Probiotic enhancers and uses thereof

Abstract

Described herein are methods for improving the inhibition profile of Streptococcus salivarius, comprising formulating S. salivarius in a composition comprising an effective amount of a saccharide supplement. Also described herein are compositions comprising Streptococcus salivarius and a saccharide supplement, methods of inhibiting microorganisms using such compositions, therapeutic formulations comprising such compositions, and methods of treating or preventing disease using such compositions and therapeutic formulations.

Description

[Technical field]

[0001] The present invention relates broadly to methods for enhancing the effectiveness of Streptococcus salivarius probiotics, and compositions useful in such methods. [Background technology]

[0002] Probiotics are live microorganisms that can provide a variety of health benefits when ingested by or administered to a subject. For example, Lactobacillus and Bifidobacterium species are well known for their use in maintaining or improving intestinal health.

[0003] The expansion of the probiotics market has increasingly focused on how to enhance the effectiveness of probiotic strains.To date, much of the focus has been on how prebiotics can change the intestinal microbiome or improve the effectiveness of probiotics that are beneficial for intestinal health.As a result of this research, various prebiotics have been identified that induce the growth or activity of intestinal probiotics.These prebiotics include non-digestible fiber compounds, resistant starch, arabinogalactan, and oligosaccharides such as inulin and galactooligosaccharides.

[0004] Prebiotics were originally described as "non-digestible food ingredients that have a beneficial effect on the host by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the colon, thereby improving human health." This definition meant that only a limited number of carbohydrates met the criteria for promoting the bacteria normally present in the colon. Since this description was conceived, prebiotics have been defined as "selectively fermented ingredients that bring about specific changes in the composition and / or activity of the gastrointestinal microflora, thereby benefiting the host's health." However, the currently accepted definition excludes the microflora that is only present in the oral cavity.

[0005] WO2016172658 describes the use of microbiome regulators, such as sugars or sugar alcohols, to improve the growth and / or colonization of beneficial bacteria in the large or small intestine. WO2004074496 describes the use of galactooligosaccharides to increase beneficial bacteria in the gastrointestinal tract. US20200030366 describes a method of treating or preventing colonization by gastrointestinal pathogenic microorganisms, including administering dietary fiber, such as inulin. The composition may also include prebiotics, including various sugars / sugars, such as sucrose, maltose, lactose, fructose, galactose, glucose, raffinose, mannose, ribose, and trehalose.

[0006] More recently, probiotics for oral application or orally targeted probiotics have been identified. S. salivarius probiotics are known for use against various oral pathogens. For example, S. salivarius strain K12 ("K12") has been documented for use in preventing or treating ear, nose, and throat infections, such as those caused by Streptococcus pyogenes (see, for example, WO 2001027143 to Blis Technologies Ltd). K12 is also known for use in treating halitosis caused by anaerobic bacteria (see, for example, WO 2005007178 to Blis Technologies Ltd). S. salivarius strain Mia (herein "M18") is known for use in the treatment of dental caries caused by Streptococcus mutans (see, e.g., WO 2003070919 to Blis Technologies Ltd).

[0007] US20190336428 describes the use of sugars selected from D-turanose, D-melezitose, D-lactitol, myo-inositol, and N-acetyl-D-mannosamine to selectively increase the growth of beneficial bacteria in the oral cavity. WO2012065811 describes a nutritional composition for children that may include probiotics and prebiotics. US20160166501 describes an oral composition comprising Lactobacillus helveticus for use in oral hygiene. The composition may also include other probiotic strains and excipients. WO2007144334 describes a composition for treating otitis media that comprises a probiotic Lactobacillus strain and a bacterial strain capable of exerting a bacteriostatic effect, such as S. salivarius K12. The composition may be in the form of an infant formula that includes lactose.

[0008] US20190343899 describes hard candy or taffy compositions containing prebiotics prepared at elevated temperatures. WO2017129639 describes infant formulas containing oligosaccharides.

[0009] Simple sugars are known to promote bacterial growth by being used as prebiotics and ingested as an energy source. When bacteria utilize these sugars, they may produce organic acids as by-products. These acids may have a weak non-selective inhibitory effect on the growth of other bacteria. Furthermore, when used in the oral cavity, the production of acidic by-products may lower the environmental pH and cause erosion of tooth enamel that may progress to caries. A more acidic environment also promotes the growth of harmful dental pathogens such as S. mutans.

[0010] There is a need to identify substitutes for common sugars to promote antimicrobial activity in probiotics without significantly affecting pH. There is also a need to identify substitutes for common sugars that enhance the colonization efficacy of probiotics to provide health benefits. There is also a need for compositions that can enhance the activity of probiotics in areas other than the gastrointestinal tract. It is an object of the present invention to go some way to meeting any one or more of these needs and / or at least to provide the public with a useful choice.

[0011] Other objects of the present invention will become apparent from the following description, which is given by way of example only.

[0012] Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters formed part of the prior art base or were common general knowledge in the art relevant to the present invention by virtue of existing prior to the priority date. Summary of the Invention

[0013] In a first aspect, the present invention provides a method for improving the inhibition profile of Streptococcus salivarius, the method comprising formulating S. salivarius in a composition comprising an effective amount of a supplemental saccharide; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0014] In a second aspect, the present invention provides a method for upregulating one or more genes in Streptococcus salivarius, comprising formulating S. salivarius in a composition comprising an effective amount of a sugar supplement; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0015] In a third aspect, the present invention provides a method for inhibiting skin, dental, oral, mucosal and / or ENT microorganisms, comprising contacting the microorganisms with a composition comprising Streptococcus salivarius and an effective amount of a saccharide; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0016] In a fourth aspect, the present invention provides a method for increasing production of one or more of a lantibiotic peptide, a bacteriocin, or a urease by Streptococcus salivarius, comprising formulating S. salivarius in a composition comprising an effective amount of a supplemental sugar; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; Methods are provided in which the supplemental sugar is galactose or raffinose, or a combination thereof, and production is increased relative to a composition lacking the supplemental sugar.

[0017] In a fifth aspect, the present invention provides a composition comprising an effective amount of a saccharide supplement for use in improving the inhibition profile of Streptococcus salivarius, the composition comprising: Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The composition is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0018] In a sixth aspect, the present invention provides a composition comprising Streptococcus salivarius K12 and raffinose in an amount of 2-3% by weight.

[0019] In a seventh aspect, the present invention provides a composition comprising Streptococcus salivarius K12 and galactose in an amount of 0.25 to 0.75% by weight.

[0020] In an eighth aspect, the present invention provides a composition comprising Streptococcus salivarius M18 and raffinose in an amount of 2-3% by weight.

[0021] In a ninth aspect, the present invention provides a composition comprising Streptococcus salivarius M18 and galactose in an amount of 0.25 to 0.75% by weight.

[0022] In a tenth aspect, the present invention provides a composition comprising Streptococcus salivarius K12, Streptococcus salivarius M18, and raffinose in an amount of 2-3% by weight.

[0023] In an eleventh aspect, the present invention provides a composition comprising Streptococcus salivarius K12, Streptococcus salivarius M18, and galactose in an amount of 0.25 to 0.75% by weight.

[0024] In a twelfth aspect, the present invention provides a composition comprising Streptococcus salivarius K12, Streptococcus salivarius M18, raffinose in an amount of 2-3% by weight, and galactose in an amount of 0.25-0.75% by weight.

[0025] In a thirteenth aspect, the present invention provides a composition comprising Streptococcus salivarius K12, raffinose in an amount of 1.2 to 2.2% by weight, and galactose in an amount of 0.7 to 1.7% by weight.

[0026] In a fourteenth aspect, the present invention provides a composition comprising Streptococcus salivarius M18, raffinose in an amount of 1.2 to 2.2% by weight, and galactose in an amount of 0.7 to 1.7% by weight.

[0027] In a fifteenth aspect, the present invention provides a composition comprising Streptococcus salivarius K12, Streptococcus salivarius M18, raffinose in an amount of 1.2 to 2.2% by weight, and galactose in an amount of 0.7 to 1.7% by weight.

[0028] In a sixteenth aspect, the present invention provides a therapeutic formulation comprising a composition according to any one of the fifth to fifteenth aspects.

[0029] In a seventeenth aspect, the present invention provides a method for treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof a composition of any one of the fifth to fifteenth aspects, or a therapeutic formulation of the sixteenth aspect.

[0030] In an 18th aspect, the present invention provides a method for inhibiting microorganisms susceptible to Blis-producing S. salivarius, comprising administering to a subject in need thereof a composition of any one of the 5th to 15th aspects, or a therapeutic formulation of the 16th aspect.

[0031] In a nineteenth aspect, the present invention relates to the use of Streptococcus salivarius and a saccharide supplement in the manufacture of a medicament, the medicament comprising: (a) the treatment or prevention of a disease or disorder; or (b) inhibition of microorganisms susceptible to Bliss-producing S. salivarius; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The present invention provides a method for the preparation of a food product comprising the steps of: (a) administering to said food a food supplement comprising ...

[0032] In a twentieth aspect, the present invention provides a composition comprising Streptococcus salivarius and an effective amount of a saccharide supplement, the composition comprising: (a) the treatment or prevention of a disease or disorder; or (b) for use in inhibiting microorganisms susceptible to Bliss-producing S. salivarius; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The composition is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0033] In a twenty-first aspect, the present invention provides a method for producing a composition comprising Streptococcus salivarius and an effective amount of a saccharide supplement, comprising: (a) combining Streptococcus salivarius with a sugar supplement; (b) mixing to produce a homogenous blend; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0034] In a twenty-second aspect, the present invention relates to the use of a composition produced by the method of the twenty-first aspect for the treatment or prevention of a disease or disorder, or for the inhibition of microorganisms susceptible to Bliss-producing S. salivarius.

[0035] The following embodiments and preferences may relate to any of the above aspects, either alone or in any combination of any two or more.

[0036] In various embodiments, the upregulated gene encodes a lantibiotic peptide or a bacteriocin.

[0037] In various embodiments, the upregulated gene encodes a class I lantibiotic peptide or a class II bacteriocin.

[0038] In various embodiments, the lantibiotic peptide is salA, salB, sal9, or a combination thereof.

[0039] In various embodiments, the lantibiotic peptide is salA, salB, or a combination thereof.

[0040] In various embodiments the bacteriocin is salQ.

[0041] In various embodiments, the upregulated genes encode subunits of the urease protein.

[0042] In various embodiments, the urease is ureC.

[0043] In various embodiments, at least one of the upregulated genes comprises or consists of a polynucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 15-22, or at least one of the upregulated genes comprises or consists of a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 23-30.

[0044] In various embodiments, at least one of the upregulated genes comprises or consists of a polynucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 15-22, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 15-22.

[0045] In various embodiments, at least one of the upregulated genes comprises or consists of a polynucleotide sequence encoding a polypeptide having at least 70% identity to any one of SEQ ID NOs:23-30, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs:23-30.

[0046] In various embodiments, a. at least one of the upregulated genes is a salA gene or a variant thereof comprising or consisting of a polynucleotide sequence having at least 70% identity to SEQ ID NO: 15 or 19, or encoding a polypeptide having at least 70% identity to SEQ ID NO: 23 or 27; b. at least one of the upregulated genes is a salB gene or a variant thereof comprising or consisting of a polynucleotide sequence having at least 70% identity to SEQ ID NO: 16 or encoding a polypeptide having at least 70% identity to SEQ ID NO: 24; c. at least one of the upregulated genes is a salQ gene or a variant thereof comprising or consisting of a polynucleotide sequence having at least 70% identity to SEQ ID NO: 17 or 21, or encoding a polypeptide having at least 70% identity to SEQ ID NO: 25 or 29; d. at least one of the upregulated genes is a sal9 gene or a variant thereof comprising or consisting of a polynucleotide sequence having at least 70% identity to SEQ ID NO:20 or encoding a polypeptide having at least 70% identity to SEQ ID NO:28; and / or e. At least one of the upregulated genes is a ureC gene or a variant thereof comprising or consisting of a polynucleotide sequence having at least 70% identity to SEQ ID NO: 18 or 22, or encoding a polypeptide having at least 70% identity to SEQ ID NO: 26 or 30.

[0047] In various embodiments, the lantibiotic peptide or bacteriocin is a class I or class II lantibiotic peptide or bacteriocin.

[0048] In various embodiments, the lantibiotic peptide is salA, salB, sal9, or a combination thereof.

[0049] In various embodiments, the lantibiotic peptide is salA, salB, or a combination thereof.

[0050] In various embodiments the bacteriocin is salQ.

[0051] In various embodiments, the method enhances production of a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs:23-30.

[0052] In various embodiments, the polypeptide has at least 75% identity to any one of SEQ ID NOs:23-30, preferably at least 80%, 85%, 90%, 95%, or 99% identity to any one of SEQ ID NOs:23-30.

[0053] In various embodiments, the methods improve the inhibition profile of S. salivarius against skin, teeth, oral cavity, mucosa, and / or ENTR microorganisms.

[0054] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganisms are selected from the group consisting of Staphylococcus aureus species, Staphylococcus intermedius species, Staphylococcus saprophyticus species, Moraxella catarrhalis species, Haemophilus influenzae species, Streptococcus pyogenes species, Pseudomonas aeruginosa species, Streptococcus mutans species, and the like. mutans species, Streptococcus pneumoniae species, Cutibacterium acnes species, Candida albicans species, Streptococcus sobrinus species, Corynebacterium species, Fusobacterium nucleatum species, Aggregatibacter actinomycetemcomitans species, Porphyromonas gingivalis species, Tannerella forsythia species, Treponema denticola species, denticola) species, P. intermedia (P.intermedia species, Prevotella species, Actinomyces viscosus species, Streptococcus equismillis species, Streptococcus dygalactiae species, Streptococcus sanguis species, Staphylococcus cohnii species, B.intermedius species, Atopobium parvulum species, Eubacterium saburreum species, Eubacterium sulci species, Parvimonas micra species, Solobacterium moorei species, Streptococcus agalactiae species, C. minutissimus species, and Propionibacterium propionicus. species, Streptococcus agalactiae species, Streptococcus dysgalactiae species, Staphylococcus simulans species, Staphylococcus xylosus species, fungi causing tinea pedis infections, species other than S. salivarius K12 or M18, Lactococcus lactis species, Staphylococcus epidermidis species, Streptococcus constellatus species, Klebsiella pneumoniae species, Acinetobacter baumanii species, or any combination of any two or more thereof.

[0055] In various embodiments, the microorganism is S. aureus A222, S. aureus 20, S. aureus 14, S. aureus 19, S. aureus A504, S. saprophyticus ATCC15305, M. catarrhalis TW1, M. catarrhalis TW2, H. influenzae TW5, S. pyogenes M76, S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes and any combination of any two or more thereof.

[0056] In various embodiments, the composition comprises at least about 0.1% by weight of S. salivarius.

[0057] In various embodiments, the composition comprises about 0.1 to about 20% by weight of S. salivarius.

[0058] In various embodiments, the composition comprises at least about 1×10 3 cfu / g of S. salivarius.

[0059] In various embodiments, the composition comprises about 1×10 3 ~Approx. 1×10 13 cfu / g of S. salivarius.

[0060] In various embodiments, the composition comprises less than about 20% by weight of supplemental sugars.

[0061] In various embodiments, the composition comprises from about 0.1 to about 20% by weight of supplemental sugars.

[0062] In various embodiments, the compositions are formulated for oral, dental, nasal, mucosal, topical, or pulmonary administration.

[0063] In various embodiments, the composition is formulated in a sustained release composition.

[0064] In various embodiments, the composition is formulated into a powder, lozenge, nasal spray, nasal gel, nose drop, oral drop, oral gel, oral spray, inhalable topical composition, chewable, melt, film, gummy, toothpaste, tooth gel, varnish, mousse, mouthwash, food (e.g., yogurt), cream, gel spray, deodorant, serum, lotion, balm, moisturizer, pessary, or suppository.

[0065] In various embodiments, the microorganism is a Streptococcus or Staphylococcus bacterium selected from the species S. aureus, S. saprophyticus, S. mutans, S. pyogenes, S. pneumoniae, and the S. salivarius strain is K12.

[0066] In various embodiments, the Streptococcus or Staphylococcus bacterium is selected from S. aureus A222, S. saprophyticus ATCC15305, S. mutans OMZ175, S. constellatus T-29, S. pyogenes 71-698, and S. pneumoniae D39, and the S. salivarius strain is K12.

[0067] In various embodiments, the supplemental sugar is raffinose and is present in the composition in an amount of 0.5-15%, or 1-12%, or 1.5-10%, or 2-7%, or 2.5-5% by weight.

[0068] In various embodiments, the supplemented saccharide is galactose and is present in the composition in an amount of 0.5-15%, or 1-12%, or 1.5-10%, or 2-7%, or 2.5-5% by weight.

[0069] In various embodiments, the bacterium is selected from the species S. pyogenes and S. pneumoniae, and the S. salivarius strain is M18.

[0070] In various embodiments, the bacterium is selected from S. pyogenes 71-698 and S. pneumoniae D39, and the S. salivarius strain is M18.

[0071] In various embodiments, the bacterium is selected from S. constellatus, S. mutans, and S. saprophyticus, and the S. salivarius strain is M18.

[0072] In various embodiments, the bacterium is selected from S. constellatus T29, S. mutans OMZ175, and S. saprophyticus ATCC15305, and the S. salivarius strain is M18.

[0073] In various embodiments, the supplemental sugar is raffinose and is present in the composition in an amount of 0.25-10%, or 0.5-8%, or 0.75-7%, or 1-6%, or 1.25-5% by weight.

[0074] In various embodiments, the supplemented sugar is galactose and is present in the composition in an amount of 0.25-10%, or 0.5-8%, or 0.75-7%, or 1-6%, or 1.25-5% by weight.

[0075] In various embodiments, the composition comprises one or more of galactose in an amount of 0.1-1%, or 0.2-0.8%, or 0.25-0.75%, or 0.5% by weight, and raffinose in an amount of 0.5-5%, or 1-4%, or 2-3%, or 2.5% by weight.

[0076] In various embodiments, the composition comprises one or more of galactose in an amount of about 1.7% by weight and raffinose in an amount of about 1.25% by weight.

[0077] In various embodiments, the composition comprises one or more of galactose in an amount of about 0.5% by weight and raffinose in an amount of about 2.5% by weight.

[0078] In various embodiments, the composition comprises raffinose in an amount of 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0% by weight.

[0079] In various embodiments, the compositions comprise galactose in an amount of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% by weight.

[0080] In various embodiments, the composition comprises raffinose in an amount of 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, or 2.2% by weight.

[0081] In various embodiments, the composition comprises galactose in an amount by weight of 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.25%, 1.3%, 1.4%, 1.5%, 1.6%, or 1.7%.

[0082] In various embodiments, the compositions further comprise one or more of a carrier; a tableting aid, including a binder or lubricant; and a flavoring agent.

[0083] In various embodiments, the therapeutic formulation is formulated for oral, dental, nasal, mucosal, topical, or pulmonary administration.

[0084] In various embodiments, the therapeutic formulation is a sustained release composition.

[0085] In various embodiments, the therapeutic formulation is a powder, lozenge, nasal spray, nasal gel, nasal drops, oral drops, oral gel, oral spray, inhalable topical composition, chewable, melt, film, gummy, toothpaste, tooth gel, varnish, mousse, mouthwash, food (e.g., yogurt), cream, gel, spray, deodorant, serum, lotion, balm, moisturizer, pessary, or suppository.

[0086] In various embodiments, the therapeutic formulation is a powder.

[0087] In various embodiments, the therapeutic formulation is a lozenge.

[0088] In various embodiments, the disease or disorder is caused by an oral, dental, mucosal, skin, or ENTR pathogen.

[0089] In various embodiments, the disease or disorder is caused by a pathogenic Streptococcus or Staphylococcus bacteria.

[0090] In various embodiments, the pathogenic Streptococcus or Staphylococcus bacteria is selected from the species S. aureus, S. saprophyticus, S. mutans, S. pyogenes, and S. pneumoniae.

[0091] In various embodiments, the disease or disorder is selected from otitis media, sore throat, dental caries, acute pharyngitis, tonsillitis, pneumonia, chronic obstructive pulmonary disease (COPD), periodontitis, gingivitis, halitosis, dental caries, sepsis, meningitis, candidiasis (oral candidiasis), vaginitis, body odor, acne, actinomycosis, psoriasis, dysentery, cellulitis, impetigo, atopic dermatitis, bacteremia, athlete's foot, soft tissue infection, erythema, hospital-acquired infection, erythema, SARS-CoV, influenza A, influenza B, and RSV, or any combination of any two or more thereof.

[0092] In various embodiments, the microorganism susceptible to Bliss-producing S. salivarius is selected from the species S. salivarius, S. epidermidis, S. constellatus, and L. lactis.

[0093] In various embodiments, the microorganism susceptible to Bliss-producing S. salivarius is selected from the species S. pyogenes, F. nucleatum, and P. gingivalis.

[0094] In some embodiments, the subject is a human. In some embodiments, the human is a child.

[0095] In some embodiments, the composition is a cosmetic product.

[0096] In some embodiments, the composition is a dietary supplement.

[0097] In various embodiments, the composition is a natural health product.

[0098] In some embodiments, the composition is a complementary medicine.

[0099] In various embodiments, the composition is a lozenge and the method for producing the composition further comprises pressing the homogenous blend to produce the lozenge.

[0100] In various embodiments, the inhibition profile of S. salivarius in the composition is improved as compared to a composition lacking supplemental sugars.

[0101] In various embodiments, the composition comprises: (a) the treatment or prevention of a disease or disorder; or (b) For use in inhibiting microorganisms susceptible to Bliss-producing S. salivarius.

[0102] The present invention may also be broadly described as consisting of any or all combinations of two or more of the parts, elements and features referred to or indicated in the specification of this application, either individually or collectively, and where a specific integer having known equivalents in the art to which the invention pertains is referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0103] Reference to a range of numbers disclosed herein (e.g., 1-10) also incorporates reference to every rational number within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also to any range of rational numbers within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and thus all subranges of every range explicitly disclosed herein are intended to be hereby expressly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be expressly set forth in this application as well.

[0104] Where patents, other external documents, or other sources of information are referenced herein, this is generally for the purpose of providing a context for discussing the features of the present invention. Unless otherwise expressly stated, reference to such external documents should not be construed as an admission that such documents or such sources are prior art in any jurisdiction or form part of the common general knowledge in the art.

[0105] Numerous modifications of the structure and a wide variety of embodiments and applications of the present invention will be suggested to those skilled in the art to which the present invention pertains without departing from the scope of the invention, which is defined in the appended claims. The disclosures and descriptions herein are purely illustrative and are not intended to be in any sense limiting.

[0106] Although the invention is broadly defined as above, those skilled in the art will appreciate that the invention is not so limited, but also includes embodiments of which the following description provides examples. [Brief description of the drawings]

[0107] The present invention will now be described with reference to the accompanying drawings.

[0108] [Figure 1]Figure 2 shows the change in size of ENTR pathogen ZOI size (mm) after a deferred antagonism assay performed with K12 as the production organism cultivated in the presence of various concentrations of galactose. The control condition was set to 0 mm (normalized) and the deviation from 0 represents the change in ZOI size (mm).

[0109] [Diagram 2] Figure 2 shows the change in size of ENTR pathogen ZOI size (mm) after a deferred antagonism assay performed with K12 as the production organism cultivated in the presence of various concentrations of raffinose. The control condition was set at 0 mm and the deviation from 0 represents the change in ZOI size (mm).

[0110] [Diagram 3] Figure 2 shows the change in size of ENTR pathogen ZOI size (mm) after a deferred antagonism assay performed with M18 as the production organism cultured in the presence of various concentrations of galactose. The control condition was set at 0 mm and the deviation from 0 represents the change in ZOI size (mm).

[0111] [Figure 4] Figure 2 shows the change in size of ENTR pathogen ZOI size (mm) after a deferred antagonism assay performed with M18 as the production organism cultivated in the presence of various concentrations of raffinose. The control condition was set at 0 mm and the deviation from 0 represents the change in ZOI size (mm).

[0112] [Diagram 5] Comparison of stimulation of K12 inhibitory effect using raffinose vs. sugars (equimolar) against the skin pathogen S. aureus A222. Raffinose vs. (a) Trimix (a mixture of the three sugars galactose, glucose, and fructose in equimolar concentrations present in raffinose); (b) individual sugars; (c) Comparison of stimulation of M18 inhibitory effect using raffinose vs. sugars (equimolar) against the skin pathogen S. aureus A222. Raffinose vs. individual sugars.

[0113] [Figure 6] Comparison of stimulation of K12 inhibitory effect using raffinose vs. saccharides (equimolar) against dental pathogen S. mutans OMZ175. Raffinose vs. (a) Trimix (a mixture of three saccharides at equimolar concentrations); (b) individual saccharides; (c) Comparison of stimulation of M18 inhibitory effect using raffinose vs. saccharides (equimolar) against dental pathogen S. mutans OMZ175. Raffinose vs. individual saccharides.

[0114] [Figure 7] Comparison of stimulation of K12 inhibitory effect using raffinose versus saccharides (equimolar) against the lower respiratory tract pathogen S. pneumoniae D39. Raffinose versus (a) trimix (a mixture of three saccharides at equimolar concentrations); (b) individual saccharides; (c) Comparison of stimulation of M18 inhibitory effect using raffinose versus saccharides (equimolar) against the lower respiratory tract pathogen S. pneumoniae D39. Raffinose versus individual saccharides.

[0115] [Figure 8] Figure 1 shows a comparison of stimulation of the K12 inhibitory effect using raffinose versus saccharides (equal weight %) against the skin pathogen S. aureus A222. Raffinose versus (a) Trimix (a mixture of three saccharides at equal weight %); (b) individual saccharides; (c) a comparison of stimulation of the M18 inhibitory effect using raffinose versus saccharides (equal weight %) against the skin pathogen S. aureus A222. Raffinose versus individual saccharides.

[0116] [Figure 9] Comparison of stimulation of K12 inhibitory effect using raffinose vs. sugars (equal weight %) against dental pathogen S. mutans OMZ175. Raffinose vs. (a) Trimix (a mixture of three sugars at equal weight %); (b) individual sugars; (c) Comparison of stimulation of M18 inhibitory effect using raffinose vs. sugars (equal weight %) against dental pathogen S. mutans OMZ175. Raffinose vs. individual sugars.

[0117] [Figure 10] Comparison of stimulation of the K12 inhibitory effect using raffinose versus sugars (equal weight %) against the ENTR pathogen S. pyogenes 71-968. Raffinose versus (a) Trimix (a mixture of three sugars at equal weight %); (b) individual sugars; (c) Comparison of stimulation of the M18 inhibitory effect using raffinose versus sugars (equal weight %) against the ENTR pathogen S. pyogenes 71-968. Raffinose versus individual sugars.

[0118] [Figure 11] Figure 1 shows a comparison of the stimulation of the K12 inhibitory effect using raffinose versus sugars (equal weight percent) on the microorganism S. constellatus T29, which is susceptible to Bliss-producing S. salivarius. Raffinose versus (a) Trimix (a mixture of three sugars at equal weight percent concentrations); (b) individual sugars; (c) a comparison of the stimulation of the M18 inhibitory effect using raffinose versus sugars (equal weight percent) on the microorganism S. constellatus T29, which is susceptible to Bliss-producing S. salivarius. Raffinose versus individual sugars.

[0119] [Figure 12] Figure 1 shows a comparison of stimulation of the K12 inhibitory effect using raffinose versus sugars (equal weight %) against the lower respiratory tract pathogen S. pneumoniae D39. Raffinose versus (a) Trimix (a mixture of three sugars at equal weight %); (b) individual sugars; (c) a comparison of stimulation of the M18 inhibitory effect using raffinose versus sugars (equal weight %) against the lower respiratory tract pathogen S. pneumoniae D39. Raffinose versus individual sugars.

[0120] [Figure 13]Figure 1 shows a comparison of stimulation for K12 inhibitory effect using raffinose versus sugars (equal weight %) against the ENTR pathogen S. pyogenes 71-698, the dental pathogens S. mutans OMZ175, S. pneumoniae D39, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the microorganism S. constellatus T29 susceptible to Bliss-producing S. salivarius. Raffinose versus trimix (a mixture of three sugars at equal weight %) and individual sugars. Comparison of stimulation for M18 inhibitory effect using raffinose versus sugars (equal weight %) against a range of pathogens and microorganisms susceptible to Bliss-producing S. salivarius. Raffinose versus individual sugars.

[0121] [Figure 14] Comparison of stimulation of K12 inhibitory effect using raffinose versus sugars (equal weight %) against microorganisms susceptible to Bliss-producing S. salivarius; (a) Raffinose versus Trimix (a mixture of three sugars at equal weight %); (b) Individual sugars.

[0122] [Figure 15] Figure 1 shows a comparison of the stimulation to M18 inhibitory effect using raffinose versus sugars (equal weight percent) against the microorganisms susceptible to Bliss-producing S. salivarius; S. salivarius #6, S. salivarius 193, S. salivarius 20P3. Raffinose versus Trimix (a mixture of three sugars at equal weight percent concentrations) and individual sugars.

[0123] [Figure 16]Figure 1 shows a comparison of stimulation to K12 inhibitory effects using raffinose versus sugars (equal wt%) against several oral and ENTR pathogens S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, and S. pyogenes M74. Raffinose versus Trimix (a mixture of three sugars at equal wt% concentrations) and individual sugars.

[0124] [Figure 17] Figure 1 shows a comparison of the stimulation to M18 inhibitory effect using raffinose versus sugars (equal weight %) against several oral and ENTR pathogens S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, and S. pyogenes M74. Raffinose versus Trimix (a mixture of three sugars at equal weight % concentrations) and individual sugars.

[0125] [Figure 18] Figure 1 shows a comparison of the stimulation to K12 inhibitory effect using raffinose versus saccharides (equal weight %) against several lower respiratory tract pathogens S. dysgalactiae Bris 2, S. dysgalactiae T277, S. pneumoniae D39, S. pneumoniae RX1, S. pneumoniae PK8, Raffinose versus Trimix (a mixture of three saccharides at equal weight %) and individual saccharides.

[0126] [Figure 19] Figure 1 shows a comparison of stimulation to M18 inhibitory effects using raffinose versus saccharides (equal weight %) against several ENTR / lower respiratory tract pathogens S. dysgalactiae Bris 2, S. dysgalactiae T277, S. pneumoniae D39, S. pneumoniae RX1, S. pneumoniae PK8. Raffinose versus Trimix (a mixture of three saccharides at equal weight % concentrations) and individual saccharides.

[0127] [Figure 20] Comparison of stimulation to K12 inhibitory effect using raffinose versus sugars (equal weight %) against several dental pathogens S. mutans OMZ175, S. mutans ATCC10449, S. mutans D10, S. mutans UA159, S. mutans FW75, A. viscosus T14, S. sanguinis K11, S. sobrinus OMZ176, Raffinose versus Trimix (a mixture of three sugars at equal weight %) and individual sugars.

[0128] [Figure 21] Comparison of stimulation to M18 inhibitory effect using raffinose versus sugars (equal weight %) against several dental pathogens S. mutans OMZ175, S. mutans ATCC10449, S. mutans D10, S. mutans UA159, S. mutans FW75, A. viscosus T14, S. sanguinis K11, S. sobrinus OMZ176. Raffinose versus Trimix (a mixture of three sugars at equal weight %) and individual sugars.

[0129] [Figure 22] Comparison of stimulation to K12 inhibitory effects using raffinose versus sugars (equal wt%) against several skin pathogens S. cornii, S. simulans, S. aureus A222, S. aureus 20, S. aureus 19, S. aureus 14. Raffinose versus trimix (a mixture of three sugars at equal wt% concentrations) and individual sugars.

[0130] [Figure 23] Comparison of stimulation to M18 inhibitory effects using raffinose versus saccharides (equal wt%) against several skin pathogens S. cornii, S. simulans, S. aureus A222, S. aureus 20, S. aureus 19, S. aureus 14. Raffinose versus trimix (a mixture of three saccharides at equal wt% concentrations) and individual saccharides.

[0131] [Figure 24]Figure 1 shows a comparison of the stimulation of dairy-free K12 inhibitory effects using raffinose versus sugars (equal weight percentages) against the ENTR pathogens S. pyogenes 71, S. pneumoniae D39698, the dental pathogen S. mutans OMZ175, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the bliss-producing S. salivarius-susceptible microorganism S. constellatus T29. Raffinose versus Trimix (a mixture of three sugars at equal weight percent concentrations) and individual sugars.

[0132] [Diagram 25] Figure 1 shows a comparison of the stimulation to dairy-free M18 inhibitory effect using raffinose versus sugars (equal weight percentages) against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, dental pathogen S. OMZ175, skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the bliss-producing S. salivarius-susceptible microorganism S. constellatus T29. Raffinose versus Trimix (a mixture of three sugars at equal weight percent concentrations) and individual sugars.

[0133] [Figure 26] Comparison of stimulation of growth of K12 in M17 broth with raffinose, galactose, trimix (a mixture of three sugars at equal weight percent concentrations) and individual sugars.

[0134] [Figure 27] Figure 1 shows a comparison of the stimulatory and inhibitory effects of K12 growth in M17 broth using raffinose versus sugars (equal weight %) against the oral and ENTR pathogens S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, and S. pyogenes M74. Raffinose versus Trimix (a mixture of three sugars at equal weight % concentrations) and individual sugars.

[0135] [Figure 28]Figure 1 shows a comparison of the stimulatory and inhibitory effects of K12 growth in M17 broth using raffinose versus sugars (equal weight %) against the ENTR pathogens S. pyogenes 71-698, S. pyogenes M74, S. pyogenes M66, S. pneumoniae D39, the dental pathogen S. mutans OMZ175, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the bliss-producing S. salivarius-susceptible microorganism S. constellatus T-29. Raffinose versus trimix (a mixture of three sugars at equal weight % concentrations) and the individual sugars glucose, fructose and galactose.

[0136] [Figure 28A] Comparison of stimulation of growth of M18 in M17 broth with raffinose, galactose, trimix (a mixture of three sugars at equal weight percent concentrations) and individual sugars.

[0137] [Figure 28B] Figure 1 shows a comparison of the stimulatory and inhibitory effects of M18 growth in M17 broth using raffinose versus sugars (equal weight %) against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, dental pathogen S. mutans OMZ175, skin pathogen S. saprophyticus ATCC15305, and the bliss-producing S. salivarius-susceptible microorganism S. constellatus T-29. Raffinose versus trimix (a mixture of three sugars at equal weight % concentrations) and the individual sugars glucose, fructose, and galactose.

[0138] [Figure 29] Magnification of K12 and M18 mucoid morphology when grown in CABCa agar supplemented with raffinose is shown compared to the CABCa control.

[0139] [Diagram 30] Photographs of the change in K12 and M18 (dairy or non-dairy) to a mucoid morphology when grown in CABCa agar supplemented with raffinose are shown in comparison to the CABCa control.

[0140] [Diagram 31] Photographs of K12 and M18 producer streaks and glass slides showing bacterial growth are shown. The dotted black oval visually indicates that more mucus is produced in CABCa agar supplemented with raffinose compared to CABCa supplemented with trimix (a mixture of three sugars at equal weight percent concentrations) and the CABCa control.

[0141] [Diagram 32] Figure 1 shows the effect of 2.5% w / w raffinose on the antibacterial activity of different S. salivarius strains against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, the dental pathogens S. sobrinus OMZ176, S. mutans OMZ175, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the microorganism S. constellatus T29 susceptible to bliss-producing S. salivarius.

[0142] [Diagram 33] Figure 1 shows the effect of 0.5% w / w galactose on the antibacterial activity of different S. salivarius strains against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, the dental pathogens S. sobrinus OMZ176, S. mutans OMZ175, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the microorganism S. constellatus T29 susceptible to Bliss-producing S. salivarius.

[0143] [Diagram 34] 1 shows the effect of raffinose, galactose, and their combinations on the antibacterial activity of S. salivarius K12 against different Gram-negative strains F. nucleatum ATCC25586, F. nucleatum FH2, F. nucleatum FH3, P. gingivalis ATCC33277, P. gingivalis W50, P. intermedia ATCC23611, pathogens responsible for halitosis.

[0144] [Diagram 35]1 shows the effect of raffinose, galactose, and their combinations on the antibacterial activity of S. salivarius M18 against different Gram-negative strains F. nucleatum ATCC25586, F. nucleatum FH2, F. nucleatum FH3, P. gingivalis ATCC33277, P. gingivalis W50, P. intermedia ATCC23611, pathogens responsible for halitosis.

[0145] [Diagram 36] Figure 1 shows the effect of raffinose, galactose and their combination on the antibacterial activity of S. salivarius K12 freeze-dried raw material against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, the dental pathogens S. sobrinus OMZ175, S. mutans OMZ176, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the microorganism S. constellatus T29 susceptible to Bliss-producing S. salivarius.

[0146] [Figure 37] Figure 1 shows the effect of raffinose, galactose, and their combinations on the antibacterial activity of S. salivarius M18 lyophilized raw material against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, M. catarrhalis TW1, the dental pathogens S. sobrinus OMZ176, S. mutans OMZ175, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the microorganism S. constellatus T29 susceptible to Bliss-producing S. salivarius.

[0147] [Figure 38]Figure 1 shows the effect of raffinose, galactose, and their combinations on the antibacterial activity of S. salivarius K12 and S. salivarius M18 freeze-dried raw materials against the ENTR pathogens S. pyogenes 71-698, S. pneumoniae D39, S. agalactiae ATCC12386, M. catarrhalis TW1, the dental pathogens S. sobrinus OMZ176, S. mutans OMZ175, the skin pathogens S. saprophyticus ATCC15305 and S. aureus A222, and the microorganisms S. constellatus T29 and L. lactis ATCC19435 susceptible to bliss-producing S. salivarius. [Figure 39] Pathogens in commercial powder preparations (split plate method): S. pyogenes 71-698, S. pyogenes 71-679, S. pyogenes H13, S. pyogenes K26, S. pyogenes H29, S. pyogenes WS02, S. mutans OMZ175, S. mutans FW75, S. sobrinus ATCC27351, S. sanguinis K11, S. agalactiae ATCC12386, A. viscosus T14, A. viscosus ATCC15987, S. constellatus T29, S. pyogenes K7, S. pneumoniae D39, S. pneumoniae PK8, S. sobrinus OMZ176, S. dysgalactiae T-148, S. dysgalactiae Bris 2 shows the effect of supplemented sugars on the inhibitory activity of S. salivarius K12 against S. dysgalactiae T277, C. auris ATCC 51966, and S. aureus 19.

[0148] [Diagram 40] A comparison of the antibacterial activity of five compositions is shown: a commercially available powder formulation (Daily Defense Junior) containing S. salivarius K12 combined with 0.5% w / w galactose; Daily Defense Junior combined with 2.5% w / w raffinose; Daily Defense Junior combined with 0.5% w / w galactose and 2.5% w / w raffinose; a commercially available infant formula combined with S. salivarius K12; and whole milk powder combined with S. salivarius K12.

[0149] [Diagram 41] Figure 1 shows the relative expression of salA in S. salivarius K12 with the following sugars added to solid medium (CABCa agar): 0.5% w / w galactose; 2.5% w / w raffinose; 0.5% w / w galactose combined with 2.5% w / w raffinose; 0.5% w / w sucrose; 2.5% w / w sucrose. Data points are means (n=3 ± SD). Data points are means (n=3 ± SD).

[0150] [Diagram 42] Figure 1 shows the relative expression of salB in S. salivarius K12 when different combinations of sugars were added to solid medium (CABCa agar): 0.5% w / w galactose; 2.5% w / w raffinose; 0.5% w / w galactose combined with 2.5% w / w raffinose; 0.5% w / w sucrose; 2.5% w / w sucrose. Data points are means (n=3 ± SD).

[0151] [Diagram 43] Figure 1 shows the relative expression of salQ in S. salivarius K12 when different combinations of supplemental sugars were added to solid medium (CABCa agar): 0.5% w / w galactose; 2.5% w / w raffinose; 0.5% w / w galactose combined with 2.5% w / w raffinose; 0.5% w / w sucrose; 2.5% w / w sucrose. Data points are means (n=3 ± SD).

[0152] [Diagram 44] Figure 1 shows the relative expression of urease (ureC) in S. salivarius K12 when different combinations of supplemented sugars were added to solid medium (CABCa agar): 0.5% w / w galactose; 2.5% w / w raffinose; 0.5% w / w galactose combined with 2.5% w / w raffinose; 0.5% w / w sucrose; 2.5% w / w sucrose. Data points are means (n=3 ± SD).

[0153] [Diagram 45]4 shows the mean % of S. salivarius K12 (of total S. salivarius) in saliva samples obtained from groups of participants using lozenges containing S. salivarius K12 with no supplemented sugars (G1), with galactose (G2), with raffinose (G3), and with raffinose and galactose (G4) 1 hour, 8 hours, and 24 hours after dissolving the first lozenge in the mouth, and 48 hours after dissolving the lozenge in the mouth daily for 7 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0154] The role of sugars, particularly digestible sugars such as monosaccharides, disaccharides, and trisaccharides, to stimulate oral bacterial growth and / or activity has not been thoroughly investigated. The present inventors sought to better understand the role of such sugars on oral probiotic performance.

[0155] The metabolism of certain monosaccharides, such as glucose, can act in some bacteria to repress the activation / transcription of certain genes, including bacteriocin genes. This effect is commonly known as catabolite repression (see Gorke & Stulke, Nature Reviews Microbiology, 2008, 6, 613-624). While evaluating whether other sugars would have a similar repressive effect, the inventors surprisingly found that supplementation of the growth medium with certain other sugars (such as lactose) had the opposite effect, increasing the inhibition spectrum of several strains of bacterial species, including but not limited to Streptococcus salivarius.

[0156] definition The following definitions are presented to better define the present invention and as a guide to those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, all technical and scientific terms used herein are to be understood to have the same meaning as would be understood by one of ordinary skill in the relevant art to which this disclosure pertains.

[0157] For example, general chemical and biological terms used herein in formulas have their ordinary meanings.

[0158] Examples of definitions of general terms in microbiology, molecular biology, and biochemistry are given in Methods for General and Molecular Microbiology, 3. rd Edition,CAReddy,et al.(eds.),ASM Press,(2008);Encyclopaedia of Microbiology,2nd ed.,Joshua Lederburg,(ed.).Academic Press,(2000);Microbiology By Cliffs Notes,I.Edward Alcamo,Wiley,(1996);Dictionary of Microbiology and Molecular Biology,Singleton et al.(2d ed.)(1994);Biology of Microorganisms 11th ed.Brock et al.,Pearson Prentice Hall,(2006);Biodiversity of Fungi: Inventory and Monitoring Methods,Mueller et al.Academic Press,(2004);Genes IX,Benjamin Lewin,Jones & Bartlett Publishing,(2007);The Encyclopaedia of Molecular Biology,Kendrew et al.(eds.),Blackwell Science Ltd., (1994); and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995). These sources also provide standard microbiological, molecular biology, and biochemistry protocols and procedures that can be used to practice the invention.

[0159] The term "comprising" as used in this specification and claims means "consisting at least in part of." When interpreting each statement in this specification and claims that contains the term "comprising," there may be other features or features preceding that term. Related terms such as "comprise," "comprised," and "comprises" should be interpreted in the same manner.

[0160] As used herein, the term "and / or" means "and" or "or," or both.

[0161] As used herein, "(s)" following a noun refers to the plural and / or singular form of the noun.

[0162] As used herein, the term "subject" refers to animals, including humans and non-human animals such as dogs, pigs, cats, horses, sheep, cows, chickens, fish, and other domestic and farm animals.

[0163] The term "treatment" as used herein refers to a subject undergoing prophylactic or therapeutic treatment. Prophylactic treatment includes treatment to prevent or reduce the likelihood or severity of infection, or to inhibit or control microbial population. Treatment may be for inhibiting or reducing microbial population in a subject. Treatment may also be provided to an infected subject to reduce the severity of the infection or its associated symptoms, or to reduce or eliminate the infection or its associated symptoms. The subject being treated may be of any age, e.g., infant, child, adolescent, adult, or elderly. When a "patient", "individual", or "host" is referred to, it is synonymous with the term "subject".

[0164] As used herein, a "supplemental saccharide" is a saccharide that improves the inhibition profile and / or mucoid properties of a bacterial species, such as S. salivarius.

[0165] As used herein, "improving the inhibition profile of Streptococcus salivarius" means enhancing the potency of inhibition and / or increasing the spectrum of inhibitory activity compared to S. salivarius in the absence of an effective amount of supplemental sugar. The improvement is observed whether the supplemental sugar is added to a composition that includes other sugars or to a composition that does not include sugar.

[0166] As used herein, the term "upregulating a gene or genes" refers to increasing the expression level of a transcribed RNA encoded by a gene, resulting in an increase in a peptide, bacteriocin, or protein. The related term "method of upregulating a gene" refers to a method of increasing the expression level of a transcribed RNA encoded by a gene, resulting in an increase in a peptide, bacteriocin, or protein. In embodiments, the method results in an increase in the expression level of a transcribed RNA encoded by a gene, resulting in an increase in expression of said peptide, bacteriocin, or protein, and / or an increase in the production of said peptide, bacteriocin, or protein.

[0167] As used herein, "improved mucoid properties" means that the bacterial colonies of S. salivarius are more mucoid (sticky mucus-like) and are better able to adhere the probiotic to a substrate, thus allowing for enhanced colony formation.

[0168] "ENTR" microorganisms are those of the ear, nose, throat, or respiratory tract, including pathogens that infect the ear (ear), nose (nose), throat, upper respiratory tract, and lower respiratory tract, as well as non-pathogenic microorganisms that colonize the ear (ear), nose (nose), throat, upper respiratory tract, and lower respiratory tract. As used herein, the term "lower respiratory tract" or "LRT" refers to the trachea, bronchi, and lungs. This term is contrasted with "upper respiratory tract" or "URT," which refers to the nose, nasal cavity, and nasopharynx.

[0169] "Salivaricin" is a bacteriocin-like inhibitor (BLIS) produced by S. salivarius strains. Thus, BLIS-producing S. salivarius is S. salivarius that produces salivaricin.

[0170] As used herein, the term "microbe" or "microorganism" refers to a bacterium, a fungus, a virus, or a combination thereof. Similarly, as used herein, the term "microbial" population, infection, disease, or condition includes bacterial, viral, and fungal populations, infections, diseases, or conditions. Microorganisms may be pathogenic or non-pathogenic (e.g., commensal).

[0171] The term "effective amount" as used herein means an amount effective to inhibit, reduce, or control microbial population, or to protect against, delay, reduce, stabilize, ameliorate, or treat microbial infection in and / or on a patient. An effective amount also refers to an amount sufficient to provide a beneficial effect to a patient. Such beneficial effects may include detectable: increased IFN-γ, decreased NF-κB-mediated cytokine response in the lung, inhibition of microbial replication, and / or decreased microbial load. The effect should be sufficient to provide a medically significant reduction in the likelihood of bacterial, fungal, or viral infection, or a medically or statistically significant reduction in the rate, extent, severity, or length of microbial infection or associated symptoms or secondary infection. Reduction in microbial survival, growth, and / or proliferation is contemplated.

[0172] In one embodiment, the term "statistically significant" as used herein refers to the likelihood that a result or relationship is caused by something other than chance. A result may be found to be statistically significant using statistical hypothesis tests known and used in the art. Statistical hypothesis tests provide a "P-value" as known in the art, which represents the probability that the measured result is due to chance alone. It is believed to be generally accepted in the art that a significance level of 5% (0.05) or less is considered statistically significant.

[0173] The phrase "inhibits the growth of microorganisms susceptible to the compositions of the present invention" and similar terms refer to the growth inhibition of at least one or more species of microorganisms susceptible to BLIS-producing streptococci strains, such as S. salivarius. The inhibition of bacterial growth can be determined by various methods, including inhibition of colony-forming units (CFU) of the target bacterial strain, as described in WO 01 / 27143. The inhibition of viral growth can be determined by various methods, including virus yield reduction assay (VYR), observation of cytopathic effect, or virucidal assay. The inhibition of fungal growth can be determined, for example, by adapting the deferred antagonism method (see Tagg and Bannister 1979; Med Microbiology 12:397) to fungi, as described in the examples. The inhibition of germ tube formation provides another measure of antifungal activity (e.g., Reynolds and Braude (1956) Clin Res Proc 4:40).

[0174] The term "contacting" as used herein refers to both direct and indirect contact between the microorganism and the Bliss composition. Indirect contact includes exposure of the microorganism to the Bliss composition in the environment, particularly in its natural environment.

[0175] As used herein, a "composition" or "BLIS composition" refers to a composition or formulation that includes S. salivarius K12, M18, or a combination thereof, and may include its BLIS-containing naturally released extracellular products, such as salivaricin or other antimicrobial agents; and optionally a carrier, diluent, or excipient. In some embodiments, the composition may be a cosmetic, a dietary supplement, a natural health product, or a complementary medicine. Dietary supplements (also known as food supplements) are foods intended to supplement the normal diet and contain concentrated nutrients that are vitamins or minerals or other substances that have nutritional or physiological effects. Natural health products include probiotics, herbal remedies, vitamins and minerals, homeopathic medicines, traditional medicines, and amino acids and essential fatty acids. Complementary medicines are medicines that have been evaluated for safety and quality, and may have been evaluated for efficacy.

[0176] The unit "cfu / g" means colony forming units per gram.

[0177] As used herein, the term "variant" refers to a gene, polynucleotide, or polypeptide sequence that differs from a specifically specified sequence by having one or more nucleotides or amino acid residues deleted, substituted, or added. Variants may be naturally occurring allelic variants or non-naturally occurring variants. Variants may be from the same species or from other species and may include homologs, paralogs, and orthologs.

[0178] In certain embodiments, variants of the polynucleotides and polypeptides disclosed herein have the same or similar biological activity as the biological activity of the polynucleotides or polypeptides disclosed herein. The term "variant" with respect to polynucleotides and polypeptides encompasses all forms of the polynucleotides and polypeptides defined herein.

[0179] Method of the invention In a first aspect, the present invention provides a method for improving the inhibition profile of S. salivarius, comprising formulating S. salivarius in a composition comprising an effective amount of a supplemental saccharide, wherein the Streptococcus salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

[0180] The potency of inhibition is the extent to which the inhibited microorganism is inhibited. The potency of inhibition can be measured by determining the size of the zone of inhibition (ZOI) compared to a control sample on an agar plate, as described in Tagg JR, Bannister LV. "Fingerprinting" beta-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors. J Med Microbiol. 1979 Nov; 12 (4): 397-411. A stronger inhibitory effect is revealed by a larger zone of inhibition. Another method for determining the potency of inhibition is a liquid assay, such as that described in Enhanced Production, Purification, Characterization and Mechanism of Action of Salivariicin 9 Lantibiotic Produced by Streptococcus salivarius NU10, PLoS One (2013) 8 (10): e77751. In the case of viruses, potency can be measured by the Selectivity Index (SI), which measures the ratio of a sample's toxic concentration to its effective biological activity concentration in cell line experiments. Greater potency is indicated by a higher SI index compared to the control. Other methods of determining the potency of inhibition will be apparent to those skilled in the art.

[0181] S. salivarius strains useful herein can be characterized at least in part by deferred antagonism assays (P-typing - inhibition of indicator strains) or by determining which salivaricins the strain produces, as described by Tagg and Bannister (1979) J. Med. Microbiol. 12:397. K12 exhibits P-type 777 (see WO2001027143), M18 exhibits P-type 677 on blood agar with calcium carbonate, and P-type 777 on trypticase soy yeast extract-calcium carbonate agar (see WO2003070919).

[0182] The extent of inhibitory activity is the number of microorganisms inhibited. In various embodiments, the S. salivarius of the present invention inhibits microorganisms or species that are not inhibited by S. salivarius in the absence of an effective amount of supplemental sugar. Applicants have unexpectedly found that supplemental sugars can not only affect the growth and inhibitory activity of S. salivarius, but can also increase the range of microorganisms on which the probiotic is active. We believe this is the first time such a change in the range of activity of a probiotic has been reported. As stated above, inhibitory activity can be measured by determining the size of the zone of inhibition compared to a control sample on an agar plate. Uninhibited species have an inhibition zone of 0 or an SI index of less than 4.9.

[0183] In one aspect, the present invention provides a composition comprising an effective amount of a supplemental saccharide for use in improving the inhibition profile of Streptococcus salivarius and S. salivarius, wherein the S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

[0184] Described herein is a method of inhibiting non-pathogenic bacteria, comprising contacting the bacteria with a composition comprising S. salivarius and an effective amount of a supplemental sugar, wherein the S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental sugar is galactose or raffinose, or a combination thereof. In various embodiments, the bacteria is selected from L. lactis, S. epidermidis, and S. constellatus.

[0185] As discussed in the Background, monosaccharides are known to promote bacterial growth by being ingested as an energy source and produce organic acid by-products, which may have a weak non-selective inhibitory effect on the growth of other bacteria. Surprisingly, the inventors have demonstrated that the improved inhibition profile of S. salivarius is not solely due to the pH effect of the supplemented sugars (see Examples 4 and 6). Without being bound by theory, the inventors believe that the improved inhibition profile is due to enhanced activity of bacteriocins or other encoded antimicrobial agents (e.g., non-ribosomal peptide synthetases (NRPS)). Furthermore, the inventors have shown that raffinose unexpectedly affects cell morphology, resulting in larger sized and more mucoid (sticky slime-like) colonies, allowing probiotics to better adhere to the substrate, thus enabling enhanced colony formation (see Example 11).

[0186] Streptococcus salivarius S. salivarius are Gram-positive bacteria that primarily colonize the human oral cavity and are the predominant commensal species. They have been extensively studied for use as probiotic bacteria. Two S. salivarius strains have been commercialized by Blis Technologies Ltd for oral and dental health under the trade names BLIS M18™ and BLIS K12™.

[0187] The range of S. salivarius strains that can be used in the methods of the present invention are known in the art. S. salivarius K12 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124, Braunschweig, Germany on October 8, 1999 and assigned the accession number DSM13084. S. salivarius M18 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124, Braunschweig, Germany on December 12, 2001 and assigned the accession number DSM14685. S. salivarius M18 is described in International Publication No. WO20030709191, which is incorporated herein by reference.

[0188] Bacteriocins are antagonists that have evolved as conserved biological tools capable of conferring a competitive advantage to the producing organism. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria to inhibit the growth of closely related species. A diverse range of bacteriocins have been identified and classified into four main classes, which are further divided into subclasses. The class of bacteriocin is determined by parameters such as the producing species, the mechanism of action, the spectrum of activity, and the degree of post-translational modifications.

[0189] BLIS K12™ produces class I and class II bacteriocins: salivaricin A (SEQ ID NOs: 15 and 23), salivaricin B (SEQ ID NOs: 16 and 24), and salivaricin Q (SEQ ID NOs: 17 and 25) (salA, salB, and salQ, respectively). BLIS M18™ produces class I and class II bacteriocins: salivaricin A (SEQ ID NOs: 19 and 27), salivaricin 9 (SEQ ID NOs: 20 and 28), and salivaricin Q (SEQ ID NOs: 21 and 29) (salA, sal9, and salQ, respectively). SalA and SalB are post-translationally modified peptide molecules that exhibit high target specificity against Gram-positive group A streptococci (i.e., Streptococcus pyogenes). SalA functions by binding to target membrane structures and disrupting the structural integrity of the membrane by forming pores in the structures, ultimately resulting in cell death. SalB functions by inhibiting enzymatic processes of target species, which leads to the prevention of important cellular processes that result in cell death. Bacteriocins with over 90% identity to salQ have also been characterized as "blpU-like cassettes" (Santagati et al., Front. Biosci. (Schol Ed) 2018, 10(2), 238-247). BLIS K12™ and BLIS M18™ also produce different versions of the salivaricin MPS, a Class III bacteriocin. Class III bacteriocins are large, unmodified, thermolabile peptide molecules that target conserved structures expressed by Gram-positive streptococci and other bacterial species. BLIS K12™ also produces an antibacterial NRPS.

[0190] Urease protein is important in regulating bacterial pH homeostasis, and when bacteria produce urease protein in the oral cavity, it has the effect of regulating the pH of the mouth and saliva to a more neutral pH rather than a more acidic pH, which is beneficial for dental health. UreC is the alpha subunit of the urease protein encoded by the ureC gene. Although the functional protein contains more subunits, protein expression can be predicted based on the ureC gene in BLIS K12™ (SEQ ID NO: 18) and BLIS M18™ (SEQ ID NO: 22).

[0191] In various embodiments, compositions useful in the present invention comprise at least 0.1% by weight of S. salivarius.

[0192] In various embodiments, the composition comprises from about 0.1 to about 20% by weight of S. salivarius, e.g., from about 0.1 to about 18% by weight, from about 0.1 to about 16% by weight, from about 0.1 to about 15% by weight, from about 0.1 to about 14% by weight, from about 0.1 to about 12% by weight, from about 0.1 to about 10% by weight, from about 0.1 to about 9% by weight, from about 0.1 to about 8% by weight, from about 0.1 to about 7% by weight, from about 0.1 to about 6% by weight, from about 0.1 to about 5% by weight, from about 0.5 to about 18% by weight, from about 0.5 to about 16% by weight, from about 0.5 to about 1 ...6% by weight, from about 0.5 to about 16% by weight, from about 0.5 to about 16% by weight, from about 0.5 to about 16% by weight, from about 1 to about 14% by weight, about 0.5 to about 12% by weight, about 0.5 to about 10% by weight, about 0.5 to about 9% by weight, about 0.5 to about 8% by weight, about 0.5 to about 7% by weight, about 0.5 to about 6% by weight, about 0.5 to about 5% by weight, about 1 to about 18% by weight, about 1 to about 16% by weight, about 1 to about 15% by weight, about 1 to about 14% by weight, about 1 to about 12% by weight, about 1 to about 10% by weight, about 1 to about 9% by weight, about 1 to about 8% by weight, about 1 to about 7% by weight, about 1 to about 6% by weight, or about 1 to about 5% by weight of S. salivarius.

[0193] In various embodiments, the composition comprises about 1×10 3 ~Approx. 1×10 13 cfu / g of S. salivarius probiotic cells. In various embodiments, the composition comprises about 1×10 4 ~Approx. 1×10 12 , about 1×10 5~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 7 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 12 , about 1×10 4 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 7 ~Approx. 1×10 10 , about 1×10 8 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 7 ~Approx. 1×10 9 cfu / g. In various embodiments, the product or composition comprises S. salivarius cells in an amount of about 1×10 9 containing S. salivarius cells in the amount of cfu / g.

[0194] In various embodiments, when multiple strains of S. salivarius are present, the composition comprises at least 0.1% by weight of each strain of S. salivarius.

[0195] In various embodiments, when multiple strains of S. salivarius are present, the composition comprises from about 0.1 to about 20% by weight of each strain of S. salivarius, e.g., from about 0.1 to about 18% by weight, from about 0.1 to about 16% by weight, from about 0.1 to about 15% by weight, from about 0.1 to about 14% by weight, from about 0.1 to about 12% by weight, from about 0.1 to about 10% by weight, from about 0.1 to about 9% by weight, from about 0.1 to about 8% by weight, from about 0.1 to about 7% by weight, from about 0.1 to about 6% by weight, from about 0.1 to about 5% by weight, from about 0.5 to about 18% by weight, from about 0.5 to about 16% by weight, from about 0.5 to about 18 ... The composition comprises about 15% by weight, about 0.5 to about 14% by weight, about 0.5 to about 12% by weight, about 0.5 to about 10% by weight, about 0.5 to about 9% by weight, about 0.5 to about 8% by weight, about 0.5 to about 7% by weight, about 0.5 to about 6% by weight, about 0.5 to about 5% by weight, about 1 to about 18% by weight, about 1 to about 16% by weight, about 1 to about 15% by weight, about 1 to about 14% by weight, about 1 to about 12% by weight, about 1 to about 10% by weight, about 1 to about 9% by weight, about 1 to about 8% by weight, about 1 to about 7% by weight, about 1 to about 6% by weight, or about 1 to about 5% by weight of each strain of S. salivarius.

[0196] In various embodiments, when multiple strains of S. salivarius are present, the composition comprises about 1×10 3 ~Approx. 1×10 13 cfu / g of each strain of S. salivarius, e.g., approximately 1 × 10 4 ~Approx. 1×10 12 , about 1×10 5 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 7 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 12 , about 1×10 4 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 7 ~Approx. 1×10 10 , about 1×10 8 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 9 , about 1×105 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 7 ~Approx. 1×10 9 cfu / g of each strain of S. salivarius.

[0197] supplementary sugars Sugars considered for testing included the monosaccharides glucose, fructose, and galactose; the disaccharides sucrose and lactose; and the trisaccharide raffinose.

[0198] Lactose is primarily milk sugar and is not a preferred ingredient for people on a dairy-free diet or who have lactose intolerance, therefore lactose was excluded from consideration.

[0199] As mentioned above, sucrose and glucose are cariogenic and generally have an inhibitory effect on antagonistic behavior (see, e.g., Reinhold & Titgemeyer, Fritz. (2002) FEMS Microbiology Letters. 209. 141-8; Jankovic & Bruckner, J Mol Microbiol Biotechnol. 2007; 12(1-2): 114-20). Therefore, these sugars were also excluded from the study.

[0200] In various embodiments, the supplemental sugar is galactose or raffinose, or a combination thereof. In various embodiments, the supplemental sugar is galactose. In various embodiments, the supplemental sugar is raffinose. In various embodiments, the supplemental sugar is a mixture of galactose and raffinose.

[0201] D-galactose is a monosaccharide sugar. Raffinose is a trisaccharide containing galactose, glucose, and fructose monomer units.

[0202] In various embodiments, the composition comprises less than 20% by weight of each supplementary saccharide. In various embodiments, the composition comprises less than 20% by weight of galactose. In various embodiments, the composition comprises less than 20% by weight of raffinose. In various embodiments, the composition comprises less than 20% by weight of each of galactose and raffinose.

[0203] In various embodiments, the composition contains 0.01 to 20% by weight of each supplementary saccharide, for example, about 0.01 to about 18% by weight, or about 0.01 to about 15% by weight, or about 0.01 to about 12% by weight, or about 0.01 to about 10% by weight, or about 0.01 to about 9% by weight, or about 0.01 to about 8% by weight, or about 0.01 to about 7% by weight, or about 0.01 to about 6% by weight, or about 0.01 to about 5% by weight, or about 0.01 to about 4% by weight, or about 0.1 to about 20% by weight, or about 0.1 to about 18% by weight, or about 0.1 to about 15% by weight, or about 0.1 to about 12% by weight, or about 0.1 to about 10% by weight, or about 0.1 to about 9% by weight, or about 0.1 to about 8% by weight, or about 0.1 to about 7% by weight, or about 0.1 to about 6% by weight, or about 0.1 to about 5% by weight, or about 0.1 to about 4% by weight, or about 0.25 to about 20% by weight, or about 0.25 to about 18% by weight, or about 0.25 to about 15% by weight, or about 0.25 to about 12% by weight, or about 0.25 to about 10% by weight, or about 0.25 to about 9% by weight, or about 0.25 to about 8% by weight, or about 0.25 to about 7% by weight, or about 0.25 to about 6% by weight %, or about 0.25 to about 5% by weight, or about 0.25 to about 4% by weight, or about 0.5 to about 20% by weight, or about 0.5 to about 18% by weight, or about 0.5 to about 15% by weight, or about 0.5 to about 12% by weight, or about 0.5 to about 10% by weight, or about 0.5 to about 9% by weight, or about 0.5 to about 8% by weight, or about 0.5 to about 7% by weight, or about 0.5 to about 6% by weight, or about 0.5 to about 5% by weight, or about 0.5 to about 4% by weight, or about 1 to about 20% by weight, or about 1 to about 18% by weight, or about 1 to about 15% by weight, or about 1 to about 12% by weight, or is about 1 to about 10% by weight, or about 1 to about 9% by weight, or about 1 to about 8% by weight, or about 1 to about 7% by weight, or about 1 to about 6% by weight, or about 1 to about 5% by weight, or about 1 to about 4% by weight, or about 1.25 to about 20% by weight, or about 1.25 to about 18% by weight, or about 1.25 to about 15% by weight, or about 1.25 to about 12% by weight, or about 1.25 to about 10% by weight, or about 1.25 to about 9% by weight, or about 1.25 to about 8% by weight, or about 1.25 to about 7% by weight, or about 1.25 to about 6% by weight, or about 1.25 to about 5% by weight, or about 1.25 to about 4% by weight, or about 2 to about 20% by weight, or about 2 to about 18% by weight, or about 2 to about 15% by weight, or about 2 to about 12% by weight, or about 2 to about 10% by weight, or about 2 to about 9% by weight, or about 2 to about 8% by weight, or about 2 to about 7% by weight, or about 2 to about 6% by weight, or about 2 to about 5% by weight, or about 2 to about 4% by weight, or about 2.5 to about 20% by weight, or about 2.5 to about 18% by weight, or about 2.5 to about 15% by weight, or about 2.5 to about 12% by weight, or about 2.5 to about 10% by weight, or about 2.5 to about 9% by weight, or about 2.5 to about 8% by weight, or about 2.5 to about 7% by weight, or about 2.5 to about 6% by weight, or about 2.5 to about 5% by weight, or about 2.5 to about 4% by weight of each supplementary sugar. .

[0204] In various embodiments, the composition contains 0.01 to 20% by weight of galactose, for example, from about 0.01 to about 18% by weight, or from about 0.01 to about 15% by weight, or from about 0.01 to about 12% by weight, or from about 0.01 to about 10% by weight, or from about 0.01 to about 9% by weight, or from about 0.01 to about 8% by weight, or from about 0.01 to about 7% by weight, or from about 0.01 to about 6% by weight, or from about 0.01 to about 5% by weight, or from about 0.01 to about 4% by weight, or from about 0.1 to about 20% by weight, or from about 0.1 to about 18% by weight, or from about 0.1 to about 15% by weight, or from about 0.1 to about 20% by weight. about 12% by weight, or about 0.1 to about 10% by weight, or about 0.1 to about 9% by weight, or about 0.1 to about 8% by weight, or about 0.1 to about 7% by weight, or about 0.1 to about 6% by weight, or about 0.1 to about 5% by weight, or about 0.1 to about 4% by weight, or about 0.25 to about 20% by weight, or about 0.25 to about 18% by weight, or about 0.25 to about 15% by weight, or about 0.25 to about 12% by weight, or about 0.25 to about 10% by weight, or about 0.25 to about 9% by weight, or about 0.25 to about 8% by weight, or about 0.25 to about 7% by weight, or about 0.25 to about 6% by weight. %, or about 0.25 to about 5% by weight, or about 0.25 to about 4% by weight, or about 0.5 to about 20% by weight, or about 0.5 to about 18% by weight, or about 0.5 to about 15% by weight, or about 0.5 to about 12% by weight, or about 0.5 to about 10% by weight, or about 0.5 to about 9% by weight, or about 0.5 to about 8% by weight, or about 0.5 to about 7% by weight, or about 0.5 to about 6% by weight, or about 0.5 to about 5% by weight, or about 0.5 to about 4% by weight, or about 1 to about 20% by weight, or about 1 to about 18% by weight, or about 1 to about 15% by weight, or about 1 to about 12% by weight, or about 1 to about 10% by weight, or about 1 to about 9% by weight, or about 1 to about 8% by weight, or about 1 to about 7% by weight, or about 1 to about 6% by weight, or about 1 to about 5% by weight, or about 1 to about 4% by weight, or about 1.25 to about 20% by weight, or about 1.25 to about 18% by weight, or about 1.25 to about 15% by weight, or about 1.25 to about 12% by weight, or about 1.25 to about 10% by weight, or about 1.25 to about 9% by weight, or about 1.25 to about 8% by weight, or about 1.25 to about 7% by weight, or about 1.25 to about 6% by weight, or about 1.25 to about 5% by weight, or about 1.25 to about 4% by weight, or about 2 to about 20% by weight, or about 2 to about 18% by weight, or about 2 to about 15% by weight, or about 2 to about 12% by weight, or about 2 to about 10% by weight, or about 2 to about 9% by weight, or about 2 to about 8% by weight, or about 2 to about 7% by weight, or about 2 to about 6% by weight, or about 2 to about 5% by weight, or about 2 to about 4% by weight, or about 2.5 to about 20% by weight, or about 2.5 to about 18% by weight, or about 2.5 to about 15% by weight, or about 2.5 to about 12% by weight, or about 2.5 to about 10% by weight, or about 2.5 to about 9% by weight, or about 2.5 to about 8% by weight, or about 2.5 to about 7% by weight, or about 2.5 to about 6% by weight, or about 2.5 to about 5% by weight, or about 2.5 to about 4% by weight of galactose. .

[0205] In various embodiments, the composition contains 0.01 to 20% by weight raffinose, e.g., from about 0.01 to about 18% by weight, or from about 0.01 to about 15% by weight, or from about 0.01 to about 12% by weight, or from about 0.01 to about 10% by weight, or from about 0.01 to about 9% by weight, or from about 0.01 to about 8% by weight, or from about 0.01 to about 7% by weight, or from about 0.01 to about 6% by weight, or from about 0.01 to about 5% by weight, or from about 0.01 to about 4% by weight, or from about 0.1 to about 20% by weight, or from about 0.1 to about 18% by weight, or from about 0.1 to about 15 ... about 12% by weight, or about 0.1 to about 10% by weight, or about 0.1 to about 9% by weight, or about 0.1 to about 8% by weight, or about 0.1 to about 7% by weight, or about 0.1 to about 6% by weight, or about 0.1 to about 5% by weight, or about 0.1 to about 4% by weight, or about 0.25 to about 20% by weight, or about 0.25 to about 18% by weight, or about 0.25 to about 15% by weight, or about 0.25 to about 12% by weight, or about 0.25 to about 10% by weight, or about 0.25 to about 9% by weight, or about 0.25 to about 8% by weight, or about 0.25 to about 7% by weight, or about 0.25 to about 6% by weight. %, or about 0.25 to about 5% by weight, or about 0.25 to about 4% by weight, or about 0.5 to about 20% by weight, or about 0.5 to about 18% by weight, or about 0.5 to about 15% by weight, or about 0.5 to about 12% by weight, or about 0.5 to about 10% by weight, or about 0.5 to about 9% by weight, or about 0.5 to about 8% by weight, or about 0.5 to about 7% by weight, or about 0.5 to about 6% by weight, or about 0.5 to about 5% by weight, or about 0.5 to about 4% by weight, or about 1 to about 20% by weight, or about 1 to about 18% by weight, or about 1 to about 15% by weight, or about 1 to about 12% by weight, or about 1 to about 10% by weight, or about 1 to about 9% by weight, or about 1 to about 8% by weight, or about 1 to about 7% by weight, or about 1 to about 6% by weight, or about 1 to about 5% by weight, or about 1 to about 4% by weight, or about 1.25 to about 20% by weight, or about 1.25 to about 18% by weight, or about 1.25 to about 15% by weight, or about 1.25 to about 12% by weight, or about 1.25 to about 10% by weight, or about 1.25 to about 9% by weight, or about 1.25 to about 8% by weight, or about 1.25 to about 7% by weight, or about 1.25 to about 6% by weight, or about 1.25 to about 5% by weight, or about 1.25 to about 4% by weight, or about 2 to about 20% by weight, or about 2 to about 18% by weight, or about 2 to about 15% by weight, or about 2 to about 12% by weight, or about 2 to about 10% by weight, or about 2 to about 9% by weight, or about 2 to about 8% by weight, or about 2 to about 7% by weight, or about 2 to about 6% by weight, or about 2 to about 5% by weight, or about 2 to about 4% by weight, or about 2.5 to about 20% by weight, or about 2.5 to about 18% by weight, or about 2.5 to about 15% by weight, or about 2.5 to about 12% by weight, or about 2.5 to about 10% by weight, or about 2.5 to about 9% by weight, or about 2.5 to about 8% by weight, or about 2.5 to about 7% by weight, or about 2.5 to about 6% by weight, or about 2.5 to about 5% by weight, or about 2.5 to about 4% by weight of raffinose. .

[0206] In various embodiments, when a mixture of galactose and raffinose is present, the composition contains 0.01 to 20% by weight each of galactose and raffinose, e.g., about 0.01 to about 18% by weight, or about 0.01 to about 15% by weight, or about 0.01 to about 12% by weight, or about 0.01 to about 10% by weight, or about 0.01 to about 9% by weight, or about 0.01 to about 8% by weight, or about 0.01 to about 7% by weight, or about 0.01 to about 6% by weight, or about 0.01 to about 5% by weight, or about 0.01 to about 4% by weight, or about 0.1 to about 20% by weight, or about 0.1 to about 20% by weight. about 18% by weight, or about 0.1 to about 15% by weight, or about 0.1 to about 12% by weight, or about 0.1 to about 10% by weight, or about 0.1 to about 9% by weight, or about 0.1 to about 8% by weight, or about 0.1 to about 7% by weight, or about 0.1 to about 6% by weight, or about 0.1 to about 5% by weight, or about 0.1 to about 4% by weight, or about 0.25 to about 20% by weight, or about 0.25 to about 18% by weight, or about 0.25 to about 15% by weight, or about 0.25 to about 12% by weight, or about 0.25 to about 10% by weight, or about 0.25 to about 9% by weight, or about 0.25 to about 8% by weight, or about 0.25 to about 7% by weight, or about 0.25 to about 6% by weight, or about 0.25 to about 5% by weight, or about 0.25 to about 4% by weight, or about 0.5 to about 20% by weight, or about 0.5 to about 18% by weight, or about 0.5 to about 15% by weight, or about 0.5 to about 12% by weight, or about 0.5 to about 10% by weight, or about 0.5 to about 9% by weight, or about 0.5 to about 8% by weight, or about 0.5 to about 7% by weight, or about 0.5 to about 6% by weight, or about 0.5 to about 5% by weight, or about 0.5 to about 4% by weight, or about 1 to about 20% by weight, or about 1 to about 18% by weight, or about 1 to about 15% by weight, or about 1 to about 12% by weight, or about 1 to about 10% by weight, or about 1 to about 9% by weight, or about 1 to about 8% by weight, or about 1 to about 7% by weight, or about 1 to about 6% by weight, or about 1 to about 5% by weight, or about 1 to about 4% by weight, or about 1.25 to about 20% by weight, or about 1.25 to about 18% by weight, or about 1.25 to about 15% by weight, or about 1.25 to about 12% by weight, or about 1.25 to about 10% by weight, or about 1.25 to about 9% by weight, or about 1.25 to about 8% by weight, or about 1.25 to about 7% by weight, or about 1.25 to about 6% by weight, or about 1.25 to about 5% by weight, or about 1.25 to about 4% by weight, or about 2 to about 20% by weight, or about 2 to about 18% by weight, or about 2 to about 15% by weight, or about 2 to about 12% by weight, or about 2 to about 10% by weight, or about 2 to about 9% by weight, or about 2 to about 8% by weight, or about 2 to about 7% by weight, or about 2 to about 6% by weight, or about 2 to about 5% by weight, or about 2 to about 4% by weight, or about 2.5 to about 20% by weight, or about It contains 2.5 to about 18% by weight, or about 2.5 to about 15% by weight, or about 2.5 to about 12% by weight, or about 2.5 to about 10% by weight, or about 2.5 to about 9% by weight, or about 2.5 to about 8% by weight, or about 2.5 to about 7% by weight, or about 2.5 to about 6% by weight, or about 2.5 to about 5% by weight, or about 2.5 to about 4% by weight of each of galactose and raffinose.

[0207] Upregulated genes In one aspect, the invention provides a method for upregulating one or more genes in Streptococcus salivarius, comprising formulating S. salivarius in a composition comprising an effective amount of a sugar supplement; Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0208] In some embodiments, the upregulated genes may comprise or consist of a polynucleotide sequence encoding a lantibiotic peptide or bacteriocin, such as a class I or class II lantibiotic peptide or bacteriocin. For example, as described above, BLIS K12™ produces the bacteriocins salivaricin A, salivaricin B and salivaricin Q, which are encoded by salA, salB and salQ, respectively (SEQ ID NOs: 15, 16 and 17, respectively). The peptide sequences of these bacteriocins are shown in SEQ ID NOs: 23, 24 and 25, respectively.

[0209] As a further example, as described above, BLIS M18™ produces the bacteriocins salivaricin A, salivaricin 9 and salivaricin Q, which are encoded by salA, sal9 and salQ, respectively (SEQ ID NOs: 19, 20 and 21, respectively). The peptide sequences of these bacteriocins are shown in SEQ ID NOs: 27, 28 and 29, respectively.

[0210] In some embodiments, the upregulated gene may comprise or consist of a polynucleotide sequence encoding a subunit of the urease protein, hi some embodiments, the upregulated gene is ureC, e.g., BLIS K12™ ureC (SEQ ID NO: 18) or BLIS M18™ ureC (SEQ ID NO: 22).

[0211] In some embodiments, at least one of the upregulated genes comprises or consists of a polynucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 15-22, or at least one of the upregulated genes comprises or consists of a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 23-30.

[0212] In some embodiments, at least one of the upregulated genes is the salA gene, the salB gene, the salQ gene, the sal9 gene, and / or the ureC gene, or a mutant of any thereof.

[0213] These genes and variants thereof can be readily identified by techniques known in the art. For example, these genes can be identified by sequence similarity to known salA, salB, salQ, sal9, or ureC genes, or to genes encoding salA, salB, salQ, sal9, or ureC proteins, using sequence alignment tools. Alternatively, protein-encoding genes can be identified by structural similarity to known salA, salB, salQ, sal9, or ureC proteins, using structural alignments.

[0214] For example, these genes can be identified by computer-based methods well known to those skilled in the art using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR, etc.). See, for example, Nucleic Acids Res. 29:1-10 and 11-16, 2001. Similarity searches retrieve and align target sequences for comparison with the sequence being analyzed (i.e., the query sequence). Sequence comparison algorithms use a scoring matrix to assign an overall score to each of the alignments.

[0215] An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]), which includes BLASTN, BLASTP, BLASTX, tBLASTN, and tBLASTX, which are publicly available from (ftp:Inc. / / ftp.ncbi.nih.gov / blast / ) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA. Use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.

[0216] Multiple sequence alignment of related sequences can be performed using the CLUSTALW (Thompson, JD, Higgins, DG and Gibson, TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22: 4673-4680), CLUSTAL Omega (Sievers et al., (2011). Molecular Systems Biology 7: 539, https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP (which uses progressive pairwise alignment) (Feng and This can be carried out using the method of the present invention (Doolittle, 1987, J. Mol. Evol. 25, 351).

[0217] Pattern recognition software applications are available to find motif or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motif and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or identical motifs in a query sequence. MAST results are provided as a series of alignments with appropriate statistics and a visual summary of the motifs found. MEME and MAST were developed at the University of California, San Diego.

[0218] PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method to identify the function of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org / prosite) contains biologically significant patterns and profiles and is designed to be used with appropriate computational tools to assign new sequences to known families of proteins or to determine which known domains are present in a sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search the SWISS-PROT and EMBL databases with a given sequence pattern or signature.

[0219] Another example of a protein domain model database is Pfam (Sonnhammer et al., 1997, A comprehensive database of protein families based on seed alignments, Proteins, 28:405-420; Finn et al., 2010, The Pfam Protein Families database', Nucl. Acids Res., 38:D211 D222). "Pfam" refers to a large collection of protein domains and protein families maintained by the Pfam Consortium and available at several sponsored worldwide websites, including pfam.xfam.org / (European Bioinformatics Institute (EMBL-EBI)). The latest release of Pfam is Pfam 35.0 (November 2021). Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignments are high-quality assignments generated by seed alignments curated using representative members of the protein family and profile hidden Markov models based on the seed alignments. (Unless otherwise stated, matches of a query protein to a Pfam domain or family are Pfam-A matches). A complete alignment for the family is then automatically generated using all identified sequences belonging to the family (Sonnhammer (1998) J. Nucleic Acids Research, 2009). 26,320-322;Bateman(2000)Nucleic Acids Research 26,263-266;Bateman(2004)Nucleic Acids Research 32,Database Issue,D138-D141;Finn(2006)Nucleic Acids Research Database Issue 34,D247-251;Finn(2010)Nucleic Acids Research Database Issue 38,D 211-222).For example, protein sequences can be queried against HMMs using HMMER homology search software (e.g., HMMER2, HMMER3, or more advanced versions, hmmer.org) by accessing the Pfam database using the above website. Significant matches that identify a query protein as being in a pfam family (or as having a particular Pfam domain) are matches whose bit scores are equal to or greater than the collection threshold for the Pfam domain. Expectation values ​​(e-values) can also be used as a criterion for including a query protein in a Pfam or for determining whether a query protein has a particular Pfam domain, with low e-values ​​(much less than 1.0, e.g., less than 0.1, or 0.01 or less) representing a low probability that the match is due to chance.

[0220] Some specific examples of known genes from S. salivarius are provided herein according to the table below. [Table 1]

[0221] In some embodiments, at least one of the upregulated genes comprises or consists of a polynucleotide sequence having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 15-22.

[0222] In some embodiments, at least one of the upregulated genes comprises or consists of a polynucleotide sequence encoding a polypeptide having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs:23-30.

[0223] In some embodiments, at least one of the upregulated genes is a polynucleotide sequence having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15 or 19. In one embodiment, the salA gene comprises or consists of the sequence shown in SEQ ID NO: 23 or 27, or encodes a polypeptide having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23 or 27.

[0224] In some embodiments, at least one of the upregulated genes is a polynucleotide sequence having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:16. In one embodiment, the salB gene comprises or consists of the sequence shown in SEQ ID NO:24 or encodes a polypeptide having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:24.

[0225] In some embodiments, at least one of the upregulated genes is a polynucleotide sequence having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17 or 21. In one embodiment, the salQ gene comprises or consists of the sequence or encodes a polypeptide having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:25 or 29.

[0226] In some embodiments, at least one of the upregulated genes is a polynucleotide sequence having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:20. In one embodiment, the sal9 gene comprises or consists of the sequence shown in SEQ ID NO:28, or encodes a polypeptide having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:28.

[0227] In some embodiments, at least one of the upregulated genes is a polynucleotide sequence having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18 or 22. ureC gene comprising or consisting of the sequence or encoding a polypeptide having at least 70% identity, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 26 or 30.

[0228] Determination of Polynucleotide Sequence Identity Polynucleotide sequence identity can be determined as follows: Compare the subject polynucleotide sequence with the candidate polynucleotide sequence using BLASTN (BLAST program suite, version 2.2.5 [Nov 2002]) of bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250), BLASTN is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized, except that filtering of low complexity parts should be turned off.

[0229] Polynucleotide sequence identity can be determined using the following unix command line parameters:

[0230] bl2seq-i nucleotideseq1-j nucleotideseq2-F Fp blastn

[0231] The parameter -FF turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and % of identical nucleotides in the line "Identities=".

[0232] Polynucleotide sequence identity can also be calculated over the entire length of overlap between candidate and subject polynucleotide sequences using global sequence alignment programs (e.g., Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). A complete implementation of the Needleman-Wunsch global alignment algorithm can be found in the Needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6, pp. 276-277), which is available from http: / / www.hgmp.mrc.ac.uk / Software / EMBOSS / . The European Bioinformatics Institute server also provides the ability to perform an EMBOSS-Needle global alignment between two sequences online at http: / www.ebi.ac.uk / emboss / align / .

[0233] Alternatively, the GAP program can be used, which calculates the optimal global alignment of two sequences without penalizing terminal gaps.GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.

[0234] Another method for calculating % polynucleotide sequence identity is based on aligning the sequences being compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5).

[0235] Variant polynucleotides or genes disclosed herein also include polynucleotides or genes that differ from the sequences disclosed herein, but that, as a result of the degeneracy of the genetic code, encode polypeptides that have similar activity to the polypeptides encoded by the polynucleotides disclosed herein. Sequence modifications that do not change the amino acid sequence of the polypeptide are "silent mutations."

[0236] The variant polynucleotides or genes disclosed herein may contain sequence modifications resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity.

[0237] Variant polynucleotides with silent mutations and conservative substitutions in the encoded polypeptide sequence can be determined using the bl2seq program publicly available from the BLAST suite of programs (version 2.2.5 [Nov 2002]) at NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ) via the tblastx algorithm as described above.

[0238] Determination of Polypeptide Sequence Identity Polypeptide sequence identity can be determined as follows: A subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (BLAST suite of programs, from version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized, except that filtering of low complexity regions should be turned off.

[0239] Polypeptide sequence identity can also be calculated over the entire length of the overlap between a candidate polypeptide sequence and a subject polypeptide sequence using a global sequence alignment program. EMBOSS-Needle (http: / www.ebi.ac.uk / emboss / align / ) and GAP (available at Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) are also suitable global sequence alignment programs for calculating polypeptide sequence identity, as described above.

[0240] Another method for calculating % polypeptide sequence identity is based on aligning the sequences to be compared using Clustal X (Jeanmougin et al. 1998, Trends Biochem. Sci. 23, 403-5).

[0241] The polypeptide variants disclosed herein also encompass variants that exhibit similarity to one or more of the specifically specified sequences, which similarity is likely to preserve the functional equivalence of the sequences that could not reasonably be expected to have arisen by chance. Such sequence similarity for polypeptides can be determined using the publicly available bl2seq program from the BLAST suite of programs from NCBI (version 2.2.5 [Nov 2002]) (ftp: / / ftp.ncbi.nih.gov / blast / ). Polypeptide sequence similarity can be examined using the following unix command line parameters:

[0242] bl2seq-i peptideseq1-j peptideseq2-F Fp blastp

[0243] The parameter -FF turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for pairs of sequences. The program finds regions of similarity between sequences, and for each such region, reports an "E-value", which is the expected number of times one might expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E-values, much smaller than 1, this is approximately the probability of such a random match.

[0244] The variant polypeptide sequence preferably has a repeat sequence of 1×10 compared to any one of the specifically specified sequences. -10 less than 1×10 -20 less than 1×10 -30 less than 1×10 -40 less than 1×10 -50 less than 1×10 -60 less than 1×10 -70 less than 1×10 -80 less than 1×10 -90 less than 1×10 -100 The E value is less than 0.01.

[0245] A variant polypeptide sequence may contain conservative substitutions of one or several amino acids of the described polypeptide sequence without significantly altering its biological activity.

[0246] Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length.

[0247] Inhibited bacteria In various embodiments, the methods enhance the inhibition profile of S. salivarius against skin, oral cavity, teeth, mucosa (e.g., oral, rectal, vaginal) and / or ENTR microorganisms, including pathogenic and other non-pathogenic microorganisms.

[0248] The microorganism may be non-pathogenic. The subject has a pre-existing microflora that is generally not harmful or may be beneficial to the subject. Examples of such non-pathogenic microflora bacteria include S. salivarius species, e.g., S. epidermidis species, L. lactis species, S. constellatus species.

[0249] In some embodiments, it may be useful to inhibit or reduce the population of such non-pathogenic microorganisms, for example, promoting colonization with bliss-producing S. salivarius such as K12 or M18.

[0250] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENT microorganisms include S. aureus species, S. intermedius species, S. saprophyticus species, M. catarrhalis species, H. influenzae species, S. pyogenes species, P. aeruginosa species, S. mutans species, S. pneumoniae species, C. acnes species, C. albicans species, S. sobrinus species, Corynebacterium species, F. nucleatum species, A. actinomycetemcomitans species, P. gingivalis species, Tannerella forsythia species, Treponema denticola species, P. intermedia species, Prevotella species, A. viscosus species, S. excisii species, The fungus causing tinea pedis infection is selected from the group consisting of S. salivarius species other than K12 or M18, S. salivarius species, S. sanguinis species, S. corni species, B. intermedius species, A. parvum species, E. subaleum species, E. sursi species, P. micra species, S. moulay species, S. agalactiae species, C. minutissimus species, P. propionicus species, S. agalactiae species, S. dysgalactiae species, S. simulans species, Xylosus species, fungi causing tinea pedis infection, S. salivarius species other than K12 or M18, L. lactis species, S. epidermidis species, S. constellatus species, or any combination of any two or more thereof.

[0251] In various embodiments, the oral and / or dental microorganisms are selected from S. intermedius, M. catarrhalis, H. influenzae, S. pyogenes, S. mutans, S. pneumoniae, F. nucleatum, A. actinomycetemcomitans, P. intermedia, Prevotella, A. viscosus, S. sobrinus, B. intermedia, A. parvum, E. sabreum, E. sursi, P. micula, S. moulay, S. agalactiae, or any combination of any two or more thereof.

[0252] In various embodiments, the skin microorganism is selected from S. aureus species, S. saprophyticus species, S. pyogenes species, C. acnes species, C. albicans species, S. cohnii species, C. minutissimus species, P. propionicus species, S. agalactiae species, S. dysgalactiae species, S. simulans species, Xylosus species, fungi causing tinea pedis infections, S. epidermidis species, or any combination of any two or more thereof.

[0253] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from S. aureus species, S. saprophyticus species, M. catarrhalis species, H. influenzae species, S. pyogenes species, P. aeruginosa species, S. mutans species, S. pneumoniae species, S. salivarius species other than K12 or M18, L. lactis species, S. epidermidis species, S. constellatus species, or a combination of any two or more thereof.

[0254] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from the group consisting of S. aureus A222, S. aureus 20, S. aureus 14, S. aureus 19, S. aureus A504, S. aureus ATCC 6538, S. saprophyticus ATCC 15305, M. catarrhalis TW1, M. catarrhalis TW2, H. influenzae TW5, S. pyogenes M76, S. pyogenes 71-698, S. pyogenes 100-120, S. aureus 102-110, S. aureus 106-110, S. aureus 108-111, S. aureus 106-111, S. aureus 108-112, S. aureus 108-113, S. aureus 108-114, S. aureus 108-115, S. aureus 108-116, S. aureus 108-117, S. aureus 108-119, S. aureus 108-120, S. aureus 108-121, S. aureus 108-122, S. aureus 108-123, S. aureus 108-124, S. aureus 108-125, S. aureus 108-126, S. aureus 108-129 ... S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, P. aeruginosa I2, P. aeruginosa ATCC27853, S. mutans OMZ175, S. mutans FW75, S. pneumoniae D39, S. pneumoniae RX1, S. pneumoniae PK8, S. extremilis Bris 2, S. dysgalactiae T277, C. acnes ATC6919, S. sanguinis K11, S. sobrinus OMZ176, S. corni, S. simulans, L. lactis T-21, S. epidermidis 11, S. epidermidis E30, S. constellatus T29, S. salivarius 6, S. salivarius 193, S. salivarius 20P3, or any combination of any two or more thereof.

[0255] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from the group consisting of S. aureus A222, S. aureus 20, S. aureus 14, S. aureus 19, S. aureus A504, S. saprophyticus ATCC15305, M. catarrhalis TW1, M. catarrhalis TW2, H. influenzae TW5, S. pyogenes M76, S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, P. aeruginosa I2, S. mutans OMZ175, S. pneumoniae D39, L. lactis T-21, S. epidermidis 11, S. constellatus T-29, S. salivarius 6, S. salivarius 193, S. salivarius 20P3, or any combination of any two or more thereof.

[0256] S. salivarius strains with antiviral activity, particularly anti-SARS-CoV2 (Covid-19) activity, are described in International Application No. PCT / NZ2021 / 050054 (Blis Technologies Ltd). Prophylactic and therapeutic compositions and methods for treating respiratory viruses are also described. Data show that S. salivarius K12, M18 have various antiviral activities against SARS-CoV2, influenza A, influenza B, and respiratory syncytial virus (RSV). Thus, the microorganisms inhibited herein also include respiratory viruses such as SARS-CoV2, influenza A, influenza B, and respiratory syncytial virus (RSV).

[0257] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENT microorganism is a Streptococcus or Staphylococcus bacterium, hi various embodiments, the Streptococcus or Staphylococcus bacterium is selected from the species S. aureus, S. saprophyticus, S. mutans, S. pyogenes, S. pneumoniae, S. constellatus, and S. salivarius. In various embodiments, the Staphylococcus bacterium is selected from S. aureus A222, S. saprophyticus ATCC15305, and the Streptococcus bacterium is selected from S. mutans OMZ175, S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, S. pneumoniae D39, S. salivarius species, such as S. salivarius 6, S. salivarius 193, S. salivarius 20P3, and S. constellatus T-29.

[0258] In various embodiments, the Streptococcus or Staphylococcus bacterium is selected from the species S. aureus, S. saprophyticus, S. mutans, S. salivarius, S. constellatus, S. pyogenes, and S. pneumoniae, S. equisimilis, S. dysgalactiae, wherein the S. salivarius is K12. In various embodiments, the Staphylococcus bacterium is selected from S. aureus A222 and S. saprophyticus ATCC15305, and the Streptococcus bacterium is selected from S. mutans OMZ175, S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, S. pneumoniae D39, S. equisimilis Bris 2, S. dysgalactiae T277, S. salivarius species, such as S. salivarius 6, S. salivarius 193, S. salivarius 20P3, and S. constellatus T-29, and the S. salivarius strain is K12.

[0259] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from S. aureus species, S. saprophyticus species, L. lactis species, S. epidermidis species, S. salivarius species, M. catarrhalis species, S. mutans species, H. influenzae species, S. pneumoniae species, S. pyogenes species, L. lactis T-21, S. epidermidis 11, S. epidermidis E30, and S. constellatus T-29, the S. salivarius strain is K12, and the supplemented sugar is galactose.

[0260] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from the group consisting of S. aureus A222, S. aureus 20, S. aureus 14, S. aureus 19, S. aureus A504, S. saprophyticus ATCC15305, L. lactis T-21, S. epidermidis 11, M. catarrhalis TW1, M. catarrhalis TW2, S. mutans OMZ175, H. influenzae, S. pneumoniae D39, S. pyogenes 71-6, ... B21, S. aureus B11, S. aureus B21, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, S. aureus B11, 98, S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, L. lactis T-21, S. epidermidis 11, S. epidermidis E30, and S. constellatus T-29, the S. salivarius strain is K12, and the supplemented sugar is galactose.

[0261] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from S. aureus, S. saprophyticus, L. lactis, S. epidermidis, the S. salivarius strain is K12, and the supplemental sugar is raffinose.

[0262] In various embodiments, the skin, oral cavity, teeth, mucosa and / or ENTR microorganism is selected from the group consisting of S. aureus A222, S. aureus 20, S. aureus 14, S. aureus 19, S. aureus A504, S. saprophyticus ATCC 15305, S. pyogenes 71-698, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, Selected from S. pyogenes M17, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, L. lactis T-21, S. epidermidis 11, S. salivarius 6, S. salivarius 193, and S. salivarius 20P3, wherein the S. salivarius strain is K12 and the supplemental sugar is raffinose.

[0263] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganism is selected from S. constellatus, S. pyogenes, S. pneumoniae, S. salivarius, S. mutans, S. saprophyticus, S. aureus, M. catarrhalis, L. lactis, H. influenzae, P. aeruginosa, the S. salivarius strain is M18, and the supplemental sugar is raffinose.

[0264] In various embodiments, the skin, oral cavity, teeth, mucosa, and / or ENTR microorganisms are selected from the group consisting of S. pyogenes M76, S. pneumoniae D39, S. mutans OMZ175, S. saprophyticus ATCC15305, S. aureus A222, M. catarrhalis TW1, M. catarrhalis TW2, L. lactis T-21, H. influenzae TW5, P. aeruginosa I2, S. pyogenes 71-69, and / or S. pyogenes 72-76. 8, S. pyogenes FF22, S. pyogenes 71-679, S. pyogenes W-1, S. pyogenes M57, S. pyogenes EMM92, S. pyogenes M66, S. pyogenes M74, S. constellatus T-29, S. salivarius 6, S. salivarius 193, and S. salivarius 20P3, wherein the S. salivarius strain is M18, and the supplemental sugar is raffinose.

[0265] In various embodiments, the bacterium is selected from the species S. pyogenes and S. pneumoniae, and the S. salivarius strain is M18. In various embodiments, the bacterium is selected from the species S. pyogenes 71-698 and S. pneumoniae D39, and the S. salivarius strain is M18.

[0266] In various embodiments, the microorganism is a virus selected from SARS-CoV2, influenza A, influenza B, and RSV.

[0267] Prevention or treatment of disease or inhibition of microorganisms In one aspect, the present invention provides a method for treating or preventing a disease or disorder, comprising administering to a subject in need thereof a composition comprising S. salivarius and an effective amount of a supplemental saccharide, wherein the S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

[0268] In one aspect, the present invention relates to the use of S. salivarius and a supplemental saccharide in the manufacture of a medicament for the treatment or prevention of a disease or disorder, wherein the S. salivarius is Streptococcus salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

[0269] In one aspect, the present invention provides a composition for use in the treatment or prevention of a disease or disorder comprising S. salivarius and an effective amount of a supplemental saccharide, wherein the S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

[0270] In one aspect, the present invention provides a composition comprising S. salivarius and an effective amount of a supplemental saccharide, wherein the S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

[0271] In various embodiments, the composition or therapeutic formulation improves the inhibition profile and / or improves the mucoid properties of S. salivarius.

[0272] In various embodiments, the disease or disorder is caused by a skin, oral cavity, dental, mucosal, or ENTR pathogen.

[0273] In various embodiments, the disease or disorder is otitis media, pharyngitis, dental caries, acute pharyngitis, tonsillitis, pneumonia, COPD, periodontal disease, gingivitis, halitosis, dental caries, sepsis, meningitis, vaginitis, body odor, acne, actinomycosis, psoriasis, erythema, nosocomial erythema, SARS-CoV2, influenza A, influenza B, and RSV, candidiasis (oral candidiasis), athlete's foot, or any combination of any two or more thereof.

[0274] In various embodiments, the disease or disorder is caused by a pathogenic bacterium.In various embodiments, the disease or disorder is caused by a pathogenic Streptococcus bacterium.

[0275] In various embodiments, the disease or disorder is otitis media, sore throat, dental caries, acute pharyngitis, tonsillitis, pneumonia, COPD, periodontal disease, gingivitis, halitosis, dental caries, sepsis, meningitis, vaginitis, body odor, acne, actinomycosis, psoriasis, erythema, cellulitis, impetigo, atopic dermatitis, bacteremia, soft tissue infection, erythema, nosocomial erythema, or any combination of any two or more thereof.

[0276] In some embodiments, the disease or disorder is caused by a pathogenic virus.

[0277] In various embodiments, the disease or disorder is SARS-CoV2, influenza A, influenza B, or RSV.

[0278] In various embodiments, the disease or disorder is caused by a pathogenic fungus (eg, a yeast or skin mycosis).

[0279] In various embodiments, the disease or disorder is candidiasis (oral candidiasis), athlete's foot (tinea pedis), or other ringworm infections.

[0280] In various embodiments, the subject is a mammal, including humans, dogs, cats, horses, sheep, cattle, and other domestic and farm animals. In some embodiments, the subject is a non-human subject. In some embodiments, the subject is a human. In various embodiments, the subject is an infant, child, or adult.

[0281] In one aspect, the present invention also relates to a method for inhibiting microorganisms susceptible to Bliss-producing S. salivarius, comprising administering to a subject in need thereof a composition of the present invention or a therapeutic formulation of the present invention. The microorganisms to be inhibited may be non-pathogenic microorganisms, such as S. salivarius, S. epidermidis, L. lactis, S. constellatus, etc. It may be desirable to reduce the population of such non-pathogenic microorganisms Salivarius to promote adhesion and colonization by the S. salivarius used in the present invention.

[0282] Raw material composition Lyoprotectants and cryoprotectants include products containing Bliss producing strains (including S. salivarius containing products) to have a protective function and maintain cell viability. Lyoprotectants have a protective function during drying, whereas cryoprotectants have a protective function during freezing. The same composition can have both functions, and these terms are used interchangeably herein unless otherwise specified. Suitable lyoprotectants or cryoprotectants are known to those skilled in the art.

[0283] In various embodiments, the lyoprotectant may be selected from sodium caseinate, peptone, skim milk powder, whey protein, trehalose, glycerol, betaine, sucrose, galactose, glucose, lactose, lactitol, mannitol, maltodextrin, sodium citrate, and combinations thereof.

[0284] In various embodiments, the lyoprotectant can be a mixture of trehalose, lactitol, and maltodextrin.

[0285] In various embodiments, the composition is dairy-free. In various embodiments, the composition is free of any dairy-derived ingredients. In various embodiments, the lyoprotectant can be a mixture of sucrose, sodium citrate, and maltodextrin or trehalose.

[0286] In various embodiments, the composition is a powder, for example prepared by mixing a lyophilized S. salivarius powder with a powder of a saccharide supplement, or by co-lyophilizing S. salivarius and a saccharide supplement. The inventors have found that the saccharide supplements investigated herein do not affect the stability of the lyophilized raw powder of S. salivarius (data not reported).

[0287] Those skilled in the art will appreciate that the composition may also include other excipients, including diluents or flow aids.

[0288] Use of the ingredient in therapeutic preparations, e.g., lozenges, etc. The composition can be formulated into a therapeutic formulation for administration by a variety of methods. A "therapeutic formulation" is a composition suitable for use in the prophylactic or therapeutic treatment of an individual in need thereof. Generally, a therapeutic formulation comprises a S. salivarius strain and a saccharide supplement as described above, as well as a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0289] In one aspect, the compositions of the invention are formulated into a therapeutic formulation. In various embodiments, the compositions or therapeutic formulations are formulated for oral, dental, nasal, ENTR, or topical administration.

[0290] In various embodiments, the therapeutic formulation is a powder, lozenge, nasal spray, nasal gel, nasal drops, oral drops, oral gel, oral spray, inhalable, aerosol, topical composition, chewable, melt, film, gummy, toothpaste, tooth gel, varnish, mousse, mouthwash, food product (e.g., yogurt), cream, gel, spray, deodorant, serum, lotion, balm, moisturizer, pessary, or suppository. Time-release or sustained release products that maintain the level of supplemental sugar in the oral cavity or ENTR are preferred in some embodiments.

[0291] Slow or sustained release products that maintain the level of glycerol or ENTR in the oral cavity are preferred in some embodiments. Such slow or sustained release products are known in the art and include multi-layer tablets, slow or fast dissolving melts, films, chewing gums, gels, mucoadhesives, or buccal adhesive delivery systems.

[0292] "Acceptable carriers, diluents, and / or excipients" refers to vehicles for delivery of S. salivarius strains or extracts to the surface of the host that are compatible with the viability of the bacterial cells or the activity of the extract. Acceptable carriers, diluents, and excipients suitable for use in the administration of viable streptococcal strains, particularly S. salivarius strains and extracts, are well known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 22nd, Gennaro, ed., 2013, Mack Publishing Co., Easton, PA) and are incorporated herein by reference. Suitable carriers are generally inert and can be either solid or liquid.

[0293] In various embodiments, the carrier is a pharma- ceutically acceptable carrier. Such pharma- ceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. A variety of pharma- ceutically acceptable carriers suitable for administration of live or lyophilized bacteria are well known in the art (see, for example, Remington's, supra; and Pharmaceutical Composition LACTINEXae (Hynson, Westcott and Dunning, Baltimore, Md., USA), a commercial preparation for oral administration of live lactobacillus). Suitable solid carriers known in the art include, but are not limited to, for example, magnesium carbonate; magnesium stearate; cellulose; talc; sugars, such as maltose, fructose, sucrose, mannitol, lactose, isomalt, maltodextrin, starch; wheat flour; oligosaccharides and skim milk, and similar edible powders.

[0294] Typical diluents are, for example, starch; lactose; mannitol; kaolin; calcium phosphate or sulfate; inorganic salts such as sodium chloride; and powdered sugar or cellulose.

[0295] Therapeutic formulations may also include excipients, such as resins; fillers; binders; lubricants; solvents; glidants; disintegrants; preservatives; buffers; flavoring agents; colorants; sweeteners; and, optionally, fragrances.

[0296] Typical binders include starch, gelatin, sugars such as lactose, fructose, glucose, etc. Natural and synthetic gums such as acacia, alginates, locust bean gum, methylcellulose, polyvinylpyrrolidine, tragacanth gum, xanthan gum, etc. are also convenient. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.

[0297] Lubricants to prevent sticking to the die during manufacturing include slippery solids such as talc, silica, magnesium and calcium stearate, polyethylene glycol, stearic acid and hydrogenated vegetable oils.

[0298] Disintegrants are substances that swell when wet and break down the composition, releasing streptococci or extracts. Disintegrants include starches, clays, celluloses, algins and gums, more specifically corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, cation exchange resins, alginic acid, guar gum, citrus pulp, carboxymethylcellulose, powdered sponge, and sodium lauryl sulfate.

[0299] For delivery to the respiratory tract, the composition can also be in a form for administration by inhalation.Inhalation products are typically in powder or micronized powder form, or in liquid form.Products can be conveniently administered using inhalers, nebulizers, atomizers, or any other approved device for delivery to the respiratory tract.Carriers for inhalable products are well known in the art, and include lactose, erythritol, sorbitol, and cyclodextrin.

[0300] The therapeutic formulation may further contain nutrients to maintain the viability and enhance the effectiveness of the bacteria in the formulation. Additional components useful in the composition are agents that selectively enhance the growth of desirable bacteria over undesirable organisms.

[0301] In various embodiments, the therapeutic formulation comprises buffers (phosphate buffer, citric acid), calcium carbonate, multivitamins, minerals (e.g., zinc, vitamins C and D), antioxidants (berries, e.g., blackcurrant, quercetin, licorice), fluoride, xylitol, yeast extracts (Saccharomyces cerevisiae or Saccharomyces Boulardii), lysates, extracts (Bifidobacterium, Lactobacillus), and / or yogurt cultures.

[0302] In various embodiments, the therapeutic formulation further comprises other enhancing agents to enhance the production or activity of the composition. In various embodiments, the enhancing agents are selected from carbohydrates, e.g., oligosaccharides such as Nutriose® FB (Roquette Freres, Restron, France), maltodextrose, and lactulose; prebiotic agents; chemicals such as reducing agents, e.g., cysteine ​​and mercaptoethanol; and ions of metals, e.g., magnesium.

[0303] Therapeutic formulations can also be formulated to contain flavoring agents, coloring agents, sweetening agents (xylitol, maltodextrin, Monk fruit extract, stevia, aspartame), taste masking agents (Smoothenol®), fragrances, or other compounds that enhance the acceptability of the product to the patient without compromising the efficacy of the product and / or enhance patient compliance. Methods of preparing therapeutic formulations for administration by inhalation are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 22nd ed., supra, incorporated herein by reference).

[0304] The topical therapeutic formulation may include other additives conventionally used in topical compositions, such as moisturizers. Those skilled in the art will further appreciate that the additives must be compatible with the viability and efficacy of the probiotics. Such additives may provide or improve the therapeutic, cosmetic, stability, and / or appearance properties of the therapeutic formulation. Examples of suitable additives include, but are not limited to, carriers (e.g., vegetable oils, triglycerides, glycerol, propylene glycol, water, saline), surfactants, dispersants, emulsifiers, inhibitory activity enhancers, buffers, antimicrobial agents, prebiotics, fragrances, antioxidants, colorants, skin protectants, anti-aging agents (hyaluronic acid, ceramides, olive squalene), antimicrobial agents, aluminum salts, inorganic pigments, odor absorbers or neutralizers, or sunscreens. Such additives may be included in the therapeutic formulation of the present invention in amounts typical of topical formulations. A variety of pharma- ceutically acceptable additives suitable for topical application of live or lyophilized bacteria are well known in the art.

[0305] It may be advantageous to formulate the composition into a sustained or extended release composition or therapeutic formulation. In various embodiments, the composition is formulated into a sustained release composition. In various embodiments, the composition is formulated into a two-part composition that provides immediate release of S. salivarius and the supplemental sugar, and sustained release of additional supplemental sugar.

[0306] Method of administration The reader will understand that the compositions and formulations of the present invention can be administered according to a wide variety of protocols for inhibiting microbial populations for both therapeutic and non-therapeutic purposes. Any protocol known in the art for administration of S. salivarius K12 and M18 can be used.

[0307] In various embodiments, the therapeutic formulation is orally administered once, twice, three times, four times, or up to 12 times daily. In various embodiments, the therapeutic formulation is orally administered via a lozenge, powder, melt, mouthwash, or toothpaste. For the treatment of halitosis, pretreatment with a mouthwash, such as chlorhexidine mouthwash, or mechanical cleaning, such as a toothbrush, is recommended.

[0308] In various embodiments, the therapeutic formulation is administered topically as frequently as needed, usually once or twice a day. In various embodiments, the composition is administered topically by cream, serum, deodorant, spray, or moisturizer. It may be advisable to pretreat the skin with water, soap, or cleaning formulation before administration.

[0309] In various embodiments, the therapeutic formulations are administered rectally or vaginally as needed, typically via a pessary or suppository, once or twice daily.

[0310] In various embodiments, the therapeutic formulations are administered via the pulmonary route, for example by a nebulizer or inhaler, as frequently as needed, typically once or twice daily.

[0311] The therapeutic formulations are useful for improving the oral health of a subject, for example, by preventing or treating any of the conditions identified in WO2001027143, WO2002070719, and WO2005007178 (noted above), all of which are incorporated herein by reference in their entireties. S. salivarius M18 is also known to help reduce dental plaque, support oral health and oral bacterial flora, reduce caries, prevent dental cavities, treat and prevent gingivitis, and treat and prevent periodontitis (Burton, JP, et al., 2013 J. Med. Microbiol. 62, 875-884; Burton, JP et al., 2013, 2013, PLoS ONE 8; Di Pierro, et al. 2015. Clin Cosmet Investig Dent.; 7:107-13; L Scariya, DVN, M Varghese, 2015. Int. J. Pharma Bio Sci. 6, 242-250).

[0312] Method of Manufacturing the Ingredients In one aspect, the invention provides a method of producing a composition comprising S. salivarius and an effective amount of a supplemental saccharide, comprising: (a) combining S. salivarius with a sugar supplement; (b) mixing to produce a homogenous blend; S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0313] In various embodiments, the Streptococcus salivarius is in the form of a powder. In various embodiments, the saccharide is in the form of a powder, such as a lyophilized powder. In various embodiments, the powder is a raw powder that includes S. salivarius and a lyoprotectant as described above.

[0314] In various embodiments, the mixing occurs in a blender. In various embodiments, the product is then packaged.

[0315] In various embodiments, excipients are added.

[0316] In various embodiments, the composition provides an improved inhibition profile of S. salivarius.

[0317] In various embodiments the compositions are for use in the treatment or prevention of a disease or disorder, including the diseases and disorders referenced above.

[0318] In various embodiments, the composition is for use in inhibiting microbial populations susceptible to Bliss-producing S. salivarius.

[0319] In one aspect, the invention relates to the use of a composition produced by a method of the invention for the treatment or prevention of a disease or disorder.

[0320] In one aspect, the invention relates to the use of a composition produced by the method of the invention to inhibit a microbial population susceptible to Bliss-producing S. salivarius.

[0321] Methods for manufacturing the formulation In one aspect, the present invention provides a method for producing a therapeutic formulation comprising a composition comprising S. salivarius and an effective amount of a saccharide supplement, comprising: a) mixing excipients; b) adding a composition comprising the S. salivarius of the present invention and a sugar supplement; c) blending to provide a therapeutic formulation; the Streptococcus salivarius is Streptococcus salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0322] The method may include further processing steps to provide a therapeutic formulation. One skilled in the art will know the excipients required depending on the route of administration and blending methods such as homogenization.

[0323] In various embodiments, the present invention provides a method of making a mouth lozenge comprising a composition comprising S. salivarius and an effective amount of a supplemental saccharide, the method comprising: a) mixing the carriers, tableting aids (e.g., binders, lubricants), and flavoring agents; b) adding a composition comprising the S. salivarius of the present invention and a sugar supplement; c) blending the mixture; and d) formulating the mixture into a lozenge to provide a lozenge; S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0324] The inventors have determined that the supplemented sugars investigated herein do not affect the stability of S. salivarius in the lozenge (data not reported).

[0325] In various embodiments, the present invention provides a method for producing an oral powder containing composition comprising S. salivarius and an effective amount of a sugar supplement, the method comprising: a) mixing the carriers, tableting aids (e.g., binders, lubricants), and flavoring agents; b) adding a composition comprising the S. salivarius of the present invention and a sugar supplement; c) blending the mixture to provide an oral powder; S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0326] In various embodiments, the present invention provides a method for preparing a topical composition comprising a composition comprising S. salivarius and an effective amount of a supplemental saccharide, the method comprising: a) mixing an oil vehicle and a dispersant; b) adding a composition comprising the S. salivarius of the present invention and a sugar supplement; c) homogenizing the mixture to provide a topical composition; S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0327] In various embodiments, the invention provides a method for preparing a pessary or suppository comprising a composition comprising Streptococcus salivarius and an effective amount of a sugar supplement, the method comprising the steps of: a) mixing a solid lipid with other excipients; b) adding a composition comprising the S. salivarius of the present invention and a sugar supplement; c) homogenizing the mixture to provide a pessary or suppository; S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0328] In various embodiments, the present invention provides a method of preparing a formulation for pulmonary administration comprising S. salivarius and an effective amount of a sugar supplement, the method comprising the steps of: a) optionally mixing a dry powder carrier with other excipients; b) adding a composition comprising the S. salivarius of the present invention and a sugar supplement; c) mixing to provide a formulation for pulmonary administration; S. salivarius is S. salivarius M18, S. salivarius K12, or a combination thereof; The method is provided wherein the supplemental sugar is galactose or raffinose, or a combination thereof.

[0329] The following non-limiting examples are offered to illustrate the present invention and are not intended to limit its scope in any way. EXAMPLES

[0330] material All the following culture media and sugars were supplied by Fort Richard Laboratories, New Zealand: CABK12 agar plates; Columbia blood agar base (CAB) bottles; Columbia agar base with 0.5% (w / v) CaCO3 (CABCa); Columbia agar base supplemented with 0.1% CaCO3 and 5% v / v sheep blood (sBaCa); Haemophilus agar plates; Mitis salivarius agar; D-galactose - BD Difco; Todd Hewitt Broth (THB) - BD Difco (used according to the manufacturer's instructions); M17 Broth - BD Difco (used according to the manufacturer's instructions but without added sugars).

[0331] All the following materials were supplied by Lab Supply Ltd, New Zealand: Calcium Carbonate (CaCO3) - PanReac Applichem; D-(+) Glucose - Monohydrate - Applichem; Ethanol (96% AR grade) - diluted with water before use to make a 70% solution; D(+)-Glucose Monohydrate - Applichem; Chloroform (Emsure, ACS, ISO, Reag. Ph Eur) - Merck; Water for molecular biology (nuclease free) - AppliChem; 10x Tris-Acetate-EDTA (TAE) buffer - Applichem; Hydrochloric acid (HCl);

[0332] D-(+) raffinose pentahydrate; D-(-) fructose (both from Sigma, New Zealand);

[0333] Chloroform (Emsure, ACS, ISO, Reag. Ph Eur)-Supelco (Sigma Aldrich, New Zealand).

[0334] The following materials were all supplied by Thermo Fisher Scientific, New Zealand: PureLink RNA Mini Kit; TRIzol reagent; Phasemaker tubes; RNaseZap; TURBO DNA-free kit; SuperScript IV VILO; PowerTrack SYBR Green master Mix; 10,000× SYBR safe DNA gel stain; MicroAmp Optical 384-well reaction plate; MicroAmp Optical adhesive film and phosphate-buffered saline (PBS)-Oxoid. [Table 2]

[0335] Phosphate Buffered Saline (PBS)-Dulbecco's A-Oxoid (supplied by Thermo Fisher Scientific, New Zealand and used according to manufacturer's instructions). AnaeroGen Sachet-Oxoid-supplied by Thermo Fisher Scientific, New Zealand.

[0336] Promega GoTaq G2 Hot start Green Master mix, supplied by Invitro Technologies, New Zealand.

[0337] Maestrogen AccuRuler 1 kb DNA RTU Ladder, supplied by Mediray, New Zealand.

[0338] Similac 360 Total Care infant formula (Abbott Global, USA) was purchased via www.Amazon.com Whole milk powder (Anchor Blue™ milk powder, Anchor, New Zealand);

[0339] S. salivarius strains K12 and M18 - supplied by Blis Technologies Ltd, New Zealand and available from Deutsche Sammlung von Mikroorganism und Zellkulturen (DSM); K12 raw product (K12 powder containing trehalose / lactitol / maltodextrin), M18 raw product (M18 powder containing trehalose / lactitol / maltodextrin); K12 and M18 dairy-free raw product (powders containing sucrose, sodium citrate, and maltodextrin lyophilization protectant); a commercial powder formulation containing K12, Daily Defence Junior (DDJ) (composition: S. salivarius K12, isomalt, maltodextrin, vanilla flavor), a commercial lozenge formulation containing K12 (Throat Guard Pro) (composition: S. salivarius K12, isomalt, tableting aid, natural flavor) - all from Blis Technologies Ltd, New Zealand); S. saprophyticus ATCC 15305, S. mutans NCTC 10449 (ATCC 25175); S. mutans UA159 (ATCC 700610); S. corneii (ATCC 29974); S. simulans (ATCC 27848); C. acnes 6919; F. nucleatum ATCC 25586; P. gingivalis ATCC 33277; P. gingivalis ATCC 53978; P. intermedia ATCC 25611; S. salivarius (ATCC 13419, ATCC 25923, and ATCC 7073); S. sobrinus ATCC 27351, S. agalactiae ATCC 12386, A. viscosus ATCC 15987, C. auris ATCC 51966 - all American Type Culture Available from the American College of Chemists Collection (ATCC); M. catarrhalis TW1 and TW2; L. lactis T-21; S. pyogenes M76; P. aeruginosa I2; H. influenzae TW5; S. aureus A222; S. mutans OMZ175; S. pneumoniae D39; S. pyogenes 71-698; S. constellations T-29; S. pyogenes FF22; S. pyogenes W-1; S. pyogenes M17; S. pyogenes M57; S. pyogenes EMM92; S. pyogenes M66; S.pyogenes M74;S. pyogenes H13;S. pyogenes K26;S. pyogenes WS02,S. dysgalactiae Bris 2;S. dysgalactiae T277;S. pneumoniae RX1;S. pneumoniae PK8;S. mutans D10;S. mutans FW75;A. viscosus T14;S. sanguinis K11;S. aureus 20;S. aureus 19;S. aureus 14;F. nucleatum FH2;F. nucleatum FH3;S. salivarius CN3410;34A;K14;H29;S. pyogenes K7;S. dysgalactiae T-148;S. aureus 19 - All Ordered from Blis Technologies Ltd; S. sobrinus OMZ176 (CCUG21020) - available from the Culture Collection University of Gothenburg (CCUG); S. pyogenes 71-679 - standard gift from Lewis Wannamaker.

[0340] Method 1 - Preparation of solid culture medium Calcium carbonate (CaCO3) was added to all solid culture media (CAB) during preparation as a buffering agent. CaCO3 was present in all prepared culture media at a concentration of 0.5% (w / v) (CABCa).

[0341] Pre-made bottles of Columbia Agar Base (CAB) agar in 180 mL capacity were ordered from Fort Richard Laboratories, New Zealand. Additional ingredients were added to these 180 mL bottles. Carbohydrate was added to separate bottles of CAB agar at 0.1%, 0.5%, 1.0%, 1.5%, and 2.0% (w / v) concentrations. The following formula was used to determine the amount of carbohydrate (and CaCO-3) to add to achieve the desired concentration of that ingredient:

[0342]

number

[0343] For example, to obtain CAB agar base containing CaCO3 and galactose at a concentration of 0.5% (w / v) using one of the pre-made Fort Richard agar bottles, the following formula is used:

[0344]

number

[0345] A precise method was used to ensure sterility of the culture medium and also to ensure that the correct amount of any added ingredients was accurately incorporated into the culture medium. The ingredients were removed from their containers using a clean spoon / spatula and weighed on a balance in a clean weigh boat, this was measured to the nearest milligram. After weighing the ingredients, they were placed into clean 30 ml containers, labelled and sealed. The pre-made CAB agar is solid when it arrives from Fort Richard and therefore a crater approximately 3 cm wide and 3 cm deep needs to be cut into the agar using a sterile scalpel to create a space for the ingredients to be poured into. After this, the ingredients are poured into the crater and 1 ml of sterile distilled water is dispensed on top to partially submerge the ingredients in the agar. The agar that was cut to create the cavity was then placed back into the crater and a magnetic stir bar was placed into the bottle. The bottle was then autoclaved at 110°C for 10 minutes. The bottle was stored in a water bath set at 50°C. Sterile Petri dishes were labeled with the appropriate information (date, contents of the plate, and added ingredients) and placed in a Class II biological safety hood with the lid removed. Before pouring the agar into the Petri dishes, the contents were thoroughly stirred for 1-2 minutes using a magnetic stir bar. As an additional sterilization measure, a Bunsen flame was ignited in the biological safety cabinet and the contents of the bottle were poured into the Petri dishes. If any air bubbles appeared during pouring, they were popped using the Bunsen flame. The agar was left to solidify for 5-10 minutes. One plate was labeled "negative control" and placed in an incubator overnight to ensure no contamination occurred during the agar preparation.

[0346] Method 2 - Deferred antagonism assay One to two producer colonies were transferred from the stock plate to 900 μL of the suspension solution using a new cotton swab. After this, the suspension was vortexed and the sample was inoculated onto solid culture medium using a new cotton swab. The inoculation was streaked diametrically across the agar plate in a 1.5 cm wide strip. The plate was then incubated. After incubation, all visible colonies were removed from the agar surface by wiping. The plate was then treated with chloroform by dispensing 2 ml of chloroform onto a 4 cm x 4 cm piece of cloth and sealing the inverted agar plate on top for 30 minutes. The plate was then air-dried for 30 minutes after the chloroform treatment. One to two indicator colonies were then transferred from the stock plate to the complementary suspension solution. The suspension was then vortexed and the sample was inoculated onto the same agar surface. These samples were streaked perpendicular to the initial producer streak.

[0347] Each indicator streak was prepared with a new suspension containing cells from the corresponding stock plate containing the indicator strain of interest. A new swab was used to transfer the indicator strain suspension sample. Results were measured by qualitatively examining the subsequent size (if any) of the zone of inhibition (ZOI) of indicator strain colony growth. A positive result is when there is a significant reduction or complete inhibition of indicator colony growth on the culture medium. A negative result is when there is no sign of a reduction in indicator strain colony density.

[0348] Method 3 - Dose-response assay CABCa agar plates containing various concentrations of sugars were prepared according to the method described in Method 1. The producer strain was transferred from the stock agar plate to 900 μL of THB using a new cotton swab. A plastic spread plater was cut to a width of approximately 1.2 cm, then dipped in ethanol and allowed to air dry to ensure sterility. A 100 μL sample of the producer strain suspension was then dispensed onto the agar surface in (approximately) 20 μL spots in a straight line diametrically across the agar surface. The spots were placed approximately 1.5 cm apart from each other. The spots were then evenly distributed across the agar surface in a 1.5 cm wide strip using a plastic spreader. This step was done to ensure that a uniform producer strain streak was inoculated onto the agar surface and provided an approximate measure of the initial inoculum concentration. Separate plastic spreaders were used for samples containing different producer strains. The samples were left immersed on the agar surface until there was no visible liquid remaining on the agar surface (approximately 20 min). After preparation of producer streaks, the deferred antagonism assay protocol was followed according to method 2. Results were recorded by measuring the size of the zone of inhibition (ZOI) in mm.

[0349] Example 1: Antagonism of ENTR microorganisms by K12 This example shows the induction of ENTR microbial antagonism by K12 and supplemental sugars.

[0350] BLIS K12™ cultured on CABCa agar plates containing galactose and raffinose at 0.1%, 1.0%, 1.5%, and 2.0% (w / v) concentrations were tested for their antimicrobial activity by deferred antagonism assay according to the protocol outlined in Method 3. The results presented in Figures 1 and 2 show the change in zone of inhibition size for ear, nose, and throat (ENT) microbial indicator strains. When K12 was cultured in the presence of various concentrations of galactose, the ZOI size for several ENTR indicator strains increased significantly from the control condition (Figure 1). M. catarrhalis TW1 and TW2 show similar changes in ZOI size across various concentrations of galactose. The change in ZOI size for TW1 then peaks (12 mm) at 1.5% galactose (w / v) and then decreases at 2.0% galactose (w / v), while the change in ZOI size for TW2 peaks (11 mm) at 2.0% galactose (w / v).The change in ZOI size for L. lactis T-21 tends to be upwards in relation to the concentration of galactose present in the culture medium.

[0351] The ZOI size of S. pyogenes M57 increases from control conditions at 0.1%, 1.0%, and 1.5% (w / v) concentrations of galactose. The change in ZOI size of M76 remains at a level (11 mm) between 1.5% and 2.0% concentrations of galactose. The concentration of galactose that produces the greatest ZOI change (on average) is 2.0% (w / v) (average ZOI change at 2% galactose = 9.5 mm).

[0352] When K12 was cultured on CABCa incorporating various concentrations of raffinose during the deferred antagonism assay, a change in ZOI size from control conditions (no carbohydrate) was shown for several ENT microbial indicator strains (Figure 2). The change in ZOI size for L. lactis from control conditions to 0.1% raffinose (w / v) was 15 mm. This large increase was followed by an increase to 16 mm at 1.0% raffinose (w / v) (indicating significantly greater inhibition), and then the ZOI size plateaued and finally decreased at 2.0% raffinose (w / v). The change in ZOI size for H. influenzae TW5 and S. pyogenes M76 also peaked at 1.5% raffinose (w / v), at 12 mm and 13 mm, respectively. The ZOI size for TW5 actually returned to the same as the control condition (0 mm) at 2% raffinose (w / v). The ZOI size of M. catarrhalis TW1 and TW2 changed very similarly between conditions (Figure 2). The change in ZOI size for each strain peaked at the end of our test range (2.0% (w / v) (13 mm). The concentration of galactose that produced the greatest ZOI size change (on average) was the 1.5% raffinose (w / v) condition (average ZOI change at 1.5% raffinose = 10.33 mm).

[0353] Example 2: Antagonism of ENTR microorganisms by M18 This example demonstrates the induction of ENTR microbial antagonism by M18 and supplemental sugars.

[0354] BLIS M18™ cultured on CABCa agar plates containing galactose and raffinose at concentrations of 0.1%, 1.0%, 1.5%, and 2.0% (w / v) were tested for their antimicrobial activity by performing a deferred antagonism assay according to the protocol outlined in Method 3. The results, presented in Figures 3 and 4, show the change in zone of inhibition size for ear, nose, and throat (ENT) microbial indicator strains.

[0355] When M18 was cultured on CABCa incorporating various concentrations of galactose, there were subsequent changes in ZOI size for several ENTR microbial indicator strains during the deferred antagonism assay (Figure 3). M. catarrhalis strains TW1 and TW2 both showed an increase in their respective zones of inhibition (12 → 23 mm and 13 → 23 mm) when galactose was incorporated into the culture medium at 1.0% and 1.5% (w / v) concentrations versus the control condition. The zones of inhibition for L. lactis and S. pyogenes M76 were both greatest when galactose was present at a 1.5% (w / v) concentration (9 mm and 14 mm, respectively). These were notable differences from their control condition ZOI sizes. All susceptible ENTR indicator strains showed a decrease in ZOI size when galactose was present at a 2.0% (w / v) concentration compared to the 1.5% (w / v) concentration condition. The condition that produced the largest average ZOI change was the 1.5% galactose (w / v) condition. Mean ZOI change in 1.5% galactose = 11.5 mm.

[0356] When M18 was cultured on CABCa agar incorporating various concentrations of raffinose, a very similar pattern emerged in ZOI size as observed under galactose conditions. However, under raffinose conditions, more ENTR indicator strains showed some inhibition and the ZOI size (average) was larger than the corresponding ZOI size under galactose conditions. The zones of inhibition for M. catarrhalis strains TW1 and TW2 showed the largest change in size from the control condition when raffinose was present at a concentration of 1.5% (w / v) (31 mm, respectively). Contrary to the trend observed under galactose conditions, the ZOI size of L. lactis was largest under 0.1% (w / v) raffinose conditions (20 mm). The ZOI size of P. aeruginosa I2 was smaller across all raffinose conditions, except for the 1.5% (w / v) concentration where the ZOI = 10 mm wide. There was no change from the control condition. The ZOI size of H. influenzae TW5 was greatest in the 1.5% (w / v) raffinose condition (26 mm). Again, the ZOI size of all susceptible ENTR indicator strains showed a significant decrease in width when raffinose was incorporated into the culture medium at a concentration of 2.0% (w / v) (Figure 4). The condition that produced the greatest ZOI change on average was the 1.5% raffinose (w / v) condition. Mean ZOI change at 1.5% raffinose = 22.16 mm.

[0357] When the same experiment was performed with K12- / - (a strain that does not harbor the bacteriocin-encoding megaplasmid), no zones of inhibition were formed for any of the ENTR indicator strains.

[0358] Example 3: Activity of raffinose compared to trimix (an equimolar mixture of three sugars) and individual sugars This example shows that the stimulatory effect of K12 and M18 is due to raffinose and not due to equimolar amounts of one or all of its individual components, i.e., raffinose is not metabolized to its individual components that have the effect. In addition, the effect of any changes in pH from the assay design that may have influenced the change in inhibitory effect was evaluated.

[0359] Dose-response assays were performed according to method 3 above, except that a 1.2 cm streak was used.

[0360] Figures 5-7 highlight the enhanced efficacy of BLIS K12 or M18 plus raffinose, trimix and individual sugars against skin, teeth, ENTR and pathogens.

[0361] Conclusion: Comparative evaluation of equimolar concentrations of raffinose versus trimixed sugars and individual sugars showed that in the presence of raffinose, K12 has better inhibitory activity against microorganisms associated with skin, teeth and the ENTR compared to an equivalent composition of three monomeric sugars. This effect appears to be mediated by enhanced production of antimicrobial molecules (such as bacteriocins or nonribosomal peptides) rather than an inhibitory effect due to a decrease in pH.

[0362] Example 4: Activity of raffinose compared to trimix (a mixture of three sugars at equal weight percent concentrations) and individual sugars This example shows that the stimulatory effect of K12 and M18 is due to raffinose and equal weight percent amounts of one or all of its individual sugars, i.e., raffinose is not metabolized to the individual sugars that have this effect.

[0363] Dose-response assays were performed according to method 3 above, except that a 1.2 cm streak was used. For each sugar tested, the pH of the producer streak was adjusted after growth of the producer to vary the acidic conditions.

[0364] Enhanced efficacy of K12 or M18+ (% w / v) containing raffinose over equal weight percent amounts of saccharides, either as trimix or individual saccharides, was seen against representative microorganisms from skin, dental, and lower respiratory tract diseases, as well as other S. salivarius (Figures 8-13). Activity is normalized to baseline controls.

[0365] Example 5: Raffinose promotes antibacterial (eg, bacteriocin or non-ribosomal peptide) activity of K12 and M18 rather than acid production. This example shows that the stimulation of greater inhibition of these microorganisms is due to increased production of antimicrobial molecules (bacteriocins or nonribosomal peptides) and not due to an associated decrease in pH to a more acidic (and therefore potentially inhibitory) environment.

[0366] METHODS: Bacterial test strains were evaluated for their ability to grow on agar equivalent to Columbia agar base without calcium carbonate at various pHs ranging from pH 4.5 to pH 7. Bacterial test strains were suspended in either Todd-Hewitt broth or the appropriate growth medium for the strain and then swabbed across the test plates at various pHs. These plates were then incubated at 37°C, 5% CO2, and bacterial growth of each test strain was monitored at 18 and 24 hours.

[0367] Results: At 24 hours, all strains tested grew at pH 7, but as the pH decreased, the number of strains able to grow decreased (Table 1). Only 3 of the 9 strains grew at pH 5.25 or below. This indicates that acid production by either K12 or M18 grown on CABCa supplemented with trimix or individual sugars may reduce or completely inhibit growth of the strains being tested. [Table 3]

[0368] Note that raffinose did not induce a significant decrease in pH (>5.25), but did induce K12 to produce inhibitory activity. The inhibitory activity of K12 observed in the presence of raffinose is due to antibacterial activity and not a pH effect.

[0369] Example 6: Activity of raffinose with K12 or M18 compared to trimix (a mixture of three sugars at equal weight percent concentration) and individual sugars This experiment shows that the inhibitory range of K12 was found to extend to species and strains that are not typically inhibited by K12 (Tables 2 and 3). K12 is induced by raffinose specifically against skin microorganisms, highlighting the potential benefit of K12 for skin indications such as impetigo and atopic dermatitis.

[0370] The method used was the same as in Example 4.

[0371] Results: Table 2 below highlights which species / strains became sensitive to inhibitory molecules (e.g. bacteriocins) from K12 incubated with raffinose (Table 2). Considering for example 2.5% raffinose, containing equal % (0.83%, each of the three sugars). Similar results were obtained for M18. [Table 4]

[0372] This effect, shown below in Table 3, was seen with the indicated percentages of raffinose and equivalent trimix (by weight %) or individual sugars. [Table 5]

[0373] K12 or M18 in the presence of raffinose showed enhanced potency when compared to equimolar or equiwt% concentrations of trimix sugars or sugars alone.

[0374] The inhibitory activity of raffinose is due to induction of bacteriocin-producing K12 or M18 rather than, or in addition to, acid production from metabolism of simple sugars (acid production in the oral cavity may cause caries and provide a safe niche for acid-tolerant bacteria such as S. mutans).

[0375] A selective dose-related inhibitory effect was observed with raffinose, suggesting that specific dose levels of raffinose are required to achieve benefit, rather than a global prebiotic effect of common sugars alone or in combination.

[0376] Specific observations: The indicator strain S. aureus A222 changed from insensitive to sensitive to K12 when 1.25, 2.5, 5, and 10% w / v raffinose was added to K12. · The indicator strains S. mutans OMZ175 and S. saprophyticus ATCC15305 changed from insensitive to sensitive to K12 when 2.5, 5, and 10% w / v raffinose was added. The indicator strain S. mutans OMZ175 changed from insensitive to sensitive to M18 when 0.5, 1.25, 2.5, and 5% w / v raffinose was added. The indicator strains S. constellatus T-29, S. aureus A222, and S. saprophyticus ATCC15305 changed from insensitive to sensitive to M18 when 1.25, 2.5, or 5% w / v raffinose was added.

[0377] Increased inhibition by K12 was seen for: S. constellatus T-29 containing 0.5-10% w / v raffinose S. pyogenes containing 0.5-10% w / v raffinose S. pneumoniae containing 0.5-10% w / v raffinose

[0378] Increased inhibition by M18 was seen for: S. pyogenes containing 0.5-10% w / v raffinose S. pneumoniae containing 0.5-10% w / v raffinose

[0379] Example 7A: Effect of raffinose concentration on the induction of inhibitory activity of K12 or M18 This example shows the effect of raffinose concentration on the enhanced inhibitory effect. Raffinose concentrations of 1.7, 2.5, 3.3, 5, and 10% are compared in Tables 4 to 7 below.

[0380] The gaps in the table indicate where no results were obtained due to test contaminants. [Table 6] [Table 7] [Table 8] [Table 9]

[0381] Raffinose concentrations of 1.7, 2.5, 3.3, and 5% were all able to inhibit at least seven of the indicator strains tested.

[0382] Inhibition of the indicator strain M. catarrhalis was seen in Table 6 but not in Table 7 where the pH of the agar was readjusted after growth of the producer strain, suggesting that the inhibition was due to acid production by the producer strain.

[0383] For most of the indicator strains, the maximum zone of inhibition was measured on agar plates supplemented with 2.5% raffinose.

[0384] Example 7B: Effect of concentration of galactose and galactose / raffinose combinations Objective: To determine the effect of the concentration of galactose on inducing the inhibitory activity of K12 and M18.

[0385] Methods: CABCa agar plates with or without 0.5, 1.25, 1.7, 2.5, 3.3, and 5% galactose were prepared by adding 0.5% (w / v) calcium carbonate to solid CAB agar in a bottle, which was then melted by autoclaving for 10 min at 110° C. After cooling, filter-sterilized sugar solution or distilled water was added and mixed, and then 20 ml was pipetted into a Petri dish.

[0386] Deferred antagonism assays were performed using K12 or M18 stock suspended in Todd Hewitt broth (THB), at approximately 1–2 × 10 6 A 1.2 cm streak containing cfu was spread in the center of a CABCa control or CABCa galactose-supplemented test plate. After 18 h of growth at 37 °C and 5% CO2, the bacterial growth was removed, the pH of the agar in the initial streak was measured and adjusted to pH 6.5-7.5 using 0.5 M sodium carbonate pH 11, and then the agar surface was sterilized using chloroform vapor. Bacterial test strains were suspended in THB and then swabbed across the plate perpendicular to the initial streak. After the plates were incubated for a further 18 h at 37 °C and 5% CO2, any zone of inhibition was measured (mm) for each test strain.

[0387] Results and conclusions: Surprisingly, only at a concentration of 0.5% w / w galactose, Blis K12 and Blis M18 showed enhanced inhibitory activity (potency) and spectrum (number of pathogenic strains) against pathogens involved in ENTR and skin infections (Table 8). This allows us to conclude that the mere presence of galactose may not be sufficient, but that a certain concentration of galactose is required to induce inhibitory activity. [Table 10] nr = no result due to contamination

[0388] Example 7C: Effect of the concentration of the combination of raffinose and galactose on the induction of the inhibitory activity of K12 or M18 This experiment aimed to determine the effect of the combined concentration of raffinose and galactose on the inhibitory activity of K12 and M18.

[0389] Method: As in Example 7B, except that CABCa agar plates were prepared with or without the following combinations of raffinose and galactose: [Table 11]

[0390] Results: As shown in Table 9, activity against various pathogens was observed for various combinations of raffinose and galactose in weight percent. [Table 12] Example 8: Activity of raffinose (2.5%) with K12 or M18 compared to trimix (a mixture of three sugars at equal weight percent concentration) and individual sugars This experiment shows that the inhibitory range of K12 or M18 was found to extend to species and strains that are typically not inhibited by K12 or M18. This concentration of raffinose was found to inhibit several ENTR, dental, and skin pathogens and S. salivarius strains.

[0391] The method used was the same as in Example 4.

[0392] Results: Figures 14-23 show the inhibitory effect of K12 or M18 on ENTR, dental, and skin pathogens, as well as other S. salivarius strains susceptible to bacteriocin-producing S. salivarius K12 or M18.

[0393] Example 9: Raffinose enhances the inhibitory activity of K12 and M18 when K12 and M18 are sourced from different fermentation processes and contain different lyoprotectant mixtures, i.e. dairy-free This experiment shows that raffinose enhances the inhibitory effect of K12 and M18, independent of the source of K12 at the ingredient supplier and the different lyoprotectant matrices in which K12 or M18 are housed.

[0394] The method used is described in Example 4. The pH of the producer streaks was adjusted after growth of the producer strain to change the acidic conditions for all test plates.

[0395] Enhanced inhibitory activity of dairy-free K12 with 2.5% w / w raffinose over equal weight percent sugars as either trimix or individual sugars was seen against representative microorganisms from skin, teeth, oral cavity, ENTR disease and other S. salivarius strains (Figures 24-25). Activity is normalized to baseline control.

[0396] Conclusion: Dairy-free K12 or M18, as well as K12 or M18, also showed enhanced inhibitory activity against various microorganisms in the presence of raffinose and galactose. In comparison, trimix or other sugars did not show similar inhibitory effects.

[0397] Example 10A: Effect of raffinose and galactose on induction of proliferation and inhibitory activity of K12 This example shows that raffinose and galactose do not contribute to greater cell numbers of K12, yet still enhance the antibacterial effect.

[0398] Methods: Growth curves were performed using 1 × 10 cells in M17 broth (control) [BD Difco #218561] or M17 broth (test) supplemented with either 2.5% raffinose [D-(+)-raffinose pentahydrate - Sigma #R0250], 2.5% trimix, 0.83% or 2.5% galactose [D-galactose - BD Difco #216310], 0.83% or 2.5% glucose [D(+)-glucose-monohydrate - Applichem #A3617], or 0.83% or 2.5% fructose [D(-)fructose - Sigma #F3510] (all w / v). 5 CFU / ml starting culture was derived using K12 stock. Broth cultures were then incubated at 37°C, 5% CO2 in air and samples were taken at the following 0, 6, and 18 hour time points and analyzed for cell number and optical density using a spectrophotometer (optical density (OD) at 600 nm). To determine cell number, 1:10 serial dilutions of culture samples were prepared in phosphate buffered saline (PBS Dulbecco A-Oxoid #BR0014G) and then 20 μl spots of each dilution were spotted in triplicate on CABK12 agar plates. These were then incubated at 37°C, 5% CO2 in air for 18 hours. Cell numbers for each culture sample at different time points were then calculated from the number of colonies grown in each of the 1:10 dilutions.

[0399] result: All sugar combinations resulted in similar cell numbers for K12, therefore they did not result in any substantial differences, except for glucose, which gave the highest cell number (Figure 26). However, despite this, glucose did not result in any higher inhibitory activity (likely due to catabolite repression). Surprisingly, K12 grown in the presence of raffinose and galactose shows higher inhibitory activity against S. pyogenes strains (Figure 27) or other pathogens (Figure 28). Raffinose in the trimix or the individual sugars fed separately did not result in any greater increase in inhibitory activity compared to the control.

[0400] Example 10B: Effect of raffinose and galactose on induction of proliferation and inhibitory activity of M18 This example shows that raffinose and galactose do not contribute to higher cell numbers of S. salivarius M18, but still enhance the antibacterial effect.

[0401] Methods: Growth curves were performed using 1 × 10 cells in M17 broth (control) [BD Difco #218561] or M17 broth (test) supplemented with either 2.5% raffinose [D-(+)-raffinose pentahydrate - Sigma #R0250], 2.5% trimix, 0.83% or 2.5% galactose [D-galactose - BD Difco #216310], 0.83% or 2.5% glucose [D(+)-glucose-monohydrate - Applichem #A3617], or 0.83% or 2.5% fructose [D(-)fructose - Sigma #F3510] (all wv). 5 CFU / ml starting cultures were derived using M18 stock. Broth cultures were then incubated at 37°C, 5% CO2 and samples were taken at the following 0, 6, and 18 hour time points and analyzed for cell count. To determine cell counts, 1:10 serial dilutions of culture samples were prepared in phosphate buffered saline (PBS Dulbecco A-Oxoid#BR0014G) and then 20 μl spots of each dilution were spotted in triplicate onto CABK12 agar plates. These were then incubated at 37°C, 5% CO2 for 18 hours. Cell counts for each culture sample at different time points were then calculated from the number of colonies grown in each of the 1:10 dilutions.

[0402] result: All sugar combinations resulted in similar cell numbers for M18 (Figure 28A). Surprisingly, M18 grown in the presence of raffinose and galactose exhibits higher inhibitory activity against S. pyogenes strains and other microorganisms (Figure 28B). Raffinose in the trimix or the individual sugars supplied separately did not result in any greater increase in inhibitory activity compared to the control.

[0403] Example 11: Cell morphology of K12 and M18 feedstocks grown either in the presence and absence of raffinose, or a trimix of the sugars galactose, glucose, or fructose, or individual sugars The objective of this experiment was to determine whether S. salivarius K12 and M18 stocks (-) produced sticky mucoid-like colonies (compared to controls) when grown on either CABCa control plates or CABCa test plates supplemented with 2.5% raffinose or 2.5% Trimix (0.83% of each sugar: galactose, glucose, and fructose) or 0.83% of the individual sugars.

[0404] S. salivarius K12 and M18, sourced from non-dairy sources, were also analyzed for morphological changes when grown on either CABCa control plates or CABCa test plates supplemented with 2.5% raffinose or 2.5% Trimix (0.83% each of the following sugars: galactose, glucose, and fructose).

[0405] Method: Columbia Blood Agar + 0.5% CaCO3 (CABCa) - 180ml bottle, 0.9g Calcium Carbonate (CaCO3) PanReac Applichem #1412121210 was added to the bottle of agar by preparing a well in the agar using a scalpel, tilting into the calcium carbonate and covering with the cut agar. The agar was then melted in an autoclave at 110°C / 10min, then cooled, mixed well and 20ml was pipetted into a Petri dish.

[0406] CABCa + raffinose (2.5% w / v) - 0.9g CaCO3 and 4.5g (2.5%) D-(+)-raffinose pentahydrate (Sigma #R0250) were added to a bottle of agar by preparing a well in the agar using a scalpel, tilting into the calcium carbonate and covering with the cut agar. The agar was then melted in an autoclave at 110°C / 10min, cooled, mixed well and then 20ml was pipetted into a Petri dish.

[0407] CABCa+ Trimix (a mixture of three sugars at equal weight percent concentration). In a 180 ml bottle, 0.9 g CaCO3 and 1.5 g (0.83%) of each sugar: D-galactose, D(+)-glucose, and D(-)-fructose were added to the bottle of agar by preparing a well in the agar using a scalpel, tilting into the calcium carbonate and carbohydrate, and covering with the cut agar. The agar was then melted in an autoclave at 110°C / 10 min, then cooled and mixed well before pipetting 20 ml into a Petri dish.

[0408] D-(+)-raffinose pentahydrate-Sigma#R0250 molecular weight=594.51g / mol

[0409] D-Galactose - BD Difco #216310 Molecular weight = 180.16 g / mol

[0410] D(+)-Glucose-monohydrate - Applichem#A 36171000 Molecular weight = 198.17g / mol

[0411] D(-) Fructose - Sigma - #F3510 Molecular weight = 180.16 g / mol

[0412] CABCa agar test plates were prepared supplemented with either individual sugars: galactose, glucose, and fructose at a concentration of 0.83% w / v, or a trimix of these sugars. CABCa test plates were also prepared supplemented with raffinose at 2.5% w / v. All test plates were supplemented with 0.5% calcium carbonate. CABCa agar plates supplemented with 0.5% w / v calcium carbonate were also prepared as control plates.

[0413] Freeze-dried raw material: K12-1.94×10 11 cfu / g, Dairy-free K12-2.4×10 11 cfu / g, M18-2.2×10 11 cfu / g, dairy-free M18-4.2×10 11 cfu / g.

[0414] Dilutions: Todd-Hewitt Broth (THB) - 30g Todd-Hewitt Broth powder (BD Difco #279240) + distilled water (1000ml), autoclaved at 121℃ / 15min.

[0415] Preparation of the production strain: 1 g of material was added to 9 ml of THB in a small stomacher bag to obtain approximately 1 × 10 10 A 1:10 dilution of cfu / ml was obtained. This was then mixed in a Stomacher apparatus for 5 minutes. A 10 -4 Serial dilutions of 1:10 were performed to cfu / ml.

[0416] Spread plate: 10 of 20 μl of raw material -4 The cfu / ml dilutions were spread onto either CABCa control or CABCa test plates. These were then incubated at 37° C., 5% CO2 for 18 hours. Plates were then visualized and photographed to assess morphological changes.

[0417] Deferral Assay Plates: For these, as before, add 10 µl of starting material to 100 µl of the -3Dilutions were prepared by dispensing as drops in a vertical line down the center of a test CABCa plate supplemented with or without sugar, and then spreading as a 1.2 cm streak down the center of the plate using a cut-down plastic spreader. Plates were incubated at 37° C., 5% CO2 for 18 hours.

[0418] The source producer streaks were photographed and then removed using a glass slide to visualize bacterial mass and morphology.

[0419] Results: Spread plates: S. salivarius K12 and M18 grown on 2.5% w / v raffinose show larger mucoid colonies than grown on CABCA control plates. K12 and M18 colonies on CABCa test plates supplemented with either trimix (a mixture of three sugars at equal weight percent concentrations) or individual sugars did not appear mucoid (Figure 29).

[0420] Close-up of K12 and M18 colonies on CABCa agar and CABCa agar supplemented with 2.5% w / v raffinose: Compared to the control, S. salivarius K12 or M18 (dairy or non-dairy) appear visually as large, sticky mucoid-like colonies. This effect was not observed with 2.5% w / v trimix (a 0.83% w / v mixture of equal weight percent concentrations of three sugars) and / or with 0.83% w / v of the individual sugars (Figure 30).

[0421] Photograph of K12 producer streaks and glass slide showing bacterial growth. The dotted black oval visually indicates the amount of slime produced (Figure 31).

[0422] Photograph of M18 producer streaks and glass slide showing bacterial growth. The dotted black oval visually indicates the amount of slime produced (Figure 31).

[0423] The K12 and M18 producers streak also appeared very mucoid when grown on CABCa supplemented with 2.5% w / v raffinose (Figure 31).

[0424] Example 12: Induction of antibacterial effect (inhibition) varies with S. salivarius strain Objective: To determine whether induction of antibacterial effects in the presence of supplemental sugars is an inherent property of all S. salivarius strains.

[0425] Methods: Preparation of solid culture medium - CAB agar plates with or without either 2.5% w / w raffinose or 0.5% w / w galactose were prepared by adding 0.5% (w / v) calcium carbonate to solid CAB agar in a bottle, then melted by autoclaving for 10 minutes at 110° C. After cooling, filter-sterilized sugar solution or distilled water was added and mixed, then 20 ml was pipetted into a Petri dish.

[0426] S. salivarius producer strain suspensions were prepared and approximately 6 colonies of each S. salivarius strain were added to separate tubes containing 1 ml of THB and mixed thoroughly.

[0427] Deferred antagonism assay - 100 μl of S. salivarius producer strain suspension was dispensed as a drop onto a CABCa plate supplemented with or without raffinose or galactose in a vertical line in the center of the test plate. The suspension was then spread as a 1.2 cm streak in the center of the plate using a cut-down plastic spreader. Plates were incubated at 37° C., 5% CO2 in air for 18 hours.

[0428] After incubation, bacterial growth was removed from the agar plates using a sterile cotton swab, and the pH of the production strain streaks was then adjusted by placing 1 cm wide filter paper strips soaked in 0.5 M sodium carbonate (pH 11) on the agar plates to buffer the acid and adjust the pH to approximately pH 6.5-7.5.

[0429] Plates were surface sterilized with chloroform vapor for 30 minutes and then air dried for 30 minutes.

[0430] Bacterial indicator suspensions were prepared by adding 3-9 colonies (depending on size) of each strain to separate tubes containing 3 ml of THB. These suspensions were then swabbed across the agar plates perpendicular to the producer strain streak, and the agar plates were then incubated for a further 18 hours at 37°C, 5% CO2 in air.

[0431] Results: S. salivarius strains were assayed, including strains obtained from ATCC. Almost all but one strain showed no inhibitory activity against pathogens involved in ENT and skin infections when grown in the presence of 2.5% w / w raffinose (Figure 32) or 0.5% w / w galactose (Figure 33). The exception was S. salivarius ATCC7073, the only other strain besides K12 and M18 that had antibacterial activity when supplemented with 2.5% w / w raffinose.

[0432] Conclusion: The induction of inhibitory activity in S. salivarius by raffinose or galactose is not an inherent property of S. salivarius. Supplementation of other S. salivarius strains with galactose does not induce the same inhibitory effect observed for K12 and M18. Except for S. salivarius strain ATCC7073, supplementation of other S. salivarius strains with raffinose does not induce the same inhibitory effect observed for K12 and M18.

[0433] Example 13: Induction of antibacterial activity in K12 or M18 against Gram-negative pathogens, including those involved in causing halitosis The aim of this experiment was to investigate the induction of inhibitory activity by galactose or raffinose in K12 or M18 against the Gram-negative bacteria F. nucleatum, P. gingivalis, and P. intermedia, which are not inhibited by K12 or M18 alone and can cause halitosis and other dental infections.

[0434] Methods: CABCa agar plates with or without 2.5% w / w raffinose, 0.5% w / w galactose, or a combination of both sugars were prepared by adding 0.5% (w / v) calcium carbonate to solid CAB agar in a bottle, then melted by autoclaving for 10 min at 110° C. After cooling, filter-sterilized sugar solution or distilled water was added and mixed, then 20 ml was pipetted into a Petri dish.

[0435] Deferred antagonism assays were performed using K12 or M18 stock suspended in THB. 6 A 1.2 cm streak containing cfu was spread in the center of a CABCa control or CABCa galactose-supplemented test plate. After 18 h of growth at 37°C and 5% CO2 in air, the bacterial growth was removed, the pH of the agar in the initial streak was measured and adjusted to pH 6.5-7.5 using 0.5 M sodium carbonate pH 11, and then the agar surface was sterilized using chloroform vapor. Bacterial test strains were suspended in THB and swabbed across the plate perpendicular to the initial streak, and the plates were then incubated for 4 days at 37°C in an oxygen-free jar containing an anaeroGen sachet. The zone of inhibition for each test strain was then measured (mm).

[0436] Results: K12 was found to inhibit various strains of gram-negative bacteria that cause halitosis when supplemented with either 2.5% w / w raffinose or a combination of 2.5% w / w raffinose and 0.5% w / w galactose (Figure 34). A solution of 0.5% w / w galactose alone did not induce antibacterial activity in K12 against any of the halitosis-related bacterial species tested.

[0437] M18 was also found to inhibit several strains of bacteria that cause halitosis when supplemented with either 2.5% w / w raffinose or a combination of 2.5% w / w raffinose and 0.5% w / w galactose (Figure 35).

[0438] Example 14: Inhibitory effect of supplementing K12 and M18 freeze-dried raw powders with galactose and raffinose Objective: To determine the inhibitory activity of K12 and / or M18 raw powder in the presence of raffinose and / or galactose

[0439] Methods: The amounts of raffinose and galactose used were calculated based on the size of the area of ​​the production strain streaked to allow for absorption of the sugars into the agar (calculated to be 4.25 g). Based on this, amounts of galactose in the amount of 0.021 g (i.e., 0.5% w / w galactose of 4.25 g agar volume), raffinose in the amount of 0.11 g (i.e., 2.5% w / w), and a combination of galactose (0.5% w / w) and raffinose (2.5% w / w) (total of 0.13 g) were weighed into sterile containers. K12 and / or M18 raw materials were suspended and diluted with 1-2 x 10 6The suspensions were diluted with sterile distilled water to a concentration of cfu / 100 μl. 100 μl of K12 only, M18 only, and K12+M18 suspensions were then spread as a 1 cm streak in the center of a CABCa agar plate using a sterile spreader. In addition, 100 μl of K12 only, M18 only, and K12+M18 suspensions were mixed with the weighed galactose and / or raffinose powders above using a sterile stir bar. The total volume of each mixture was pipetted with a wide-bore tip into the center of a CABCa agar plate and spread as a 1 cm streak in the center using a sterile spreader. All plates were incubated lid-up at 37° C., 5% CO2 in air for 18 hours. The bacterial growth was then removed using a microscope slide, the pH of the agar in the producer streak area was measured, and the plate was surface sterilized with chloroform vapor after adjusting to pH 6.5-7.5 using 0.5 M sodium carbonate (pH 11). The indicator bacterial test strain was suspended in 3 ml of sterile THB and swabbed across the plate perpendicular to the producer streak area. The plate was incubated for a further 18-24 hours at 37°C, 5% CO2 in air. The zone of inhibition for each test organism was then measured in mm using a ruler. The amount of raffinose and galactose used was calculated based on the size of the producer streak area to allow for absorption into the agar (calculated to be 4.25 g).

[0440] Results: Both galactose (0.5% w / w) and raffinose (2.5% w / w) and their combination were found to induce inhibitory activity in the lyophilized raw powders of K12 (Figure 36) or M18 (Figure 37) or the combination of K12 and M18 (Figure 38).

[0441] Example 15: Effect of raffinose and galactose on the induction of inhibitory activity in a commercial powder formulation (Daily Defense Junior) containing S. salivarius K12 Objective: To evaluate the efficacy and safety of the commercially available powder formulation Daily Defense Junior (composition: S. salivarius K12 (1.25 × 10 9To compare the inhibitory effect of S. salivarius K12 in 100% ethanol (cfu / 0.8g), isomalt, maltodextrin, vanilla flavoring, with the added supplemental sugars raffinose and galactose.

[0442] Methods: The test formulations were prepared as follows. 1. Control: A commercially available powder formulation containing S. salivarius K12 (Daily Defense Junior) 2. Galactose 0.5% w / w was added to the commercial powder and mixed thoroughly to obtain a homogenous mixture. 3. Raffinose 2.5% w / w was added to the commercial powder and mixed thoroughly to obtain a homogenous mixture.

[0443] The following method was used to measure the inhibitory effect of S. salivarius K12 in the context of powder formulations. Exactly 40 mL of molten CAB agar (CABCa) at 50° C. containing 0.5% calcium carbonate was poured onto an agar plate (120×120 mm). After the agar was cooled and solidified, it was split down the middle and one half of the agar gel was removed. The other half was left on the plate as blank agar marked as side “A”. Approximately 0.8 g of each combination (DDJ powder with (1) galactose, (2) raffinose, or (3) a combination of raffinose and galactose) was mixed with 1 mL of sterile distilled water and vortexed to generate a homogenous suspension. 100 μl of the suspension was set aside to spread on the surface of the agar (producer strain B side). The remaining suspension was then mixed with 20 mL of molten CABCa agar, and the mixture was poured onto the empty half of the agar plate to form the producer strain “B” side. 100 μL of the reserved suspension was spread on the surface of side B and the plates were incubated for 18 hours at 37°C and 5% CO2 in air. After incubation, the bacterial growth on the producer B side was removed. The pH of the producer side was measured and adjusted to 6.5-7.5 by immersing the agar in a solution of 0.5 M sodium carbonate pH 11. The plate surface was sterilized with chloroform vapor. The bacterial indicator strains were suspended in THB and streaked across the agar plate from left (blank agar A side) to right (producer B side) using a sterile cotton swab. The plates were again incubated for 18 hours at 37°C and 5% CO2 in air. The zone of inhibition for each indicator strain was measured (in mm) using a ruler. The results were also read using the naked eye to determine the presence of small colonies or complete zones of inhibition.

[0444] Results: Figure 39 shows that the inhibitory activity of S. salivarius K12 was improved in powders containing raffinose (2.5% w / w), galactose (0.5% w / w), or a combination thereof (raffinose 2.5% w / w + galactose 0.5% w / w) compared to a commercial powder containing K12 alone (control).

[0445] Example 16: Properties of formulations from US Patent Application Publication No. 20190343899(A1) Objective: To determine the manufacturing conditions of the prior art formulation from US Patent Application Publication No. 20190343899.

[0446] Method: The formulation was prepared according to the instructions in Example 1 of Prior Art D1. Briefly, the liquid ingredients were mixed together, slowly added to the solid ingredients, and heated to about 100° C. on a hot plate until melted. The mixture was then allowed to cool to about 60° C. until completely solidified.

[0447] Results: The formulation described in Example 1 of US20190343899 was prepared by melting the ingredients to prepare a formulation with the consistency of hard candy or taffy. High temperatures (approximately 100°C) were required to melt the ingredients. Due to the high heat, S. salivarius could not be added to the formulation because temperatures above 50°C are harmful to the probiotics. For this reason, it was not possible to add probiotics to the formulation at the melting stage (above approximately 60°C) without complete loss, especially heat-sensitive probiotics such as S. salivarius.

[0448] During cooling, the consistency of the formulation, which allowed the incorporation of the probiotics into the formulation with sufficient homogeneity, was only maintained at temperatures above 60° C. This temperature was still too high to add the probiotics without causing cell death, which occurs from about 50° C. and above.

[0449] Example 17: Comparative Example - Formulation from WO2017129639(A1) Methods: A powdered infant nutritional product (infant formula) with a composition similar to that of Example 1 of WO2017129639 was purchased (Similac 360 Total Care (Abbott Global). This product contains vitamins, minerals, lactose, five human oligosaccharides and whole milk powder.

[0450] To determine the effect of supplemental sugars on the induction of inhibitory activity in S. salivarius K12 in formula, S. salivarius K12 and supplemental sugars were added to infant formula as follows. 1. Control: S. salivarius K12 was added to infant formula powder and mixed thoroughly to obtain approximately 1.25 × 10 9 A homogenous mixture containing cfu / g of S. salivarius K12 was obtained. 2. Galactose 0.5% w / w: S. salivarius K12 was added to infant formula powder and 0.5% w / w galactose and mixed thoroughly to obtain approximately 1.25 x 10 9 A homogenous mixture containing cfu / g of S. salivarius K12 was obtained. 3. Raffinose 2.5% w / w: S. salivarius K12 was added to infant formula powder and 2.5% w / w raffinose and mixed thoroughly to obtain approximately 1.25×10 9 A homogenous mixture containing cfu / g of S. salivarius K12 was obtained.

[0451] The induction of inhibitory activity was measured in the same manner as in Example 15.

[0452] Results: Surprisingly, the addition of galactose or raffinose to the control formulation of Example 17 showed reduced inhibitory activity compared to the control (Table 10).

[0453] Example 18: Comparison of antibacterial properties of powder formulations Methods: The antibacterial activity of several compositions was tested as follows. 1. Control formulation of Example 17; 2. Whole milk powder: S. salivarius K12 was added to whole milk powder (Anchor Blue™ Milk powder, Anchor, New Zealand) and mixed thoroughly to obtain approximately 1.25×10 9 A homogenous mixture containing cfu / g of S. salivarius K12 was obtained.

[0454] The induction of inhibitory activity of the powder formulation was measured in the same manner as in Example 15.

[0455] Results: Surprisingly, these two formulations induced no or less significant inhibitory activity compared to the three formulations of Example 15 containing the commercially available S. salivarius K12 Daily Defense Junior powder product supplemented with raffinose, galactose, and their combination (Figure 40). [Table 13] A=Infant formula 2 of Example 17 containing 0.5% w / w galactose, B=Infant formula 3 of Example 17 containing 2.5% w / w raffinose, - symbol indicates reduced activity compared to control formulation 1 of Example 17.

[0456] Example 19: Upregulation of beneficial genes Objective: To investigate the upregulation of genes encoding antibacterial molecules and any other beneficial genes in S. salivarius K12 that may lead to improved antibacterial efficacy in the presence of galactose or raffinose.

[0457] Methods: CABCa agar plates with or without 2.5% w / w raffinose, 0.5% w / w galactose, combinations of 2.5% w / w raffinose with 0.5% w / w galactose, 0.5% w / w glucose, or 2.5% w / w glucose were prepared by adding 0.5% (w / v) calcium carbonate to solid CAB agar in a bottle, then melted by autoclaving for 10 min at 110° C. After cooling, filter-sterilized sugar solution or distilled water was added and mixed, then 20 ml was pipetted into a Petri dish.

[0458] Approximately 1×10 8 A stock suspension of cfu / ml of S. salivarius K12 was prepared in PBS. 100 μl of this suspension was spread evenly on the control and test agar plates. The plates were incubated at 37° C., 5% CO2 for approximately 19 hours.

[0459] Bacterial growth from each plate was collected using a sterile cotton swab and resuspended in 1 ml of PBS in a screw-cap tube. The bacterial cells were then pelleted by centrifugation at 13000 rpm for 1 min at 4°C. The supernatant was removed and the cell pellet was resuspended in 1 ml of TRIzol reagent. The bacterial cell suspension was transferred to a 2 ml screw-cap tube containing 0.1 mm zirconia / silicon beads. The tubes were then vortexed at maximum speed for 5 min to bead beat and lyse the bacterial cells. The tubes were placed on ice for 1 min and then re-bead beat again on the vortex for 5 min. The tubes were then frozen at -20°C.

[0460] The lysed bacterial suspension was thawed and 0.2 ml of chloroform was added to each tube. The tubes were incubated for 2-3 minutes, kept on ice and also mixed manually by inverting the tubes frequently. The beads were then allowed to settle and then the upper liquid suspension was transferred to the phase formation tube. The tubes were incubated on ice for 5 minutes with manual mixing by inverting the tubes frequently. The tubes were then centrifuged at 12000 rpm, 4°C for 15 minutes, separating into a lower (red) phenol-chloroform phase, an interphase, and an upper (clear) aqueous phase.

[0461] 560 μl of the clear upper aqueous phase was transferred to a new Eppendorf tube and then frozen again at -20°C.

[0462] The lab bench and other equipment used for RNA extraction were treated with RNaseZap to remove any possible RNases.

[0463] RNA was extracted from TRIzol thawed suspensions using a Thermofisher PureLink RNA mini kit according to the manufacturer's instructions for extracting RNA from TRIzol samples as follows.

[0464] 600 μl of 70% ethanol was added to each tube of thawed lysed bacterial suspension. Approximately 600 μL of sample was transferred to a spin cartridge in a collection tube.

[0465] Centrifuge at 12000xg for 15 seconds at room temperature. The flow-through was discarded and the spin cartridge was reinserted into the same collection tube. A final 600μl of sample was added and the spin column was centrifuged again. The flow-through was discarded and the spin cartridge was reinserted into the same collection tube.

[0466] 700 μL of Wash Buffer I was added to the spin cartridge. Centrifuge at 12000×g for 15 seconds at room temperature. The flow-through and collection tube were discarded. The spin cartridge was inserted into a new collection tube.

[0467] 500 μL of Wash Buffer II was added to the spin cartridge.

[0468] Centrifuge at 12000 x g for 15 seconds at room temperature. The flow-through was discarded and the spin cartridge was reinserted into the same collection tube. Another 500 μL of Wash Buffer II was added to the spin cartridge and centrifuged at 12000 x g for 1 minute at room temperature to dry the membrane. The collection tube was discarded and the spin cartridge was inserted into a new Eppendorf tube.

[0469] 100 μL of RNase-free water was added to the center of the spin cartridge. It was incubated at room temperature for 1 minute. The spin cartridge was centrifuged in an Eppendorf tube at room temperature for 2 minutes at ≧12000×g to elute the RNA.

[0470] The concentration of the eluted RNA was measured using a Nanodrop.

[0471] The extracted RNA was then DNase treated to remove any contaminating DNA using the Thermo Fisher TURBO-DNA Free kit as follows.

[0472] 50 μl reactions containing less than 50 μg of RNA were prepared. The same concentration of RNA was added to each sample reaction to standardize the amount of RNA across all samples, calculated from the Nanodrop RNA concentration. The reactions in Eppendorf tubes were mixed with the required amount of RNA, 5 μl Turbo DNase buffer, 2 μl Turbo DNase, and then the volume was made up to 50 μl with nuclease-free water while keeping all reagents on ice. A no-RNA control sample was prepared to use as a blank when measuring the concentration of DNase-treated RNA samples using the Nanodrop. The Eppendorf tubes were incubated at 37°C for 30 minutes. The reactions were then inactivated by the addition of 10 μl of DNase inactivation reagent and mixed thoroughly. The Eppendorf tubes were incubated at room temperature for 5 minutes, inverting 2-3 times to mix the reagents during incubation. The samples were centrifuged at 10,000 × g for 1.5 minutes. The supernatant was then transferred to a new Eppendorf tube, which was also centrifuged, and the supernatant was again transferred to a new Eppendorf tube. The concentration of the DNase-treated RNA samples was then measured using a Nanodrop, using a non-RNA control sample as the blank.

[0473] The DNase treated RNA samples were then checked for DNA contamination, which generated a DNA band of approximately 500 pb in size by PCR using SalB primers.

[0474] PCR reactions were prepared in 0.2 ml PCR tubes for each DNase-treated RNA sample, a positive control sample of DNA extracted from S. salivarius K12, and a negative control sample containing only nuclease-free water. 25 μl reactions were prepared by mixing 12.5 μl of GoTaq G2 Hot start green Mastermix, 1 μl of SalB forward primer, 1 μl of SalB reverse primer, 1 μl of either RNA or DNA sample, and nuclease-free water to a volume of 25 μl.

[0475] PCR amplification consisted of an initial denaturation at 94° C. for 15 min, followed by 30 cycles of the following: denaturation at 95° C. for 30 s; annealing at 40° C. for 30 s; extension at 73° C. for 30 s. After the 30 cycles, 92° C. for an additional 2 min.

[0476] After PCR amplification, 0.5 cm thick 2% agarose / 1x TAE gels containing 1x SYBR safeDNA gel stain and 1.5 mm wide combs were loaded into wells with either 10 μl of each DNase-treated RNA sample or 5 μl of AccuRuler 1 kb DNA RTU ladder to determine band size of any visualized bands.

[0477] No DNA bands were detected in the DNase treated samples, confirming that any DNA contamination had been removed.

[0478] The RNA samples were then converted to cDNA using superscript IV vilo master mix according to the manufacturer's instructions as follows.

[0479] 20 μl reactions were prepared in 0.2 ml PCR tubes accommodating up to 2.5 μg RNA, and the amount of RNA added was normalized so that all samples had the same concentration of RNA in the tube. To each tube, 4 μl of superscript IV vilo master mix was added and the volume brought to 20 μl with nuclease-free water. A duplicate set of reactions was prepared containing the same concentration of RNA for each sample, but with 4 μl of superscript IV vilo No RT control added, and the volume was also brought to 20 μl with nuclease-free water.

[0480] The PCR tubes were then placed in a PCR machine and the following incubations were performed: 25° C. for 10 min (primer annealing), 50° C. for 10 min (to reverse transcribe the RNA), and 85° C. for 5 min (to inactivate the enzyme).

[0481] cDNA samples from K12 sources grown on CABCa control plates and CABCa supplemented with various sugars were then analyzed by qPCR for the levels of gene expression of the following genes: salA, salB, salQ, and ureC.

[0482] 10 μl qPCR reactions were prepared containing 2 μl cDNA, 5 μl SYBR green master mix, 0.5 μl forward primer, 0.5 μl reverse primer, and nuclease-free water to a volume of 10 μl. The dilution of cDNA in each qPCR was optimized for each primer set.

[0483] The qPCR method consisted of the following cycles: hold step: 50°C for 2 min, followed by 95°C for 10 min; PCR step: 95°C for 15 s, followed by 60°C for 1 min; melt curve step: 95°C for 15 s, 60°C for 1 min, followed by 95°C for 15 s.

[0484] To analyze the relative expression levels, -ΔΔCt The relative fold gene expression levels were determined by comparing various sugars with the CABCa plate control using the method. The reference gene used for this analysis was gyrA.

[0485] Results: Both salA and salB genes were upregulated in K12 when either 2.5% w / w raffinose, 0.5% w / w galactose or a combination of both were added compared to the no sugar control (Figures 41 and 42). Interestingly, 0.5% w / w galactose appeared to cause the greatest increase in gene expression of salA (about 12000-fold) and salB (641-fold), followed by 2.5% w / w raffinose (about 5000-fold and 241-fold) and then the combination (about 2400-fold and 160-fold). Upregulation of these genes did not occur to the same extent when the control sugar glucose was added at either 0.5% w / w (about 50-fold and 23-fold) or 2.5% w / w (5.2-fold and 0.8-fold) concentrations.

[0486] Furthermore, salQ was upregulated when either 2.5% w / w raffinose (93-fold), 0.5% w / w galactose (205-fold), or a combination of both (45-fold) was added compared to the no sugar control (Figure 43). However, salQ also appeared to be upregulated when 2.5% w / w glucose (81-fold) was added, indicating that this may be a general sugar effect.

[0487] When either 2.5% w / w raffinose, 0.5% w / w galactose, or a combination of both was added to the medium, there was an upregulation of urease (ureC), with expression increasing by 234-, 224-, and 191-fold, respectively (Figure 44). 0.5% w / w and 2.5% w / w glucose also resulted in a moderate increase in urease expression by 22- and 96-fold, respectively.

[0488] Example 20: Changes in the levels of K12 colonization in the oral cavity of healthy human volunteers Objective: To determine whether the addition of galactose and / or raffinose to a commercial S. salivarius K12 lozenge formulation (Throat Guard Pro, Blis Technologies, New Zealand) alters K12 colonization levels when ingested once daily for 7 days.

[0489] Methods: A double-blind, randomized, controlled colonization pilot study with no crossover is conducted in healthy adult humans to evaluate the colonization efficacy of lozenges containing S. salivarius K12 (approximately 2.5 million cfu / lozenge) without galactose or raffinose (control G1, containing S. salivarius K12, isomalt, tableting aids, and natural flavors, Blis Technologies, New Zealand) and lozenges additionally containing raffinose 2.5% w / w (G2), galactose 0.5% w / w (G3), and a combination of galactose 0.5% w / w and raffinose 2.5% w / w (G4).

[0490] Lozenges G2-G4 were prepared by blending S. salivarius K12, isomalt, tableting aids, and natural flavors with raffinose 2.5% w / w (G2); galactose 0.5% (G3); and galactose 0.5% w / w and raffinose 2.5% w / w (G4). Each blend was then pressed into a tablet press to obtain lozenges. Each of the four groups of lozenges was formulated to contain approximately 2,500,000,000 cfu / lozenge.

[0491] Participants were enrolled if they were healthy, practiced good oral hygiene, were between 18 and 80 years old, were not undergoing antibiotic therapy, had no immunocompromised condition or history of autoimmune disease, and had no allergies or sensitivities to dairy products. According to the inclusion criteria, a total of 20 participants were recruited and divided into four groups. Test group: K12 lozenge (2.5 Bcfu / lozenge) G1: K12 lozenge (control) (n=5) G2: K12 lozenge containing raffinose (2.5% w / w) (n=5) G3: K12 lozenge containing galactose (0.5% w / w) (n=5) G4: K12 lozenge containing raffinose (2.5% w / w) and galactose (0.5% w / w) (n=5)

[0492] Participants were asked to gargle with mouthwash (first night only) and wait one hour before collecting a saliva sample (pre-study sample). Participants were then asked to slowly dissolve one lozenge in their mouth. Participants were then asked to collect additional saliva samples 1 hour, 8 hours, and 24 hours after taking the lozenge. Participants were then asked to take one lozenge per night for six more nights and to collect a final saliva sample 48 hours after the last dose.

[0493] Microbial Sampling and Analysis: During and at the end of the study, tubes containing saliva samples for each time point and each participant were collected and stored in a freezer (-20°C) until analysis.

[0494] The saliva samples were -4 Serial dilutions (multiple resuspensions of 100 μL samples in 900 μL PBS) were used to inoculate Mitis-Salivariu agar plates (Streptococcus salivarius selective medium) using 50 μL of inoculum per plate. Plates were incubated for 24 h at 37 °C and 5% CO2 in air. After incubation, K12 or M18 colonies were differentiated by their inhibitory activity against specific indicator strains I1 (Micrococcus luteus T-18) and I3 (Streptococcus constellatus T-29). Suspensions of indicator strain I1 were made by adding one colony to 3 ml of THB, and I3 suspensions were made by adding four colonies to 3 ml of THB. Indicator strains were swabbed onto blood agar plates (sBaCa) covering the entire surface of the agar. Using toothpicks, S. salivarius-like colonies grown from saliva samples on Mitis salivarius agar were spiked onto pre-seeded indicator lawns of I1 and then I3 and incubated for 24 h at 37°C and 5% CO2 in air. Colonies with zones of inhibition for both I1 and I3 were identified as presumptive positive K12, as they indicate activity of the salA and salB genes.

[0495] result Figure 45 shows that the mean % of S. salivarius K12 (of total S. salivarius) in saliva samples from groups of participants using lozenges containing the supplemented sugars galactose (G2), raffinose (G3), and raffinose and galactose (G4) was higher than the mean % of the control group of S. salivarius K12 alone (control G1). Thus, the presence of supplemented sugars increased the colonization efficiency of S. salivarius K12 in the oral cavity.

[0496] In the supplemented sugar groups, raffinose (G3) showed the greatest increase in colonization compared to pre-test levels for all sample points, followed by galactose (G2) and the combination of raffinose and galactose (G4). The % of S. salivarius K12 in the total S. salivarius population remains higher than in the raffinose group (G3). Levels in the raffinose group remained higher than pre-test even after cessation of lozenge intake, suggesting improved persistence of S. salivarius K12 with supplemented sugar.

[0497] It is not intended that the scope of the present invention be limited to only the above-described examples, as will be appreciated by those skilled in the art, many variations are possible without departing from the scope of the present invention as set forth in the appended claims. [Table 14] [Table 15]

Claims

1. A method for improving the inhibition profile of Streptococcus salivarius, comprising formulating said S. salivarius in a pharmaceutical composition containing an effective amount of a supplementary saccharide, wherein said method does not include a therapeutic method, wherein said Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof, and wherein said supplementary saccharide is galactose or raffinose, or a combination thereof.

2. A method for upregulating one or more genes in Streptococcus salivarius, comprising formulating said S. salivarius in a pharmaceutical composition containing an effective amount of a supplementary saccharide, wherein said Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof, and wherein said supplementary saccharide is galactose or raffinose, or a combination thereof.

3. The method according to claim 2, wherein said upregulated gene encodes a lantibiotic peptide, bacteriocin, or a subunit of an urease protein.

4. a) wherein said lantibiotic peptide is salA, salB, sal9 or a combination thereof, or b) wherein said bacteriocin is salQ, or said subunit of the urease protein is ureC. The method according to claim 3.

5. The method according to any one of claims 2 to 4, wherein at least one of the up-regulated genes comprises or consists of a polynucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 15 to 22, or wherein at least one of the up-regulated genes comprises or consists of a polynucleotide sequence encoding a polypeptide having at least 70% sequence identity to any one of SEQ ID NOs: 23 to 30. **Claim 6** The method according to any one of claims 1 to 5, which improves the inhibition profile of S. salivarius against skin, teeth, oral cavity, mucosa, and / or ENT microorganisms. **Claim 7** The skin, oral cavity, teeth, mucosa, and / or ENT microorganisms are S. aureus (Staphylococcus aureus) species, S. intermedius species, S. saprophyticus (Staphylococcus saprophyticus) species, M. catarrhalis (Catalase coccus) species, H. influenzae species, S. pyogenes (Streptococcus pyogenes) species, P. aeruginosa (Pseudomonas aeruginosa) species, S. mutans, S. pneumoniae (Streptococcus pneumoniae) species, C. acnes (Acne bacteria) species, C. albicans (Candida) species, S. sobrinus species, Corynebacterium species, F. nucleatum species, A. actinomycetemcomitans species, P. gingivalis species, Tannerella forsythia species, Treponema denticola species, P. intermedia species, Prevotella species, A. viscosus species, S. equismillis species, S. sanguis species, S. cohnii species, B. intermedius species, A. parvulum species, E. saburreum species, E. sulci species, P. micra species, S. moorei species, C. minutissimus species, P. propionicas species, S. agalactiae species, S. dysgalactiae species, S. simulans species, S. xylosus species, fungi causing tinea pedis infection, S. salivarius species, species other than K12 or M18, L. lactis (L.the method according to claim 6, selected from the species Lactococcus lactis, S. epidermidis (Staphylococcus epidermidis), S. constellatus, K. pneumoniae, A. baumanii, or any combination of two or more thereof..

8. A pharmaceutical composition for inhibiting skin, dental, oral, mucosal, and / or ENT microorganisms by contacting the microorganisms, the pharmaceutical composition comprising Streptococcus salivarius and an effective amount of a supplemental saccharide, wherein the Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

9. Use of Streptococcus salivarius and a supplemental saccharide in the manufacture of a medicament, wherein the medicament is (a) for the treatment or prevention of a disease or disorder caused by an oral, dental, mucosal, skin, or ENT pathogen, or (b) for inhibiting microorganisms sensitive to S. salivarius producing halitosis, wherein the Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof, and the supplemental saccharide is galactose or raffinose, or a combination thereof.

10. a) The microorganism is selected from Streptococcus or Staphylococcus bacteria selected from the species S. aureus (Staphylococcus aureus), S. saprophyticus (Staphylococcus saprophyticus), S. mutans, S. pyogenes (Streptococcus pyogenes), S. pneumoniae (Streptococcus pneumoniae); the S. salivarius strain is K12, or b) The microorganism is selected from the species S. pyogenes (Streptococcus pyogenes), S. pneumoniae (Streptococcus pneumoniae), S. constellatus, S. mutans, and S. saprophyticus (Staphylococcus saprophyticus); the S. salivarius strain is M18, The pharmaceutical composition according to claim 8.

11. a) The microorganism is selected from Streptococcus or Staphylococcus bacteria selected from the species S. aureus, S. saprophyticus, S. mutans, S. pyogenes, S. pneumoniae, the S. salivarius strain is K12, or b) The microorganism is selected from the species S. pyogenes (Streptococcus pyogenes), S. pneumoniae (Streptococcus pneumoniae), S. constellatus, S. mutans, and S. saprophyticus (Staphylococcus saprophyticus); the S. salivarius strain is M18, The use according to claim 9.

12. A pharmaceutical composition for the treatment or prevention of diseases or disorders caused by oral, dental, mucosal, skin, or ENT pathogens, comprising Streptococcus salivarius and an effective amount of a supplemental saccharide, the Streptococcus salivarius is Streptococcus salivarius M18, Streptococcus salivarius K12, or a combination thereof, A pharmaceutical composition, wherein the supplementary saccharide is galactose or raffinose, or a combination thereof.

13. Use according to claim 9, wherein the disease or disorder is caused by pathogenic Streptococcus or Staphylococcus bacteria.

14. A pharmaceutical composition according to claim 12, wherein the disease or disorder is caused by pathogenic Streptococcus or Staphylococcus bacteria.

15. Use according to claim 9 or 13, wherein the disease or disorder is selected from otitis media, pharyngolaryngitis, dental caries, acute pharyngitis, tonsillitis, pneumonia, COPD, periodontal membrane disease, gingivitis, halitosis, dental caries, sepsis, meningitis, candidiasis (oral candidiasis), vaginitis, body odor, acne, actinomycosis, psoriasis, erythema, cellulitis, impetigo, atopic dermatitis, bacteremia, tinea pedis including athlete's foot, soft tissue infection, erythema, nosocomial infection, erythema, SARS-CoV, influenza A, influenza B, and RSV, or any combination of any two or more thereof.

16. A pharmaceutical composition according to claim 12 or 14, wherein the disease or disorder is selected from otitis media, pharyngolaryngitis, dental caries, acute pharyngitis, tonsillitis, pneumonia, COPD, periodontal membrane disease, gingivitis, halitosis, dental caries, sepsis, meningitis, candidiasis (oral candidiasis), vaginitis, body odor, acne, actinomycosis, psoriasis, erythema, cellulitis, impetigo, atopic dermatitis, bacteremia, tinea pedis including athlete's foot, soft tissue infection, erythema, nosocomial infection, erythema, SARS-CoV, influenza A, influenza B, and RSV, or any combination of any two or more thereof.

17. The pharmaceutical composition or medicament is a) containing about 0.1 to about 20% by weight of each S. salivarius, and / or b) containing about 1×10³ to about 1×10¹³ cfu / g of each S. salivarius. The use according to any one of claims 9, 11, 13 or 15, or the method according to any one of claims 1 to 7.

18. The pharmaceutical composition is a) about 0.1 to about 20% by weight of each S. thermophilus, and / or The pharmaceutical composition according to any one of claims 8, 10, 12, 14 or 16.

19. The pharmaceutical composition or medicament contains about 0.1 to about 20% by weight of each supplementary saccharide, the use according to any one of claims 9, 11, 13, 15 or 17, or the method according to any one of claims 1 to 7.

20. The pharmaceutical composition contains about 0.1 to about 20% by weight of each supplementary saccharide, the pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16 or 18.

21. The pharmaceutical composition or medicament contains galactose in an amount of 0.1 to 1% by weight and raffinose in an amount of 0.5 to 5% by weight, the use according to any one of claims 9, 11, 13, 15, 17 or 19, or the method according to any one of claims 1 to 7.

22. The pharmaceutical composition contains galactose in an amount of 0.1 to 1% by weight and raffinose in an amount of 0.5 to 5% by weight, the pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16, 18 or 20.

23. The pharmaceutical composition or medicament contains S. thermophilus which is Streptococcus salivarius K12, and the supplementary saccharide which is raffinose in an amount of 1 to 4% by weight, the use according to any one of claims 9, 11, 13, 15, 17, 19 or 21, or the method according to any one of claims 1 to 7.

24. The pharmaceutical composition contains S. thermophilus which is Streptococcus salivarius K12, and the supplementary saccharide which is raffinose in an amount of 1 to 4% by weight, the pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16, 18, 20 or 22. **Claim 25**: The use according to any one of claims 9, 11, 13, 15, 17, 19, 21, or 23, or the method according to any one of claims 1 to 7, wherein the pharmaceutical composition or the medicament further comprises one or more of a carrier; a tabletting aid comprising a binder or a lubricant; and a flavoring agent. **Claim 26**: The pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16, 18, 20, 22, or 24, wherein the pharmaceutical composition further comprises one or more of a carrier; a tabletting aid comprising a binder or a lubricant; and a flavoring agent. **Claim 27**: The use according to any one of claims 9, 11, 13, 15, 17, 19, 21, 23, or 25, or the method according to any one of claims 1 to 7, wherein the pharmaceutical composition or the medicament is formulated for oral, dental, nasal, mucosal, topical, or pulmonary administration. **Claim 28**: The pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, wherein the pharmaceutical composition is formulated for oral, dental, nasal, mucosal, topical, or pulmonary administration. **Claim 29**: The use according to any one of claims 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, or the method according to any one of claims 1 to 7, wherein the pharmaceutical composition or the medicament is formulated as a sustained-release composition. **Claim 30**: The pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28, wherein the pharmaceutical composition is formulated as a sustained-release composition. **Claim 31** The use according to any one of claims 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, or the method according to any one of claims 1 to 7, wherein the pharmaceutical composition or the medicine is a powder, a lozenge, a nasal spray, a nasal gel, a nasal drop, an oral drop, an oral gel, an oral spray, an inhalable, topical composition, a chewable, a melt, a film, a gummy, a toothpaste, a toothpaste gel, a varnish, a mousse, a mouthwash, a food (e.g., yogurt), a cream, a gel, a spray, a deodorant, a beauty liquid, a lotion, a balm, a moisturizer, a pessary, or a suppository. **Claim 32** A pharmaceutical composition according to any one of claims 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, wherein the pharmaceutical composition is a powder, a lozenge, a nasal spray, a nasal gel, a nasal drop, an oral drop, an oral gel, an oral spray, an inhalable, topical composition, a chewable, a melt, a film, a gummy, a toothpaste, a toothpaste gel, a varnish, a mousse, a mouthwash, a food (e.g., yogurt), a cream, a gel, a spray, a deodorant, a beauty liquid, a lotion, a balm, a moisturizer, a pessary, or a suppository.

Images