Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/005—Antimicrobial preparations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/24—Lactobacillus brevis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/25—Lactobacillus plantarum

Definitions

• the present invention relates to the fields of medicine, microbiology and nutrition and, particularly, to a novel combination of probiotic Lactobacillus strains and compositions useful in the field of oral health.
• Dental plaque-related diseases particularly gingivitis, periodontitis and caries, represent a major part of the global burden of oral diseases.
• Periodontal diseases are largely caused by specific gram-negative anaerobic bacterial infections, leading to the initial destruction of the soft connective tissue and, subsequently, to the disruption of the underlying alveolar bone and ligament supporting the teeth.
• the bacterial species Porphyromonas gingivalis has been implicated as a major etiologic agent in the development and progression of periodontitis.
• Other species also contributing to gingival inflammation are Treponema denticola, Prevotella denticola and Fusobacterium nucleatum. Based on the World Health
• Dental caries also known as tooth decay
• Steptococcus mutans is one of a few specialized organisms equipped with receptors that help for better adhesion to the surface of teeth, thus being an early coloniser of the dental surface and the most significant contributor to caries.
• the growth and metabolism of this pioneer species creates an acidic environment in the mouth which causes the highly mineralized tooth enamel to be vulnerable to decay.
• halitosis is caused by a number of volatile compounds which are derived from the bacterial degradation of sulphur-containing amino acids.
• the implicated bacteria mostly Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas intermedia, and Treponema denticoia
• This affection has a significant impact - personally and socially - on those who suffer from it, and is estimated to be the third-most-frequent reason for seeking dental aid, following tooth decay and periodontal disease.
• Oral bacteria form a biofilm (dental plaque) on all hard and soft oral tissues which is considered to be the principal etiologic agent in the pathological conditions of the mouth.
• the accumulation of bacteria within the biofilm facilitated by poor oral health maintenance, predisposes to allogenic shifts in the microbial community, leading to the onset of periodontal inflammation and caries formation, as well as contributing to halitosis.
• Yeasts, and particularly, Candida albicans may also be the cause of disorders in the oral cavity.
• the elderly are vulnerable to Candida infection provoked by chronic diseases, medication, poor oral hygiene, reduced salivary flow, or the impairment of the immune system. Even though the colonization by Candida may be asymptomatic, heavy growth usually leads to local candidiasis, with various types of mucosal lesions and symptoms.
• pathogenic microorganisms is a promising means for controlling oral infections.
• the use of probiotics for oral health applications has been scarcely studied.
• very few commercial products containing probiotics are being marketed which incorporate such a health applications.
• Prodentis® from BioGaia.
• Prodentis is a chewing gum that contains a probiotic L. reuteri ATCC 55730 strain and has
• Lactobacillus sp. vary greatly in their adherent capacity to saliva-coated surfaces in a test model system mimicking oral cavity conditions (Stamatova I, et al. "In vitro evaluation of yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to saliva-coated surfaces”. Oral Microbiol Immunol, 2009, vol. 24, p. 218-223).
• Streptococcus salivarius K12 is another commercial probiotic intended for use in the oral cavity.
• S. salivarius K12 was isolated from the saliva of a healthy child and has been shown to perform in vitro antimicrobial activity against various bacterial species incriminated in the etiology of halitosis (Burton JP, et al. "Preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodor parameters". J Appl Microbiol, 2006, vol. 100, p. 754-764).
• Probiotics have therefore a potentiality to provide beneficial effects in the oral cavity, provided that suitable probiotic strains are identified.
• suitable probiotic strains are identified.
• the latter acquires special relevance while contemplating the use of oral lactobacilli, since certain oral lactobacilli have been described as cariogenic due to their high acidogenic potential which favours the degradation of hard tissues, such as enamel and dentine.
• the present invention provides a composition comprising Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480.
• Lactobacillus brevis CECT 7480 it is understood the Lactobacillus brevis strain deposited in the Spanish Type Culture Collection under the accession number CECT 7480, as well as mutant or derivative microorganisms that have been obtained by techniques known in the state of the art using the deposited strain as starting material, such mutants or derivatives at least retaining the herein described relevant features and advantages of the strain Lactobacillus brevis CECT 7480.
• the strains of the present invention have the advantage that they are particularly useful as probiotics.
• probiotic is recognised in the state of the art as a microorganism which, when administered in adequate amounts, confers a health benefit to the host.
• a probiotic microorganism must fulfil several requirements related to lack of toxicity, viability, adhesion and beneficial effects. These probiotic features are strain-dependent, even among bacteria of the same species. Therefore, it is important to find those strains that have a better performance in all probiotic requirements.
• the strains of the invention are useful as oral probiotics, i.e.
• Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 display a significant inhibitory activity against a broad number of pathogens of the oral cavity that are implicated in the development of oral disorders, such as gingivitis, periodontitis, caries and halitosis, while displaying minimal antagonism against common commensal strains of the human oral flora.
• these two strains show a lack of inhibitory activity between them, thus allowing their combined use in a single formula.
• the combination of these strains into a single formula i.e.
• the composition of the invention has the advantage of displaying a higher antagonistic activity against oral pathogens as compared to the activity of the individual strains used separately.
• the strains of the invention display a cooperative activity against oral pathogens and are especially useful when used in combination.
• antagonising microorganisms that are implicated in pathological conditions in the oral cavity such as Streptococcus mutans, Porphyromonas gingivalis, Treponema denticola, Prevotella denticola and Fusobacterium nucleatum, these strains have the effect of altering the oral microbiological profile to a healthier profile, thereby benefitting oral health conditions.
• the strains of the composition of the invention are good oral probiotics since, in addition to a strong antagonistic activity, they exhibit a good ability to colonise the oral cavity.
• Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 are able to grow in the presence of lysozyme and hydrogen peroxide.
• these strains also have a good ability to adhere to oral tissues.
• the strains of the invention have the advantage of displaying a high ability to form aggregates. This is important because it enables said strains to inhibit or reduce dental plaque by interfering with pathogens biofilm formation. It is known that lactic acid bacteria probiotic strains with
• aggregation activity can inhibit or reduce dental plaque formation by pathogenic bacteria, which is the result of the aggregation of pathologic bacteria among each other and also with other microorganisms.
• pathogenic bacteria which is the result of the aggregation of pathologic bacteria among each other and also with other microorganisms.
• the biofilm formed by oral pathogens on hard and soft oral tissues is considered to be an important etiologic agent in the pathological conditions of the mouth, leading to the onset of periodontal inflammation, caries formation and halitosis.
• the formation of aggregates by the strains of the invention is increased when both strains are combined into a composition, meaning that the strains are more effective in displacing pathogenic bacteria when combined into a single formula.
• strains CECT 7481 and CECT 7480 display a particularly low acidification profile.
• Lactobacillus species like most lactic acid bacteria, are characterised by a high production of volatile acids as a result of fermentation of sugars in the human diet.
• the acidogenic property of these bacteria can be a possible side-effect in the oral cavity, since it raises the risk of caries.
• lactobacilli have been considered as being cariogenic. Therefore, the reduced acid production displayed by the Lactobacillus strains forming the composition of the invention renders them particularly suitable for health applications in the oral cavity.
• EFSA European Food Safety Authority
• the inventors have found that these strains do not display any significant resistance to antibiotics of human and/or veterinary importance (ampicillin, gentamicin, streptomycin, erythromycin, tetracycline, clindamycin, and chloramphenicol), thus precluding the risk of a potential transfer of antibiotic resistance to pathogenic species.
• the strains of the invention have a better performance for all parameters relevant for an oral probiotic when compared with commercial strains which are known in the art oral probiotics.
• the new strains are more resistant to oral conditions, have greater ability to form aggregates, higher (and wider) antagonistic activity, better adhesion and/or lower acidification profile than Streptococcus salivarius K12 and Lactobacillus reuteri ATTC 55730 and Lactobacillus brevis CD2. Protocols for determining each one of said properties are included below. Additionally, the combination of both strains of the invention into a single composition generally results in a cooperative performance of the strains in relevant functionalities. Thus a composition comprising both strains is particularly appropriate for use as an oral probiotic.
• composition of the first aspect of the invention is useful as medicament. Particularly, when the composition comprising Lactobacillus plantarum CECT 7481 and
• Lactobacillus brevis CECT 7480 is administered, it is useful in the prevention and/or treatment of disorders in the oral cavity and, preferably, for the prevention and/or treatment of disorders caused by pathogens in the oral cavity.
• the beneficial effect of the strains forming the composition of the invention is the result of improving the microbiological profile of the oral cavity to yield a healthier oral flora. Growth in the oral cavity of these beneficial bacteria induces an environmental pressure that inhibits the growth of common and/or opportunistic pathogenic
• This environmental pressure is derived from the competition for adhesion sites and nutrients, the production of antimicrobial compounds and the displacement of pathogens by aggregation of the probiotic bacteria.
• probiotic bacteria One further item to take into consideration is the ability of probiotic bacteria to modulate the immune response.
• the mechanisms by which probiotics modulate immunity have been broadly studied on gastrointestinal structures. Probiotic species have shown their ability to alter the balance of proinflammatory and anti-inflammatory cytokines secreted by epithelial cells. Probiotics also regulate immune responses by enhancing innate immunity and modulating pathogen-induced inflammation via toll-like receptor-regulated signalling pathways. The enhancement of local immune responses, as well as of systemic immune responses by probiotics can offer new opportunities for probiotics in preventing infections at peripheral mucosal surfaces, such as those in the oral cavity.
• the invention provides a composition comprising the strains of this invention for use as a medicament.
• the invention provides the composition as defined in the first aspect of the invention for use in the prevention and/or treatment of a disorder from the oral cavity that is caused by oral pathogens in an animal, including a human.
• this aspect can be formulated as the use of the composition as defined in the first aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of a disorder from the oral cavity that is caused by oral pathogens in an animal, including a human.
• the invention also provides a method for the prevention and/or treatment of a disorder from the oral cavity that is caused by oral pathogens in an animal, including a human, comprising administering to said animal in need thereof the composition as defined in the first aspect of the invention.
• disorder from the oral cavity which is caused by oral pathogens is used herein in its broadest meaning as any derangement or abnormality that can be found in the oral cavity which has been caused by an oral pathogen such as bacteria, viruses or yeasts. Such disorder can be a serious
• compositions are used for the treatment and/or prevention of a dental plaque related disorder.
• the dental plaque related disorder is selected from the group consisting of caries, sensitive teeth, gingivitis and periodontitis.
• composition is used for the treatment and/or prevention of halitosis.
• composition is used for the treatment and/or prevention of candidiasis.
• composition according to the invention that comprises the strains of the invention can be formulated as edible, cosmetical or pharmaceutical products.
• Said composition can comprise, in addition to the strains of the invention, one or more other active agents and/or cosmetically acceptable excipients (in the case of a cosmetical composition), pharmaceutically acceptable excipients (in the case of a pharmaceutical composition) or adequate edible ingredients (in the case of an edible composition).
• the composition of the invention further comprises one or more active agents.
• the additional active agent or agents are other probiotic bacteria which are not antagonic to the strains forming the
• the additional active agent or agents are suitable for treating and/or preventing halitosis, candidiasis, caries, sensitive teeth and/or periodontal diseases.
• the strains may be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture which has been post-treated, and alone or together with suitable carriers or ingredients. Prebiotics could be also added.
• the composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of powders, tablets, film preparations, solutions, aerosols, granules, lozenges, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tincture or fluid extracts or in a form which is particularly suitable for oral administration.
• the invention provides a pharmaceutical composition that contains the composition of the invention together with pharmaceutically acceptable excipients.
• the pharmaceutical product may be prepared in any suitable form which does not negatively affect the
• pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (either a human or non-human animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• a subject either a human or non-human animal
• Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.
• compositions of the invention are useful for the amelioration and/or prevention of the symptoms produced by these disorders. Such symptoms include, but are not limited to, bad breath and stained teeth.
• cosmetically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with human skin without undue toxicity, incompatibility, instability and allergic response, among others.
• Each "cosmetically acceptable” carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the cosmetic formulation.
• Suitable carriers, excipients, etc. for cosmetic formulations can be found in standard texts.
• the strains of the invention can be also included in a variety of edible products, such as milk products, yogurt, curd, cheese (e.g. quark, cream, processed, soft and hard), fermented milk, milk powder, milk based fermented product, ice-cream, a fermented cereal based product, milk based powder, a beverage, a dressing, and a pet food.
• edible product is used herein in its broadest meaning, including any type of product, in any form of presentation, which can be ingested by an animal, but excluding cosmetical, pharmaceutical and veterinary products.
• meat products e.g.
• liver paste, frankfurter and salami sausages or meat spreads can be used as an ingredient in other food products.
• Particularly interesting edible products are functional foods and infant formulas.
• an edible composition which contains the composition of the invention together with other edible ingredients.
• edible ingredients refers to ingredients which are fit to be eaten, i.e. to be used as food, by an animal, preferably but not limited to humans, cattle or pet animals.
• probiotic bacterial compositions such as the one disclosed herein, are considered as dietary supplements.
• Dietary supplements also known as food supplements or nutritional supplements, provide beneficial ingredients that are not usually ingested in the normal diet.
• dietary supplements are considered as food products, but sometimes they are defined as drugs, natural health products, or nutraceutical products. In the sense of the present invention, dietary supplements also include nutraceuticals. Dietary
• the composition of the invention is a dietary supplement.
• the composition according to the invention is a dietary supplement, it can be administered as such, can be mixed with a suitable drinkable liquid, such as water, yoghurt, milk or fruit juice, or can be mixed with solid or liquid food.
• the dietary supplement can be in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, pastilles, sweets, bars, syrups and corresponding administration forms, usually in the form of a unit dose.
• the dietary supplement comprising the composition of the invention is administered in the form of tablets, lozenges, capsules or powders, manufactured in conventional processes of preparing dietary supplements.
• the products comprising the composition of the invention are meant for use in oral health applications, either by preventing or treating an oral disorder, or by ameliorating the symptoms derived from these disorders.
• another aspect of the present invention provides an oral care product comprising the composition as mentioned above, together with pharmaceutically excipients, or cosmetically acceptable excipients, or other edible ingredients.
• the composition is an oral product, which in the ordinary course of usage, is not intentionally swallowed for purposes of systemic administration of particular therapeutic agents, but is rather retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and/or oral tissues for purposes of oral activity.
• Non limiting examples of such products are toothpastes, dentifrices, tooth powders, topical oral gels, mouth rinses, denture products, mouth sprays, chewing gums, dental floss or dental tapes.
• the oral composition may be a single phase oral composition or may be a combination of two or more oral compositions.
• the oral care product is a chewing gum, a tooth-paste, a mouth spray, a lozenge or an oral dispersible tablet.
• the oral care products are in the form of lozenges or oral dispersible tablets.
• the oral care products of the present invention may also comprise other orally active agents, such as teeth whitening actives, including bleaching or oxidizing agents like peroxides, perborates, percarbonates, peroxyacids, persulfates, metal chlorites, and combinations thereof. Teeth colour modifying substances may also be considered among the oral care actives useful in the present invention.
• the oral care products may additionally comprise flavouring compounds such as menthol.
• the strains forming the composition of the invention are preferably in the form of viable cells.
• the strains of the invention can also be in the form of non-viable cells such as killed cultures or compositions containing beneficial factors produced by Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480. This could include thermally killed micro-organisms or micro-organisms killed by exposure to altered pH, sonication, radiation or subjection to pressure.
• non-viable cells product preparation is simpler, cells may be incorporated easily into commercial products and storage requirements are much less limited than viable cells.
• the strains can be in any concentration ratio suitable for the intended use.
• the strains can be in a concentration ratio which is comprised between of 3:1 and 1 :3 ⁇ Lactobacillus plantarum CECT 7481 : Lactobacillus brevis CECT 7480).
• the concentration ratio is 1 : 1 .
• the strains of the invention are included in the composition in an effective amount for the required use.
• an effective amount is the amount of colony forming units (cfu) for each strain in the composition that is high enough to significantly modify the condition to be treated in a positive way but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment.
• An effective amount of said probiotic microorganism will be determined by the skilled in the art and will vary with the particular goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disorder, and the final formulation.
• the strain or strains are present in an amount from aboutI O 5 cfu/g to about 10 12 cfu/g, preferably in an amount from about 10 7 cfu/g to about 10 11 cfu/g.
• colony forming unit (“cfu") is defined as number of bacterial cells as revealed by microbiological counts on agar plates.
• the composition of the invention is an oral care product comprising between 10 7 -10 10 cfu/g. Dietary supplements usually contain probiotic strains in an amount ranging from 10 5 and 10 12 cfu/g.
• the composition of the invention is a dietary supplement comprising between 10 7 -10 10 cfu/g.
• the strains of the invention are produced by cultivating the bacteria in a suitable medium and under suitable conditions.
• the strains can be cultivated alone to form a pure culture, or as a mixed culture together with other microorganisms, or by cultivating bacteria of different types separately and then combining them in the desired proportions.
• the cell suspension is recovered and used as such or treated in the desired manner, for instance, by concentrating or freeze-drying, to be further employed in the preparation of the products.
• the probiotic preparation is subjected to an immobilisation or encapsulation process in order to improve the shelf life.
• immobilisation or encapsulation of bacteria are known in the art.
• the strains forming part of the composition of the invention are incorporated to an oral care product as encapsulated bacteria.
• Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 are effective not only when combined in a single composition, but also when used on their own, or in two different compositions administered simultaneously, sequentially or separately after a certain period of time.
• one of the strains can be prescribed to be used together with the other strain for oral health in order to prevent or treat oral disorders, particularly disorders that are produced by oral pathogens, more preferably dental-plaque related disorders and/or halitosis and/or candidiasis.
• one more aspect of the invention provides Lactobacillus plantarum CECT 7481 .
• Lactobacillus brevis CECT 7480 provides Lactobacillus brevis CECT 7480.
• the strains of the invention are described herein for the first time and are therefore new.
• both strains, Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 have a different pulsed field gel electrophoresis pattern than closely related commercial lactobacilli strains, such as Lactobacillus plantarum 299v, Lactobacillus plantarum VSL#3, Lactobacillus casei VSL#3 and Lactobacillus casei DN 1 14.001 .
• Some documents of the state of the art describe compositions comprising strains of Lactobacillus plantarum and Lactobacillus brevis. However, the composition of the invention is new with respect to said compositions.
• KR100780030 discloses a fermented soy milk comprising strains of Lactobacillus brevis and Lactobacillus plantarum.
• the disclosed strains were isolated from kimchi, a meal consisting in fermented vegetables that is typical from Korea.
• the strains of the invention were isolated from children saliva in a South American developing region, where kimchi is not consumed.
• the L brevis and L plantarum strains of the invention have a completely different origin, meaning that either Lactobacillus plantarum CECT 7481 or Lactobacillus brevis CECT 7480 are the same as the referred Korean strains.
• KR100866504 discloses a fermented red ginseng using Lactobacillus plantarum P2 (microbial deposition number
• KCTC1 1391 BP KCTC1 1391 BP
• Lactobacillus brevis M2 microbial deposition number KCTC1 1390BP
• strain L brew ' s CD2 is different form Lactobacillus brevis CECT 7480. As shown in TABLE 4, when combining the strain Lactobacillus plantarum CECT 7481 with L brew ' s CD2 an antagonistic effect is observed with respect to the ability to form aggregates. In contrast, the combination of Lactobacillus plantarum CECT with Lactobacillus brevis CECT 7480 results in a greater ability to form aggregates. Additionally, it has been demonstrated that L brew ' s strain of the invention has a significantly lower acidification profile as compared with commercial L brew ' s CD2 (see TABLE 5).
• Lactobacillus brevis CECT 7480 is not only different to L brew ' s CD2, but also better suited for oral health applications. Altogether, while having different effects, the combination of the invention is different from that disclosed in WO 2006/080035.
• FIG. 1 Pulsed field electrophoresis patterns of S / ' -l (A, B) and Sma-l (C, D) restricted genomic DNA of: 1 , Lactobacillus plantarum CECT 7481 (F2096); 2, Lactobacillus plantarum 299V; 3, Lactobacillus plantarum VSL#3; 4,
• Lactobacillus brevis CECT 7480 (13141 ); 5, Lactobacillus casei VSL#3; 6, Lactobacillus casei DN 1 14.001 .
• M stands for molecular marker.
• Novel strains F2096 and 12141 were isolated from saliva from 0-5 year-old children from a tropical South American developing region. Saliva was dissolved in PBS buffer (pH 7.4), aliquoted and plated on MRS (Man Rogosa Sharp, Sigma-Aldrich Chem, Spain) agar supplemented with 10 pg/ml vancomycin (SIGMA). Strains were cultured under microaerophilic conditions (5% CO2) at 37 °C. Once grown, isolated strains were stored by lyophilisation in PBS 0.1X with 15% skim milk powder.
• Strains F2096 and 13141 were identified as Lactobacillus plantarum and Lactobacillus brevis, respectively (see section 2 below). Both strains were deposited in the Spanish Type Culture Collection (Universitat de Valencia, Campus de Burjassot, Edif. de Investigacion, 46100 Burjassot, Valencia, Spain). Lactobacillus plantarum (strain F2096) was deposited on 21 .01 .2009 and given accession number 7481 . Lactobacillus brevis (strain 13141 ) was deposited on 18.02.2009 and given accession number 7480. Both deposited strains are viable and keep all their features related to their deposit.
• strain F2096 corresponds to Lactobacillus plantarum CETC 7481 , and strain 13141 to Lactobacillus brevis CECT 7480.
• Pediococcus acidilactici F2019 (herein after referred to as P. acidilactici), Lactobacillus paracasei 13152 (herein after referred to as L paracasei) and Pediococcus pentosaceus 154 (herein after referred to as P. pentosaceus) were isolated from human saliva as explained above.
• Streptococcus salivarius K12 was isolated from commercial BLIS K12® in TBS (tryptic soy broth) and cultured in aerobic conditions at 37 °C.
• Lactobacillus reuteri ATCC 55730 strain was isolated from commercial Reuteri Drops®, Lactobacillus plantarum 299v from Poviva®, Lactobacillus brevis CD2 from Inersan®, Lactobacillus plantarum VSL#3 and Lactobacillus casei VSL#3 from VSL#3®, and
• Lactobacillus casei DN 1 14.001 from Actimel® All of the latter strains were isolated on MRS, and cultured at 37 °C with 5% CO2.
• Potentially pathogenic strains Porphyromonas gingivalis CIP 103683, Fusobacterium nucleatum CIP 104988, Treponema denticola CIP 103917 and Prevotella denticola CIP 104478T were obtained from Institut Pasteur, and cultured following provider's instructions. Streptococcus mutans of clinical origin was cultured on Brain Heart Agar at 37 °C and 5% CO2. Identification of the strains was performed by sequencing.
• the strains of the invention were grown overnight on MRS medium (pH 6.4) at 37 °C in an atmosphere containing 5% CO2. Bacteria were further harvested, washed and resuspended in pre-lysis buffer (480 ⁇ EDTA 50 mM pH 8.0; 120 ⁇ lysozyme 10 mg/ml), and further incubated at 37 °C for 60 min. DNA was extracted using Wizard genomic DNA purification kit (Promega). After centrifugation of the pre-treated bacteria at 14000 g for 2 min to remove the supernatant, the Promega's protocol was followed. In brief, bacteria were resuspended in Nuclei Lysis Solution and incubated at 80 °C for 5 min, then cooled to room temperature. Cell lysates were incubated in RNase solution at 37 °C for 60 min and proteins were precipitated by adding the Protein
• Precipitation Solution and vortexing at high speed Samples were cooled down and centrifuged at 15000 g for 3 min. The supernatants containing the DNA were transferred to clean 1 .5 ml microfuge tubes and mixed with 600 ⁇ of isopropanol by inversion. DNA was collected by centrifugation at 15000 g for 2 min and carefully pouring off the supernatant. DNA samples were washed with 600 ⁇ of 70% ethanol by gently inverting the tube several times. Ethanol was removed by aspiration, after centrifugation at 15000 g for 2 min. Finally, the DNA pellet was resuspended in 100 ⁇ of Rehydration Solution by incubating at 65 °C for 1 h. Samples were stored at 2-8 °C.
• the 16S rRNA was amplified by PCR using the universal primers Eub27f and Eub1492r, which produce a fragment nearly full-sequence of 16S (more than 1000 nucleotides) (TABLE 1 ). Then, the DNA obtained as explained above was washed using the kit Quiaquick (Quiagene).
• Genus Identification was carried out using the Ribosomal Database Project tool (Wang Q, et al., "Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy", Appl Environ Microbiol, 2007, vol. 73, p. 5261 -5267). Species identification was performed by comparison of the obtained sequence with 16S sequences of known organisms from both RefSeq data base (http://www.ncbi.nlm.nih.gov/RefSeq/) by means of a BLASTN, and from Ribosomal Database Project (http://rdp.cme.msu.edu/, J.R. Cole et aA, "The Ribosomal Database Project (RDP-II): introducing myRDP space and quality controlled public data", Nucl. Acids Res. 2007, vol. 35, p. 169-172).
• RDP Rivibosimal Database Project
• Lactobacillus plantarum species Lactobacillus plantarum species.
• Pulse-field electrophoresis was carried out using CHEF DRIII apparatus (BioRad Laboratories). Inserts were loaded in a 1 % agarose gel (SeaKem ME agarose, FMC BioProducts, ME, USA). TABLE 2 describes electrophoresis conditions for each enzyme. DNA molecular weight markers were Lambda ladder PFG Marker and Low Range PFG Marker (New England Biolabs). After electrophoresis, gels were stained with ethidium bromide and UV using GelDoc System (BioRad). TABLE 2. Electrophoresis conditions for Sfi- ⁇ and Sma- ⁇ restricted genomic DNA from F2096 and 13141 strains.
• Said mixed cultures contained an equal amount of each of their constituent strains and the same total bacterial concentration as the single-strained cultures, i.e. 10 8 cfu/ml.
• a fixed volume of each of the single-strain cultures and mixed cultures was plated uniformly and grown to confluence at the appropriate temperatures in a 5% CO2 incubator. Then, uniformly sized cylinder sections of confluent agar plates were placed loan-to-loan over the pathogen plate and incubated overnight at 37 °C.
• GAA growth inhibitory activity
• both P2096 and 13141 have a broad inhibition pattern against oral pathogens. This is especially relevant for P.
• the combination of F2096 and 13141 in a mixed culture displays a higher antagonistic activity against oral pathogens as compared to the activity of the individual strains used separately as single cultures -it is to note that the concentration of each strain in the mixed culture is half of that in the single cultures, therefore the antagonistic effect of the combination is higher than the sum of the individual effects for these strains-.
• This synergistic effect does not occur when each of the strains were combined with another inhabitant of the oral cavity, P. acidilactici.
• the strains of the invention display a synergistic activity against oral pathogens and are especially useful when used in a single formula.
• %AC Percent aggregation capacity
• the ability to form aggregates is important for a probiotic strains with oral health applications because it enables said strains to inhibit or reduce dental plaque by interfering with pathogens biofilm formation.
• Mean aggregation capacity values for the new strains and their combination among them or with P. acidilactici or L. brevis CD2 are shown in TABLE 4 as compared to control strains. These results clearly demonstrate that the new isolates showed very good aggregation activity, significantly higher than both commercial controls S. salivarius K12 and L. reuteri ATTC 55730. Further, aggregate formation by the strains of the invention is increased when both strains are combined in a mixed culture, meaning that the strains are more effective in displacing pathogenic bacteria when combined into a single composition.
• the ability of the new strains to produce acid when growing in culture media supplemented with different sugars present in human diet was evaluated.
• the strains were grown during 18 h at 37°C and 5% CO2 in the following media: MRS and minimal medium supplemented with 4% glucose, 4% fructose, 4% lactose or 4% sucrose.
• Minimal medium contained 2 g/L peptone water, 2 g/L yeast extract, 0.1 g/L NaCI, 0.04 g/L K 2 HP0 4 , 0.04 g/L KH 2 P0 , 0.01 g/L MgS0 *7H 2 0, 0.01 g/L CaCI 2 *6H 2 0, 2 g/L NaHC0 3 , 0.05 g/L hemina
• low values correspond to highly acidogenic strains.
• High acid production is an undesirable side effect for oral probiotics, since it promotes caries formation.
• the acid production value for F2096 and 13141 and several probiotic control strains grown on different sugars can be seen in TABLE 5, together with the values obtained for commercial strains.
• MM stands for minimal medium.
• strains F2096 and 13141 in different sugars results in a remarkably poor acid production as compared with other bacterial strains that had also been isolated from the oral cavity, namely, L. paracasei, P. pentosaceus and P. acidilactici.
• the strains of the invention are also less acidogenic than commercial oral probiotic S. salivarius K12.
• strain 13141 has a lower acidification profile when compared to L. reuteri ATTC 55730, which is known to be particularly low acidogenic and is being marketed as an anti-cariogenic probiotic. It is also noteworthy that L. brevis strain of the invention 13141 has a significantly lower acidification profile as compared with commercial L. brevis CD2. Therefore, strains F2096 and 13141 have a low acidification profile, thus being suitable for oral health applications.
• strains were grown in the medium which contains glucose (0.5% w/v), fructose (0.5% w/v), yeast extract (1 % w/v), meat extract (1 % w/v), eukaryotic cells (200 cells/ml) and pectin (0.5% w/v) during 48 h at 37°C and 5% C02.
• the strains receive a production of malodor value between 1 and 5, where 1 is absence of odor and 5 is a very unpleasant odor.
• % SV [(ODtf-ODto (with supplement)) / (OD t f-OD t o (without supplement))]* 100, where OD is optical density, and tf and to are final time and initial time, respectively.
• OD optical density
• New strains F2096 and 13141 showed better resistance to oral stress than commercial strains S. salivarius K12 and L. reuteri ATCC 55730.
• the adhesion measurement was performed by the addition of 3.3 ml of radiolabeled-! 0 8 cfu of each strain to either 1 .05*0,5 cm-fragments of pig tongue or pre-incubated Caco-2 cells or 50 mg of HA beads (80 m of diameter) in ELISA plates. After 45 min, supernatants were carefully removed. HA beads (together with adhered bacteria) were recovered by centrifugation. The tongue or Caco-2 cells together with the adhering bacteria were scrapped from the ELISA wells. Finally, recovered bacteria were lysed to calculate specific radioactivity (cpm/cfu).
• strains F2096 and 13141 have global better adhesion capacity to oral tissue when compared with oral probiotic strain S. salivarius K12.
• the ability to adhere to oral tissues is a relevant feature for strains to be used as probiotics since it gives these bacteria a competitive advantage when competing for adhesion sites against pathogens and favoring permanence in the oral cavity.
• strains that display good adhesion to oral tissues contribute to displace pathogens from the oral cavity and promote a healthier oral flora.
• Antibiotic susceptibility for strains F2096 and 12141 was studied following the technical guidance given by the European Food Safety Authority (EFSA) ("Update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance". The EFSA Journal, 2008, vol. 732, p. 1 -15). Culture conditions for the strains were as follows: growth on surface of MRS agar plates containing EFSA recommended antibiotic concentrations at 37 °C and 5% CO2. B) Results
• Tetracyclin 32 none 8 none chloramphenicol 8 none 4 none n.r. , not recommended by EFSA BIBLIOGRAPHIC REFERENCES

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