EA042202B1 - ANTISEPTIC-RESISTANT LACTIC BACTERIA - Google Patents

Description

As explained above, antiseptics are generally known to have a broad spectrum of antimicrobial activity. This allows you to kill many types of bacteria and fungi, but they also have a negative effect on symbiotic or probiotic bacteria. In fact, it is known that trace amounts of antiseptics can be detected after their administration, which also compromises the use of probiotics even after the administration of an antiseptic. Therefore, the use of lactic acid bacteria, especially as a probiotic treatment, has never been considered possible in combination with antiseptics.

The present inventors surprisingly found that strains of Pediococcus have a greater ability to tolerate antiseptics, thus it is possible to introduce these strains when antiseptics are used for therapeutic or cosmetic purposes. Without wishing to be bound by a particular theory, it is believed that by the principle of competitive exclusion, which favors harmless bacteria if they attach before pathogenic strains attach, administration of Pediococcus in combination with antiseptics may be particularly effective in protecting against colonization by opportunistic pathogens.

Therefore, in one aspect of the present invention, a Pediococcus strain is provided for use in combination with an antiseptic, said Pediococcus strain having the ability to tolerate the presence of at least one antiseptic selected from the group consisting of

a) triclosan;

b) an essential oil containing at least one individual component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate; And

c) a single component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate, wherein the Pediococcus strain is considered to have the ability to tolerate said antiseptic when the inhibition halo, as determined by the disk diffusion assay method, observed for the Pediococcus strain, less than the Porphyromonas gingivalis inhibition halo, wherein said antiseptic is at a concentration sufficient to observe Porphyromonas gingivalis inhibition, and the Pediococcus strain and the antiseptic are formulated for separate, sequential, simultaneous, or mixture administration.

This first aspect can alternatively be formulated as the use of a Pediococcus strain in combination with an antiseptic, said Pediococcus strain having the ability to tolerate the presence of at least one of the antiseptics described above.

Antiseptic-resistant strains of lactic acid bacteria.

Preferably, the strain of Pediococcus, which is discussed in this application, was an isolated strain of lactic acid bacteria. One of skill in the art will understand that in this context, isolated refers to a strain of lactic acid bacteria that has been isolated from the natural environment, i.e. it is not present in its natural environment, hence it is free from other organisms and substances present in its natural environment.

The term tolerance in this application refers to the ability, either inherited or not, of a microorganism to survive under temporary exposure to an antiseptic, which is often achieved by slowing down a vital process in the bacterium. The ability to tolerate is usually understood in the literature as the ability of microorganisms to survive without changing the minimum inhibitory concentration (MIC). However, it can also be understood as a reduced sensitivity of a microorganism to an antimicrobial agent, which is characterized by an increase in the minimum inhibitory concentration (MIC), or as a situation in which the antimicrobial agent no longer prevents microbial growth.

The term antiseptic in this application refers to an antimicrobial agent that destroys or inhibits the growth of microorganisms in or on living tissue. The term antimicrobial

- 5 042202 of the present invention has the usual meaning in this field, namely, an agent that kills microorganisms or inhibits their growth. They can be further grouped according to the microorganisms against which they primarily act, for example against bacteria (antibacterial agents) and against fungi (antifungal agents). Antiseptics are generally distinguished from antibiotics because antibiotics tend to travel through the lymphatic system to kill bacteria within the body, and from disinfectants, which kill microorganisms found on non-living objects.

Based on the detailed description of the assays given in Examples 2-3, one skilled in the art will be able to repeat said assay in the usual manner to objectively determine whether a particular strain has the ability to tolerate antiseptics. In particular, Example 2 provides a method for determining whether a given strain is capable of tolerating a specified antiseptic, wherein the antiseptic tolerance is expressed as an inhibition halo determined by a disk diffusion assay method. For example, a strain of Pediococcus is considered to be able to tolerate said antiseptic when the inhibition halo as determined by disk diffusion analysis observed for the Pediococcus strain is less than the inhibition halo of Porphyromonas gingivalis, while said antiseptic is at a concentration sufficient to observe inhibition. P. gingivalis.

In particular embodiments, a Pediococcus strain will be considered to be capable of tolerating said antiseptic when the inhibition halo diameter determined by the disc diffusion assay for the Pediococcus strain is at least 1, 2, 3, or 4 mm smaller than the diameter determined for P. gingivalis. In a more specific embodiment, the inhibition halo diameter observed for Pediococcus is at least 4 mm smaller than the inhibition halo diameter determined for P. gingivalis.

The strains of Pediococcus proposed in the present invention are characterized by the ability to tolerate at least one of the antiseptics described in the first aspect. However, it is important to note that in addition to this ability, these strains can also be resistant to other appropriate antiseptics, as shown in the examples. This means that the property described in the first aspect can be considered a key property or marker for identifying a group of Pediococcus strains useful for the purposes of the present invention.

In the examples, various strains are provided that meet the requirements for having this ability to tolerate antiseptics better than P. gingivalis. In addition to these strains, it is quite possible to identify and isolate other strains from the LAB population with the same ability to tolerate antiseptics using the method described in detail. The examples also demonstrate that the ability to tolerate antiseptics is useful for the purposes of the present invention. Thus, although some specific strains with this useful property have been investigated and identified, there is no reason to limit the scope of the present invention to these strains, since all the steps of the method for obtaining other useful strains are exhaustively described in this application. Therefore, according to the present invention also proposed a set of strains other than those used in the examples, which have the same property. It is important to note that not all KSD strains will be able to tolerate antiseptics; thus, the present invention provides a method for detecting them.

It is likewise pertinent to note that the use of the disk diffusion assay method and the assay conditions described in Example 2 do not limit the scope of the present invention. This assay is just one of the assays suitable for testing the ability of strains to tolerate antiseptics. The concentrations and quantities of antiseptics will be selected by a specialist to test the ability of strains to tolerate antiseptics, depending on the specific antiseptic to be tested. In particular, the concentrations and amounts of antiseptics to be applied will be those necessary to observe P. gingivalis inhibition.

Triclosan (CAS Registry Number: 3380-34-5) (TCS) is a diphenyl ether and can be called 2,4,4'-trichloro-2'-hydroxydiphenyl ether, 5-chloro-2-(2,4dichlorophenoxy )phenol or Triclosanum. The molecular formula of TCS is C12H7CI3O2 and the molecular weight of this chemical is 289.55. Most commercially produced TCS is over 99% pure and is available in solid form as a white to off-white crystalline powder with a subtle aromatic odor. Triclosan is effective against many types of bacteria and some types of fungi, preventing bacteria from multiplying and/or eventually causing cell death. It penetrates the bacterial cell wall and targets a variety of cytoplasmic and membrane sites, including RNA synthesis and the production of various biomolecules.

Essential oils are also used as alternative antiseptics, particularly for oral hygiene. Essential oils (EOs) in this application are oils derived from vegetable or animal sources, or their synthetic equivalents, and they consist of complex mixtures of several components, such as monoterpene and sesquiterpene hydrocarbons, mono-terpene and sesquiterpene alcohols, complex ethers, ethers, aldehydes, ketones, oxides and the like.

The individual components of essential oils are compounds that can be isolated from the oil (natural) or can be completely or partially chemically synthesized.

Menthol may also be referred to as 2-isopropyl-5-methylcyclohexanol or 5-methyl-2-(1methylethyl)cyclohexanol and is an organic compound made synthetically or derived from field mint, peppermint or other mint oil.

Eucalyptol is a cyclic ether and monoterpenoid, which is especially rich in essential oils of plants from the genus Eucalyptus. Eucalyptol is also known by various synonyms: 1,8-cineol, kayaputol, 1,8-epoxy-para-menthane, 1,8-oxido-para-menthane, eucalyptol, 1,3,3-trimethyl-2oxabicyclo[2,2 ,2]octane, cineol.

Thymol (also known as 2-isopropyl-5-methylphenol) is a naturally occurring monoterpene phenolic derivative of cymene, isomeric to carvacrol, found in thyme oil and isolated from Thymus vulgaris (common thyme) and various other plant species as a white crystalline substance with a pleasant aromatic odor and strong antiseptic properties.

Methyl salicylate is an organic ester produced naturally by many plant species, especially wintergreens. Methyl salicylate is also commonly referred to as methyl 2hydroxybenzoate, wintergreen oil, wintergreen oil, or birch oil.

In one embodiment, the Pediococcus strain further has the ability to tolerate a bisguanide antiseptic.

Biguanides are a class of chemically related compounds known for their bactericidal properties. These compounds include the antiseptics alexidine and chlorhexidine. Alexidine may also be referred to as 1-[K'-[6-[[amino-[[K'-(2ethylhexyl)carbamimidoyl]amino]methylidene]amino]hexyl]carbamimidoyl]-2-(2-ethylhexyl)guanidine. Chlorhexidine is a cationic polybiguanide and may also be referred to as (1E)-2-[6[[amino-[(E)-[amino-(4-chloroanilino)methylidene]-amino]methylidene]amino]hexyl]-1- [amino-(4chloroanilino)methylidene]guanidine.

In particular, the Pediococcus strain has the ability to tolerate chlorhexidine gluconate.

The present inventors have also found that Pediococcus strains may also have the ability to inhibit (ie, antagonize) the growth of bacterial pathogens, especially oral pathogens. This allows Pediococcus strains to be particularly useful in avoiding pathogen colonization and even inhibiting their growth, thus improving the microbiota profile.

Thus, in one embodiment, the Pediococcus strain further has the ability to antagonize at least one pathogen selected from the group consisting of Fusarium nucleatum, Treponema denticola, and Prevotella denticola. In particular, the Pediococcus strain has the ability to antagonize at least two of these pathogens. In particular, the Pediococcus strain has the ability to antagonize F. nucleatum, T. denticola and P. denticola.

In one particular embodiment, the Pediococcus strain further has the ability to antagonize Tannerella forsythia.

In one particular embodiment, the Pediococcus strain further has the ability to antagonize Porphyromonas gingivalis.

Based on the detailed description of the assay in this application (see example 4 assay for antagonistic activity), one skilled in the art will be able to repeat the assay in the usual manner to objectively determine whether the strain antagonizes the pathogens of interest. The descriptions and conditions of said antagonistic activity assay do not limit the scope of the present invention. This assay is an assay suitable for testing the ability of strains of interest to antagonize pathogens.

In a particular embodiment, the Pediococcus strain is a Pediococcus acidilactici strain. In particular, the Pediococcus strain is Pediococcus acidilactici deposited with the Spanish Type Culture Collection under accession number CECT 8633.

Thus, in one aspect of the present invention, there is provided a strain belonging to the species Pediococcus acidilactici deposited in the Spanish Type Culture Collection under accession number CEST 8633.

In another aspect of the present invention, a composition is provided that contains from 10 ⁴ to 10 ¹² cfu/g of viable cells of at least one strain of Pediococcus, while the Pediococcus strain has at least the properties (i.e., the ability to tolerate antiseptics) described in the first aspect.

Compositions according to the present invention may contain a single strain or be a combination of different strains of LSD belonging to the same or different species. In a particular embodiment, the Pediococcus strain of the present invention is in combination with a Lactobacillus plantarum strain and/or a Lactobacillus brevis strain. In particular, the composition containing strains of Pediococcus according to the present invention further comprises a strain

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Lactobacillus plantarum deposited with the Spanish Type Culture Collection under Accession No.

CEST 7481, and a strain of Lactobacillus brevis deposited with the Spanish Type Culture Collection under accession number CEST 7480.

It is clear that by using these deposited strains as starting material, a person skilled in the art will be able in the usual way using conventional mutagenesis or re-isolation techniques to obtain additional variants or mutants that retain or improve the respective properties and advantages of these strains described in this application, constituting the composition according to the present invention. Thus, the present invention also provides variants of the strains described in this application. As used herein, the term strain variant or mutant refers to any naturally occurring or engineered strain derived from a parent strain X predominantly by a mutation that retains an antiseptic tolerance similar to that of said strain.

For example, the 16S rRNA gene sequence of the strain variant proposed in this application may be approximately 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence 16S rRNA SEQ ID NO: 1 of the strain described in this application.

In another embodiment, the degree of relatedness between the altered and the original strains is defined as the degree of similarity obtained from the analysis of the genomes of the original and altered strains using pulsed field gel electrophoresis (FGE) using one or more restriction endonucleases. The degree of similarity obtained using the GEPP can be measured using the Dice similarity coefficient. In specific embodiments, the Dice similarity ratio between the variant and the parent strain is about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.5%, about 99.6% , about 99.7%, about 99.8%, about 99.9%, about 99.99%, about 99.999%, about 99.9999%, about 99.99999%, about 99.999999% or more, but less than 100%.

In one particular embodiment, mutants are generated using recombinant DNA technology. In another embodiment, the implementation of the mutants was obtained using non-specific mutagenesis. Thus, in another aspect of the present invention, there is provided a method for producing a mutant of Pediococcus acidilactici, wherein said strain is a CECT 8633 strain, said method comprising using said deposited strain as a starting material and carrying out mutagenesis, wherein the resulting variant or mutant is further at least the ability of said deposited strain to tolerate antiseptics is maintained or increased.

In a specific embodiment, the strains were fermented in an artificial environment and post-fermented to produce bacterial cells, and the resulting bacterial cells were in liquid medium or in solid form. Specifically, the post-treatment was selected from the group consisting of drying, freezing, lyophilization, fluid bed drying, spray drying, and liquid cooling, and lyophilization was specifically selected.

The strains according to the present invention were obtained by cultivation (or fermentation) of bacteria in a suitable artificial environment and under suitable conditions. The expression "artificial environment for microorganisms" should be understood as an environment containing natural substances and optionally synthetic chemicals, such as the polymer polyvinyl alcohol, which can reproduce some of the functions of sera. Commonly suitable artificial media are nutrient broths that contain elements including a carbon source (eg, glucose), a nitrogen source (eg, amino acids and proteins), water, and salts necessary for bacterial growth. Growth media may be in liquid form, or often may be mixed with agar or other gelling agent to form a solid medium. These strains can be cultivated separately to obtain a pure culture, or as a mixed culture with other microorganisms, or by cultivating different types of bacteria separately and then combining them in desired ratios. After cultivation, and depending on the final composition, the strains can be used as purified bacteria, or alternatively, a bacterial culture or cell suspension can be used, either as is or after suitable post-treatment. In the present description, the term biomass refers to a culture of bacterial strains obtained after cultivation (or fermentation, since this term is synonymous with the term cultivation).

The term post-treatment in the context of the present invention should be understood as any treatment carried out on the biomass in order to obtain well-storable bacterial cells. The purpose of the post-treatment is to reduce the metabolic activity of the cells in the biomass and thus slow down the rate of reactions that are detrimental to the cells. As a result of post-treatment, the bacterial cells may be in solid or liquid form. In solid form, the bacterial cells to be stored may be in the form of a powder or granules. In any case, both solid and liquid forms containing bacterial cells are not present in nature,

- 8 042202 therefore do not occur in nature, as they are obtained as a result of an artificial process (s) of subsequent processing. Post-treatment processes in particular embodiments may require the use of one or more of the so-called post-treatment agents. In the context of the present invention, the term post-treatment agent refers to a compound that is used to carry out the post-treatment processes described herein. Post-treatment agents should include, without limitation, dehydrating agents, bacteriostatic agents, cryoprotective agents (cryoprotectants), excipients (also known as lyoprotectants), carrier material (also known as fill material), etc., used either alone, or in combination.

There are two main approaches to reducing the metabolic activity of bacterial cells and therefore two approaches to post-treatment. The first is to reduce the rate of all chemical reactions, which can be done by lowering the temperature by refrigeration or freezing using cold stores, mechanical freezers and liquid nitrogen freezers. As an alternative, reducing the rate of all chemical reactions can be achieved by adding substances that inhibit the growth of bacterial cells, namely a bacteriostatic agent, abbreviated as b/static.

The second approach to post-treatment is to remove water from the biomass, this process may include the sublimation of water using a lyophilizer. Suitable techniques for removing water from the biomass are drying, lyophilization, spray drying, or fluid bed drying. Post-treatments that allow a solid form to be obtained can be drying, freezing, lyophilization, fluid bed drying, or spray drying.

The post-treatment is preferably lyophilization, which involves the removal of water from the frozen bacterial suspensions by sublimation under reduced pressure. This process consists of three stages: pre-freezing the product to obtain a frozen structure, primary drying to remove most of the water and secondary drying to remove bound water. Given the purpose and expected variability of industrial processes for the production and isolation of freeze-dried bacterial cultures, the last of these is usually included in some amount of an inert excipient, also known as a lyoprotectant. Its role is to standardize the content of live probiotic bacteria in the product. Commercially available lyophilized cultures use the following inert excipients: sucrose, sucrose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharmaceutically acceptable non-hygroscopic excipients. Optionally, other stabilizing or cryoprotective agents, such as ascorbic acid, are also used to form a viscous paste that is lyophilized. In any case, the material thus obtained can be ground to a suitable size, including powder.

The use of lactic acid bacteria in combination with an antiseptic.

It has been described above that the Pediococcus strains of the present invention are capable of tolerating antiseptics and are therefore suitable for use in combination with antiseptics. Examples of antiseptics include, without limitation, triclosan, plant extracts and essential oils, biguanides, and cetylpyridine.

In one particular embodiment, the Pediococcus strain is used in combination with striclosan.

In another specific embodiment, the Pediococcus strain is used in combination with essential oils. Examples of the latter include, but are not limited to: cinnamon oil, basil oil, bergamot oil, clary sage oil, ylang ylang oil, orange flower oil, sandal tree oil, frankincense oil, ginger oil, peppermint oil, lavender oil, absolute Jasmine Oil, Bourbon Geranium Oil, Spearmint Oil, Clove Oil, Patchouli Oil, Rosemary Oil, Rosewood Oil, Sandalwood Oil, Tea Tree Oil, Vanilla Oil, Lemongrass Oil, Cedarwood Oil, Balsam Oils, Tangerine Oil, Cypress Oil tulipidus oil, japonica oil, ginkgo oil, eucalyptus oil, lemon oil, orange oil, sweet orange oil, pomegranate seed oil, manuka oil, citronella oil, wintergreen oil and calendula oil. In a more specific embodiment, the Pediococcus strain is used in combination with an essential oil selected from the group consisting of peppermint oil, eucalyptus oil, thyme oil, and wintergreen oil.

In one particular embodiment, the Pediococcus strain is used in combination with the individual components of the essential oils. Examples of individual components of essential oils include, but are not limited to: thyme, oregano, curcumin, 1-citronellol, alpha-amylcinnamic aldehyde, lyral, geraniol, farnesol, hydroxycitronellal, isoeugenol, eugenol, camphor, eucalyptol, linalool, citral, thymol, limonene and menthol. Additional examples of individual components include sesquiterpenoid compounds, which may be active compounds in essential oils. Sesquiterpenoid compounds containing 15 carbon atoms are biosynthetically formed from three 5-carbon isoprene units. Sesquiterpenoid compounds include, but are not limited to, farnesol, nerolidol, bisabolol, apriton, chamazulene, santolol, zingiberol, carotol, and caryophyllene. In a more specific embodiment, the Pediococcus strain is used in combination with a single essential oil component selected from the group consisting of menthol, eucalyptol, thymol, and methyl salicylate.

In one particular embodiment, the Pediococcus strain is used in combination with biguanides, in particular alexidine or chlorhexidine. Alexidine and chlorhexidine are used mainly in the form of salts. The chlorhexidine salts of the present invention include, but are not limited to, chlorhexidine palmitate, chlorhexidine diphosphanilate, chlorhexidine digluconate, chlorhexidine diacetate, chlorhexidine dihydrochloride, chlorhexidine dichloride, chlorhexidine dihydroiodide, chlorhexidine diperchlorate, chlorhexidine dinitrate, chlorhexidine sulfate, chlorhexidine persulfate, chlorhexioacidine sulfite, chlorhexidine dihydrochloride chlorhexidine phosphate, chlorhexidine difluorophosphate, chlorhexidine diformate, chlorhexidine dipropionate, chlorhexidine diiodobutyrate, chlorhexidine di-n-valerate, chlorhexidine dicaproate, chlorhexidine malonate, chlorhexidine succinate, chlorhexidine malate, chlorhexidine tartrate, chlorhexidine dimonoglycolate, chlorhexidine monodiglycolate, chlorhexidine monodichloroglycolate chlorhexidine hydroxyisobutyrate, chlorhexidine diglucoheptonate, chlorhexidine diisothionate, chlorhexidine dibenzoate, chlorhexidine dicinnamate, chlorhexidine dimandelate, chlorhexidine diisophthalate, di-2-hydroxynaphthoa tons of chlorhexidine and chlorhexidine embonate. In a more specific embodiment, the Pediococcus strain is used in combination with chlorhexidine, in particular chlorhexidine gluconate.

These strains and antiseptics according to the present invention can be formulated for separate, sequential, simultaneous administration or administration as a mixture.

Administration schedules and forms will be determined by one skilled in the art depending on the disease to be treated. The introduction of antiseptics and lactic acid bacteria can be carried out separately (waiting some time between the introduction of the Pediococcus strain and the antiseptic), sequentially (one after the other), in parallel (simultaneously) or as a mixture (together). In one embodiment, said antiseptic and said strains of the present invention are administered over a time period of at most 12 hours. In particular, said time period is at most 6 hours. In particular, said time period is at most 1 hour. In particular, said time period is at most 1 hour. , the specified period of time lasts no more than 10 minutes. In a more specific embodiment, the treatment regimen is based on the simultaneous administration of an antiseptic and said strains. One common administration schedule is to administer an antiseptic and then administer the Pediococcus strain of the present invention. In specific embodiments, the antiseptic and the Pediococcus strain are administered at the same time. When the antiseptic and lactic acid bacteria are administered to a patient at the same time, they may be administered as separate forms or as part of the same composition. When the products are administered in separate dosage forms, said dosage forms may be in the same or different containers.

In a particular drug administration schedule, the subject in need of is asked to rinse their mouth with a mouth rinse containing an antiseptic (eg, chlorhexidine at a concentration of 0.12% (w/v)) for 30-60 seconds. The mouthwash is then spit out and an oral tablet containing lactic acid bacteria is introduced into the mouth and allowed to slowly melt.

The administration may be continuous or intermittent, as deemed appropriate by the attending physician, in particular taking into account any change in the disease state or any undesirable side effects. Continuous administration refers to the administration of the composition in a continuous manner, while intermittent administration refers to treatment that is carried out intermittently.

In non-limiting embodiments, the Pediococcus strains and compositions of the present invention are used by a dentist for emergency treatment, and the patient can be maintained on oral hygiene products such as chewing gums, toothpaste, mouthrinse, mouth spray, lozenges, or dispersible oral tablets containing the self-film-forming powder composition of the present invention or lactic acid bacteria with conventional excipients. The dentist may administer, for example, by applying the compositions of the present invention with a syringe, in particular with a blunt needle. Other non-limiting examples of professional administrations are the application of a dental splint containing a gel comprising the compositions of the present invention, or the application of the composition in the form of a lacquer to the surfaces of the teeth with a brush. In applications such as halitosis, periodontitis, gingivitis or caries, the compositions of the present invention can also be applied by the patient himself.

In one specific embodiment, the implementation of the antiseptic is in a concentration in the range from 0.05 to 0.5% (by weight). In particular, the concentration of the antiseptic is in the range of 0.05 to 0.2% (by weight). In particular, the concentration is 0.05%, 0.1%, 0.12% or 0.2% (by weight).

In one aspect of the present invention, a set of parts is provided, comprising

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a) the first test tube containing an antiseptic; And

b) a second tube containing at least a strain of Pediococcus.

In a particular embodiment, the kit further comprises instructions for use, in particular containing information on dosage and/or administration schedules.

Products for personal hygiene.

In one aspect of the present invention, personal care products are provided which contain the strains and compositions of the present invention, together with suitable excipients and/or carriers. The term personal care products refers to products that are used for personal care and/or appearance enhancement (cosmetic purposes).

One skilled in the art will appreciate that personal care products in this context contain an effective amount of the strains of the present invention. The effective amount of Pediococcus strains will be determined by one of skill in the art and will vary depending on the particular goal to be achieved, the age and physical condition of the patient being treated, the severity of the underlying disease, and the final composition. For example, in oral health products, the strain or strains are present in an amount from about 105 cfu/g to about 10 ¹² cfu/g, in particular in an amount from about 10 ⁷ cfu/g to about 10 ¹¹ cfu/g. The term colony forming units (CFU) refers to the number of bacterial cells found in microbiological counts on agar plates. In a particular embodiment, the composition contains 10 ⁷ -10 ¹⁰ CFU/g.

The term excipient in the present description is understood in a broad sense, including any natural or synthetic substance included in the composition together with the active ingredient of a pharmaceutical product, veterinary product, drug, dietary supplement, health food product and personal care product. The most appropriate excipient(s) to use will depend on the final formulation.

Non-limiting examples of personal care products in which the present invention may be used include bar soap, liquid soap (e.g., hand soap), hand sanitizer (including rinse-off and leave-on alcohol-based and water-based hand sanitizers). ), cotton buds and pads, shaving cream, talcum powder, toilet paper, pre-surgery skin disinfectant, wet paper, refreshing wipes, disinfectant wipes, body wash, acne treatment products, antifungal cream for diaper rash, antifungal cream for skin, shampoo, conditioner, cosmetic deodorant, antimicrobial creams, body lotion, hand cream, topical cream, aftershave lotion, skin tonic, oral hygiene products, sunscreen lotion, and baby products such as, but not limited to listed, cleansing wipes, baby shampoo, baby soap and diaper cream To.

The present invention may also be applied to wound care products such as, but not limited to, wound healing ointments, creams and lotions, wound dressings, burn wound cream, bandages, tape, and tape strips for seamless wound bridging, and such. medical products such as medical overalls, bonnets, face masks and shoe covers, surgical drops, etc.

In one particular embodiment, the personal care product is a topical cream or gel.

In various non-limiting embodiments of the present invention, the personal care product containing the compositions of the present invention may further comprise a thickening and/or gelling agent such as stearyl alcohol, cationic hydroxyethyl cellulose (Ucare; JR30), hydroxypropyl methylcellulose, hydroxypropyl cellulose (Klucel), chitosan pyrrolidone carboxylate (Kytamer), behenyl alcohol, zinc stearate, emulsifying waxes including but not limited to Incroquat and Polawax, acrylic acid addition polymer, resin such as Carbopol (R) ETD™ 2020, guar gum, gum arabic, acrylate/stearet copolymer -20 methacrylate, agar, algin, alginic acid, ammonium acrylate copolymers, ammonium alginate, ammonium chloride, ammonium sulfate, amylopectin, attapulgite, bentonite, C915 alcohols, calcium acetate, calcium alginate, calcium carrageenan, calcium chloride, caprylic alcohol, carbomer 910 , carbomer 934, carbomer 934R, carbomer 940, k arbomer 941, carboxymethylhydroxyethylcellulose, carboxymethylhydroxypropyl guar, carrageenan, cellulose, cellulose gum, cetearyl alcohol, cetyl alcohol, cornstarch, damar, dextrin, dibenzylidene sorbitol, dihydrogenated ethylene tallowamide, ethylene diolamide, ethylene distearamide, gelatin, guar gum, guarpropylonium chloride, guarhydroxymonium chloride hyaluronic acid, silicon hydroxide, hydroxybutylmethylcellulose, hydroxyethylcellulose, hydroxyethylethylcellulose, hydroxyethylstearamidemonoisopropanolamine (MIPA), isocetyl alcohol, isostearyl alcohol, karaya gum, kelp, lauryl alcohol, locust bean gum, magnesium aluminum silicate, magnesium silicate, magnesium trisilicate, methoxy-PEG copolymer 22/dodecyl glycol, methylcellulose, microcrystalline cellulose, montmorillonite, myristyl alcohol, oatmeal, oleyl alcohol, palm kernel

- 11 042202 alcohol, pectin, PEG-2M, PEG-5M, polyacrylic acid, polyvinyl alcohol, potassium alginate, potassium aluminum polyacrylate, potassium carrageenan, potassium chloride, potassium sulfate, potato starch, propylene glycol alginate, sodium acrylate/vinyl alcohol copolymer, carboxymethyl dextran sodium, sodium carrageenan, sodium cellulose sulfate, sodium chloride, sodium polymethacrylate, sodium aluminum silicate, sodium sulfate, stearalkonium bentonite, stearalkonium hectorite, stearyl alcohol, tallow alcohol, triethanolamine hydrochloride (TEA), gum tragacanth, tridecyl alcohol, magnesium aluminum tromethamine silicate, wheat flour, wheat starch, xanthan gum, abietyl alcohol, acrylinolic acid, aluminum behenate, aluminum caprylate, aluminum dilinoleate, aluminum salts such as aluminum distearate and isostearates, beeswax, behenamide, butadiene/acrylonitrile copolymer, C29-70 acid, behenate calcium, calcium stearate, candelilla wax, carnauba wax, ceresin, cholesterol , Cholesterol Hydroxystearate, Coconut Alcohol, Copal, Diglyceryl Stearate Malate, Dihydroabietyl Alcohol, Dimethyl Lauramine Oleate, Dodecanoic Acid/Cetearyl Alcohol/Glycol Copolymer, Erucamide, Ethylcellulose, Glyceryl Triacetyl Hydroxystearate, Glyceryl Triacetyl Ricinolate, Glycol Dibehenate, Glycol Dioctanoate, Glycol Distearate, C14 Hexanediol Disterate, Hydrogenated Polymers olefin, hydrogenated castor oil, hydrogenated cottonseed oil, hydrogenated lard, hydrogenated menhaden oil, hydrogenated palm kernel glycerides, hydrogenated palm kernel oil, hydrogenated palm oil, hydrogenated polyisobutene, hydrogenated soybean oil, hydrogenated tallow amide, hydrogenated tallow glyceride, hydrogenated vegetable glyceride, vegetable oil, Japanese wax, jojoba oil wax fraction, lanolin alcohol, shea butter, lauramide, methyl dehydroabietate, hy drogenated methyl resinate, methyl resinate, methylstyrene/vinyl toluene copolymer, microcrystalline wax, lignite sour wax, lignite wax, myristyl leucosanol, myristyl loctadecanol, octadecene/maleic anhydrine copolymer, octyl dodecyl stearoyl stearate, oleamide, oleostearin, downy palm leaf wax, oxidized paraffin, polyethylene, o,zo brazilite гидрогенизированный резинат пентаэритритила, тетраоктаноат пентаэритритила, резинат пентаэритритила, тетраабиетат пентаэритритила, тетрабегенат пентаэритритила, тетраолеат пентаэритритила, тетрастеарат пентаэритритила, сополимер офтальмический сополимер ангидрида/глицерина/глицидилдеканоата, офтальмический тримеллитовый/гликолевый сополимер, полибутен, полибутилентерефталат, полидипентен, полиэтилен, полиизобутен, полиизопрен, поливинилбутираль , Polyvinyl Laurate, Propylene Glycol Diaprilate, Propylene Glycol Dicocoat, Propylene Glycol Diisononanoate, Propylene Glycol Dilaurate, Propylene Glycol Dipelargonate, Propyle Distearate glycol, propylene glycol diundecanoate, PVP/eicosene copolymer, PVP/hexadecene copolymer, rice bran wax, stearalkonium bentonite, stearalkonium hectorite, stearamide, diethanolamine (DEA)-stearamide distearate, diisobutyl adipate (DIBA)-stearamide stearate, monoethanolamine (MEA)-stearate stearamide, stearone, stearyl erucamide, stearyl stearate, stearyl stearoyl stearate, synthetic beeswax, synthetic wax, trihydroxystearin, triisononanoin, triisostearin, triisostearyl trilinoleate, trilaurin, trilinoleic acid, trilinolein, trimyristin, triolein, tripalmitin, tristearin, zinc laurate, zinc laurate, zinc, zinc resinate and mixtures thereof. Gelling agents which are used in the carriers may be natural gelling agents such as natural gums, starches, pectins, agar and gelatin. Often gelling agents are based on polysaccharides or proteins. Their examples include, but are not limited to, guar gum, xanthan gum, alginic acid (E400), sodium alginate (E401), potassium alginate (E402), ammonium alginate (E403), calcium alginate (E404, brown algae polysaccharides), agar (E406, a polysaccharide derived from red algae), carrageenan (E407, a polysaccharide derived from red algae), locust bean gum (E410, natural gum from locust bean seeds), pectin (E440, a polysaccharide derived from apple or citrus fruit) and gelatin (E441, obtained by partial hydrolysis of animal collagen).

In various non-limiting embodiments of the present invention, the strains and compositions used in the personal care product may additionally contain a surfactant. The surfactant may be an anionic surfactant, a cationic surfactant, an ampholytic surfactant, or a nonionic surfactant. Examples of nonionic surfactants include polyethoxylates, fatty alcohols (e.g., cetet-20 (polyethylene oxide cetyl ether containing an average of approximately 20 ethylene oxide units), and other bridge(R) nonionic surfactants available from ICI Americas, Inc. (Wilmington, Delaware)), cocamidopropyl betaine, alkylphenols, fatty acid esters of sorbitol, sorbitan, or polyoxyethylene sorbitan. Suitable anionic surfactants include ammonium lauryl sulfate and lauryl ether sulfosuccinate. In various non-limiting embodiments of the present invention, the personal care product may contain water.

In various non-limiting embodiments of the present invention, the strains and compositions used in the personal care product may further comprise a hydrogel, including, for example, a compound such as hydroxypropyl methyl cellulose, cationic hydroxyethyl cellulose (U-care polymers), ethyl cellulose, hydroxypropyl cellulose, hydroxymethyl cellulose , carboxymethyl cellulose, polyethylene oxide (polyox resins), and chitosan pyrrolidone carboxylate (Kytomer

PC).

The strains and compositions of the present invention are also intended for use in maintaining oral health. Accordingly, in one particular embodiment, the personal care product is an oral hygiene product comprising the strains or compositions of the present invention together with pharmaceutical excipients or cosmetically acceptable excipients or other edible ingredients. It should be clear that the compositions according to the present invention will be there in an effective amount. The term oral hygiene product refers to products that are used to keep the mouth and teeth clean to prevent dental problems, most commonly tooth cavities, gingivitis, periodontal (gum) disease, and bad breath. In this sense, an oral hygiene product is not intentionally ingested for systemic administration of specific therapeutic agents, but instead is left in the oral cavity for a time sufficient to contact substantially all tooth surfaces and/or oral tissues in order to be active in the oral cavity. . Non-limiting examples of such products are toothpastes, dentifrices, tooth powders, topical oral gels, mouthwash, denture products, mouth sprays, chewing gums, dental floss, dental tapes, grinding powder, polishing pastes, dental varnishes, fissure sealants, filling materials, oral cream or gel, lozenge, lozenges, orodispersible tablet or strip, or powder that can be sprinkled directly into the mouth.

In a particular embodiment, the oral hygiene product is selected from the group consisting of toothpaste, oral gel, oral spray, mouthrinse, or orodispersible tablet.

Oral hygiene products may additionally contain flavoring and taste masking agents. Non-limiting examples of these agents for use in oral hygiene products include cinnamaldehyde, eugenol, eucalyptol, menthol, N-ethyl-para-menthane-3-caboxamide, anethole, peppermint oil, spearmint oil, and field mint oil.

Oral care products may optionally contain humectants, gelling agents, abrasives, fluoride sources, desensitizing agents, flavoring agents, colorants, sweeteners, preservatives, structurants, surfactants, antitartar agents, and antiplaque agents.

Oral care products may also contain other oral active agents such as tooth whitening actives, including whitening or oxidizing agents such as peroxides, perborates, percarbonates, peroxyacids, persulfates, metal chlorites, and combinations of these agents. Tooth discoloring agents may also be considered among the oral hygiene active agents useful in the present invention.

The composition of toothpastes is well known to those skilled in the art. It is preferred to use non-ionic (eg, sugar-fatty acid esters) or amphoteric (eg, coconut-derived betaines) surfactants in toothpaste formulations, since anionic surfactants have a negative effect on sensitive gingival epithelial tissue. In the case of toothpastes, the use of sodium bicarbonate to neutralize acidity in the oral cavity is also particularly preferred. In addition, toothpastes may contain thickening agents such as xanthan gum, abrasive silicas and other auxiliary agents in addition to agents commonly used in the toothpaste industry. Preferably the lactic acid bacteria are encapsulated or otherwise protected for inclusion in a toothpaste formulation.

It is known to those skilled in the art that a mouthrinse, mouthrinse, dental rinse, oral rinse, or mouthrinse in this application refers to a liquid composition that is passively held in the mouth or rolled around the mouth by contraction of the muscles of the mouth. cavities and/or head movements and which can be used to rinse the mouth by tilting the head back and gurgling fluid at the back of the mouth. Generally, mouth rinses are an antiseptic solution designed to reduce the microbial load in the mouth, although other mouth rinses may be given for other purposes, such as analgesic, anti-inflammatory, or antifungal effects. In addition, some mouthwashes act as saliva substitutes to neutralize acid and keep the mouth moist in xerostomia (dry mouth). In addition to water, polyhydroxylated compounds such as glycerol or glycols (eg propylene glycol, nonionic surfactants, etc.) and other adjuvants can be included in rinses to improve appearance, taste and preservation.

- 13 042202

Sprays are compositions the same or similar to mouth rinses, but distributed in spray bottles for easy application of the dose needed to lubricate and protect the mouth without the need for subsequent rinsing.

Oral gels contain polymers that allow direct, stable application to the oral cavity. With respect to these polymers, for the purposes of the present invention, it is preferable to use a combination of polymers, generally known as polycarbophil and carbomer, because they keep the gel structure stable for a very long time under extreme temperature conditions. These gels may also contain some natural, non-cariogenic sweetener such as sorbitol.

In another embodiment, the personal care product is a cosmetic product that contains strains of Pediococcus or compositions of the present invention along with cosmetically acceptable excipients. Cosmetic products or cosmetics are products intended to be applied to the human body in order to cleanse, enhance beauty, promote attractiveness or change appearance without affecting the structure or function of the body. Cosmetics can also provide therapeutic benefits.

The term cosmetically acceptable refers to compounds, materials, compositions, and/or dosage forms that, within sound medical judgment, are 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 that it is compatible with the other ingredients of the cosmetic composition. Suitable carriers, auxiliaries, etc. for cosmetic formulations can be found in the standard literature.

Non-limiting examples of cosmetic products include cleansing pads, colognes, cotton swabs, cotton pads, eyeliner, tissue paper, hair tweezers, lip gloss, lipstick, lotion, makeup product, nail files, lipstick, perfume, razors , shaving cream, moisturizer, talcum powder, toilet paper and wet wipes (including but not limited to liquid or powder foundation, liquid or solid eyeliner, mascara, cream eye shadow, tinted powder, powder compact dry or wet type, etc.).

Pharmaceutical products and biologically active food additives.

In one aspect, the Pediococcus strains and compositions of the present invention are formulated as pharmaceutical products. The term "pharmaceutical product" in the present description is understood in a broad sense, including any composition that contains the active ingredient, in this case, the strains according to the present invention preferably in the form of a composition, and optionally at least one antiseptic, together with pharmaceutically acceptable excipients. This term is not limited to drugs.

The term pharmaceutically acceptable is known in the art and includes compounds, materials, compositions, carriers, vehicles, and/or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with tissues of a subject (e.g., human) without causing undue toxicity, irritation. , allergic response, or other problem or complication consistent with an acceptable benefit/risk ratio. Each carrier, excipient, etc. must also be acceptable in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, auxiliaries, etc. can be found in the standard pharmaceutical literature. Non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; ethers such as ethyl oleate and ethyl laurate; agar; buffer substances such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions and other non-toxic compatible substances used in pharmaceutical formulations.

Excipients are selected without limitation from the group including fillers/diluents/bulking agents, binders, release agents, disintegrants, coatings, anti-caking agents, antioxidants, lubricants, sweeteners, flavors, colors, tensides, and other classes of pharmaceutically and veterinarily acceptable excipients. substances.

Fillers are selected without limitation from the group including inulin, oligofructose, pectin, modified pectins, microcrystalline cellulose, lactose, starch, maltodextrin,

- 14 042202 sucrose, glucose, fructose, mannitol, xylitol, non-crystallizing sorbitol, calcium carbonate, dicalcium phosphate, other inert inorganic and organic pharmacologically acceptable excipients and mixtures of these substances. In the oral suspension dosage form, excipients or diluents are selected from the group consisting of vegetable oil, oleic acid, oleyl alcohol, liquid polyethylene glycol, other pharmacologically acceptable inert liquids, or mixtures of these substances.

Binders are used in solid dosage forms, for example, to hold the ingredients together in a tablet, to ensure that tablets and granules can be produced with the required mechanical force, and to give volume to low active dose tablets. Binders in solid dosage forms such as tablets are lactose, sucrose, corn (maize) starch, modified starches, microcrystalline cellulose, modified cellulose (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxyethyl cellulose), other water-soluble cellulose ethers, polyvinylpyrrolidone (PVP). ), also known as povidone, polyethylene glycol, sorbitol, maltitol, xylitol, and dibasic calcium phosphate; other suitable pharmacologically acceptable binders or mixtures of these substances.

Release agents are used to reduce adhesion between powder (granules) and press surfaces and thus prevent sticking to tablet presses. They are also used to help protect tablets from sticking together. The most commonly used release agent is magnesium stearate.

As disintegrants and super disintegrants in solid dosage forms such as tablets and capsules, the following substances are used, without limitation: cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose and formaldehyde-casein, other suitable pharmacologically acceptable disintegrant and super disintegrant or their mixtures.

Coatings, in the case of solid dosage forms such as tablets and capsule-filling granules, protect ingredients from spoilage by moisture in the air, allow larger, unpleasant-tasting tablets to be swallowed more easily, and/or, in the case of enteric coatings, allow intact passage through highly acidic environments. gastric juice (pH approximately 1) and allow release in the duodenum or ileum (small intestine). Most film-coated tablets are film-coated with a cellulose ether, hydroxypropyl methylcellulose (HPMC). Other coating materials are sometimes used, for example synthetic polymers and copolymers such as polyvinyl acetate phthalate (PVAF); methyl acrylate/methacrylic acid copolymers; methyl methacrylate/methacrylic acid copolymers; shellac, zein corn protein, or other polysaccharides; waxes or wax-like substances such as beeswax, stearic acid; higher fatty alcohols such as cetyl or stearyl alcohol; hard paraffin; glycerol monostearate; glycerol distearate; or combinations thereof. The capsules are coated with gelatin or hydroxypropyl methylcellulose.

Enteric coatings control the rate of drug release and determine in which part of the digestive tract the drug will be released. Materials used to form enteric coatings include fatty acids, waxes, shellac, plastics, vegetable fibers and mixtures thereof, also in combination with the other coatings mentioned above.

An anti-caking agent is an auxiliary substance that is added to powdered or granular materials to prevent the formation of lumps (caking) and to facilitate packaging, transportation and consumption. As anti-caking agents in solid dosage forms such as tablets, capsules or powders, the following agents are used: magnesium stearate, colloidal silicon dioxide, talc, other pharmacologically acceptable anti-caking agents or mixtures thereof.

Lubricants are used in solid dosage forms, in particular tablets and capsules, to prevent ingredients from aggregating with each other and to prevent sticking to tablet presses or capsule filling devices, and also in hard capsules. As lubricants in tablets or hard gelatin capsules, lubricants such as talc or silicon are most commonly used, and fats such as vegetable stearin, magnesium stearate or stearic acid, and mixtures thereof.

Sweeteners are added to improve the taste of ingredients, especially in solid dosage forms such as chewable tablets, as well as in liquid dosage forms such as cough syrup. Sweeteners can be selected from artificial, natural or synthetic or semi-synthetic sweeteners; non-limiting examples of sweeteners are aspartame, acesulfame potassium, cyclamate, sucralose, saccharin, sugars, or any mixtures thereof.

Flavoring additives can be used to mask off-tasting active ingredients in any dosage form. Flavoring agents can be natural (eg fruit extract) or artificial. For example, to improve the taste

- 15 042202 (1) bitter product: mint, cherry or anise can be used;

(2) salty product: peach or apricot or licorice root can be used;

(3) sour food: raspberries; and (4) a very sweet product: vanilla.

With the exception of excipient class excipients, the composition of the present invention may contain other pharmacologically active substances or nutrients, including, but not limited to, vitamins such as vitamin D (calciferol) in a pharmaceutically acceptable chemical form, in the form of a salt or derivative; minerals in a pharmaceutically and nutritionally acceptable chemical form; and L-amino acids. The preparation of formulations according to the present invention is within the purview of the average person skilled in the art and will depend on the final dosage form. For example, without limitation, when the final dosage form is an oral solid form such as tablets, capsules, powder, granules, oral suspension, etc. the process of obtaining solid dosage forms of the composition includes homogenization (1) active(s) ingredient(s), including at least one or more probiotic bacteria after processing according to the present invention in an effective amount; with (2) one or more excipients to form a homogeneous mixture, to which, for example, lubricants such as magnesium stearate or other lubricants are added as required to form the final powder dosage form. This homogeneous powder is filled into conventional gelatine capsules or alternatively into gastro-resistant capsules. In the case of tablets, they are produced by direct compression or granulation. In the first case, a homogeneous mixture of active ingredients and suitable excipients, such as anhydrous lactose, non-crystallizing sorbitol and other excipients, is obtained. In the second case, tablets are obtained by processing the mixture in granular form. The granules are obtained by the process of granulating the active ingredients of the formulation with suitable fillers, binders, disintegrants and a small amount of purified water. The granules thus obtained are sieved and dried until a water content of <1% by weight is reached.

With respect to the process for preparing liquid dosage forms (e.g., oral suspensions), it involves homogenizing the active ingredient(s) of the formulation, including at least one or more probiotic bacteria after treatment according to the present invention in an effective amount in an inert liquid diluent ( excipient) such as various vegetable oils such as sunflower, soybean or olive oil; oleic acid; oleyl alcohol; liquid polyethylene glycols such as PEG 200, PEG 400 or PEG 600; or other inert pharmacologically acceptable liquids. This process further includes treating the homogeneous mixture with one or more processes selected from the group consisting of (1) stabilizing the composition by adding suspension stabilizers such as beeswax, colloidal silicon dioxide, etc. and homogenization;

(2) sweetening the formulation by adding a sweetener and homogenizing;

(3) flavoring the composition by adding flavoring agent and homogenization.

Such formulations may also contain other adjuvants or ingredients commonly used in the art.

The specified pharmaceutical product may have different forms or names depending on the way the product is approved, as well as depending on the country. For example, a drug is a specific pharmaceutical product. The health food product is another specific pharmaceutical product. The terms medical food or product for special medical purposes are used in some countries to refer to a specially formulated product intended for the dietary treatment of a disease in which there are special nutritional needs that cannot be met by a normal diet alone. They are defined in regulations such as the FDA regulation Orphan Drug Act Amendment in the United States, 1988, and EU Commission Directive 1999/21/EC in Europe. Health food products are distinct from the broader category of dietary supplements and from conventional foods that are known to be beneficial to health. Thus, in a particular embodiment, the strains of the present invention are included in a health food product.

Often strains of Pediococcus and compositions such as described in this application are considered biologically active food supplements. A dietary supplement, also known as a dietary supplement or nutritional supplement, is considered another specific pharmaceutical product. It is a dietary supplement preparation or product derived from ingredients commonly found in foods that provide nutrients or beneficial ingredients that are not normally consumed with a normal diet or cannot be consumed in sufficient quantities. For the most part, dietary supplements are considered food, but sometimes they are defined as medicines, natural

- 16 042202 health or nutraceutical products. Within the meaning of the present invention, dietary supplements also include nutraceuticals. Dietary supplements are usually sold over the counter, ie. without an appointment. If the dietary supplement takes the form of a pill, capsule, tablet or powder, then it contains the same excipients that are used in medicines. The dietary supplement, however, may also take the form of a food product that is fortified with certain nutrients (eg a bar or yogurt). Thus, in a particular embodiment, the strain-containing formulation of the present invention takes the form of a dietary supplement. The dietary supplement may be administered as is, may be mixed with suitable drinkable liquids such as water, yogurt, milk or fruit juice, or may be mixed with solid or liquid food. As used herein, a dietary supplement may be in the form of tablets or lozenges, pills, capsules, granules, powders, suspensions, sachets, sweets, bars, syrups and appropriate administration forms, usually as a single dose.

The strains of the present invention can also be included in various food products such as dairy products (yogurt, cheese, fermented milk, milk powder, dairy based fermented product, ice cream, cereal based fermented product, milk based powder), bread, bars , spreads, biscuits and cereals, drink, various types of oils or dressing. The term food product is used in this application in its broadest sense, including any type of product, in any form of presentation that can be ingested by an animal, but excluding pharmaceutical and veterinary products. Examples of other foodstuffs are meat products, chocolate spreads, fillers and coatings, chocolate, confectionery, baked goods, sauces and soups, fruit juices, and coffee creamers. Food products of particular interest are dietary supplements and infant formulas. The food product preferably contains a carrier material such as oatmeal, lactic fermented foods, resistant starch, dietary fiber, carbohydrates, proteins and glycosylated proteins. In a particular embodiment, the strains of the present invention are encapsulated or coated.

In another aspect of the present invention, a solid composition is provided comprising a cryoprotectant; lyophilized biomass containing at least one lactic acid bacterium according to the present invention; and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is preferably selected from emulsion, gel, paste, granules, powder and gum. In additional aspects, the present invention provides an oral hygiene product, pharmaceutical composition, edible product, dietary supplement and cosmetic composition containing an effective amount of the composition specified in the above aspect. In a particular embodiment, the oral hygiene product is a chewing gum, toothpaste, oral spray, lozenge, or orodispersible tablet. In a particular embodiment, the pharmaceutical composition, comestible, or dietary supplement is a lozenge or orodispersible tablet.

Medical applications and methods of use.

In one aspect of the present invention, strains of Pediococcus and compositions of the present invention are provided for use as medicines. This first aspect may alternatively be formulated as a probiotic treatment method comprising administering, if necessary, an effective amount of a composition comprising at least one isolated strain of lactic acid bacteria described in the first aspect of the present invention. The term probiotic, as used herein, refers to live microorganisms that, when administered in sufficient amounts, confer a health benefit on the host.

In particular, by providing several beneficial effects in the human host, the strains of the present invention are useful in the treatment of diseases or conditions associated with dysbiosis or caused by pathogens. Dysbiosis (also referred to as dysbacteriosis) in this application is a term for microbial imbalance or maladjustment on the surface or within the body, such as microbiota disturbance. For example, a part of the human microbiota, such as skin flora, gut flora, vaginal flora, oral flora, can be disrupted in such a way that there is a lack of symbiotic dominant species, and the number of normally displaced or suppressed species increases to fill the empty space. The loss of beneficial microbes, the proliferation of pathobionts, and the loss of diversity are events that include dysbiosis. Dysbiosis can be caused by circumstances as diverse as repeated and inappropriate exposure to antibiotics or antiseptics. Thus, in one aspect of the present invention, strains of the present invention useful in combination with antiseptics are provided for use in the prevention and/or treatment of dysbiosis or conditions associated with dysbiosis.

In another aspect, compositions of the present invention are provided for use in the prevention and/or treatment of dysbiosis or conditions associated with dysbiosis.

- 17 042202

The strains of the present invention are particularly useful for dental hygiene. Thus, in one aspect of the present invention, compositions and strains of the present invention are provided for use in combination with antiseptics for use in the prevention and/or treatment of an oral disease in an animal, including a human. Alternatively, this aspect can be formulated as the use of strains or compositions according to the present invention for the manufacture of a medicinal product for the prevention and/or treatment of diseases of the oral cavity of an animal, including a human. Also alternatively, the present invention also provides a method for preventing and/or treating an oral disease in an animal, including a human, comprising administering to said animal in need thereof the strains or compositions described in the present invention.

Specifically, oral disease is caused by oral pathogens. Illustrative, non-limiting examples of an oral disease that is caused by a pathogen in the oral cavity are plaque-related diseases, caries, gingivitis, periodontitis, candidiasis, herpes, and ulcers, as well as halitosis, stained teeth, sensitive teeth, peri-implant diseases, among others.

In one embodiment, Pediococcus strains and compositions thereof are used to treat and/or prevent periodontal disease, including, but not limited to, gingivitis, periodontitis, and peri-implant disease. In particular, the periodontal disease is gingivitis or periodontitis.

In an additional embodiment, Pediococcus strains and compositions thereof are used to treat and/or prevent candidiasis.

Periodontal therapy usually involves first the mechanical removal of subgingival plaque by scraping or ultrasound; under the action of this mechanical removal, the bacteria in the biofilm are released and washed away. This treatment to date uses antiseptic compositions that are designed to prevent re-colonization by Gram-negative bacteria. The disadvantage of regular and widespread use of compositions containing antiseptics, however, is the side effects of these products and a dangerous effect on the symbiotic microbiota.

In another embodiment, the strains and compositions of the present invention are useful in the treatment of skin lesions. Examples of skin lesions include, but are not limited to, fungal nail infections; superficial skin lesions (eg, erysipelas, open wound infections, acne, abscess, abscess, eczema, dermatitis, contact dermatitis, hypersensitivity, contact lesions, bedsores, and diabetic lesions).

Screening methods.

In one aspect of the present invention, a method for screening and isolating strains of lactic acid bacteria is provided, comprising the following steps:

i) analysis of the ability to tolerate antiseptics of individual lactic acid bacteria from a pool of candidate lactic acid bacteria strains; and ii) selecting and isolating strains of lactic acid bacteria having a better ability to tolerate antiseptics than that of Porphyromonas gingivalis.

In one embodiment, antiseptic tolerance is expressed as an inhibition halo as determined by a disk diffusion assay.

In one specific embodiment, the ability to tolerate antiseptics includes the following steps:

i) evenly spreading a suspension of lactic acid bacteria in phosphate buffered saline at 0.1 M with an optical density equivalent to McFarland standard No. 3 onto the culture medium in a dish to obtain a confluent layer;

ii) placing a 6 mm diameter cellulose disk in contact with the growth medium;

iii) adding an antiseptic to the cellulose disc at a concentration that inhibits the growth of Porphyromonas gingivalis;

iv) overnight incubation at 37°C under anaerobic or microaerophilic conditions;

v) measurement in millimeters of zones of inhibition after 24 or 48 hours by placing the dish on a flat ruler;

vi) expressing the results as an inhibition halo.

The person skilled in the art will be able to select the growth medium for steps i) to ii) in the usual way according to the nutrient requirements of the strain of lactic acid bacteria to be tested.

In one embodiment, a method for screening and isolating lactic acid bacteria is used to screen and isolate a strain of lactic acid bacteria for personal care. In a specific embodiment, this method is for screening and isolating lactic acid bacteria for oral hygiene.

In one embodiment, the pool of candidate lactic acid bacteria strains that are used to analyze the ability to tolerate antiseptics includes strains of lactic acid bacteria

- 18 042202 of the genus Lactobacillus or Pediococcus. In particular, candidate strains are strains

Pediococcus, in particular strains of Pediococcus acidilactici.

Brief description of the figures

Fig. 1. The ability of bacteria to tolerate triclosan expressed as halo inhibition (GIng) in millimeters (mm) as determined by disk diffusion analysis. Lactobacillus brevis CD2 (L.b. Cd2); Pediococcus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8633 (P.a. CECT 8633); Pediococcus pentosaceus CECT 8330 (P.p. CECT8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12); and Porphyromonas gingivalis.

Fig. 2. The ability of bacteria to tolerate thymol, expressed as halo inhibition (GIng) in millimeters (mm), as determined by disk diffusion analysis. Lactobacillus brevis CD2 (L.b. CD2); Pediococcus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8633 (P.a. CECT 8633); Pediococcus pentosaceus CECT 8330 (P.p. CECT8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12); and Porphyromonas gingivalis.

Fig. 3. The ability of bacteria to tolerate Listerine Original® (Listerine Original®), expressed as halo inhibition (Ging) in millimeters (mm), determined using a disk diffusion method of analysis. Lactobacillus brevis CD2 (L.b. CD2); Pediococcus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8633 (P.a. CECT 8633); Pediococcus pentosaceus CECT 8330 (P.p. CECT8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12); and Porphyromonas gingivalis.

Fig. 4. The ability of bacteria to tolerate Listerine Zero® (Listerine Zero®), expressed as halo inhibition (Ging) in millimeters (mm), determined using a disk diffusion method of analysis. Lactobacillus brevis CD2 (L.b. CD2); Pediococcus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8633 (P.a. CECT 8633); Pediococcus pentosaceus CECT 8330 (P.p. CECT8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12); and Porphyromonas gingivalis.

Fig. 5. The ability of bacteria to tolerate chlorhexidine, expressed as halo inhibition (GIng) in millimeters (mm), determined using a disk diffusion assay method. Lactobacillus brevis CD2 (L.b. Cd2); Pediococcus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8633 (P.a. CECT 8633); Pediococcus pentosaceus CECT 8330 (P.p. CECT8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12); and Porphyromonas gingivalis.

Fig. 6. The ability of bacteria to tolerate chlorhexidine gluconate, expressed as halo inhibition (GIng) in millimeters (mm), as determined by disk diffusion analysis. Lactobacillus brevis CD2 (L.b. CD2); Pediococcus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8633 (P.a. CECT 8633); Pediococcus pentosaceus CECT 8330 (P.p. CECT8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12); and Porphyromonas gingivalis.

Fig. 7. Halos of inhibition observed for Pediococcus acidilactici CECT8633, Streptococcus salivarius K12 (S.S.) and Porphyromonas gingivalis (P.g.) around 6 mm cellulose discs soaked in 20 µl of 0.12% (v/v) chlorhexidine gluconate.

Fig. Fig. 8. Growth kinetics of Streptococcus salivarius K12 in the absence (control) or in the presence of various concentrations of Listerine Zero® (K - control without antiseptic; 1:1 - not diluted; 1:2 - 2-fold dilution; 1:4 - 4-fold dilution; 1:8 - 8-fold dilution). x-axis = time (t) in hours (h).

Fig. 9. Growth kinetics of Streptococcus oralis in the absence (control) or in the presence of various concentrations of Listerine Zero® (K - control without antiseptic; 1:1 - not diluted; 1:2 - 2-fold dilution; 1:4 - 4-fold dilution ; 1:8 - 8-fold dilution). x-axis = time (t) in hours (h).

Fig. 10. Growth kinetics of Lactobacillus brevis CD2 in the absence (control) or in the presence of various concentrations of Listerine Zero® (K - control without antiseptic; 1:1 - not diluted; 1:2 - 2-fold dilution; 1:4 - 4-fold dilution; 1:8 - 8-fold dilution). x-axis = time (t) in hours (h).

Fig. 11. Growth kinetics of Pediococcus pentosaceus CECT8330 in the absence (control) or in the presence of various concentrations of Listerine Zero® (K - control without antiseptic; 1:1 - not diluted; 1:2 - 2-fold dilution; 1:4 - 4-fold dilution; 1:8 - 8-fold dilution). x-axis = time (t) in hours (h).

Fig. 12. Growth kinetics of Pediococcus acidilactici AB134 in the absence (control) or in the presence of various concentrations of Listerine Zero® (K - control without antiseptic; 1:1 - not diluted; 1:2 - 2-fold dilution; 1:4 - 4-fold dilution; 1:8 - 8-fold dilution). x-axis = time (t) in hours (h).

Fig. 13. Growth kinetics of Pediococcus acidilactici CECT8633 in the absence (control) or in the presence of various concentrations of Listerine Zero® (K - control without antiseptic; 1:1 - not diluted; 1:2 - 2-fold dilution; 1:4 - 4-fold dilution; 1:8 - 8-fold dilution). x-axis = time (t) in hours (h).

Fig. 14. Survival rate (SV) of strains of lactic acid bacteria, expressed as a percentage (mm), determined using analysis in a liquid medium. Lactobacillus brevis CD2 (L.b.CD2); Pediococ- 19 042202 cus acidilactici AB134 (P.a. AB134); Pediococcus acidilactici CECT 8630 (P.a. CECT 8630); Pediococcus pentosaceus CECT 8330 (P.p. CECT 8330); Streptococcus oralis; Streptococcus salivarius K12 (S.s. K12).

Fig. 15. Antagonistic activity of Streptococcus salivarius K12, Lactobacillus reuteri ATCC

55730 and Pediococcus acidilactici CECT8633, expressed as growth inhibition (GI) in millimeters, against oral pathogens Fusobacterium nucleatum, Treponema denticola and Prevotella denticola.

Fig. 16. Reduction in the number of tooth surfaces for which the worst gingival index score was observed (CI=3) compared to baseline. The black bar indicates the group of subjects after a standard oral hygiene procedure (placebo group); the white bar denotes the group of subjects after standard oral hygiene plus probiotic (probiotic group). Y-axis=number of tooth surfaces with a gingival index of 3 (number of overlays, with CI=3).

Fig. 17. Change in the concentration of pathogens Tannerella forsythia and Porphyromonas gingivalis (A conc. = concentration at the end of the study - concentration at the beginning of the study, expressed as a logarithm). Black bars indicate the group of subjects after a standard oral hygiene procedure (placebo group); white bars indicate the group of subjects after standard oral hygiene plus probiotic (probiotic group).

Examples

Example 1 Isolation and Identification of Pediococcus acidilactici CECT8633.

Candidate lactic acid bacteria were isolated from fresh feces of 0-9 year old children. Samples were dissolved in PBS buffer (pH 7.4), aliquoted and plated on MRS medium supplemented with various combinations of antibiotics. The strains were cultured under microaerophilic conditions (5% CO ₂ ) at 37 or 30°C. The incubation time depended on the growth rate, but usually lasted from 24 hours to 3 days. Isolation of individual strains was carried out in the same selection medium and then Gram staining was performed to obtain a primary identification. After the isolated strains had grown, they were preserved by lyophilization in 0.1 x PBS with 15% skimmed milk powder.

Identification at the species level was performed by sequencing the 16S rRNA gene. The bacteria were grown overnight on MRS medium (pH 6.4) at 37°C in an atmosphere containing 5% CO ₂ . The bacteria were then harvested, washed, resuspended in pre-lysis buffer (480 µl EDTA, 50 mM, pH 8.0; 120 µl lysozyme, 10 mg/ml) and further incubated at 37° C. for 60 min. DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega). After centrifuging the pretreated bacteria at 14,000 g for 2 min to remove the supernatant, the Promega protocol was followed. Briefly, bacteria were resuspended in nuclear lyse 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 shaking at high speed. Samples were cooled and centrifuged at 15,000 g for 3 min. Supernatants containing DNA were transferred to clean 1.5 ml microcentrifuge tubes and mixed with 600 μl of isopropanol by inversion. DNA was collected by centrifugation at 15,000 g for 2 min and the supernatant was carefully discarded. 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 15,000 g for 2 min. Finally, the DNA pellet was resuspended in 100 μl of rehydration solution by incubation at 65°C for 1 hour. Samples were stored at 2-8°C.

16S rRNA was amplified by PCR using universal eubacteria primers Eub27f and Eub1492r. The DNA obtained as explained above was then washed using the Quiaquick kit (Quiagene). Four consecutive sequencing reactions were performed for each sample in a 3130 Genetic Analyzer (Applied Biosystems) using the BigDye version 3.1 kit using primers 27F, 357F, 907R and 1492R. Data collection was performed and chromatograms were generated using DNA sequence analysis software Sequencing Analysis version 5.2 (Applied Biosystems) and verified by visual analysis with Chromas (Technelysium Pty Ltd.) and BioEdit (Ibis Biosciences).

Genus identification was performed using the Ribosomal Database Project tool and species identification was performed by comparing the resulting sequence with 16S sequences of known organisms from both the RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/) via BLASTN and from the Ribosomal Database Project (http://rdp.cme.msu.edu/).

The CECT8633 strain was identified as Pediococcus acidilactici and deposited on May 26, 2014 in the Spanish Type Culture Collection (Universitat de Valencia, Campus de Burjassot, Edif. de Investigation, 46100 Burjassot, Valencia, Spain). The deposited strain is viable and retains all of its properties related to the deposit.

The 16S rRNA sequence obtained for strain CECT8633 corresponds to the sequence of SEQ ID NO: 1.

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Example 2. The ability of strains of lactic acid bacteria to tolerate antiseptics (disk diffusion method of analysis).

LAB strains with putative probiotic properties, namely L. brevis, P. acidilactici, P. pentosaceus, S. oralis and S. Salivarius, were plated on agar plates with appropriate growth media and incubated to obtain isolated colonies. Porphyromonas gingivalis DSM20709 was used as an example of pathogens associated with periodontal pathologies. The media and atmospheric conditions used for each strain were the most suitable for their proper growth and are shown in Table 1. 1.

Table 1

Nutrient media and growth conditions for analyzed bacteria

Strain Wednesday T ^a Time (h) Atmosphere L. brevis CD2 MRS 37°C 24 hours 5% CO ₂ P. acidilactici CECT8633 MRS 37°C 24 hours 5% CO ₂ P. acidilactici AB134 MRS 37°C 24 hours 5% CO ₂ R. pentosaceus CECT8330 MRS 37°C 24 hours 5% CO ₂ S. oralis BHI 37°C 24 hours 5% CO ₂ S. salivarius K12 BHI 37°C 24 hours 5% CO ₂ P. gingivalis Sheep blood agar 37°C 48 h Anaerobiosis

For each microorganism, isolated colonies were used to obtain a suspension in phosphate-buffered saline 0.1 M (PBS) with an optical density equivalent to McFarland standard No. 3. The suspension was used to obtain a confluent layer of the microorganism on the appropriate nutrient medium (Table 1) by evenly inoculating the microorganism using a sterile spatula.

Immediately after seeding the dishes with strains, 6 mm cellulose discs were placed in them. The discs were impregnated with various common antiseptics according to Table. 2.

table 2

Antiseptics used to study the ability of lactic acid bacteria to tolerate them

Antiseptic for mouthwash Solvent Volume Concentration (% v/v or mg/mL) Chlorhexidine Dimethylformamide 5 µl 0.12% 0.06% 0.03% Chlorhexidine gluconate FBI 20 µl 0.12% 0.06% 0.03% Triclosan EtOH 5 µl 0.2% 0.1% 0.05% Listerine Zero® FBI 20 µl — Diff. 1:2 Diff. 1:4 Listerine Original® FBI 20 µl — Diff. 1:2 Diff. 1:4 Thymol EtOH 5 µl 1.2 mg/ml 0.6 mg/ml 0.3 mg/ml

The volume used was the maximum that the disk allowed without spreading to the medium. It was confirmed that the solvent had no inhibitory effect against bacteria in the amount used. Microorganisms were allowed to grow in accordance with the conditions indicated in table. 1 and the inhibition halo (in millimeters) was measured after 24 or 48 hours according to the respective optimal microbial growth conditions. The results were obtained in triplicate.

In FIG. 1 shows the inhibition observed for each microorganism in the presence of triclosan at various concentrations. The strains of the pathogen P. gingivalis, as well as Streptococcus sp. inhibited by triclosan. However, neither Lactobacillus brevis nor strains belonging to the genus Pediococcus were inhibited. Similarly, thymol inhibited the growth of P. gingivalis and strains of the Streptococcus species at the highest concentration tested, 1.2 mg/ml, but did not inhibit the growth of Lactobacillus brevis or strains of Pediococcus (FIG. 2). The same effect was observed when the discs were soaked with 20 microliters of undiluted Listerine Original® (with alcohol) (FIG. 3). P. gingivalis and Streptococcus strains were also inhibited by Listerine Zero® and its corresponding 2-fold dilution (FIG. 4). The ability of L. brevis to tolerate the presence of Listerine Zero® was greater than that of P. gingivalis and Streptoccocus, as no inhibition was observed in the presence of 4- and 2-fold Listerine Zero® dilutions. However, in contrast to triclosan, thymol and Listerine Original®, the presence of undiluted Listerine Zero® had an inhibitory effect on L brevis. None of the Pediococcus strains were inhibited by the presence of Listerine Zero®, as was observed

- 21 042202 for Listerine Original®.

In FIG. Figures 5 and 6 show that chlorhexidine and chlorhexidine gluconate were the oral rinse antiseptics with the strongest antimicrobial activity, as all strains were inhibited by the highest concentration tested (0.12%, v/v). Similar to other mouthwash ingredients, P. gingivalis and Streptococcus spp. were the strains that showed the highest sensitivity to chlorhexidine, as they were inhibited by any of the tested concentrations. L. brevis was inhibited at 0.12% and 0.06%, but not at 0.03%, and therefore showed a higher chlorhexidine tolerance than P. gingivalis and Streptococcus spp. Strains of Pediococcus species turned out to be LAB that showed the greatest resistance to chlorhexidine. The inhibition halos observed around chlorhexidine-soaked discs were 4, 3, and 2 mm for Pediococcus pentosaceous CECT8330, P. acidilactici CECT8633, and P. acidilactici AB134, respectively, while the P. gingivalis inhibition halo was 9 mm. Consequently, strains of Pediococcus acidilactici were particularly resistant to chlorhexidine. Similarly, strains of Pediococcus appeared to be the strains that showed the highest resistance to chlorhexidine gluconate. In FIG. 7. clearly illustrates the smaller inhibition halo observed for P. acidilactici CECT8633 compared to S. salivarius K12 and P. gingivalis in the presence of 0.12% chlorhexidine gluconate.

In conclusion, it was shown that the inhibitory effect of antiseptics was higher for the pathogen P. gingivalis, as well as for probiotics from Streptococcus species, followed by Lactobacillus species. Probiotics from species of the genus Pediococcus were particularly resistant to antiseptics and especially probiotics belonging to the species Pediococcus acidilactici.

Example 3. The ability of strains of lactic acid bacteria to tolerate antiseptics (analysis in a liquid medium).

In order to establish whether the presence of mouthrinses affects the growth kinetics of LAC, the inhibitory effect of Listerine Zero® in liquid media was investigated. Overnight cultures of probiotic bacteria were prepared as described in Example 2. The isolated colonies were used to obtain a bacterial suspension in sterile 0.1 M PBS until an optical density corresponding to the McFarland standard of 0.5 was reached (approximately 1.5E + 08 cfu / ml ). 100-fold dilutions were then prepared to give a concentration of approximately 1.5E+06 CFU/mL. Listerine Zero® was filtered through a 0.22 micrometer filter and 4 consecutive 2-fold dilutions were prepared.

Liquid growth media (140 μl) suitable for the proper growth of each microorganism according to the table. 1 (Example 2) was pipetted into 96-well microplates. Then 40 µl of the mouthrinse dilution was added, followed by 20 µl of the bacterial suspension containing 1U+06 cfu/ml. Also received blank controls without probiotic and positive controls without ingredients (except PBS). Bacterial growth was assessed by determining the optical density (OD) at 550 nm for the wells containing the probiotic after 24 and 48 hours and subtracting the resulting OD for the wells containing the same concentration of mouthrinse but without the probiotic. The survival rate was calculated after 48 hours using the following formula:

(OPp - OPb) - (OPo - OPob)

Survival rate (%) = ------------------*100 where OD _n is the optical density of a well inoculated with probiotic but not containing mouthrinse (positive control),

OD _b is the optical density of the well without probiotic and without mouthrinse,

OD _o is the optical density of the well with probiotic and mouthrinse, and

ODb is the optical density of the well without probiotic and with mouthrinse.

The conditions were tested in triplicate.

In FIG. 8-13 shows the growth kinetics of probiotic strains at various concentrations of Listerine Zero®. The results confirm that Listerine reduced the growth of Streptococcus strains at all concentrations tested. Only S. salivarius K12 was able to grow at 1:4 and 1:8 dilutions, but its growth was greatly reduced compared to the positive control (probiotic without mouthrinse). S. oralis was only able to grow when the dilution of Listerine mouthrinse reached 8 times its original concentration. However, even at this concentration, no growth was observed after 24 h and the amount of probiotic was approximately 2 times lower than for the positive control after 48 h. The growth rate of L. brevis was lower than for Streptococcus strains, as no growth was 48 hours after seeding the medium. However, it was confirmed that the ability of L brevis CD2 to tolerate mouthwash was higher than that of Streptococcus strains, as it was only completely inhibited when mouthwash was not present.

- 22 042202 diluted. The concentration of L. brevis CD2 when 2-, 4-, and 8-fold dilutions were present in the medium was reduced by approximately 50%.

Pediococcus strains were found to be the probiotics found to have the highest ability to tolerate mouthrinse. The presence of undiluted Listerine® completely inhibited the growth of P. pentosaceus CECT8330, P. acidilactici AB134 and P. acidilactici CECT8633 after 24 hours. However, P. acidilactici AB134 and P. acidilactici CECT8633 were the only bacteria that were able to grow in the presence of this rinse oral cavity after 48 hours.

In FIG. 14 summarizes the survival rates of the various strains at 48 hours. It can be clearly seen that only Pediococcus acidilactici strains CECT7780 and CECT8633 were able to grow in the presence of undiluted mouthrinse and that L. brevis CD2 and especially P. pentosaceus CECT8330 were also able to grow when the mouthwash was diluted 2 times.

Strain survival rate.

Streptococcus has always been lower than that of other probiotic strains at all indicated concentrations.

Example 4 Antagonistic activity against periodontal pathogens.

The antagonistic ability of P. acidilactici CECT8633 against pathogen species largely associated with gingivitis, periodontitis and halitosis, namely Fusobacterium nucleatum, Treponema denticola and Prevotella denticola, was investigated. Pathogens were obtained from the Pasteur Institute Collection (deposit codes CIP104988, CIP103919 and CIP104478T, respectively).

P. acidilactici CECT8633 was grown on MRS plates at 37°C and 5% CO ₂ until confluent. Cylindrical sections of confluent bacteria 6 mm in diameter were placed on M20 dishes inoculated with F. nucleatum and P. denticola or M71 dishes inoculated with T. denticola. The medium in M20 plates contained 30 g tryptone, 20 g yeast extract, 0.5 g cysteine hydrochloride, 5 g glucose, and 25 ml hemin solution (0.1 g hemin chloride, 4 ml triethanolamine and 96 ml distilled water per liter. Medium in M71 cups contained 15 g tryptone, 5 g yeast extract, 8 g sodium dihydrogen phosphate, 1 g ammonium carbonate, 0.5 g cysteine hydrochloride, 0.03 ml thioglycolic acid, 0.05 ml Tween 80 and 250 ml horse serum per The plates were incubated overnight at 37° C. under anaerobic conditions Growth inhibition activity (GI) was calculated by subtracting the cylinder diameter (DC, mm) from the zone of inhibition diameter (ZZI, mm) as follows: GI=(GZI- DC)/2 The results were compared with S. salivarius K12 (BLIS Technologies Ltd., New Zealand) and L. reuteri ATCC 55730 (GumPeriobalance® (BioGaia, Sweden) grown under the same conditions as P. acidilactici CECT8633.

The results are shown in FIG. 15. P. acidilactici CECT8633 was able to inhibit the growth of the three pathogens tested, in contrast to S. salivarius and L. reuteri, which inhibited two of the three pathogens tested. Moreover, P. acidilactici CECT8633 showed greater ability to inhibit the growth of F. nucleatum and T. denticola than commercially available controls.

Example 5 Clinical Efficacy

A double-blind, randomized, placebo-controlled clinical trial was conducted to evaluate the safety of LSD strains and their efficacy as probiotics to improve parameters associated with gingivitis.

Subjects included in this clinical trial met all inclusion and exclusion criteria. Inclusion criteria: age 18-55; men or women (balanced groups); non-smokers (never smoked or smoked for at least 6 months prior to the test); subjects with naturally occurring gingivitis as defined by mean gingival index (CI)>1.3; without loss of interproximal contact >2 mm in most areas. Exclusion criteria: carious lesions and/or insufficient restoration of the maxillary lateral incisors, first or second premolars and first molars or adjacent teeth, preventing the removal of plaque in areas of interest; subjects currently undergoing dental treatment; subjects currently undergoing orthodontic therapy or wearing occlusal splints; subjects suffering from any systemic disease or condition that may affect the response of gum tissues or the ability to exercise sufficient control over dental plaque (pregnancy, diabetes, quantitative and / or qualitative disorders in polymorphonuclear cells, other diseases of the immune system, etc.); subjects taking medications that may interfere with gum response; subjects taking antibiotics or antiseptics within 2 months before the study; subjects who used probiotic oral health products within 2 months prior to the study; history of hypersensitivity or allergy to any component of medicinal products.

On day 0, professional oral hygiene (calculus removal and polishing) was performed on all subjects. Provided with fluoride toothpaste and also asked to brush twice a day to standardize normal oral hygiene.

Subjects were randomly assigned to one of the following two treatment groups.

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Table 3

Investigated Treatments

PROBIOTIC PLACEBO

Composition Lactobacillus Probiotic Blend Trehalose: 125 mg (per tablet): plantarum CECT7481, Guar gum (E-412): Lactobacillus brevis CECT7480 and 80.00 mg Pediococcus acidilactici CECT8633): 125 mg Sorbitol: 691.20 mg Guar gum (E-412): 80.00 Hydrogenated mg cottonseed oil: 18.32 Sorbitol: 691.20 mg mg hydrogenated oil Mint aromatic cotton: 18.32 mg Mint flavor: 20.00 mg additive: 20.00 mg Dose: 2 tablets/day 2 tablets/day Route of administration: Oral Oral Medicinal Orally dispersible Cavity dispersible form: pills mouth pills

Neither the patients nor the performers knew which treatment was assigned to each subject. The dispersible tablets had the same appearance, and the boxes containing them included the study code and the corresponding subject identification number. The researcher gave participants boxes containing pills that looked the same. The treatment assigned to each volunteer (A or B) was recorded in a dated register. Randomization was disclosed only at the end of the study after analysis of the results, or if any significant event was observed that forced unmasking. Subjects consumed the respective product for 6 weeks. The orodispersible tablets were allowed to dissolve slowly in the mouth after brushing each tooth (twice daily). After consumption of the product, participants were not allowed to brush their teeth or consume any food or drink (other than water) for 1 hour after consumption of the tablet. Subjects were required to record pill intake by completing a daily follow-up questionnaire. It was returned to the researcher along with boxes of unused pills to examine each patient's adherence to therapy.

The primary endpoint was the gingival index (CI) measured by the modified method of Loe and Silness (1963) without bleeding on the instrument [Loe H. et al., Periodontal disease in pregnancy, Acta Odontologica Scandinavica, 1963, vol. 21, p. 533-551]. The mean CI was calculated by dividing the sum of all scores for each tooth by the total number of teeth examined.

On day 0 (at the start of the study), gingival index (CI) was determined before professional oral hygiene (calculus removal and polishing). The same indicator was determined after 6 weeks.

On day 0 (start of study) and week 6 (end of study), just prior to clinical examination, supragingival plaque samples were collected one from each quadrant (maxillary left-right/mandibular left-right) and pooled. Supragingival plaque was collected using sterile paper pins placed for 10 s. Paper pins with samples were combined and transferred to a microfuge tube containing 200 µl of TE buffer (10 mM Tris-(hydroxymethyl)-methylamine chloride, 1 mM ethylenediaminetetraacetic acid) and stored at -20°C until analysis. Real-time PCR was performed using Taqman probes with specific primers for six putative periodontal pathogens (Aggregatibacter actinomycetemcomitans; Porphyromonas gingivalis; Tannerella forsythia; Fusobacterium species; Streptococcus oralis; Campylobacter rectus).

Primary and secondary measures were analyzed using SAS. All analyzes were performed in accordance with the criteria of all randomized patients according to the prescribed treatment (RTP) and patients without violations of the protocol (PP). The results were tested using the Shapiro-Wilk test for normality. If the results were parametrically scattered, differences in primary and secondary measures between groups were determined using analysis of variance (ANOVA). In order to clarify the exact nature of the differences between groups, a retrospective analysis was performed (eg, Duncan's multi-rank test). If results were scattered non-parametrically, scores were compared using non-parametric tests (Wilcoxon for pairwise comparisons and Mann-Whitney for independent data). In all of the hypothesis testing criteria, equality between treatment groups was taken as the null hypothesis and rejected when P-values were lower than 0.05. This meant that such differences were considered significant differences when the α-error

- 24 042202 or Type I error was <0.05.

Fifty-nine patients were assessed for eligibility and randomly assigned to probiotic (n=30) or placebo (n=29) groups. Characteristics of patients at the start of the study are presented in Table. 4.

Table 4 Demographic characteristics of the study population

Probiotic N=30 Control N=29 Total N=59 Age (years) p-value = 0.7805 (Wilcoxon rank sum test) N 29 29 58 N absent 1* 0 1 Average (St. Deviation) 30.91 (12.20) 32.50 (13.62) 31.71 (12.84) CI 95% (mean) (26.27, 35.55) (27.33, 37.68) (28.33, 35.08) Range (min, max) (17.76, 55.33) (17.70, 65.70) (17.70, 65.70) Median 26.26 28.20 27.49 MKI (Q1.Q3) (21.47, 38.36) (21.34, 37.45) (21.45, 38.36) Floor p-value = 0.7106 (chi-square test) Women 19(63.3%) 17(58.6%) 36 (61.0%) Men 11 (36.7%) 12 (41.4%) 23 (39.0%)

★Missing results, but within selection criteria ranges

94.2% of patients took at least 75% of the drug, 89.7% in the probiotic group and 100.0% in the control group. No differences were observed in adherence to therapy between the groups.

At week 6, the number of distinct tooth surfaces with a CI score of 3 (number of distinct CI scores=3) was lower in the probiotic group than in the control group (n=0 (0.0%) vs. n=5 (0, 2%), p-value=0.0418).

The magnitude of change between start of study and endpoint (start of study - week 6) was also significantly higher in the probiotic group than in the control group (standard care) (P<0.01, regardless of the type of analysis: GRP or PP) (Fig. 16).

Treatment with probiotic tablets correlated with a significant reduction in levels of Tannerella forsythia as well as Porphyromonas gingivalis (FIG. 17).

In conclusion, probiotic tablets were able to reduce the number of areas where severe inflammation was present compared to professional oral hygiene alone in patients with gingivitis. The use of the probiotic also made a significant microbiological contribution, as it was associated with a decrease in the number of T. forsythia and P. gingivalis, pathogens widely known to adversely affect the course of gingivitis, which showed the strongest correlation with the number of lesions with severe inflammation among pathogenic bacteria. , the number of which was measured in the study population.

Example 6 Formulation in the form of orodispersible tablets.

The orodispersible tablets had the following composition:

125 mg Pediococcus acidilactici CECT8633;

.3 mcg of cholecalciferol (vitamin D3);

mg mint flavor;

mg guar gum;

689.53 mg sorbitol;

9.35 siloid;

9.35 magnesium stearate.

This composition is suitable for slow dissolution in the oral cavity. The probiotic concentration at the expiration date is 1E+09 CFU per tablet.

Alternatively, the tablet contains Lactobacillus plantarum CECT7481, Lactobacillus brevis CECT7480 in addition to Pediococcus acidilactici CECT8633.

Example 7 Formulation in the form of an oral gel.

The vials contain 500mg of powder containing the following ingredients: Pediococcus acidilactici CECT8633 (minimum probiotic concentration at expiration date is 6E+09 CFU/vial) Chlorophyllin E141ii (4%, by weight) Guar gum (5.2% by weight).

- 25 042202

Example 8 Composition in the form of chewing gums.

Chewing gums contain the following composition:

1340.68 mg gum base (Health in Gum PWD-03);

125 mg Lactobacillus plantarum CECT7481, Lactobacillus brevis CECT7480 and Pediococcus acidilactici

CEST8633 (ratio 1:1:1);

31.52 mg encapsulated flavor;

47.28 mg powder flavoring;

12.61 mg liquid flavor;

24.0 mg magnesium stearate;

15.75 mg silica;

1.89 mg aspartame;

1.26 mg acesulfame K.

Example 9 Composition in the form of a toothpaste.

Toothpaste consists of:

125 mg Pediococcus acidilactici CECT8633;

0.12 chlorhexidine digluconate;

g xylitol;

0.5 g alpha-tocopherol acetate;

100 g of excipient.

Alternatively, toothpaste has the following composition:

Pediococcus acidilactici CECT8633 until a final concentration of 1E+09 CFU is reached at the expiration date;

0.04 g of glycyrrhetinic acid;

0.16 g sodium fluoride;

g PEG-12;

0.8 g zeolite;

5.6 g silicon;

1.6 g polyglyceryl-5 laurate;

0.8 g xylitol;

0.48 g menthol;

0.4 g sodium saccharin;

0.4 g sodium benzoate;

0.24 g of eucalyptus spherical;

0.08 g cyclodextrin;

0.96 g hydroxypropyl cellulose;

30.56 g butylene glycol.

Example 10 Mouthwash formulation.

Mouthwash consists of a combination of ingredients present in the delivery cap and in the vial. The delivery caps contained 100 mg of lyophilized probiotic strain CECT8633 (1-2 U+09 CFU per delivery cap), magnesium stearate (2.2 mg) (herein referred to as, for example, anti-caking agent and release agent) and silica ( 0.5 mg) (referred to in the present description as an anticaking agent). The tube contained 10 ml of mouthwash containing 0.092% eucalyptol, 0.042% menthol, 0.06% methyl salicylate, 0.064% thymol, and 27% ethanol (w/v).

Pressing on the lid releases the probiotic into the liquid contained in the tube. The composition is stirred and then used as a mouth rinse for 1 minute.

Example 11 Fermentation and post-treatment of lactic acid bacteria.

The post-treatment biomass used as raw material was obtained through a manufacturing process that included fermentation and freeze-drying steps. The mother starter was obtained by inoculation of 80 ml of the MRS nutrient medium with strains of P. acidilactici. The inoculated medium was fermented at 37° C. under microaerophilic conditions for 48 hours. The resulting culture was used to inoculate 600 L of MRS medium at a ratio of approximately 1-2% starter per total volume of medium. The resulting medium was cooled at 3-5°C and centrifuged to remove most of the water present in the broth. Cryoprotectants were added to the concentrated culture and the resulting mixture was lyophilized at -45°C.

Claims (12)

1. The use of a strain of Pediococcus as a probiotic in combination with at least one antiseptic, while the specified strain of Pediococcus has the ability to tolerate the presence of at least one antiseptic selected from the group consisting of a) triclosan; b) an essential oil containing at least one individual component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate; And c) a single component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate, said strain of Pediococcus being considered to have the ability to tolerate said antiseptic when the inhibition halo, as determined by disk diffusion assay, is observed for said strain Pediococcus, less than the inhibition halo for Porphyromonas gingivalis, wherein said antiseptic is present at a concentration sufficient to establish inhibition of Porphyromonas gingivalis, and wherein said Pediococcus strain and said antiseptic are formulated for separate, sequential, simultaneous administration or administration as a mixture, and said Porphyromonas gingivalis is Porphyromonas gingivalis DSM20709, and said Pediococcus strain is a Pediococcus acidilactici strain, wherein said Pediococcus strain is used in preventing or treating an oral condition. 2. Use according to claim 1, characterized in that said strain of Pediococcus acidilactici is a strain of Pediococcus acidilactici deposited in the Spanish Type Culture Collection under accession number CEST 8633. 3. Use according to any one of claims 1 and 2, characterized in that said strain of Pediococcus is in combination with at least one antiseptic selected from the group consisting of triclosan; an essential oil containing at least one individual component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate; a separate component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate; and biguanides. 4. Use according to claim 3, characterized in that said strain of Pediococcus is in combination with at least one antiseptic selected from the group consisting of triclosan and an essential oil containing at least one individual component selected from the group consisting of menthol, eucalyptol, thymol and methyl salicylate. 5. Use according to any one of claims 1 to 4, characterized in that said antiseptic is formulated as a personal care product together with suitable excipients and/or carriers and said Pediococcus strain is formulated as a personal care product or pharmaceutical product together with acceptable excipients and/or carriers. 6. Use according to claim 5, characterized in that said personal care product is an oral hygiene product. 7. Use according to claim 6, characterized in that said oral hygiene product is a toothpaste, oral gel, oral spray, mouthwash or an orodispersible tablet. 8. Use according to any one of claims 1 to 7, characterized in that said antiseptic and said Pediococcus strain are administered separately (waiting a period of time between the introduction of the Pediococcus strain and the antiseptic), while said period of time lasts no more than 1 hour. 9. Use according to claim 8, characterized in that the subject in need of treatment is instructed to rinse the mouth with a mouthwash containing an antiseptic for 30-60 seconds, after which the mouthwash is spit out, and then placed in the mouth an oral tablet containing the Pediococcus strain and allow it to dissolve slowly. 10. Use according to any one of claims 1 to 9, characterized in that said oral condition is caused by oral pathogens. - 28 042202 11. A strain belonging to the species Pediococcus acidilactici deposited in the Spanish Type Culture Collection under accession number CECT 8633 for use as a probiotic, said strain having the ability to tolerate the presence of at least one antiseptic. 12. A method for producing a Pediococcus acidilactici mutant, said method comprising mutagenesis of a strain of Pediococcus acidilactici deposited in the Spanish Type Culture Collection under accession number CECT 8633, wherein the resulting mutant retains or increases at least the ability of said deposited strain to tolerate the presence of at least one antiseptic.