, CECT 8904, CECT 8905 and CECT8906, respectively.

[0178] Example 3. Strain genotype analysis

[0179] Strain genotyping was performed by random amplified polymorphic DNA (RAPD) to confirm that the four Pediococcus strains deposited were distinct from each other. RAPD was performed as described by Nigatu et al. 1998. The RAPD profiles of the strains are depicted in Figure 6 In the study, the four strains were confirmed to be different.

[0180] Example 4: Survival rates for oral conditions

[0181] The survival of the strains in the oral cavity was investigated by evaluating their tolerance to different concentrations of agents known to reduce bacterial survival, such as hydrogen peroxide (HP) and lysozyme. A total of 50 lactic acid bacteria candidates were evaluated and compared with commercial probiotic strains used as controls, namely Streptococcus salivarius K12 (Blis Technologies, New Zeland) and Lactobacillus reuteri DSM17938 (Biogaia, Sweden).

[0182] Probiotic candidates and Lactobacillus reuteri DSM17938 were grown in Man Rogosa Sharpe agar medium (MRSa) at 37°C and microaerobic conditions (5% CO2) for 18-24 hours. Streptococcus salivarius K12 was grown under the same conditions, but Brain Hearth Infusion medium (BHI) was used instead of MRS. A bacterial suspension with an optical density corresponding to McFarland standard 0.5 (approximately 1E+08 CFU / mL) was prepared in 0.1M phosphate buffered saline (PBS) using the isolated colonies. The bacterial suspension was then diluted 2-fold in MRS or BHI liquid medium. 200 microliters of the resulting dilution were inoculated on a 96-well microplate, to which 50 μl of a solution containing lysozyme or HP in PBS was added. The concentration of lysozyme tested was 1×10 6 and 5×10 6 U / mL (the final concentration in each well was 2×10 5 and 1×10 6 U / mL), and the concentration of HP was 5mM and 25mM (the final concentrations in the wells were 1mM and 5mM, respectively). The microplate was incubated at 37°C under microaerobic conditions (5% CO2) for 6 hours. Bacterial growth was monitored by measuring the absorbance at 625nm. The growth percentage was calculated by comparing the increase in bacterial strain growth observed in the presence of lysozyme or HP compared to the growth in the absence of these agents (positive control) using the following formula:

[0183]

[0184] OD LHis the optical density of the wells containing microorganisms and lysozyme or HP,

[0185] OD C- is the average optical density of three wells with the same amount of lysozyme but without microorganisms,

[0186] OD C+ is the average optical density of three wells inoculated with bacteria but without lysozyme or HP (positive control).

[0187] result:

[0188] Seven of the fifty probiotic candidates did not grow even in the control MRS medium (without supplementation). Therefore, these strains were discarded as potential candidates. The remaining 43 were ranked according to their ability to grow in the presence of the highest concentrations of lysozyme and HP tested. The results are shown in Table 1 and are expressed as the mean of the percentage of survival compared to the growth of the same strains in medium without supplementation of lysozyme or HP.

[0189] Table 1: Bacterial tolerance to lysozyme and hydrogen peroxide concentrations.

[0190]

[0191]

[0192] Abbreviations: ni = no inhibition (growth 100%); HP = hydrogen peroxide.

[0193] The top 25 bacteria showed the highest tolerance to lysozyme and HP, were considered the best candidates and were selected for subsequent in vitro tests to evaluate their probiotic properties. As can be seen, all strains showed good tolerance to high concentrations of lysozyme, showing survival rates of no less than 86%, which was similar to that of the commercial controls. The LAB candidates also showed good tolerance to HP, with values ​​above 84% at a concentration of 5 mM HP. These results are quite comparable to the survival rates of the commercial controls Lactobacillus reuteri DSM17938 and Streptococcus salivarius K12 (53.4 and 80.5%, respectively).

[0194] Example 5: Use of guar gum as a gelling agent with prebiotic effect

[0195] The ability of strains to use guar gum and increase their growth was investigated in vitro. For this purpose, the growth of probiotic candidates in artificial saliva supplemented with guar gum was compared with the growth of each probiotic candidate in artificial saliva without supplementation. Artificial saliva contained 1 g / L 'Lab-lemco' powder (Oxoid, Basingstoke, UK), 2 g / L yeast extract (Oxoid), 5 g / L protease peptone (Oxoid), 2.5 g / L porcine gastric mucin (Sigma Chemicals, Poole, UK), 35 g / L sodium chloride (BDH Chemicals, Poole, UK), 0.2 g / L calcium chloride (BDH), 0.2 g / L potassium chloride (BDH) in distilled water. Artificial saliva was supplemented with guar gum (Genox Pharma, Barcelona, ​​Spain) to a final concentration of 0.5% (w / v). Artificial saliva without a gel component was also prepared to compare the effect of guar gum on bacterial growth in the absence of supplemented medium. After autoclaving, 1.25 mL of 40% urea was added per liter of artificial saliva medium. 200 microliters of the prepared different culture media were pipetted into a 96-well plate. This was followed by the addition of 20 microliters of a suspension of the probiotic candidate standardized to 1E+07 CFU / mL in PBS. The same amount of PBS without bacterial inoculum was used as a negative control. The culture plates were incubated anaerobically at 37°C for 24 hours, and bacterial growth was monitored by measuring the optical density at 625 nm. The ability of the probiotic candidate to grow using guar gum was calculated according to the following formula:

[0196] ΔGrowth = (ΔDO gp -ΔDO g0 )-(ΔDO sp -ΔDO s0 )

[0197] Where ΔDO gp is the difference between the optical density at 625 nm after 24 hours compared to 0 hours in the wells supplemented with guar gum and inoculated with the probiotic candidate;

[0198] ΔDO g0 is the difference in optical density at 625 nm at 24 h compared to 0 h in wells supplemented with guar gum but containing PBS instead of LAB;

[0199] ΔDO sp is the difference between the optical density at 625 nm at 24 hours compared to 0 hours in wells not supplemented with guar gum and inoculated with the probiotic candidate; and

[0200] ΔDO s0 is the difference in optical density at 625 nm at 24 hours compared to 0 hours in wells not supplemented with guar gum and containing PBS instead of LAB.

[0201] The results were compared with those obtained with the commercial probiotics Lactobacillus reuteri DSM 17938, Lactobacillus brevis CD2, Streptococcus salivarius K12 and with the pathogens Fusobacterium nucleatum and Porphyromonas gingivalis.The experiments were performed in duplicate.

[0202] result

[0203] The ability to use guar gum as a nutrient and enhance growth compared to unsupplemented artificial saliva is described in Figure 1 In. The influence of guar gum on LAB growth depends highly on the tested bacterial strain. Compared to the saliva without supplementation, although guar gum enhances the growth of some bacterial strains, it has a deleterious effect on the growth of other bacterial strains. Bacteria strains PERI1, PERI2, PERI3, PERI4, F3163, I1003, I1005, I3028, I3030, I3140, I3142A, I3145 and I3153 benefit from the addition of guar gum. PERI1 is the bacterial strain showing the highest performance. In the control strain tested, Lactobacillus brevis CD2 and Streptococcus salivarius K12 benefit from the addition of guar gum, but the influence of guar gum on the growth of these bacterial strains is lower than other LAB candidates, and other LAB candidates are PERI1, PERI2, PERI3, F3163, I1003 and 3142A for example. Notably, in the case of Fusobacterium nucleatum and Porphyromonas gingivalis where the concern was to avoid undesirable growth of the pathogens, the effect of guar gum was negligible.

[0204] Example 6: Using hydroxyethyl cellulose (HEC) as Contains prebiotics Adhesive

[0205] As explained above in Example 5 with respect to guar gum, the strains were analyzed for their ability to use HEC to enhance their growth.

[0206] result

[0207] The effect of HEC on the growth of probiotics compared with non-artificial saliva is shown in Figure 2 The growth of a few strains was enhanced by the addition of HEC. The strains that benefited significantly from the use of HEC in the gel were PERI4, I1005, and I3142A. Other strains, including commercial strains, had poor ability to use this ingredient.

[0208] Example 7: Use of other gelling agents as prebiotics Ingredients

[0209] The potential use of other gelling agents to increase the growth of probiotics was also investigated using the same method as described above. Specifically, sodium alginate (SA) and methylcellulose (MC) were used as potential gelling agents with prebiotic effects.

[0210] result

[0211] The effect of SA supplementation on bacterial growth compared with unsupplemented artificial saliva is depicted in Figure 3 Different strains were able to use SA as a nutrient to increase their growth, including PERI, PERI2, PERI3, PERI4, I1003, I1005, I3028, I3030, I3130, I3142A, I3145, and I3153. In contrast, Fusobacterium nucleatum and Porphyromonas gingivalis had a low ability to use SA for growth. The results of MC are presented in Figure 4 None of the pathogenic and control strains were able to use MC to increase growth. In contrast, different probiotic strains benefited from MC supplementation, including F2008A, PERI2, F2043, PERI4, I1003, I1005, I3028, I3061, I3118, I3140, and I3142A.

[0212] Example 8: Antagonism against Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia.

[0213] Probiotic candidates were evaluated for their antagonistic activity against bacteria that are abnormally abundant in patients presenting with peri-implantitis. Specifically, the pathogen strains were Porphyromonas gingivalis DSM-20709, Fusobacterium nucleatum DSM 20482, and Prevotella intermedia DSM-20706 DSM 8324. Lactobacillus reuteri DSM 17938, Lactobacillus brevis CD2 ( VSL Pharmaceuticals, Inc., USA) and Streptococcus salivarius K12 (Bliss Technology, New Zealand) were used as commercial controls. The ability of LAB candidates to inhibit pathogen growth was determined using the Campbell protocol. Briefly, probiotic candidates and Lactobacillus controls were uniformly inoculated in MRS agar plates and grown to confluence at 37°C and 5% CO2 for 24 hours. Streptococcus salivarius K12 was grown under the same conditions, but BHI medium was used.

[0214] The pathogen strains were cultured overnight. Suspensions were prepared using isolated colonies of these pathogens in phosphate buffered saline (PBS) medium and uniformly smeared on solid medium suitable for their growth: Fusobacterium nucleatum and Prevotella intermedia were inoculated in blood agar and Porphyromonas gingivalis was inoculated in Schaeder Anaerobe sheep blood agar. Next, the 6 mm diameter cylindrical segments of the confluent agar plates of the tested LAB candidates were placed lane-to-lane on the culture plates inoculated with the pathogens, so that the culture plates inoculated with the pathogens faced the growth agar side of one of the cylindrical segments and the non-growth side of the other cylindrical segment. The culture plates were incubated at 37°C under anaerobic conditions for 48 hours. Next, the inhibition zone was measured by placing the agar plates flat on a ruler and measuring the halos where the pathogen growth was inhibited (partially or completely). The growth inhibitory activity (GI) was then calculated by subtracting the cylinder diameter (CD) from the inhibition zone diameter (IZD), which was measured in millimeters. The final inhibitory activity was calculated as the average of the GI values ​​of the two cylinder segments mentioned above for each probiotic strain, that is, the average of duplicates. All experiments were performed in duplicate.

[0215] result

[0216] The antagonistic activities of different probiotic candidates are detailed in Table 2. Strains PERI3, PERI4 and F3166 were the three strains that showed the greatest activity against Fusobacterium nucleatum and showed higher activity than Lactobacillus brevis CD2, Lactobacillus reuteri DSM 17938 and Streptococcus salivarius K12. Candidates F1031 and PERI2 were the candidates that showed the highest activity against Prevotella intermedia. Several strains were also effective in inhibiting Porphyromonas gingivalis. Among them, PERI1, F2006, PERI2, F3163, I1003, I3143, I3145 and I3153 showed higher activity than the commercial control used for comparison purposes.

[0217] Table 2. Inhibitory activity against Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis (results are expressed as mean ± SD in mm)

[0218]

[0219] Example 9: Antagonism against Bacillus actinomycetemcomitans .

[0220] The antagonistic activity of probiotic candidates against Bacillus actinomycetemcomitans was studied in liquid culture medium. Probiotic candidates and Lactobacillus controls were grown overnight in MRS liquid culture medium at 37°C under microaerobic conditions (5% CO2). Staphylococcus salivarius K12 was grown under the same conditions, but using BHI medium. The culture was centrifuged and the supernatant was filtered through 0.22 microns. 20 microliters of the filtered supernatant were added to a 96-well microplate containing 160 ml of BHI medium. Finally, 20 ml of a suspension of Bacillus actinomycetemcomitans standardized to 1E+05 CFU / mL in PBS was added to the wells and incubated at 37°C for 24 hours under microaerobic conditions (5% CO2). Bacillus actinomycetemcomitans was monitored by measuring the absorbance at 625 nm. The inhibitory ability of the probiotic supernatant was determined by comparing the growth of Bacillus actinomycetemcomitans when supplemented with the probiotic supernatant with its growth when not supplemented (negative control) using the following formula:

[0221]

[0222] in,

[0223] DO C The optical density at 625 nm of the wells corresponding to the negative control and containing 160 μl BHI medium + 20 μl A. actinomycetemcomitans suspension + 20 μl MRS or BHI,

[0224] DO B corresponds to the blank and is the optical density at 625 nm of the wells containing 160 μl BHI medium + 40 μl MRS or BHI, and

[0225] DO P The optical density at 625 nm of the wells corresponding to the probiotic candidate and containing 160 μl BHI medium + 20 μl Bacillus actinomycetemcomitans suspension + 20 μl probiotic supernatant.

[0226] All experiments were performed in duplicate.

[0227] result

[0228] The inhibitory activity of probiotic candidates against Bacillus actinomycetemcomitans is described in Figure 5 Among the different strains, PERI3 showed the greatest activity, being able to reduce the growth of Bacillus actinomycetemcomitans by 77.9%. This activity was significantly higher than that of commercial controls such as Lactobacillus brevis CD2, Lactobacillus reuteri DSM17938 and especially Streptococcus salivarius K12 (45.6%, 38.8% and 2.42%, respectively).

[0229] Example 10: Ability to form aggregates

[0230] The ability of bacteria to aggregate automatically is considered to be the first step necessary for the formation of biofilms and can be used as a feature for evaluating the potential biofilm-forming ability of strains. Biofilm formation creates a protective barrier that can reduce the adhesion of pathogens to oral surfaces. The ability of probiotic candidates PERI, PERI2, PERI3, PERI4, I3142A, I1005, I3030 and I3145 to form aggregates was evaluated. Lactobacillus brevis CD2 and Streptococcus salivarius K12 were used as controls. The strains were grown overnight in MRS medium (or BHI for Streptococcus salivarius K12) at 37°C and microaerobic conditions (5% CO2). After this stage, the culture was centrifuged at 1000g for 5 minutes, the supernatant was discarded and the centrifugal block was washed twice with PBS. Finally, PBS was added until a probiotic suspension with an optical density equal to McFarland turbidimetric standard 1 (approximately 3E+08CFU / mL) was obtained. Transfer 3 mL of the suspension into a spectrophotometer cuvette and monitor the optical density at 620 nm for 3 h and 6 h. Determine the autoaggregation capacity at this time interval by using the following formula:

[0231]

[0232] where DO0 is the net absorbance of the bacterial suspension at 620 nm at the start of the test (time 0), and

[0233] DO t is the net absorbance of the bacterial suspension at 620 nm at 3 or 6 hours.

[0234] result

[0235] The percentages of aggregation at 3 and 6 hours are summarized in Table 3. The probiotic candidate PERI4 was the strain that exhibited the highest auto-aggregation ability, while candidates I3030 and S. salivarius K12 exhibited the lowest activity.

[0236] Table 3: Aggregation percentage of probiotic candidates

[0237]

[0238] Example 11: Preparation of a powdered Reweight Probiotic gel

[0239] 500 g of freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 4E+10 cfu / g, 200 g of guar gum and 300 g of hydroxyethylcellulose are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a screw cap. After adding 2.5 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel is formed. The final dose of probiotics is 1E+10 cfu / vial. After gel reconstitution, the concentration of guar gum in the gel is 4% and hydroxyethylcellulose is 6%.

[0240] Example 12: Preparation of a powdered Reweight Probiotic gel

[0241] 535 g of freeze-dried powder containing Pediococcus CECT 8904 and Pediococcus CECT 8905 at 3.75E+10 cfu / g, 150 g of sodium alginate, 15 g of calcium acetate and 300 g of hydroxyethylcellulose are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a septum and an aluminum capsule. After adding 2.5 mL of water, preferably deionized or distilled water, with a syringe and shaking by hand, a reconstituted gel is formed. The final dose of probiotics is 1E+10 CFU / vial. After gel reconstitution, the concentration of alginate in the gel is 3% and hydroxyethylcellulose is 6%.

[0242] Example 13: Preparation of a powdered Reweight Probiotic gel

[0243] 700 g of freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 2.9E+10 cfu / g, 200 g of guar gum and 100 g of polyvinyl pyrrolidone are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a septum and an aluminum capsule. After adding 2.5 mL of water, preferably deionized or distilled water, with a syringe and shaking by hand, a reconstituted gel is formed. The final dose of probiotics is 1E+10 cfu / vial. After gel reconstitution, the concentration of guar gum in the gel is 4% and polyvinyl pyrrolidone is 2%.

[0244] Example 14: Preparation of a reconstitutable probiotic gel in powder form for implant application

[0245] 500 g of freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 4E+10 cfu / g and 500 g of guar gum are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a screw cap. After adding 6 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel is formed. The final dose of probiotics is 1E+10 cfu / vial. After gel reconstitution, the concentration of guar gum in the gel is 4%.

[0246] Example 15: Preparation of a reconstitutable probiotic gel in powder form for dental application

[0247] 600 g of freeze-dried powder containing Pediococcus CECT 8903 at 3.35E+10 cfu / g, 100 g of guar gum and 300 g of hydroxyethylcellulose are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a screw cap. After adding 2.5 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel is formed. The final dose of probiotics is 1E+10 cfu / vial. After gel reconstitution, the concentration of guar gum in the gel is 2% and hydroxyethylcellulose is 6%.

[0248] Example 16: Preparation of a reconstitutable probiotic gel in powder form for dental application

[0249] 590 g of freeze-dried powder containing Pediococcus CECT 8903 at 3.4E+10 cfu / g, 100 g of sodium alginate, 10 g of calcium acetate and 300 g of hydroxyethylcellulose are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a screw cap. After adding 2.5 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel is formed. The final dose of probiotics is 1E+10 cfu / vial. After gel reconstitution, the concentration of alginate in the gel is 2% and hydroxyethylcellulose is 6%.

[0250] Example 17: Preparation of a reconstitutable probiotic gel in powder form for dental application

[0251] 280 g of freeze-dried powder containing Pediococcus CECT 8906 at 7E+10 cfu / g and 720 g of hydroxyethylcellulose are mixed and homogenized. 0.5 g of this powder blend is introduced into a glass vial provided with a screw cap. After adding 6 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel is formed. The final dose of probiotics is 1E+10 cfu / vial. After gel reconstitution, the concentration of hydroxyethylcellulose in the gel is 6%.

[0252] Example 18: Preparation of a reconstitutable probiotic gel in powder form for dental application

[0253] 600 g of freeze-dried powder containing 4E+10 cfu / g of Lactobacillus brevis CD2, 100 g of guar gum and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend was introduced into a glass vial provided with a screw cap. After adding 2.5 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel was formed. The final dose of probiotics was 1E+10 cfu / vial. After the gel was reconstituted, the concentration of guar gum in the gel was 2% and the hydroxyethylcellulose was 6%.

[0254] Example 19: Preparation of a reconstitutable probiotic gel in powder form for dental application

[0255] 590 g of freeze-dried powder containing 4E+10 cfu / g of Lactobacillus brevis CD2, 100 g of sodium alginate, 10 g of calcium acetate and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend was introduced into a glass vial provided with a screw cap. After adding 2.5 ml of water, preferably deionized or distilled water and shaking manually, a reconstituted gel was formed. The final dose of probiotics was 1E+10 cfu / vial. After gel reconstitution, the concentration of alginate in the gel was 2% and hydroxyethylcellulose was 6%.

[0256] Example 20: Preparation of a reconstitutable probiotic gel in powder form for dental application

[0257] 700 g of freeze-dried powder containing Streptococcus salivarius K12 at 2.9E+10 cfu / g, 200 g of guar gum and 100 g of polyvinyl pyrrolidone were mixed and homogenized. 0.5 g of this powder blend was introduced into a glass vial provided with a septum and an aluminum capsule. After adding 2.5 mL of water, preferably deionized or distilled water, with a syringe and shaking by hand, a reconstituted gel was formed. The final dose of the probiotics was 1E+10 cfu / vial. After the gel was reconstituted, the concentration of guar gum in the gel was 4% and polyvinyl pyrrolidone was 2%.

[0258] Example 21. Use of reconstitutable probiotic gel in patients with peri-implantitis

[0259] The crown was removed and the patient was given local anesthesia. The area was cleaned and subgingival plaque was mechanically removed. Chlorhexidine 0.12% was given and, after that, a saline solution was given. The reconstructed gel of Example 11 was obtained by adding 2.5 mL of sterile water to a freeze-dried powder containing 4E+10 cfu / g of Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906, 200 g of guar gum and 300 g of hydroxyethylcellulose and mixing vigorously for 1 minute. The mixture was allowed to stand at room temperature for a period of 1 to 10 minutes and the mixture was administered to the peri-implantation bag using a syringe and a blunt tip needle, with the blunt tip located near the bottom of the bag and the product injected until the solution reached the upper gingival edge. Then, after the needle was withdrawn from the bag, a saline solution wash and air spray (for about 10 seconds) were applied to the treated area. The crown was then placed in place. The patient was advised not to brush his teeth within 6 hours after treatment.

[0260] Example 22. Use of reconstitutable probiotic gel to prevent dental caries in patients

[0261] Clean the teeth with a toothbrush and remove any thick plaque or debris. The teeth to be treated are slightly air-dried and isolated with a cotton roll to prevent re-contamination by saliva. A small amount (0.5 ml) of the gel prepared according to Example 15 is dispensed to the teeth with the aid of a brush. The patient is instructed to avoid brushing his teeth for the rest of the day.

[0262] Example 23. Use of a reconstitutable probiotic gel to prevent dental caries in a patient.

[0263] Clean teeth with a toothbrush and remove thick plaque or residue. Reconstitute a self-filming composition containing 50 mg of probiotic strains, 120 mg of guar gum and 360 mg of hydroxyethylcellulose with 6 mL of water and then draw with a syringe. Allow the gel to stand in the syringe for 1 minute and then evenly distribute it in the oral splint. Subsequently, the splint is then used in the mouth and removed after 5 minutes. Provide the patient with more vials containing the self-filming composition and instruct the patient to self-administer the reconstituted gel every 48 hours according to the same procedure, preferably after brushing teeth at night, before going to bed. Instruct the patient not to brush teeth, eat or drink beverages after applying the gel.

[0264] Example 24: Efficacy study of reconstitutable probiotic gel in animal models.

[0265] The efficacy of probiotic gel for preventing mucositis and peri-implantitis was studied in beagledogs as animal models. All procedures were performed under the supervision of veterinarians. Animals were pre-anesthetized by intramuscular injection of acepromazine (0.12%-0.25mg / kg), buprenorphine (0.01mg / kg) and medetomidine (35lg / kg) into the quadriceps femoris. An intravenous catheter (diameter 22 or 20) was inserted into the cephalic vein, and propofol was infused at a slow constant infusion rate at a rate of 0.4mg / kg / min. Conventional dental infiltration anesthesia (articaine 40mg, 1% epinephrine) was given at the surgical site. The second premolar (PM2) and the first molar (M1) of the two quadrants of the mandible were used as experimental sites. The teeth were cut open with a tungsten carbide drill and the roots were removed with forceps without damaging the remaining bone wall. A sulcus incision was made along the vestibule and lingual areas next to the alveoli, and the tissue was separated so that the alveolar crest hard tissue wall could be seen. After two months of healing of the site, 8 implants were placed on the alveolar crest and healed for another two months with a healing cup. After two months of healing, a ligature was placed around each abutment. Oral gel was also given around the implants. Five dogs were treated with a reconstructed liquid gel containing 4% guar gum, 6% hydroxyethylcellulose and 4 CFU / mL of a probiotic mixture consisting of Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 (1:1:1). One of the animals was treated with the same gel without probiotics. The animals were then fed a soft diet to induce plaque accumulation and cause peri-implant inflammation and bone loss. Additional ligatures were placed around the aforementioned individuals and implants every two weeks.

[0266] All post-operative healing was normal, and no exposure or secondary wound healing was observed. Experimental peri-implantitis resulted in signs of inflammation and bone loss. In general, animals treated with probiotic gels showed less pronounced tissue loss, inflammatory response, probing depth, mucosal indentation, and bleeding on probing than animals treated with gels without probiotics. Thus, probiotic treatment improved clinical signs associated with peri-implantitis.

[0267] Example 25: Studying the rheological properties of gelling agents and bioadhesives

[0268] Study the stickiness and adhesion of different agents. Study the following compositions:

[0269] - Sodium alginate in a concentration ranging from 2 to 8% (w / v) in water, with or without a certain concentration (0.02-0.2%) of calcium acetate.

[0270] - Guar gum in a concentration ranging from 1 to 5% (w / v) in water.

[0271] - Methylcellulose in a concentration ranging from 1 to 5% (w / v) in water.

[0272] - Hydroxyethylcellulose in a concentration ranging from 1 to 6% (w / v) in water.

[0273] - Sodium carboxymethylcellulose in a concentration ranging from 1 to 3% (w / v) in water.

[0274] Table 4. Tack and Adhesion Capabilities:

[0275] Reagents viscosity Adhesion Observations Sodium alginate high Very low Agglomeration at high concentrations Guar Gum Very high Very low Good solubility Methylcellulose Low Very low Form foam under stirring Hydroxyethyl Cellulose Very low Very high Good solubility Carboxymethyl cellulose Very low Low Good solubility

[0276] The viscosity and adhesiveness imparted to the film-forming composition, depending on the agent used, offers different possibilities depending on the clinical application of the gel. Combinations with sodium alginate, and especially guar gum with hydroxyethylcellulose, are considered good candidates for forming gels combining properties such as high viscosity and high adhesiveness.

[0277] References

[0278] Patent Literature

[0279] JP20100053062, Sunstar Inc., March 11, 2010.

[0280] Non-patent literature

[0281] Albertini, M. et al. “Assessment of periodontal and opportunistic flora in patients with peri-implantitis”. Clinical Oral Implants Research, 2014, Vol. 00, pp. 1-4

[0282] Da Silva, ESC et al. "Microbial diversity of peri-implantitis biofilm by Sanger Sequencing". Clin Oral Implants Research, 2013, Vol. 0, pp. 1-8

[0283] Persson, GR et al. "Cluster of Bacteria Associated with Peri-implantitis". Clinical Implant Dentistry and Related Research, 2013, Vol. 0, pp. 1-11.

[0284] Ata-Ali, J. et al. "Peri-implantitis: Associated microbiota and treatment". Med Oral Patol Oral Cir Bucal. 2011, Vol. 16, pp. 937-43.

[0285] L. et al. "Side effects of chlorhexidine mouth washes". Scand J Dent Res, 1971, Vol. 73, pp. 119-125

[0286] Slots, J. et al. "Antibiotics in periodontal therapy: advantages and disadvantages." J Clin Periodontol, 1990, Vol. 17, pp. 479-493

[0287] Szkaradkiewicz, AK et al. "Effect of Oral Administration Involving a Probiotic Strain of Lactobacillus reuteri on Pro-Inflammatory Cytokine Response in Patients with Chronic Periodontitis". Arch Immunol Ther Exp (Warsz) 2014, Vol. 62, pp. 495-500

[0288] Teughels, W. et al. “Clinical and microbiological effects of Lactobacillus reuteri probiotics in the treatment of chronic periodontitis: a randomized placebo-controlled study” J of Clinical Periodontology 2013, Vol. 40, pp. 1025-35

[0289] Hallstroem, H. et al. "Effect of probiotic lozenges on inflammatory reactions and oral biofilm during experimental gingivitis". Acta Odontologica Scandinavica, 2013, Vol. 71, pp. 828-833

[0290] Flichy-Fernandez, AJ, et al. “The effect of orally administered probiotic Lactobacillus reuteri-containing tablets in peri-implant mucositis: a double-blind randomized controlled trial.” J Periodont Res 2015, Epub ahead of print

[0291] Toiviainen, A. et al., "Impact of orally administered lozenges with Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 on the number of salivarymutans streptococci, amount of plaque, gingival inflammation and the oral microbiome in healthy adults." Clin Oral Investig, 2015, Vol. 19, pp. 77-83.

[0292] Maekawa, T. et al. "Topical treatment with probiotic Lactobacillus brevis CD2 inhibits experimental periodontal inflammation and bone loss" J Periodont Res 214, Vol. 44, pp. 785-791

[0293] Yanine, N. et al. "Effects of probiotics in periodontal diseases: a systematic review". Clinical oral investigations, 2013, Vol. 17, pp. 1627-34.

[0294] Andreoletti, O. et al. "The maintenance of the list of QPS microorganisms intentionally added to food or feed. Question number: EFSA-Q-2008-006". The EFSA Journal, 2008, Vol. 923, pp. 1-48

[0295] Weisburg, WG et al. "16S ribosomal DNA amplification for phylogenetic study". J Bacteriology, 1991, Vol. 173, pp. 697-703.

[0296] Muyzer, G. et al. "Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology". Antonie van Leeuwenhoeck, 1998, Vol. 73, pp. 127-141

[0297] Nigatu, A. et al. "Randomly amplified polymorphic DNA (RAPD) for discrimination of Pediococcus pentosaceus and Pediococcus acidilactici and rapid grouping of Pediococcus isolates" Letters in Applied Microbiology, 1998, Vol. 26, pp. 412-6

Claims

1. A self-film-forming composition in the form of a powder that forms a film on its own under stirring in the presence of a liquid medium, the composition comprising: (i) at least one gelling agent in powder form, (ii) at least one bioadhesive agent in powder form, and (iii) at least one lactic acid bacteria strain in powdered form, wherein (i), (ii) and (iii) are in a single container or in separate containers; wherein the self-film forming composition is administered topically; wherein the (i) at least one gelling agent is in an amount for providing viscosity to the composition and is selected from the group consisting of: (a) starch, (b) glue, (c) seaweed polysaccharide, (d) a polysaccharide selected from the group consisting of pectin and maltodextrin, (e) a cellulose derivative, (f) a polypeptide selected from the group consisting of gelatin, collagen and casein; and wherein said (ii) at least one bioadhesive agent is in an amount for providing adhesiveness to said composition, and Selected from the group consisting of: (a) gum, (b) seaweed polysaccharide, (c) cellulose derivative, (d) polysaccharide selected from the group consisting of pectin and maltodextrin, and (e) polymer selected from the group consisting of acrylate-based polymers, vinyl-based polymers and cationic polysaccharides.

2. The self-film forming composition in powder form according to claim 1, wherein the gelling agent and the bioadhesive agent have no bactericidal effect on the at least one lactic acid bacterium.

3. The self-film forming composition in powder form according to claim 2, wherein the gelling agent and the bioadhesive agent have no bacteriostatic effect on the at least one lactic acid bacterium.

4. The self-film forming composition in powder form according to claim 3, wherein the gelling agent or the bioadhesive agent has a prebiotic effect on the at least one lactic acid bacterium.

5. The self-film forming composition according to any one of claims 1 to 4, wherein the gelling agent is selected from the group consisting of gums and seaweed polysaccharides and the bioadhesive is selected from the group consisting of cellulose derivatives and vinyl-based polymers.

6. The self-film forming composition according to any one of claims 1 to 5, wherein the lactic acid bacteria strain belongs to the genus Pediococcus.

7. The self-film forming composition according to claim 6, wherein the Pediococcus strain is selected from the group consisting of the strain deposited under accession number CECT 8903, strain CECT 8904, strain CECT 8905, strain CECT 8906.

8. A self-film-forming composition in the form of a powder that forms a film on its own under stirring in the presence of a liquid medium, the composition comprising: (i) at least one agent selected from the group consisting of a gelling agent and a bioadhesive agent in powder form, and (ii) at least one Pediococcus strain in powdered form, wherein (i) and (ii) are in a single container or in separate containers.

9. The self-film forming composition according to claim 8, wherein the Pediococcus strain is selected from the group consisting of the strain deposited under accession number CECT 8903, strain CECT 8904, strain CECT 8905, strain CECT 8906.

10. A process for preparing a reconstituted formulation comprising mixing, under stirring, a self-film-forming composition in powder form according to any one of claims 1 to 9 with a liquid medium.

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