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    • 专利摘要:
    probiotic composition for oral health. The present invention relates to a composition comprising an effective amount of lactobacillus plantarum cect 7481 and lactobacillus brevis cect 7480. Said strains exhibit various functional properties that make them suitable for their use in improving oral health. such properties not only include good antagonistic properties against oral pathogens, but also the ability to colonize the oral cavity and a low acidification profile. the invention also provides the use of said composition as a probiotic and/or a medicine for oral hygiene applications as well as products containing said composition.
    公开号:BR112013003761B1
    申请号:R112013003761-0
    申请日:2011-08-17
    公开日:2021-06-01
    发明作者:Jordi CUNÉ CASTELLANA
    申请人:Ab-Biotics S.A.；
    IPC主号:
    专利说明:

    [001] The present invention relates to the fields of medicine, microbiology and nutrition, and particularly to a new combination of probiotic Lactobacillus strains and compositions useful in the field of oral health. TECHNICAL BACKGROUND
    [002] Dental plaque-related diseases, particularly gingivitis, periodontitis, and caries, represent a major part of the global burden of oral disease.
    [003] Periodontal diseases (gingivitis and periodontitis) are basically caused by specific gram-negative anaerobic bacterial infections, leading to the initial destruction of the soft connective tissue and, later, to the disturbance of the underlying alveolar bone and ligament that supports the teeth. The bacterial species Porphyromonas gingivalis has been implicated as a major etiologic agent in the development and progression of periodontitis. Other species that also contribute to gingival inflammation are Treponema denticola, Prevotella denticola and Fusobacterium nucleatum. Based on research from the World Health Organization, most children have signs of gingivitis, and among adults, the early stages of periodontal disease are highly prevalent. For example, in Europe, approximately 15 to 35% of the adult population suffer from this multifactorial disease.
    [004] Dental caries (also known as tooth decay) is a disease in which bacterial processes damage the rigid structure of the tooth. Steptococcus mutans is one of a few specialized organisms equipped with receptors that aid in better adhesion to the tooth surface, thus being a primary colonizer of the tooth surface and the most significant contributor to caries. The growth and metabolism of this pioneer species creates an acidic environment in the mouth that makes highly mineralized tooth enamel vulnerable to decay.
    [005] In addition to the above, an additional oral disorder is believed to affect a large proportion of the population: halitosis. Also referred to as bad breath, halitosis is caused by several volatile compounds that are derived from bacterial degradation of sulfur-containing amino acids. The bacteria involved (mostly Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas intermedia, and Treponema denticola) are located in stagnant areas in the oral cavity, such as the dorsal surface of the tongue, periodontal pockets, and interproximal areas. This disease has a significant impact - personally and socially - on those who suffer from it, and is estimated to be the third most frequent reason to seek dental help, after tooth decay and periodontal disease.
    [006] Oral bacteria form a biofilm (dental plaque) in all soft and hard oral tissues which is considered to be the main etiological agent in pathological conditions of the mouth. The accumulation of bacteria within the biofilm, facilitated by poor oral health maintenance, predisposes to allogeneic shifts in the microbial community, leading to the onset of periodontal inflammation and caries formation, as well as contributing to halitosis.
    [007] Yeasts, and particularly Candida albicans, can also be the cause of disorders in the oral cavity. Elderly people are vulnerable to Candida infection caused by chronic illness, medication, poor oral hygiene, reduced salivary flow, or impaired immune system. Although Candida colonization can have strong asymptomatic growth it usually leads to local candidiasis, with various types of mucosal lesions and symptoms.
    [008] Modifying the pathogenic potential of the microbiota within the oral cavity would be an interesting strategy in the fight against these disorders. In this direction, the introduction of probiotic lactobacilli to partially replace pathogenic microorganisms is a promising means to control oral infections. However, when compared to gastrointestinal diseases, the use of probiotics for oral health applications has only been studied. At present, very few commercial products containing probiotics are being sold that incorporate such health applications.
    [009] One of these products is Prodentis® from BioGaia. Prodentis is a gum containing a probiotic strain L. reuteri ATCC 55730 and has been shown to reduce gingivitis in a clinical trial (Twetman S, et al. "Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory mediators in gingival crevicular fluid ". Acta Odontol Scand, 2009, vol. 67, p. 19-24). This same strain, L. reuteri ATCC 55730, has been reported to exert strong antagonistic activity against cariogenic Streptococcus mutans (Caglar E, et al." Salivary mutans streptococci and lactobacilli levels after ingestion of the probiotic bacterium Lactobacillusjeuteri ATCC 55730 by straws or tablets" Acta Odontol Scand, 2009, vol. 64, p. 314-318). However, little is known about the impact of L. reuteri ATCC 55730 on other oral pathogens. Furthermore, L. reuteri has been isolated from the intestine, not the oral cavity, and it is not known whether this strain has the ability to form biofilms or otherwise colonize this environment in order to have a long-lasting effect. It has been shown that Lactobacillus sp. it varies greatly in its ability to adhere to saliva-coated surfaces in a test model system that mimics the conditions of the oral cavity (Stamatova I, et al. "In vitro evaluation of yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to saliva-coated surfaces ". Oral Microbiol Immunol, 2009, volume 24, p. 218-223).
    [0010] 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 was shown to perform in vitro antimicrobial activity against several bacterial species accused 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, volume 100, p. 754-764). However, the beneficial effects of this strain are limited to improving halitosis symptoms.
    [0011] Probiotics therefore have a potency to provide beneficial effects in the oral cavity, provided that suitable probiotic strains are identified. In this attempt, it is necessary to consider the putative benefits of the host, but also the safety of the strain, as well as possible adverse effects in the oral cavity. The latter acquires special relevance considering the use of oral lactobacilli, since certain oral lactobacilli have been described as cariogenic due to their high acidogenic potential that favors the degradation of hard tissues, such as enamel and dentin.
    [0012] Despite advances in the field of oral probiotics, it is clear from the above mentioned that new probiotic strains are needed which, having a broad spectrum of benefits in the oral cavity, do not present adverse effects. SUMMARY OF THE INVENTION
    [0013] The inventors have isolated new strains of the human oral microbiota. These strains, Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480, exhibit several functional properties that make them suitable for use in improving oral health. Such properties include not only good antagonistic properties against oral pathogens, but also the ability to colonize the oral cavity and a low acidification profile. As discussed below, it has also been found that when both strains are used in a unique combination, the health benefits in the oral cavity are notable.
    [0014] Thus, in a first aspect the present invention provides a composition comprising Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480.
    [0015] It is clear that using the strains deposited as starting material, one skilled in the art can routinely, by conventional mutagenesis or re-isolation techniques, obtain additional mutants or derivatives thereof that preserve or enhance the relevant attributes described in this application and the advantages of strains that form the composition of the invention. Such mutants or derivatives can be genetically modified or naturally occurring. The person skilled in the art will decide on the appropriate method to be employed to determine the functional activities of the strains. Examples of possible methods for measuring these activities are shown in the examples below.
    Thus, "by Lactobacillus plantarum CECT 7481" is understood the strain of Lactobacillus plantarum deposited in the Spanish Type Culture Collection under accession number CECT 7481, as well as mutant microorganisms or derivatives that were obtained by techniques known in the state of technique using the deposited strain as starting material, such mutants or derivatives at least maintaining the relevant attributes described in this application and advantages of the strain Lactobacillus plantarum CECT 7481. By "Lactobacillus brevis CECT 7480" is understood the strain of Lactobacillus brevis deposited in the Spanish Type Culture Collection under accession number CECT 7480, as well as mutant microorganisms or derivatives that were obtained by techniques known in the state of the art using the deposited strain as starting material, such mutants or derivatives at least maintaining the relevant attributes in this application described and advantages of the Lactobacillus brevis CECT 7480 strain.
    The strains of the present invention have the advantage that they are particularly useful as probiotics.
    [0018] The term "probiotic" is recognized in the state of the art as a microorganism that, when administered in adequate amounts, confers a health benefit to the host. A probiotic microorganism must meet several requirements related to lack of toxicity, viability, adhesion and beneficial effects. These probiotic attributes are strain-dependent, even among bacteria of the same species. Therefore, it is important to find those strains that perform best for all probiotic requirements.
    Preferably, the strains of the invention are useful as oral probiotics, i.e., probiotics for enhancing oral health. Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 have been found to exhibit significant inhibitory activity against a large number of oral cavity pathogens that are involved in the development of oral disorders such as gingivitis, periodontitis, caries and halitosis, exhibiting minimal antagonism against strains common diners of the human oral flora. Furthermore, these two strains show a lack of inhibitory activity between them, thus allowing their combined use in a single formula. Furthermore, as can be seen in the examples below, the combination of these strains in a single formula (ie the composition of the invention) has the advantage of exhibiting a higher antagonistic activity against oral pathogens compared to the activity of the individual strains used separately . Thus, the strains of the invention exhibit cooperative activity against oral pathogens and are especially useful when used in combination.
    [0020] By antagonism of microorganisms that are involved 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 changing the oral microbiological profile to a more profile healthy, thereby benefiting oral health conditions.
    [0021] However, the unique ability of a bacterial strain to antagonize oral pathogens is not sufficient to ensure a probiotic effect in the oral cavity. The strains of the composition of the invention are good oral probiotics then, in addition to a strong antagonistic activity, as they exhibit a good ability to colonize the oral cavity. As can be seen in example 7 below, Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480 are able to grow in the presence of lysozyme and hydrogen peroxide. Advantageously, these strains also have a good ability to adhere to oral tissues.
    [0022] Furthermore, the strains of the invention have the advantage of exhibiting a high capacity to form aggregates. This is important because it allows said strains to inhibit or reduce dental plaque by interfering with pathogen biofilm formation. It is known that probiotic strains of lactic acid bacteria with aggregating activity can inhibit or reduce the formation of dental plaque by pathogenic bacteria, which is the result of aggregation of pathological bacteria with each other and with other microorganisms. As mentioned above, biofilm formed by oral pathogens in oral hard and soft tissues is considered to be an important etiological agent in pathological conditions of the mouth, leading to the onset of periodontal inflammation, caries formation and halitosis. Aggregate formation by the strains of the invention is increased when both strains are combined in one composition, meaning that the strains are more effective in displacing pathogenic bacteria when combined in a single formula.
    [0023] Surprisingly, the inventors have also found that strains CECT 7481 and CECT 7480 exhibit a particularly low acidification profile. Lactobacillus species, like most lactic acid bacteria, are characterized by a high production of volatile acids as a result of the fermentation of sugars in the human diet. However, the acidogenic property of these bacteria can be a possible side effect in the oral cavity, as it increases the risk of caries. In fact, many lactobacilli were considered to be cariogenic. Therefore, the reduced acid production exhibited by the Lactobacillus strains that form the composition of the invention makes them particularly suitable for health applications in the oral cavity.
    [0024] Above the beneficial properties discussed above, asceps of the invention have the advantage of producing no or very low amounts of off-flavour compounds such as volatile sulfur compounds, valeric acid, butyric acid and putrescine. This is also of relevance when applied to the composition of the invention in the oral cavity.
    [0025] Additionally, as they correspond to the strains for use as probiotics, the strains that form the composition of the invention belong to a bacterial species that has "Qualified Presumption of Safety" (QPS) status, as defined by the European Food Safety Authority (EFSA) . Furthermore, the inventors found that these strains do not exhibit any significant resistance to antibiotics of human and/or veterinary importance (ampicillin, gentamicin, streptomycin, erythromycin, tetracycline, clindamycin and chloramphenicol), thus preventing the risk of a potential transfer of resistance to antibiotics for pathogenic species.
    [0026] Considering the above, the strains of the invention have a better performance of all parameters relevant to an oral probiotic when compared to commercial strains that are known in oral probiotics in the art. As shown in the examples below, the new strains are more resistant to oral conditions, have greater ability to form aggregates, higher (and broader) 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 of said properties are included below. Additionally, the combination of both strains of the invention in a single composition generally results in a cooperative performance of the strains in relevant functionalities. Thus a composition comprising both strains is particularly suitable for use as an oral probiotic.
    [0027] By exerting various beneficial effects on the human host, the composition of the first aspect of the invention is useful as a medicine. 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 .
    [0028] Without wishing to be bound by theory, the beneficial effect of the strains that form the composition of the invention is the result of improving the microbiological profile of the oral cavity to produce a healthier oral flora. The growth of these beneficial bacteria in the oral cavity induces an environmental pressure that inhibits the growth of common pathogenic and/or opportunistic microorganisms. This environmental pressure is derived from the competition for adhesion sites and nutrients, the production of antimicrobial compounds and the displacement of pathogens by the aggregation of probiotic bacteria.
    [0029] An additional item to consider is the ability of probiotic bacteria to modulate the immune response. The mechanisms by which probiotics modulate immunity have been extensively studied in gastrointestinal structures. Probiotic species have shown their ability to alter the balance of pro-inflammatory and anti-inflammatory cytokines secreted by epithelial cells. Probiotics also regulate immune responses by enhancing innate immunity and modulating pathogen-induced inflammation through Toll-like receptor-regulated signaling pathways. The enhancement of local immune responses as well as systemic immune responses by probiotics may offer new opportunities for probiotics in preventing infections on peripheral mucosal surfaces, such as those in the oral cavity.
    [0030] Consequently, in a third aspect, the invention provides a composition comprising the strains of this invention for use as a medicine.
    [0031] In a fourth aspect, 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 of the oral cavity that is caused by oral pathogens in an animal, including a human being. Alternatively, this aspect may 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 of the oral cavity which is caused by oral pathogens in an animal, including a human being. .
    [0032] The invention also provides a method of preventing and/or treating a disorder of the oral cavity that is caused by oral pathogens in an animal, including a human administration, comprising administering to said animal in need thereof, the composition as defined in the first aspect of the invention.
    [0033] The term "disorder of the oral cavity that is caused by oral pathogens" is used in this application in its broadest meaning as any derangement or abnormality that can be found in the oral cavity that has been caused by an oral pathogen such as bacteria, viruses or yeast. Such a disorder can be a serious pathological condition as well as a trivial condition or discomfort. Illustrative non-limiting examples of "disorder of the oral cavity that is caused by an oral pathogen" are caries, gingivitis, periodontitis, candidiasis, herpes and ulcers, as well as halitosis, stained teeth, sensitive teeth, among others.
    [0034] In an embodiment of the fourth aspect of the invention, the composition is used for the treatment and/or prevention of a disorder-related dental plaque. Preferably, the dental plaque-related disorder is selected from the group consisting of caries, sensitive teeth, gingivitis and periodontitis.
    [0035] In another embodiment of the fourth aspect of the invention, the composition is used for the treatment and/or prevention of halitosis.
    [0036] In a further embodiment of the fourth aspect of the invention, the composition is used for the treatment and/or prevention of candidiasis.
    [0037] The composition according to the invention comprising the strains of the invention can be formulated as edible, cosmetic or pharmaceutical products. Said composition may comprise, in addition to the strains of the invention, one or more other cosmetically acceptable active agents and/or excipients (in the case of a cosmetic composition), pharmaceutically acceptable excipients (in the case of a pharmaceutical composition) or suitable edible ingredients (in the case of a cosmetic composition). case of an edible composition). In a particular embodiment of the invention, the composition of the invention further comprises one or more active agents. Preferably, the additional active agent or agents are other probiotic bacteria that are not antagonistic to the strains that make up the composition of the invention. More preferably, the additional active agent or agents are suitable for treating and/or preventing halitosis, candidiasis, caries, sensitive teeth and/or periodontal diseases. Depending on the formulation, the strains can be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture that has been post-treated, and alone or together with suitable vehicles or ingredients. Prebiotics can also be 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, tablets, pills, suspensions, emulsions, capsules, syrups, liquids , elixirs, extracts, tincture or fluid extracts or in a form that is particularly suitable for oral administration.
    [0039] In other aspects, the invention provides a pharmaceutical composition that contains the composition of the invention together with pharmaceutically acceptable excipients. In this sense, the pharmaceutical product can be prepared in any suitable form that does not negatively affect the bioavailability of the strains that form the composition of the invention. The selection of the most appropriate excipients and methods of formulation in view of the particular purpose of the composition is within the limits of the person skilled in the art of pharmaceutical technology.
    [0040] The term "pharmaceutically acceptable" as used in this application refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a individual (a human or non-human animal) without excessive toxicity, irritation, allergic response, or other problem or complication, proportionate to a reasonable benefit/risk ratio. Each vehicle, excipient, etc. it must also be "acceptable" in that it is compatible with other ingredients in the formulation. Suitable vehicles, excipients etc. can be found in standard pharmaceutical texts.
    [0041] Another aspect of the invention provides a cosmetic composition which contains the composition of the invention together with cosmetically acceptable excipients. In preventing and/or treating oral disorders caused by pathological bacteria, the composition of the invention is useful for ameliorating and/or preventing the symptoms produced by these disorders. Such symptoms include, but are not limited to, bad breath and stained teeth.
    [0042] The term "cosmetically acceptable" refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human skin without excessive toxicity, incompatibility, instability and allergic response, among others. Each carrier, "cosmetically acceptable" excipient, etc., must also be "acceptable" in that it is compatible with other ingredients of the cosmetic formulation. Suitable vehicles, excipients, etc. for cosmetic formulations can be found in standard texts.
    [0043] The strains of the invention can also be included in a variety of edible products, such as milk products, yogurt, rennet, cheese (for example, quark, cream, processed, soft and hard), fermented milk, milk powder, fermented milk-based product, ice cream, a fermented cereal-based product, a powdered milk-based product, a beverage, a topping and a pet food. The term "edible product" is used in this application in its broadest meaning, including any type of product, in any form of presentation, that can be ingested by an animal, but excluding cosmetic, pharmaceutical and veterinary products. Examples of other edible products are meat products (eg liver pate, German sausage and salami sausages or meat pate), chocolate spreads, fillings (eg truffle, cream) and toppings, chocolate, sweets (by example, caramel, candy, fondados or milk candy), baked products (cakes, sweets), sauces and soups, fruit juices and coffee whiteners. Forages for animal feed are also included within the scope of the invention. The compositions of the invention can also be used as an ingredient in other food products. Particularly interesting edible products are functional foods and infant formula.
    [0044] Consequently, in another aspect of the invention, an edible composition is provided containing the composition of the invention together with other edible ingredients.
    [0045] The term "edible ingredients" refers to ingredients that are capable of being consumed, that is, being used as food, by an animal, preferably, but not limited to, humans, livestock or pets.
    [0046] Often, probiotic bacterial compositions, such as those described in this application, are considered to be dietary supplements. Dietary supplements, also known as food supplements or nutritional supplements, provide beneficial ingredients that are not normally ingested in the normal diet. Predominantly, dietary supplements are considered food products, but are sometimes defined as pharmaceuticals, natural health products, or nutraceuticals. In connection with the present invention, dietary supplements also include nutraceuticals. Dietary supplements are usually sold "over the counter", that is, without a prescription.
    [0047] In a preferred embodiment, the composition of the invention is a dietary supplement.
    [0048] If the composition according to the invention is a dietary supplement, it can be administered as such, it can be mixed with a suitable drinkable liquid, such as water, yogurt, milk or fruit juice, or it can be mixed with the solid food or liquid. In this context, the dietary supplement may be in the form of tablets, pills, capsules, tablets, granules, powders, suspensions, sachets, lozenges, candies, bars, syrups and corresponding administration forms, usually in the form of a unit dose. Preferably, the dietary supplement comprising the composition of the invention is administered in the form of tablets, tablets, capsules or powders, manufactured in conventional processes for preparing dietary supplements.
    [0049] As can be derived from the above, the products comprising the composition of the invention are intended for use in oral health applications, preventing or treating an oral disorder, or ameliorating symptoms derived from these disorders. Accordingly, another aspect of the present invention provides an oral care product comprising the composition as mentioned above, together with pharmaceutically or cosmetically acceptable excipients, or other edible ingredients.
    [0050] With regard to the present invention, it may be that the composition is an oral product, which in the ordinary course of use, is not intentionally swallowed for the purpose of systemic administration of particular therapeutic agents, but is somewhat retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and/or oral tissues for the purposes of oral activity. Non-limiting examples of such products are toothpastes, dentifrices, dental powders, topical oral gels, mouthwashes, denture products, mouth sprays, chewing gum, dental floss or dental tapes. The oral composition can be a single phase oral composition or it can be a combination of two or more oral compositions.
    [0051] In one embodiment, the oral care product is a gum, a toothpaste, a mouthwash, a tablet, or an oral dispersible tablet. Preferably, the oral care products are in the form of tablets or oral dispersible tablets.
    [0052] The oral care products of the present invention may also comprise other orally active agents, such as actives for tooth whiteners, including bleaching or oxidizing agents such as peroxides, perborates, percarbonates, peroxyacids, persulfates, metal chlorides and combinations thereof. Tooth color modifying substances can also be considered among the oral care actives useful in the present invention. Oral care products can additionally comprise flavoring compounds such as menthol.
    [0053] The strains forming the composition of the invention are preferably in the form of viable cells. However, the strains of the invention may also be in the form of non-viable cells, such as dead cultures or compositions containing beneficial factors produced by Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480. This may include thermally killed microorganisms or dead microorganisms by exposure to altered pH, sonication, radiation or subjection to pressure. With non-viable cells product preparation is simpler, cells can be easily incorporated into commercial products and storage requirements are much less limited than viable cells.
    [0054] When used in the form of the composition of the invention, the strains can be in any proportion of concentration suitable for the intended use. For example, the strains can be in a concentration ratio that is comprised between 3:1 and 1:3 (Lactobacillus plantarum CECT 7481: Lactobacillus brevis CECT 7480). Preferably, the concentration ratio is 1:1. Furthermore, the strains of the invention are included in the composition in an amount effective for the required use.
    [0055] The term "effective amount" as used in this application 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 (in a reasonable benefit/risk ratio), within the scope of sound medical judgment. An effective amount of said probiotic micro-organism will be determined by one 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. For example, in oral care products, the strain or strains are present in an amount of approximately 105 cfu/g to approximately 1012 cfu/g, preferably in an amount of approximately 107 cfu/g to approximately 1011 cfu/g. The term "colony forming unit" ("cfu") is defined as the number of bacterial cells as revealed by microbiological counts on agar plates. In a particular embodiment, the composition of the invention is an oral care product comprising between 107-1010 cfu/g.
    Dietary supplements typically contain probiotic strains in an amount ranging from 105 and 1012 cfu/g. In a particular embodiment, the composition of the invention is a dietary supplement comprising between 107-1010 cfu/g.
    [0057] The strains of the invention are produced by culturing the bacteria in a suitable medium and under suitable conditions. The strains can be grown alone to form a pure culture, or as a mixed culture together with other microorganisms, or culturing the bacteria of different types separately and then combining them in the desired proportions. After culturing, the cell suspension is recovered and used as such or treated in the desired manner, for example, by concentration or vacuum freezing, to be used further in the preparation of the products. Sometimes the probiotic preparation undergoes an immobilization or encapsulation process in order to improve shelf life. Various techniques for immobilizing or encapsulating bacteria are known in the art. In a particular embodiment, the strains that are part of the composition of the invention are incorporated into an oral care product as encapsulated bacteria.
    [0058] As will be evident to the person skilled in the art, Lactobacillusplantarum CECT 7481 and Lactobacillus brevis CECT 7480 are effective not only when combined in a single composition, but also when used alone, or in two different compositions administered simultaneously, sequentially or separately after certain period of time. In addition, the person skilled in the art will understand that one of the strains may be prescribed to be used in conjunction with another strain for oral health in order to prevent or treat oral disorders, particularly disorders that are produced by oral pathogens, most preferably disorders related to dental plaque and/or halitosis and/or candidiasis.
    [0059] Consequently, one more aspect of the invention provides Lactobacillus plantarum CECT 7481.
    [0060] Finally, another aspect of the invention provides Lactobacillusbrevis CECT 7480.
    [0061] The strains of the invention are described in this application for the first time and are therefore new. As shown in Figure 1, both strains, Lactobacillus plantarum CECT 7481 and Lactobacillus brevis CECT 7480, have a different pulsed field gel electrophoresis model than closely related commercial lactobacilli strains such as Lactobacillus plantarum 299v, Lactobacillus plantarum VSL# 3, Lactobacillus casei VSL#3 and Lactobacillus casei DN 114.001.
    [0062] Some prior art documents describe compositions comprising strains of Lactobacillus plantarum and Lactobacillus brevis. However, the composition of the invention is new with respect to said compositions.
    [0063] Especially, KR100780030 describes a fermented soy milk comprising strains of Lactobacillus brevis and Lactobacillus plantarum. The strains described were isolated from kimchi, a meal consisting of fermented vegetables that is typical of Korea. Instead, the strains of the invention were isolated from the saliva of children in a developing South American region where kimchi is not consumed. Thus, the L. brevis and L. plantarum strains of the invention have a completely different origin, meaning that Lactobacillus plantarum CECT 7481 or Lactobacillus brevis CECT 7480 are the same as said Korean strains.
    [0064] Another Korean patent application, KR100866504, describes a red ginseng fermented using Lactobacillus plantarum P2 (microbial deposit number KCTC11391BP) and/or Lactobacillus brevis M2 (microbial deposit number KCTC11390BP). These strains were isolated from ginseng. Again, the Korean strains described have a completely different origin than the strains of the invention, as they were isolated from a plant that is not consumed in South American developing regions. This makes it impossible for Lactobacillus plantarum CECT 7481 or Lactobacillus brevis CECT 7480 to be the same as the aforementioned Korean strains.
    [0065] International patent applications WO2005/082157 and WO02/39825 again describe combinations of L. brevis and L. plantarum strains. The L. brevis LBR01 strain described in the composition of WO2005/082157 was isolated from mustard pickles in Taiwan. L. brevis C21 described in the compositions of WO 02/39825 was isolated from Mongolian tofu. As above, according to the completely different origins of these L. brevis strains it is impossible for them to be the same as the strain of the present invention. Furthermore, the described strains of L. brevis LBR01 and L. brevis C21 do not appear to be publicly accessible.
    [0066] Furthermore, WO 2006/080035 describes a gynecological composition which contains an unspecified strain of Lactobacillus brevis CD2 and Lactobacillus plantarum, together with a strain of L. salivarius. However, the L. brevis CD2 strain is the different form of Lactobacillus brevis CECT 7480. As shown in TABLE 4, when combining the Lactobacillus plantarum CECT 7481 strain with L. brevis 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, the L. brevis strain of the invention was shown to have a significantly lower acidification profile compared to commercial L. brevis CD2 (see TABLE 5). In this way, Lactobacillus brevis CECT 7480 is not only different from L. brevis CD2 but also better suited for oral hygiene applications. Altogether, having different effects, the combination of the invention is different from that described in WO 2006/080035.
    [0067] Finally, WO 02/018542 mentions that Lactobacillus strains frequently found in Cheddar cheese include strains of L. brevis and L. plantarum and patent application FR2448865 (page 10, lines 11-17) describes a composition containing L. plantarum to which L. brevis can be added. However, these documents do not describe a composition containing a specific L. brevis strain and a specific L. plantarum strain. The composition of the invention comprising a specific L. brevis strain, i.e. Lactobacillus brevis CECT 7480, and a specific L. plantarum strain, i.e. Lactobacillus plantarum CECT 7481 is therefore new.
    [0068] Throughout the description and claims the word "comprises" and its variations are not intended to exclude other technical attributes, additives, components or steps. Additional objects, advantages and attributes of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practicing the invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described in this application. The following examples and drawings are provided by way of illustration, and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
    [0069] Figure 1. Sfi-I (A, B) and Sma-I (C, D) restricted genomic DNA pulsed field electrophoresis models of: 1, Lactobacillus plantarum CECT 7481 (F2096); 2, Lactobacillus plantarum 299V; 3,Lactobacillus plantarum VSL#3; 4, Lactobacillus brevis CECT 7480 (I3141); 5, Lactobacillus casei VSL#3; 6, Lactobacillus casei DN114.001. M stands for molecular marker.EXAMPLES
    [0070] The following sections describe the characterization of the strains of the invention and their specific probiotic attributes regarding oral health applications. Isolation of microorganisms
    [0071] The new strains F2096 and I3141 were isolated from the saliva of children aged 0 to 5 years from a tropical South American developing region. Saliva was dissolved in PBS buffer (pH 7.4), aliquoted and plated on MRS agar (Man Rogosa Sharp, Sigma-Aldrich Chem, Spain) supplemented with 10 μg/ml vancomycin (SIGMA). The strains were cultivated under microaerophilic conditions (CO2 5%) at 37°C. Once the strains were cultivated, isolated were stored by lyophilization in PBS 0.1X with 15% skimmed milk powder.
    [0072] Strains F2096 and I3141 have been identified as Lactobacillusplantarum and Lactobacillus brevis, respectively (see section 2 below). Both strains have been deposited with the Spanish Type Culture Collection (Universitat de València, Campus de Burjassot, Edif de Investigación, 46100 Burjassot, València, Spain). Lactobacillus plantarum (strain F2096) was deposited on 01.21.2009 and given accession number 7481. Lactobacillus brevis (strain I3141) was deposited on 02.18.2009 and given accession number 7480. Both deposited strains are viable and maintained with all its attributes related to your deposit.
    [0073] As used below, strain F2096 corresponds to Lactobacillus plantarum CETC 7481, and strain I3141, to Lactobacillus brevis CECT 7480.
    Pediococcus acidilactici F2019 (hereinafter referred to as P.acidilactici), Lactobacillus paracasei I3152 (hereinafter referred to as L. paracasei) and Pediococcus pentosaceus I54 (hereinafter referred to as P. pentosaceus) were isolated from human saliva as explained above. Streptococcus salivarius K12 was isolated from commercial BLIS K12® in TBS (tryptone soy broth) and cultivated under aerobic conditions at 37°C. The strain of Lactobacillus reuteri ATCC 55730 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 114,001 of Actimel®. All the last strains were isolated in MRS, and grown at 37°C with 5% CO2. Potentially pathogenic strains of Porphyromonas gingivalis CIP 103683, Fusobacterium nucleatum CIP 104988, Treponema denticola CIP 103917 and Prevotella denticola CIP 104478T were obtained from the Institut Pasteur, and grown following the supplier's instructions. Streptococcus mutans of clinical origin was cultivated in Brain Heart Agar at 37°C and 5% CO2. The identification of strains was performed by sequencing. Taxonomic characterization of strains 2.1. Genetic identification of genus and speciesMethods
    The strains of the invention were grown overnight in MRS medium (pH 6.4) at 37°C in an atmosphere containing 5% CO2. Bacteria were also collected, washed and resuspended in pre-lysis buffer (480 μl of 50 mM EDTA pH 8.0; 120 μl of 10 mg/ml lysozyme), and further incubated at 37°C for 60 min. DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). After centrifuging the pretreated bacteria at 14000 g for 2 min to remove the supernatant, the Promega protocol was followed. In a nutshell, the bacteria were resuspended in the Nucleus Lysing 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 Protein Precipitation Solution and centrifuged at high speed. The samples were cooled and centrifuged at 15000 g for 3 min. Supernatants containing the DNA were transferred to purify 1.5 ml microcentrifuge tubes and mixed with 600 µl of isopropanol by inversion. DNA was collected by centrifugation at 15000 g for 2 min and carefully discarding the supernatant. DNA samples were washed with 600 µl 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 precipitate was resuspended in 100 μl of Rehydration Solution by incubation at 65°C for 1 h. Samples were stored at 2-8°C.
    [0076] The 16S rRNA was amplified by PCR using the universal primers Eub27f and Eub1492r, which produce a fragment close to the complete 16S sequence (more than 1000 nucleotides) (TABLE 1). Then, the DNA obtained as explained above was washed using the Quiaquick kit (Quiagene).
    [0077] Four consecutive sequencing reactions were performed for each sample on a 3130 Genetic Analyzer (Applied Biosystems) using BigDye v.3.1 kit, using the primers shown in TABLE 1. Data collection and chromatograms were constructed using the DNA Sequence program Analysis v.5.2 (Applied Biosystems) and verified the visual analysis with Chromas (Technelysium Pty Ltd.) and BioEdit (Ibis Biosciences).
    [0078] Genus identification was performed 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, volume 73, p. 5261-5267). Species identification was performed by comparing the sequence obtained with sequences from 16 of known organisms both from the RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/) using a BLASTN, as well as from the Ribosomal Database Project(http://rdp.cme.msu.edu/, JR Cole et al., "The Ribosomal Database Project (RDP-II): introducing myRDP space and quality controlled public data", Nucl. Acids Res.^ 2007, volume 35, p. 169-172).TABLE 1. Primers used for amplification and sequencing of the 16S gene
    B) Results
    [0079] The RDP tool (Ribosimal Database Project) identified the strain F2096 as belonging to the species Lactobacillus plantarum and strain I3141 as belonging to the species Lactobacillus plantarum. 2.2. Strain GenotypingMethods
    [0080] The characterization was performed by genomic digestion and pulsed-field gel electrophoresis. The F2096 and I3141 strains were submitted to a previously described protocol (Rodas AM, et al." Polyphasic study of wine Lactobacillus strains: taxonomic implications", Int J Syst Evol Microbiol, 2005, vol. 55, p. 197-207). The commercial strains, Lactobacillus plantarum 299V, Lactobacillus plantarum VSL#3, Lactobacillus casei VSL#3 and Lactobacillus casei DN 114.001 were also included in the assay as control strains. All strains were grown on MRS agar plates and incubated at 37°C, 5% CO2 for 18 h. Cells were collected and washed 3 times in 8 ml of PET (10 mM Tris pH 7.6, 1 M NaCl), then centrifuged at 6000 rpm for 10 min. The precipitates were resuspended in 700 µl of lysis buffer (6 mM Tris, 1M NaCl, 0.1 M EDTA, 0.5% SLS, 0.2% deoxycholic acid; 1 mg/ml lysozyme; 40 U/ml mutanolysin; 20 (g/ml RNase) An equal volume of 1.6% low melting point agarose (FMC BioProducts, Rockland, ME, USA) was added to the resuspended cells and allowed to solidify at 4°C for 1 h The inserts were transferred to 2 ml of lysis buffer II (0.5 M EDTA pH 9.2, 1% N-lauryl sarcosine and 1 mg/ml pronase) and incubated at 50°C for 48 h. were washed at room temperature with TE buffer (10 mM Tris, 1 mM EDTA pH 8.0) Total DNA digestion was performed by restriction enzymes Sfi-I and Sma-I (Roche Diagnostics).
    Pulsed field electrophoresis was performed using the CHEF DRIII apparatus (BioRad Laboratories). Inserts were loaded into 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 labeled with ethidium bromide and UV using the GelDoc System (BioRad). TABLE 2. Sfi-I and Sma-I electrophoresis conditions restricted genomic DNA from I3141 and F2096 strains.

    [0082] As shown in Figure 1, the pulsed-field electrophoresis Sfi-I eSma-I restriction models of the F2096 strain differed from those for the commercial Lactobacillus plantarum 299v and Lactobacillus plantarum VSL#3 strains, while the restriction models of I3141 differed from those of closely related commercial Lactobacillus casei strains. Thus, it can be concluded that strains F2096 and I3141 are new strains. Ability to antagonize pathogensMethods
    [0083] In order to assess whether strains F2096 and I3141 showed antagonistic activities, a Campbell protocol was performed using agar plates seeded with bacterial pathogens in Oxoid medium. The pathogens used in this study were selected from those commonly present in the human oral cavity (see TABLE 1). Briefly, F2096, I3141 and P. acidilactici, another strain isolated from human saliva, were cultured overnight, each under the specific conditions mentioned above. After incubation, cultures were standardized to 108 cfu/ml and the following mixed cultures were prepared: F2096 + I3141, F2096 + P. acidilactici, and I3141 + P. acidilactici. Said mixed cultures contained an equal amount of each of their constituent strains and the same total bacterial concentration as the single strain cultures, i.e., 108 cfu/ml. A fixed volume of each of the single strain cultures and mixed cultures was plated evenly and grown to confluence at the appropriate temperatures in a 5% CO2 incubator. Then, uniformly sized cylindrical sections sorted from confluent agar plates were placed complement-to-complement on the pathogen plate and incubated overnight at 37°C.
    [0084] The following day, zones of inhibition were measured by placing the agar plate on a flat ruler. The antagonistic properties of the strains were measured as growth inhibitory activity (GIA), which was calculated by subtracting the cylinder diameter (CD) from the inhibition zone diameter (IZD) and dividing this difference by two following the formula GIA = (IZD- CD)/2. The inhibition capacities of the strains of this invention were compared with that of the commercial oral probiotic strains Streptococcus salivarius K12 and Lactobacillus reuteri ATCC 55730.B) Results
    [0085] The growth inhibitory activities of the F2096 and I3141 strains are shown in TABLE 3. The results are the average of experiments in triplicate. TABLE 3. Growth inhibitory activity against oral pathogens
    NI, no inhibition
    [0086] As can be seen from the present results, both P2096 and I3141 have a broad model of inhibition against oral pathogens. This is especially relevant for P. gingivalis, as a preliminary study has shown that antagonistic activity against this pathogen is rare (data not shown). Furthermore, it is notable that the combination of F2096 and I3141 in a mixed culture exhibits a higher antagonistic activity against oral pathogens compared to the activity of the individual strains used separately as single cultures - it should be noted that the concentration of each strain in the mixed culture is half of that in single cultures, so the antagonistic effect of the combination is higher than the sum of the individual effects for these strains. This synergistic effect did not occur when each of the strains was combined with another inhabitant of the oral cavity, P. acidilactici. Thus, the strains of the invention exhibit synergistic activity against oral pathogens and are especially useful when used in a single formula.
    When compared to commercial probiotics Streptococcus salivarius K12 and Lactobacillus reuteri ATCC 55730, the strains of the invention have a significantly higher antagonistic capacity. Finally, F2096 and I3141 exhibited minimal antagonism against each other or the common commensal strains of human oral flora. 4. Formation of aggregatesMethods
    [0088] The ability to form aggregates was evaluated by monitoring the decrease in optical density at 620 nm of nocturnal cultures due to aggregate formation and precipitation. The percent aggregation capacity (%AC) value was obtained using the following formula: %AC = (1- (ODtf/ODt0)/100, where ODtf and ODt0 are the optical density in final and initial time, respectively. initials was adjusted so that all cultures contained an equivalent number of cfu/ml. Combined cultures were prepared by mixing an appropriate amount of single strains to obtain the desired total cfu/ml containing 50% of each strain.B) Results
    [0089] The ability to form aggregates is important for probiotic strains with oral hygiene applications because it allows said strains to inhibit or reduce dental plaque by interfering with biofilm formation of pathogens.
    [0090] The mean aggregation capacity values of the new strains and their combination between them or with P. acidilactici or L. brevis CD2 are shown in TABLE 4 when compared to control strains. These results clearly demonstrate that the new isolates showed very good aggregation activity, significantly higher than both the commercial controls S. salivarius K12 and L. reuteri ATTC 55730. Furthermore, aggregate formation by the strains of the invention is increased when both strains strains are combined in a varied culture, meaning the strains are more effective in displacing pathogenic bacteria when combined into a single composition. This cooperation is, however, not present when the strains of the invention are separately combined with other strains with good aggregation capacity, such as P. acidilactici and L. brevis CD2. Furthermore, by combining the strains of the invention with L. brevis CD2, an antagonistic effect is observed, meaning that the strains of the invention are less effective in displacing pathogenic bacteria when combined with L. brevis CD2.TABLE 4. Aggregation capacity (% )
    5. Acid production Methods
    [0091] The ability of the new strains to produce acid when growing culture media supplemented with the different sugars present in the human diet was evaluated. The strains were cultivated for 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. The minimal medium contained peptone 2 g/L, water, yeast extract 2 g/L, NaCl 0.1 g/L, K2HPO4 0.04 g/L, KH2PO4 0.04 g/L, MgO4*7H2O 0.01 g/L, CaCl2*6H2O 0.01 g/L, NaHCO3 2 g/L, hemine 0.05 g/L (dissolved in a few drops of NaOH 1mol/L), cysteine HCl 0.5 g/L, salts of bile 0.5 g/L, Tween 80 2 g/L, and 10 μl vitamin K1 (all components obtained from Sigma-Aldrich Chem, Spain). Cultures were adjusted to pH 7 with HCl. The pH and number of viable cells (cfu/ml) were measured at the end of the incubation time. The production of the acid value of each culture medium was obtained by the following formula: PA value = pH * log (cfu/ml).B) Results
    [0092] According to the formula represented above, the low values correspond to highly acidogenic strains. High acid production is an undesirable side effect of oral probiotics as it promotes caries formation. The acid production value of F2096 and I3141 and several probiotic control strains grown on different sugars can be seen in TABLE 5, together with the values obtained for commercial strains. MM means minimal medium.TABLE 5. Acid production

    [0093] These results demonstrate that the growth of strains F2096 and I3141 on different sugars result in a remarkably poor acid production when compared to other bacterial strains that had also been isolated from the oral cavity, ie, L. paracasei, P. pentosaceus and P. acidilactic. The strains of the invention are also less acidogenic than commercial oral probiotic S. salivarius K12. Additionally, strain I3141 has a lower acidification profile compared to L. reuteri ATTC 55730, which is known to be particularly low acidogenic and is being sold as an anticariogenic probiotic. It is also notable that the L. brevis strain of the invention 13141 has a significantly lower acidification profile compared to commercial L. brevis CD2. Therefore, strains F2096 and I3141 have a poor acidification profile, thus being suitable for oral hygiene applications.6. Production of Bad Flavor Volatile Compounds Methods
    [0094] The production of volatile bad aroma compounds by the new strains was determined by growing in a culture medium that resembles the human diet through a sensory evaluation. Briefly, the strains were grown in a medium containing glucose (0.5% w/v), fructose (0.5% w/v), yeast extract (1% w/v), meat extract (1% w/v v), eukaryotic cells (200 cells/ml) and pectin (0.5% w/v) for 48 h at 37°C and 5% CO2. The strains receive a production of a bad aroma value between 1 and 5, where 1 is the absence of odor and 5 is a very unpleasant odor.B) Results
    [0095] The production of bad aroma compounds is very undesirable for an oral probiotic. However, the strains of the invention did not produce any unpleasant odor at all growing in a culture medium similar to the human diet.TABLE 6. Production of unpleasant odors
    7. Survival to oral conditionsMethods
    [0096] The survival of the strains in the oral cavity was studied by subjecting them to oral stress conditions. 5*107 cfu of each bacterial strain were inoculated in 96-well culture plates in 200 µl of MRS medium, in the case of F2096, I3141 and L. reuteri ATCC 55730, or Tryptone Soy Broth (TSB, Oxoid) in the case of S . salivarius K12. Cultures were supplemented with physiological concentrations of lysozyme (Sigma-Aldrich Chem, Spain) or hydrogen peroxide (Sigma-Aldrich Chem, Spain). The plates were incubated at 37°C and 5% CO2 for six hours. Bacterial growth was quantified by measuring optical density at 620 nm. The bacterial growth value was obtained by comparison with the growth achieved by the same strain in the MRS pattern of medium without supplements.B) Results
    [0097] A percentage overall survival value (%SV) was calculated as follows: %SV = [(ODtf-ODt0 (with the supplement)) / (ODtf-ODt0 (without the supplement))] * 100, where OD is the optical density, and tf and t0 are the final time and the initial time, respectively. The following values are the average of scores in triplicate: TABLE 7. Survival to oral conditions

    [0098] The new strains F2096 and I3141 showed better resistance to oral stress than the commercial strains S. salivarius K12 and L. reuteri ATCC 55730.8. Ability to adhere to oral tissuesMethods
    [0099] In vitro adhesion assay of F2096, I3141, and commercial control strain to pig tongue, Caco-2 cells (to simulate gum) and hydroxyapatite (HA) coated beads (to simulate teeth) was performed. Each strain was incubated overnight with 10 µL/ml 5-[3H]thymidine (1.0 µCi/ml, Amersham Biosciences, UK). The preparations were centrifuged and the precipitates resuspended in PBS buffer at a concentration of 108 cfu/ml. The tritium signal incorporated into the microorganisms is calculated from the initial tritium signal and the supernatant signal in a scintillation reader (Wallac 1410). The ratio between this number (signal incorporated into the biomass) and the total number of microorganisms in the culture results in cpm/cfu (signal/bacteria).
    The measurement of adhesion was performed by adding 3.3 ml of 108 radio-labeled cfu from each strain to 1.05*0.5 cm fragments of pig tongue or pre-incubated Caco-2 or 50 cells mg of HA beads (80 µm in diameter) on ELISA plates. After 45 min, supernatants were carefully removed. HA beads (along with adhered bacteria) were recovered by centrifugation. Tongue or Caco-2 cells together with adherence bacteria were discarded from the ELISA wells. Finally, recovered bacteria were lysed to calculate specific radioactivity (cpm/cfu).
    [00101] All assays were performed in triplicate and results expressed as the number of bacteria adhered per cm2. Since S. salivarius exhibited the best results from the most investigated properties (see above for results), this strain was used as a control strain in the adhesion assay.B) ResultsTABLE 8. Adhesion to oral tissues

    [00102] These results demonstrate that the F2096 and I3141 strains have the best overall adhesion capacity to oral tissue when compared to the oral probiotic strain S. salivarius K12. The ability to adhere to oral tissues is a relevant attribute of strains to be used as probiotics as it gives these bacteria a competitive advantage by competing for adhesion sites against pathogens and favoring permanence in the oral cavity. As a result, strains that exhibit good adhesion to oral tissues help to displace pathogens from the oral cavity and promote healthier oral flora.9. Antibiotic susceptibilityMethods
    [00103] The antibiotic susceptibility of F2096 and I3141 strains was studied after 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". EFSA Journal, 2008, vol. 732, p. 1-15). The culture conditions of the strains were as follows: growth on the surface of MRS agar plates containing antibiotic concentrations recommended by EFSA at 37°C and 5% CO2.B) Results
    [00104] The growth of strains in media containing antibiotics is shown in TABLE 9. The concentrations of each antibiotic to be analyzed as recommended by EFSA are given in mg/ml. No indicated value of antibiotic concentrations is specified by EFSA of the species L. brevis, therefore, antibiotic resistance in strain I3141 of L. brevis was analyzed using the recommended concentrations for obligate heterofermentative lactobacilli, group to which this species belongs.
    [00105] The results indicate that F2096 and I2141 do not show any resistance to antibiotics. They are, therefore, suitable for human consumption.TABLE 9. Antibiotic resistance of strains F2096 and I3141.
    nr, not recommended by EFSA REFERENCES BIBLIOGRAPHICASTwetman S, et al. "Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory mediators in gingival crevicular fluid". Odontol Scand Acta, 2009, vol. 67, p. 19-24. Caglar E, et al. "Salivary mutans streptococci and lactobacilli levels after ingestion of the probiotic bacterium Lactobacillusjeuteri ATCC 55730 by straws or tablets". Odontol Scand Acta, 2009, vol. 64, p. 314-318. 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. 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. 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.Ribosomal Database Project: http://rdp.cme.msu.edu/JR Cole et al., "The Ribosomal Database Project (RDP-II): introducing myRDP space and quality controlled public data", Nucl. Acids Res., 2007, vol. 35, p. 169-172. Wheels AM, et al. "Polyphasic study of wine Lactobacillus strains: taxonomic implications", Int J Syst Evol Microbiol, 2005, vol. 55, p. 197-207."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. BUDAPEST TREATY ON INTERNATIONAL RECOGNITION OF THE MICROORGANISM DEPOSIT FOR PATENT PROCESSING PURPOSES INTERNATIONAL FORMAT
    Where Rule 6.4(d) applies, such date is the date on which status with the International Depositary Authority was obtained.
    权利要求:
    Claims (15)
    [0001]
    1. Composition, characterized in that it comprises Lactobacillus plantarum deposited at the Spanish Type Culture Collection (CECT) under accession number CECT 7481 and Lactobacillus brevis deposited at the Spanish Type Culture Collection (CECT) under accession number CECT 7480.
    [0002]
    2. A probiotic product, characterized in that it comprises a composition as defined in claim 1.
    [0003]
    3. Use of a composition as defined in claim 1, characterized in that it is for the manufacture of a medicine to prevent or treat a disorder in the oral cavity that is caused by oral pathogens in an animal, including a human, in which the oral cavity disorder is: (i) a dental plaque-related disorder, wherein the dental plaque-related disorder is selected from the group consisting of gingivitis, periodontitis, caries, or sensitive teeth; (ii) halitosis; or (iii) candidiasis.
    [0004]
    4. Pharmaceutical composition, characterized in that it comprises a composition, as defined in claim 1, together with at least one pharmaceutically acceptable excipient.
    [0005]
    5. Pharmaceutical composition according to claim 4, characterized in that it is a dietary supplement.
    [0006]
    6. Edible product, characterized in that it comprises a composition, as defined in claim 1, and at least one edible ingredient.
    [0007]
    7. Edible product according to claim 6 characterized by the fact that it is a dietary supplement.
    [0008]
    8. Cosmetic composition, characterized in that it comprises a composition, as defined in claim 1, together with cosmetically acceptable excipients.
    [0009]
    9. Oral care product, characterized in that it comprises an effective amount of the pharmaceutical composition, as defined in claim 4, or the edible product, as defined in claim 6, or the cosmetic composition, as defined in claim 8.
    [0010]
    10. Oral care product according to claim 9, characterized in that it is a gum, a toothpaste, a mouthwash, a tablet or an oral dispersible tablet.
    [0011]
    11. Use of a strain of Lactobacillus plantarum deposited at the Spanish Type Culture Collection (CECT) under accession number CECT 7481, characterized by the fact that it is for the manufacture of a composition.
    [0012]
    12. Use of a Lactobacillus brevis strain deposited at the Spanish Type Culture Collection (CECT) under accession number CECT 7480, characterized by the fact that it is for the manufacture of a composition.
    [0013]
    13. Composition, characterized in that it comprises: (a) an amount of 105 cfu/g to 1012 cfu/g of a Lactobacillus plantarum strain deposited with the Spanish Type Culture Collection (CECT) under accession number CECT 7481; and (b) at least one pharmaceutically acceptable excipient, at least one edible ingredient and/or at least one cosmetically acceptable excipient.
    [0014]
    14. Composition, characterized in that it comprises: (a) an amount of 105 cfu/g to 1012 cfu/g of a Lactobacillus brevis strain deposited at the Spanish Type Culture Collection (CECT) under accession number CECT 7480; and (b) at least one pharmaceutically acceptable excipient, at least one edible ingredient and/or at least one cosmetically acceptable excipient.
    [0015]
    15. Composition, characterized in that it comprises a strain of Lactobacillus plantarum deposited at the Spanish Type Culture Collection (CECT) under the accession number CECT 7481, and a strain of Lactobacillus brevis deposited at the Spanish Type Culture Collection (CECT) under the number Accession No. CECT 7480, for use in preventing or treating a disorder in the oral cavity that is caused by oral pathogens in an animal, including a human.
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    同族专利:
    公开号 | 公开日
    CN103298924B|2016-02-03|
    CN103298924A|2013-09-11|
    EP2606155B1|2017-10-18|
    WO2012022773A9|2012-05-24|
    RU2013111767A|2014-09-27|
    MX2013001656A|2013-03-20|
    EP2420580A1|2012-02-22|
    AU2011290705A1|2013-02-07|
    US20150273000A1|2015-10-01|
    US20130209374A1|2013-08-15|
    ES2655913T3|2018-02-22|
    DK2606155T3|2018-01-22|
    JP5879349B2|2016-03-08|
    AU2011290705B2|2014-09-11|
    EP2606155A1|2013-06-26|
    WO2012022773A1|2012-02-23|
    RU2584610C2|2016-05-20|
    MX346458B|2017-03-22|
    BR112013003761A2|2016-05-31|
    US9192634B2|2015-11-24|
    US9511102B2|2016-12-06|
    JP2013535226A|2013-09-12|
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    法律状态:
    2017-10-24| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-01-16| B25G| Requested change of headquarter approved|Owner name: AB-BIOTICS S.A. (ES) |
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-05-02| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|
    2019-05-14| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2020-06-23| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-04-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-06-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/08/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP10173286.5|2010-08-18|
    EP10173286A|EP2420580A1|2010-08-18|2010-08-18|Probiotic composition for oral health|
    PCT/EP2011/064173|WO2012022773A1|2010-08-18|2011-08-17|Probiotic composition for oral health|
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