Novel streptococcus salivarius strains and oral compositions comprising same

Abstract

The present invention relates to a new S. salivarius strain (S. salivarius G7, KCCM13161P) having antibacterial, antifungal, anti-inflammatory activity and caries inhibiting activity, and a composition for prevention or improvement of oral diseases, oral probiotic comprising the same. Specifically, the new S. salivarius strain (S. salivarius G7, KCCM13161P) can prevent, treat and improve periodontal disease by stronger antibacterial activity and anti-inflammatory activity against a strain inducing periodontal disease; and prevent, treat and improve the occurrence of dental caries by stronger antibacterial activity and biofilm generation inhibiting ability against a strain inducing dental caries; and by inhibiting halitosis occurring based on oral diseases, thereby being able to contribute to improvement of oral health.

Description

Technical Field

[0001] This invention relates to novel Streptococcus salivans strains with antibacterial, antifungal, anti-inflammatory, and caries-inhibiting activities, as well as oral compositions containing the same. Background Technology

[0002] Infectious diseases of the oral cavity can be divided into gingival and dental diseases, and both are related to a biofilm formed by a variety of bacteria. In healthy individuals, the biofilm is formed by a balance regulated by a high ratio of symbiotic bacteria. However, when this balance is disrupted, and certain bacteria proliferate, oral diseases can result.

[0003] It is known that *Actinomyces israelii* and *Actinomyces naeslundii* are representative pathogens of gingivitis, while *Streptococcus mutans* and *Streptococcus sobrinus* are cariogenic bacteria (Howell A. et al., 1962; Loesche WJ. et al., 1978). These bacteria, due to their acid-producing and acid-resistant characteristics, can induce dental caries (Conrads G. et al., 2014). Furthermore, *Enterococcus faecalis* is the dominant species of human enterococci, and based on its antibiotic resistance, it is involved in oral diseases such as root canal infection, periodontitis, and peri-implantitis (Dahlen G. et al., 1993; Rams TE. et al., 2013).

[0004] On the other hand, some strains can be utilized as probiotics for oral use (Li X., 2021; Hale JDF., et al., 2022). These bacteria suppress surrounding bacteria by producing bacteriocins during the competition for habitat. *Streptococcus salivarius* also produces several lantibiotics similar to bacteriocins, such as salivaricin A, salivaricin B, salivaricin 9, and salivaricin G3216-18 (Wescombe PA., et al., 2006). Streptococcus salivarius is a Gram-positive facultative anaerobic bacterium that lives on the surface of human mucous membranes as a symbiotic bacterium (Aas JA., et al., 2005). It plays an important ecological role in forming a barrier against pathogens and reducing adhesion and colony formation (Wescombe PA., et al., 2006). Therefore, the possibility of using it as a probiotic for oral use is constantly being raised.

[0005] Furthermore, recent research papers have shown that harmful bacteria in the oral cavity have adverse effects on systemic diseases (lupus, systemic sclerosis, inflammatory bowel disease, dementia, etc.). Moreover, there is a growing consensus that maintaining the balance of oral microbiota not only affects oral health but also overall health.

[0006] It is understood that most systemic diseases occur due to the disruption of the balance of the normal flora in the human body or the invasion and proliferation of external microorganisms. Therefore, compared with the use of antibiotics that inhibit microbial growth, the use of oral probiotics that can maintain the microbial ecosystem while also having antibacterial effects can help promote public health.

[0007] Therefore, there is an urgent need to develop oral probiotics with broad antibacterial activity.

[0008] Existing technical documents

[0009] Patent documents

[0010] (Patent Document 0001) Korean Patent Publication No. 10-2022-0139280, Oral Composition Containing a Novel Lactobacillus Plantarum Strain, published on October 14, 2022.

[0011] (Patent Document 0002) Korean Patent No. 10-2198552, a composition containing Lactobacillus fermentum OK strain derived from human oral cavity for improving, preventing or treating halitosis or tooth decay, authorized on December 29, 2020.

[0012] (Patent Document 0003) Korean Patent Publication No. 10-2023-0017372, Composition containing superoxide dismutase and / or Bacillus spores and its use in improving oral health, published on February 3, 2023.

[0013] (Patent Document 0004) Korean Patent No. 10-2198553, a composition containing Lactobacillus gasseri HHuMIN D of human oral cavity for improving, preventing or treating halitosis or tooth decay, was granted on December 29, 2020.

[0014] Non-patent literature

[0015] (Non-patent document 0001) Howell A, Stephan RM, Paul F. Prevalence of Actin-omycesisraelii, A. naeslundii, Bacterionema matruchotii, and Candida albicans inselected areas of the oral cavity and saliva. J Dent Res 1962; 41: 1050-1059.

[0016] (Non-patent literature 0002) Loesche WJ, Syed SA. Bacteriology of human experimental gingivitis: effect of plaque and gingivitis score. Infect Immun 1978; 21:830-839.

[0017] (Non-patent document 0003) Conrads G, de Soet JJ, Song L, Henne K, Sztajer H, Wagner-Dobler I, et al., Comparing the cariogenic species Streptococcus sobrinus and S.mutans on whole genome level. J Oral Microbiol 2014;6:26189.

[0018] (Non-patent literature 0004) Dahlen G. Role of suspected periodontopathogens in microbiological monitoring of periodontitis. Adv Dent Res 1993; 7:163-174.

[0019] (Non-patent document 0005) Rams TE, Feik D, Mortensen JE, Degener JE, Winkelhoff AJ. Antibiotic susceptibility of periodontal Enterococcus faecalis. JPeriodontol 2013;84:1026-1033.

[0020] (Non-patent document 0006) Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 2005; 43:5721-5732.

[0021] (Non-patent document 0007) Wescombe PA, Burton JP, Cadieux PA, Klesse NA, Hyink O, Heng NC, et al. Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek 2006; 90: 269-280.

[0022] (Non-patent document 0008) Li

[0023] (Non-patent document 0009) Hale JDF, Jain R, Wescombe PA, Burton JP, Simon RR, TaggJR. Safety assessment of Streptococcus salivarius M18 a probiotic for oral health. Benef Microbes 2022;13:47-60.

[0024] (Non-Patent Literature 0010) Wescombe PA, Upton M, Renault P, Wirawan RE, Power D, Burton JP, Chilcott CN, Tagg JR. Salivaricin 9, a new lantibiotic produced by Streptococcus salivarius. Microbiology 2011; 157:1290-1299. Summary of the Invention

[0025] The problem the invention aims to solve

[0026] The object of this invention is to provide a novel strain of Streptococcus salivarius with high antibacterial, antifungal and anti-inflammatory effects, as well as an oral composition containing the strain.

[0027] means for solving problems

[0028] To address the aforementioned problems, this invention provides a novel Streptococcus salivarius (S. salivarius G7, KCCM13161P) strain with antibacterial, antifungal, and anti-inflammatory activities.

[0029] The antibacterial activity includes antibacterial activity against the following strains: *Aggregatibacter actinomycetemcomitans*, *Actinomyces israelii*, *Actinomyces naeslundii*, *Enterococcus faecalis*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Prevotella intermedia*, *Bacteroides intermedius*, *Prevotellanigrescens*, *Streptococcus mutans*, *Streptococcus sobrinus*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Actinomyces viscosus*, and *Escherichia coli*. It is one or more of the following bacteria, but not limited to: coli and Staphylococcus aureus.

[0030] In this invention, *Aggregatibacter actinomycetemcomitans* is one of the representative bacteria inducing gingivitis, a Gram-negative, facultative anaerobic, non-motile bacterium associated with localized aggressive periodontitis. In rare cases, *A. actinomycetemcomitans* causes non-oral infections (such as endocarditis) and acts as an exotoxin, producing leukotoxin (LT) and cytolethal distending toxin (Cdt). The exotoxin LT destroys cells by forming perforations in the protoplasmic membranes of monocytes, neutrophils, and a subset of lymphocytes, thereby suppressing the immune system. Furthermore, Cdt is a DNA-degrading enzyme that inhibits cell division in various cell types, including epithelial cells, gingival fibroblasts, and lymphocytes, and induces apoptosis in a subset of cells where cell division is inhibited. As an endotoxin, it can secrete lipopolysaccharide (LPS), thereby inducing the production of pro-inflammatory cytokines (IL-1, TNF) in macrophages and inducing bone resorption and collagen destruction.

[0031] In this invention, *Actinomyces israelii* is commensal in the vagina, colon, and oral cavity of normal individuals, and can induce gingivitis as an opportunistic pathogen that can cause infectious diseases when an individual's immunity is low. Furthermore, actinomycosis is most commonly caused by *Actinomyces israelii* and can be treated with antibiotics.

[0032] In this invention, *Actinomyces naeslundii*, a Gram-positive rod-shaped bacterium found in the oral cavity of women and humans with bacterial vaginosis, is associated with various periodontal diseases, including tooth decay, and particularly predisposes to gingivitis. Furthermore, *A. naeslundii* is also a cause of actinomycosis, forming edematous and purulent abscesses, and may also be accompanied by tissue fibrosis. *A. naeslundii* commonly infects the oral cavity, face, and neck, but in rare cases can also cause infections of the chest, abdomen, pelvis, and central nervous system.

[0033] In this invention, *Enterococcus faecalis*, a Gram-positive commensal bacterium residing in the gastrointestinal tract of humans and other mammals, is one of the representative bacteria causing periapical inflammation. It is known to be found in teeth that have undergone root canal treatment, typically with a carrier rate ranging from 30% to 90%. For teeth that have undergone root canal treatment and subsequently become infected, the likelihood of carrying *E. faecalis* is approximately nine times higher than during the initial infection. In rare cases, it is also known to induce endocarditis, sepsis, urinary tract infections (UTIs), meningitis, and other infections in humans. Furthermore, it exhibits multidrug resistance (MDR), showing resistance to widely used antibiotics such as aminoglycosides, aztreonam, cephalosporins, clindamycin, semi-synthetic penicillins like nafcillin and oxacillin, and trimethoprim / sulfamethoxazole.

[0034] In this invention, *Fusobacterium nucleatum*, a Gram-negative bacterium symbiotic with the human oral cavity, is one of the representative bacteria causing periodontitis. Its ability to aggregate with other bacterial species in the oral cavity makes it a major factor in the formation of periodontal plaque. Furthermore, many studies have shown its association with premature birth and colorectal cancer.

[0035] In this invention, *Porphyromonas gingivalis*, a non-motile, Gram-negative, rod-shaped, anaerobic, pathogenic bacterium, is one of the representative bacteria that induce periodontitis. *P. gingivalis* has been found in the oral cavity, upper digestive tract, respiratory tract, and colon of patients with periodontitis, and is also reportedly associated with Alzheimer's disease and rheumatoid arthritis.

[0036] In this invention, *Prevotella intermedia* (Bacteroides intermedius) is a Gram-negative obligate anaerobic pathogen involved in periodontal infections, including gingivitis and periodontitis, and is commonly found in acute necrotizing ulcerative gingivitis. Because *Prevotella intermedia* uses steroid hormones as growth factors, it is more common in pregnant women and can also be isolated from women with bacterial vaginosis.

[0037] In this invention, *Prevotella nigrescens* is a Gram-negative bacterium and is part of the normal oral flora. It is known to be one of the bacteria that induce opportunistic periodontitis, and is associated not only with periodontal disease but also with nasopharyngeal and intra-abdominal infections and carotid atherosclerosis.

[0038] In this invention, "Actinomyces viscosus" is a Gram-positive facultative anaerobic bacterium that inhabits the oral cavity of 70% of adults. *A. viscosus* causes periodontal disease in animals and can cause not only dental calculus and root caries in humans, but also endocarditis and, in rare cases, lung infections.

[0039] In this invention, "Streptococcus mutans," also known as cariogenic bacteria, is a major pathogen of tooth decay, along with lactobacilli. Streptococcus mutans induces tooth decay, leading to tooth discoloration and rot.

[0040] In this invention, *Streptococcus sobrinus*, a Gram-positive, non-motile anaerobic bacterium, combines with *Streptococcus mutans* to promote dental caries. In particular, *Streptococcus sobrinus* has a closer relationship with dental caries prevalence than *Streptococcus mutans*.

[0041] In this invention, *Streptococcus pneumoniae* is a type of streptococcus that exhibits Gram-positive and hemolytic activity. It does not cause symptoms in healthy individuals, but in children and the elderly with weakened immune systems, it is a major cause of pneumonia and meningitis, and can induce upper respiratory tract infections.

[0042] In this invention, *Streptococcus pyogenes*, as a pyogenic streptococcus, is a Gram-positive aerobic bacterium. *S. pyogenes* parasitizes the mucous membranes of the throat and genitals, rectum, and skin, and can cause various diseases such as streptococcal pharyngitis, rheumatic fever, rheumatic heart disease, and scarlet fever, and can also induce upper respiratory tract diseases.

[0043] In this invention, "Escherichia coli" is a representative Gram-negative pathogenic bacterium. When people eat food containing Escherichia coli or come into contact with infected animals, they will become infected, which can induce abdominal cramps, nausea, vomiting, etc.

[0044] In this invention, *Staphylococcus aureus*, as a representative Gram-positive pathogenic bacterium, is a pathogen that causes purulent diseases and food poisoning. *Staphylococcus aureus* is present in the nose, respiratory system, and skin, and food poisoning caused by *Staphylococcus aureus* is a type of toxin-induced food poisoning resulting from the ingestion of toxins produced by bacteria that proliferate in food.

[0045] In this invention, the antifungal activity may be antifungal activity against Candida albicans, but is not limited thereto.

[0046] In this invention, *Candida albicans*, as an opportunistic pathogenic fungus, is a common member of the human gut microbiota. It is present in 40-60% of the gastrointestinal tract and oral cavity of healthy adults, but transforms into pathogenic strains in immunocompromised individuals, thereby causing candidiasis. Candidiasis is the third or fourth most common pathogenic infection worldwide, and in particular, it can cause not only skin and genital infections but also oral and esophageal infections. Systemic infections can be treated with amphotericin B, echinocandins, or fluconazole.

[0047] The novel *Streptococcus salivarius* strain of this invention (S. salivarius G7, KCCM13161P) exhibits 4 to 8 times higher antibacterial efficacy compared to the conventional *Streptococcus salivarius* K12 and *Streptococcus salivarius* ATCC 7073 (type strain).

[0048] The novel *Streptococcus salivarius* G7 (KCCM13161P) strain of this invention exhibits a higher halitosis-inhibiting effect compared to the conventional *Streptococcus salivarius* K12 and *Streptococcus salivarius* ATCC 7073 (type strain). The *Streptococcus salivarius* G7 (accession number KCCM13161P) culture medium of this invention can significantly inhibit halitosis based on oral diseases by reducing the concentrations of hydrogen sulfide (H2S) and emulsified methyl (CH3SH) in *Porphyromonas gingivalis* culture medium, wherein hydrogen sulfide (H2S) and emulsified methyl (CH3SH) are volatile sulfur compounds that can induce halitosis.

[0049] The present invention provides a composition for the prevention or improvement of oral diseases, wherein the composition comprises one or more of the following: a novel *Streptococcus salivarius* strain (*S. salivarius* G7, KCCM13161P*) with antibacterial, antifungal, and anti-inflammatory activities; its lysate; its concentrate; its dried product; its culture; its extract; its heat-treated culture-dried product; and its fraction.

[0050] The oral disease may be selected from one or more of the following groups, but is not limited to: gingivitis, periodontitis, dental caries, halitosis, sensitive teeth, oral candidiasis, upper respiratory tract disease, and periapical disease.

[0051] The oral diseases can be caused by pathogenic bacteria or fungal infections of the oral cavity or upper respiratory tract. The compositions of the present invention can prevent or improve the oral diseases through antibacterial and anti-inflammatory activities.

[0052] The composition for the prevention or improvement of oral diseases can be commercialized in a manner selected from, but not limited to, toothpaste, mouthwash, chewing gum, confectionery, caramel, jelly, oral spray, oral ointment, oral cleaner, oral rinse and teeth whitening agent.

[0053] This invention provides an oral probiotic composition, wherein the composition comprises a novel *Streptococcus salivarius* strain (*S. salivarius* G7, KCCM13161P) or a culture thereof possessing antibacterial, antifungal, and anti-inflammatory activities. The oral probiotic composition may be selected from, but is not limited to, one or more dosage forms selected from tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, chewing gums, and candies. Examples of carriers, excipients, and diluents that may be included in the probiotics include: lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. When formulating, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidifiers, etc., can be used as diluents or excipients.

[0054] The present invention provides a pharmaceutical composition for the prevention or treatment of oral diseases, wherein the pharmaceutical composition comprises one or more of the following: a novel *Streptococcus salivarius* strain (*S. salivarius* G7, KCCM13161P*) with antibacterial, antifungal, and anti-inflammatory activities; its lysate; its concentrate; its dried product; its culture; its extract; its heat-treated culture-dried product; and its fractions.

[0055] The oral disease may be selected from one or more of the following groups, but is not limited to: gingivitis, periodontitis, dental caries, halitosis, sensitive teeth, oral candidiasis, upper respiratory tract disease, and periapical disease.

[0056] For the total weight of the overall pharmaceutical composition, the amount of the novel *Streptococcus salivarius* (S. salivarius G7, KCCM13161P) strain, its lysate, its concentrate, its dried product, its culture, its extract, its heat-treated culture-dried product, and its fractions added is preferably from 0.001% to 50% by weight, more preferably from 0.001% to 40% by weight, and most preferably from 0.001% to 30% by weight.

[0057] The pharmaceutical composition can be formulated into oral or topical dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, and aerosols using conventional methods. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, which are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to using excipients alone, lubricants such as magnesium stearate and talc can also be used.

[0058] The dosage of the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. However, determining the dosage based on these factors is feasible for those skilled in the art, and the typical dosage range is 1 × 10⁻⁶. 6 CFU / day to approximately 1×10 11 CFU / day. The preferred dosage is 1×10⁻⁶ CFU / day. 8 CFU / day up to 1×10 11 CFU / day, with a more preferred dosage of 1×10⁻⁶ CFU / day. 8 CFU / day up to 1×10 10 CFU / day. For administration, it can be administered once daily or in several divided doses. The stated dosage is not intended to limit the scope of the invention in any way. The pharmaceutical compositions of the present invention can be administered to mammals such as mice, livestock, and humans via various routes.

[0059] This invention provides a health food for the prevention or improvement of oral diseases, wherein the composition comprises one or more of the following: a novel Streptococcus salivarius (S. salivarius G7, KCCM13161P) strain with antibacterial, antifungal, and anti-inflammatory activities; its lysate; its concentrate; its dried product; its culture; its extract; its heat-treated culture-dried product; and its fractions.

[0060] With respect to the health functional food, the amount of the novel *Streptococcus salivarius* (G7, KCCM13161P) strain, its lysate, its concentrate, its dried product, its culture, its extract, its heat-treated culture-dried product, and its fraction added relative to the total weight of the whole pharmaceutical composition is preferably from 0.001% to 50% by weight, more preferably from 0.001% to 30% by weight, and most preferably from 0.001% to 10% by weight.

[0061] The functional health foods include tablets, capsules, powders, pills, liquids, etc., and as foods in which the extracts of the present invention can be added, such as various food categories, beverages, chewing gum, tea and vitamin complexes, health functional foods, etc.

[0062] Invention Effects

[0063] This invention relates to a novel *Streptococcus salivarius* (S. salivarius G7, KCCM13161P) strain with antibacterial, antifungal, and anti-inflammatory activities, and to compositions containing it for the prevention or improvement of oral diseases. Specifically, the novel *Streptococcus salivarius* (S. salivarius G7, KCCM13161P) strain can prevent, treat, and improve periodontal disease through its strong antibacterial and anti-inflammatory activities against strains that induce periodontal disease, and can inhibit halitosis caused by oral diseases, thereby contributing to improved oral health. Attached Figure Description

[0064] Figure 1 The 16S rRNA sequence of Streptococcus salivarius G7 (accession number: KCCM13161P) is shown (SEQ ID NO: 1).

[0065] Figure 2The diagram shows a phylogenetic diagram of *Streptococcus salivarius* G7 (accession number: KCCM13161P), including the following strains: *Streptococcus thermophilus* ATCC 19258; *Streptococcus vestibularis* ATCC 49124; *Streptococcus australis* Al-1; *Streptococcus peroris* JCM10158; *Streptococcus lactarius* MV1; *Streptococcus saliviloxodontae* NUM 6306; and *Streptococcus intermedia* JCL 19287. loxodontisalivarius JCL 19287); Streptococcus equinus JMC 7879; Streptococcus lutetiensis CIP 106849.

[0066] Figure 3 These are the experimental results of the antibacterial activity of Streptococcus salivarius G7 (accession number: KCCM13161P) against gingivitis pathogens (i.e., Actinobacillus actinomycetemcomitans, Actinobacillus israelii, and Actinobacillus naeslundii) and periapical inflammatory pathogens (i.e., Enterococcus faecalis).

[0067] Figure 4 These are the results of an antibacterial experiment on the antimicrobial activity of Streptococcus salivarius G7 (accession number: KCCM13161P) against periodontal pathogens (i.e., Fusobacterium nucleatum, P. gingivalis, P. intermedia, and P. nigrescens).

[0068] Figure 5These are the experimental results of the antibacterial activity of Streptococcus salivarius G7 (accession number: KCCM13161P) against caries-causing bacteria (i.e., Actinomyces viscosus, Streptococcus mutans, and Streptococcus sobrinus) and upper respiratory tract pathogens (i.e., Streptococcus pneumoniae and Streptococcus pyogenes).

[0069] Figure 6 These are the results of antibacterial experiments on Streptococcus salivarius G7 (accession number: KCCM13161P) against representative Gram-negative pathogens (i.e., Escherichia coli) and Gram-positive pathogens (i.e., Staphylococcus aureus).

[0070] Figure 7 These are the experimental results of the antifungal activity of Streptococcus salivarius G7 (accession number: KCCM13161P) against oral candidiasis-inducing fungi (i.e., Candida albicans).

[0071] Figure 8 The virulence of cell lysates containing cell surface substances from *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention was confirmed. (A: TNF-α mRNA expression; B: IL-8 mRNA expression; C: TNF-α production; D: IL-8 production)

[0072] Figure 9 This is a graph illustrating the effect of *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention on inflammatory cytokines induced by *Porphyromonas gingivalis* LPS. (A: TNF-α mRNA expression; B: IL-8 mRNA expression; C: TNF-α production; D: IL-8 production)

[0073] Figure 10 This is a graph illustrating the effect of *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention on inflammatory cytokines induced by *T. forsythia* LPS. (A: TNF-α mRNA expression; B: IL-8 mRNA expression; C: TNF-α production; D: IL-8 production)

[0074] Figure 11 This is a graph illustrating the effect of *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention on inflammatory cytokines induced by *F. alocis* LTA. (A: TNF-α mRNA expression; B: IL-8 mRNA expression; C: TNF-α production; D: IL-8 production)

[0075] Figure 12 This is a flow cytometry analysis showing the inhibitory effect of *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA on LPS and LTA cell adhesion based on the present invention. (A: *Porphyromonas gingivalis* LPS binding; B: *T. forsythia* LPS binding; C: *F. alociformis* LTA binding)

[0076] Figure 13 This is a graph illustrating the inhibitory effect of the salivary streptococcus G7 (S. salivarius G7; accession number: KCCM13161P) LTA according to the present invention on CD14 and LBP adhesion of periodontitis-associated bacterial material. (A, B, C: Inhibition of CD14 adhesion of Porphyromonas gingivalis, T. forsythia, and F. alocis; D, E, F: Inhibition of LBP adhesion of Porphyromonas gingivalis, T. forsythia, and F. alocis)

[0077] Figure 14 This is a graph showing the effect of the culture medium of *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention on the concentrations of volatile sulfur compounds H2S and CH3SH derived from *Porphyromonas gingivalis*.

[0078] Figure 15 This is a graph showing the effect of the *Streptococcus salivarius* G7 strain (accession number: KCCM13161P) of the present invention on the concentrations of volatile sulfur compounds H2S and CH3SH derived from *Porphyromonas gingivalis*.

[0079] Figure 16These are confocal laser microscope 3D images of cariogenic biofilms of *Streptococcus salivarius* G7 (accession number: KCCM13161P) with and without treatment according to the present invention. (Green represents live bacteria, red represents dead bacteria)

[0080] Figure 17 This is a graph showing the total number of bacteria, Streptococcus mutans, and Streptococcus salivarius G7 (accession number: KCCM13161P) in cariogenic biofilms at treatment concentrations according to the present invention. Detailed Implementation

[0081] The preferred embodiments of the present invention are described in detail below. However, the present invention is not limited to the embodiments described herein, and may be embodied in other forms. The content described herein is provided to fully convey the spirit of the present invention.

[0082] <Example 1: Preparation of Streptococcus salivarius strain>

[0083] The *Streptococcus salivarius* G7 (accession number: KCCM13161P) of this invention was isolated from subgingival samples of healthy Korean men, identified and characterized, and registered with the Korean Collection for Oral Microbiology (KCOM) (Streptococcus salivarius KCOM 2137). The strain was confirmed to be particularly superior to conventional *Streptococcus salivarius* in terms of antibacterial, anti-inflammatory, caries-inhibiting, and halitosis-inhibiting activities. Therefore, the patent is deposited at the Korean Center for Microbial Collection (KCCM, Korea Seed Culture Association) (address: Yurim Building, 45 Hongje-nae 2G, Seodaemun-gu, Seoul, South Korea (03641)) (accession number: KCCM13161P, deposit date: April 21, 2023).

[0084] The 16S rRNA sequence (SEQ ID NO: 1) of *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention is shown in [image / image / etc.]. Figure 1 Based on this, a phylogenetic analysis was conducted, and the results were presented in [the table / document / etc.]. Figure 2 middle.

[0085] As a comparative strain, Streptococcus salivarius K12 was isolated and identified from commercially available products, while Streptococcus salivarius ATCC 7073 (type strain) was purchased from the American Type Culture Collection (ATCC).

[0086] <Example 2: Evaluation of antibacterial and antifungal activity>

[0087] 2.1 Bacterial Culture

[0088] A total of 12 oral microorganisms were used for the antimicrobial tests of the strains, as shown in Table 1. These microorganisms consisted of Gram-positive and Gram-negative bacteria associated with oral diseases, representative antimicrobial test strains, and fungi. For bacteria associated with dental caries, *Streptococcus mutans* ATCC 25175 and *S. soborinus* ATCC 27607 were cultured using trypticase soy broth (TSB; BD Biosciences, San Jose, California, USA), and for *Actinomyces naeslundii* ATCC 12104, brain heart infusion (BHI; BD Biosciences) liquid medium was used. As bacteria associated with gingivitis and periodontitis, *Actinomyces israelii* ATCC 12102 and *Aggregatibacteractinomycetemcomitans* ATCC 43718 were cultured using BD Bioscience liquid medium. Furthermore, *Fusobacterium nucleatum* ATCC 25586, *Porphyromonas gingivalis* ATCC 33277, *Prevotella intermedia* ATCC 25611, and *Prevotella nigrescens* ATCC 33563 were cultured using BHI liquid medium supplemented with heme (1 μg / ml) and vitamin K (0.2 μg / ml) under anaerobic conditions (H2 5%, CO2 5%) at 37°C. Cultures were performed under anaerobic conditions (H2 5%, CO2 10%, N 285%). For bacteria associated with periapical inflammation (i.e., Enterococcus faecalis ATCC 29212), cultures were performed using BHI liquid medium at 37°C under anaerobic conditions (H2 5%, CO2 10%, N 285%). For fungi (i.e., Candida albicans ATCC 10231), cultures were performed using TSB under aerobic conditions.For bacteria associated with upper respiratory tract diseases (i.e., *Streptococcus pyogenes* (ATCC 12344) and *Streptococcus pneumoniae* (ATCC 49416), culture was performed under aerobic conditions using Toddhewitt broth (BD Bioscience). Finally, for representative Gram-positive and Gram-negative bacteria (*Staphylococcus aureus* (ATCC 23235) and *Escherichia coli* (ATCC 53868), culture was performed under aerobic conditions using TSB. For the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of this invention, culture was performed using M17 (BD Bioscience) liquid medium.

[0089] [Table 1]

[0090]

[0091] 2.2 Antibacterial test

[0092] Antimicrobial testing was performed using methods recommended by the Clinical & Laboratory Standards Institute (CSIS). Bacteria cultured in liquid medium were centrifuged at 4000 × g for 5 minutes, and clean liquid medium was added. Bacterial counts were determined using a bacterial counting chamber. For the bacterial suspension being tested, clean medium was used, with the anaerobic bacteria adjusted to 1.5 × 10⁻⁶. 6 The bacterial count / ml and the levels of aerobic and facultative anaerobic bacteria were adjusted to 1.0 × 10⁻⁶. 6 The number of samples per ml was used for experiments.

[0093] 180 μl of each culture medium was added to each well of a 96-well plate. In the first column, 20 μl of the prepared *Streptococcus salivarius* G7 (accession number: KCCM13161P), *Streptococcus salivarius* K12, and *Streptococcus salivarius* ATCC 7073 were added, followed by serial dilutions at 2-fold increments. The plates were then incubated in an anaerobic incubator for 24 or 36 hours. Bacterial growth was analyzed by measuring absorbance at 660 nm using a spectrophotometer, and the results are shown below. Figures 3 to 6 middle.

[0094] like Figure 3As shown, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention exhibits higher antibacterial activity against gingivitis pathogens such as *A. actinomycetemcomitans*, *A. israelii*, and *A. naeslundii*, and periapical inflammatory pathogens such as *Enterococcus faecalis*. *A. actinomycetemcomitans* causes gingivitis and also periodontitis during adolescence. Regarding the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention, for *A. actinomycetemcomitans*, growth was inhibited in a 16-fold diluted culture medium, and completely inhibited in an 8-fold diluted culture medium. Furthermore, for *A. israelii*, growth was also inhibited in an 8-fold diluted culture medium, and completely inhibited in a 4-fold diluted culture medium. And for *A. naeslundii*, growth was inhibited in an 8-fold diluted culture medium, and completely inhibited in a 4-fold diluted culture medium. For Enterococcus faecalis, its growth was significantly inhibited in a 16-fold diluted culture medium, and its growth was completely inhibited in an 8-fold diluted culture medium. These results indicate that it has more than 8 times the antibacterial activity compared to conventional strains.

[0095] like Figure 4As shown, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention exhibits superior antibacterial activity against periodontitis pathogens, namely *Fusobacterium nucleatum*, *P. gingivalis*, *P. intermedia*, and *P. nigrescens*, compared to *Streptococcus salivarius* (i.e., *Streptococcus salivarius* ATCC7073 (type strain) or *Streptococcus salivarius* K12) known in the prior art. Regarding *Streptococcus salivarius* G7 (accession number: KCCM13161P), significant inhibition of the growth of *F. nucleatum*, *P. gingivalis*, *P. intermedia*, and *P. nigrescens* was observed in pre-cultured media diluted 16-fold; and complete inhibition was observed in pre-cultured media diluted 8-fold. These results indicate that it possesses more than 8 times the antibacterial activity compared to conventional strains.

[0096] like Figure 5 As shown, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention exhibits superior antibacterial activity against dental caries pathogens (A. viscosus, *Streptococcus mutans*, and *Streptococcus sobrinus*) and upper respiratory tract pathogens (*Streptococcus pneumoniae* and *Streptococcus pyogenes*) compared to *Streptococcus salivarius* (i.e., *Streptococcus salivarius* ATCC7073 (type strain) or *Streptococcus salivarius* K12) known in the prior art. Regarding *Streptococcus salivarius* G7 (accession number: KCCM13161P), significant inhibition of the growth of *A. viscosus*, *S. mutans*, *S. sobrinus*, *S. pneumoniae*, and *S. pyogenes* was observed in pre-cultured media diluted 16-fold; and complete inhibition was observed in pre-cultured media diluted 8-fold. These results indicate that it possesses more than 8 times the antibacterial activity compared to conventional strains.

[0097] like Figure 6As shown, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of this invention exhibits superior antibacterial activity against representative Gram-negative pathogens (i.e., *Escherichia coli*) and Gram-positive pathogens (i.e., *Staphylococcus aureus*) compared to *Streptococcus salivarius* (i.e., *Streptococcus salivarius* ATCC7073 (type strain) or *Streptococcus salivarius* K12) known in the prior art. Specifically, for *Streptococcus salivarius* G7 (accession number: KCCM13161P), significant inhibition of the growth of *Escherichia coli* and *Staphylococcus aureus* was observed in 8-fold diluted culture media; and complete inhibition was observed in 4-fold diluted culture media. These results indicate that it possesses more than 4 times the antibacterial activity compared to conventional strains.

[0098] 2.3 Antifungal activity test

[0099] The antifungal activity test against Candida albicans (the pathogenic fungus of oral candidiasis) was performed in the same manner as the antimicrobial test described above, and the results are presented in [the table / document / etc.]. Figure 7 middle.

[0100] like Figure 7 As shown, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention exhibits superior antifungal activity against *C. albicans* compared to *Streptococcus salivarius* (i.e., *Streptococcus salivarius* ATCC7073 (type strain) or *Streptococcus salivarius* K12) known in the prior art. Specifically, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention showed significant inhibition of *C. albicans* growth in a culture medium diluted 8-fold; and complete inhibition in a culture medium diluted 4-fold. These results indicate that it possesses more than 4 times the antifungal activity compared to conventional strains.

[0101] Example 3: Evaluation of anti-inflammatory activity

[0102] The anti-inflammatory effect of *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention was confirmed using gingival fibroblasts. As shown in Table 2 below, lipopolysaccharide (LPS) derived from *Porpyromonas gingivalis*, lipopolysaccharide (LPS) derived from *Tannerella forsythia*, or lipoteichoic acid (LTA) derived from *Filifactor alocis* were used as inflammatory inducers.

[0103] [Table 2]

[0104]

[0105]

[0106] Porphyromonas gingivalis LPS, T. forsythia LPS, and F. alocis LTA bind to TLR4 and TLR2 on oral cell membranes, respectively, initiating signal transduction and inducing inflammatory responses. For LPS or LTA to bind to TLR4 and TLR2, CD14 and lipopolysaccharide-binding protein (LBP) are required. For Streptococcus salivarius LTA, competitive inhibition of LPS and LTA binding to periodontal bacteria to CD14 and LBP prevents their binding to the TLRs, thereby inhibiting the inflammatory-inducing activity of LPS and LTA.

[0107] The gingival fibroblast cell line HGF-1 was seeded in 12-well polystyrene plates and cultured to approximately 80% confluence. The culture medium was removed, and 1 ml of DMEM medium supplemented with 2% human serum (Sigma-Aldrich Co., San Jose, CA, USA) and penicillin / streptomycin (Hyclone) was added. Subsequently, *Streptococcus salivarius* G7 (accession number: KCCM13161P) of this invention was destroyed using sonication, and the resulting cell lysate was treated with 100 μg of LPS or LPA as an inflammatory inducer, followed by 12 hours of culture. TNF-α and IL-8 were then analyzed using an ELISA kit. Furthermore, total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA), and the purity and concentration of the extracted RNA were determined using a Nanodrop spectrophotometer (Invitrogen). Using 1 μg of total RNA, cDNA was synthesized using the Maxime RT premix method (Intron Biotech, Gyeonggi Province), and inflammation-related cytokines were analyzed using a real-time quantitative PCR instrument system (AB 7500 Real-time PCR instrument system, Applied Biosystems). Figures 8 to 11 The primer information for the inflammatory cytokines used in this experiment is shown in Table 3 below.

[0108] [Table 3]

[0109]

[0110] Figure 8The virulence of cell lysates containing cell surface material from *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention was confirmed. For the human gingival fibroblast cell line HGF-1, the following were utilized: lipopolysaccharides (Ec LPS) derived from *Escherichia coli*, lipoteichoic acid (Ss-type LTA) derived from the *S. salivarius* type strain ATCC 7073, surface proteins (SP) derived from the *S. salivarius* type strain ATCC 7073 (Ss-type SP), LTA (SS G7 LTA) derived from *S. salivarius* G7 (accession number: KCCM13161P) according to the present invention (accession number: KCCM13161P), and SP (SS G7) derived from *S. salivarius* G7 (accession number: KCCM13161P) according to the present invention. SP) was used for treatment, and the production levels of TNF-α and IL-8 mRNA and protein were shown in the figure. Figure 8 In the middle. For example Figure 8 As shown, the production of TNF-α and IL-8 mRNA and protein increased only when treated with LPS (Ec LPS) derived from Escherichia coli, while LTA or SP from the Salivarius species did not act as virulence factors.

[0111] Figure 9 This is a graph illustrating the effect of the present invention's *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA on inflammatory cytokines induced by *Porphyromonas gingivalis* LPS. (See graph for details.) Figure 9As shown, treatment of the gingival fibroblast cell line HGF-1 with *Porphyromonas gingivalis* LPS (Pg LPS) increased the production of TNF-α and IL-8 mRNA and protein. However, treatment with both the *Streptococcus salivarius* type strain and *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA inhibited the production of TNF-α and IL-8 mRNA and protein. In particular, compared with the *Streptococcus salivarius* type strain, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention significantly inhibited the production of TNF-α and IL-8 mRNA and protein.

[0112] Figure 10 This is a graph illustrating the effect of the present invention's *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA on *T. forsythia* LPS-induced inflammatory cytokines. (See graph for details.) Figure 10 As shown, treatment of the gingival fibroblast cell line HGF-1 with Forsakenella LPS (Tf LPS) increased the production of TNF-α and IL-8 mRNA and protein. However, treatment with both the *Streptococcus salivarius* type strain and the *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of this invention inhibited the production of TNF-α and IL-8 mRNA and protein. In particular, compared with the *Streptococcus salivarius* type strain, the *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of this invention significantly inhibited the production of TNF-α and IL-8 mRNA and protein.

[0113] Figure 11 This is a graph illustrating the effect of *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention on inflammatory cytokines induced by *F. alocis* LTA. (See graph for details.) Figure 11As shown, treatment of the gingival fibroblast cell line HGF-1 with *F. alocis* LTA (FaLTA) increased the production of TNF-α and IL-8 mRNA and protein. However, treatment with both the *S. salivarius* type strain and the *S. salivarius* G7 (accession number: KCCM13161P) LTA of this invention inhibited the production of TNF-α and IL-8 mRNA and protein. In particular, compared with the *S. salivarius* type strain, the *S. salivarius* G7 (accession number: KCCM13161P) LTA of this invention significantly inhibited the production of TNF-α and IL-8 mRNA and protein.

[0114] The above results confirm that the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention can inhibit inflammation induced by periodontal pathogens.

[0115] <Example 4: Evaluation of the ability of bacteria associated with periodontitis to inhibit cell adhesion>

[0116] 4.1 Flow Cytometry Analysis

[0117] Based on the above results, the efficacy of the *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention was evaluated to inhibit the adhesion of LPS or LTA derived from periodontal pathogens to cells. After fluorescent substances were adhered to *Porphyromonas gingivalis* LPS, *T. forsythia* LPS, and *F. alocis* LTA, human gingival fibroblasts (HGF-1 cells) were treated with LTA derived from the *Streptococcus salivarius* type strain (ATCC7073) (Ss type LTA) or the *Streptococcus salivarius* G7 (accession number: KCCM13161P) LTA of the present invention, and cultured. The fluorescence intensity of the cells was then confirmed by flow cytometry and presented. Figure 12 middle.

[0118] LPS (1 mg) derived from *Porphyromonas gingivalis* and *T. forsythia*, and LTA derived from *F. alocis*, were reacted with cold sodium metaperiodate at 4°C for 30 minutes. The reaction solution was then added to a standard RC dialysis membrane tubing (Spectrum Laboratories Inc., Ranch Dominaquez, CA, USA), and dialyzed for 12 hours in 100 mM sodium acetate (pH 5.5). The solution containing LPS and LTA was then mixed with 10 mM Alexa Fluor prepared by dissolving KCl in 200 mM. TM -488 hydrazide (Alexa Fluor) TM The mixture was prepared by mixing with 488 hydrazide (Invitrogen, Carlsbad, CA, USA) and incubating at room temperature for 6 hours in coupling buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2). After dialyzing three times over 12 hours in 1 L of endotoxin-free water, the fluorescently labeled LPS and LTA were transferred to new tubes and stored at -20°C.

[0119] After that, Alexa Fluor will be used. TM Alexa 488-labeled LPS or LTA was used to treat THP-1 cell lines (human mononuclear cell lines) with various concentrations of Streptococcus salivarius G7 surface extract or in the absence of Streptococcus salivarius G7 surface extract at 37°C for 1 hour. Cells were then obtained by centrifugation at 500×g for 3 minutes in a CO2 incubator and washed three times with cold Dulbecco's phosphate-buffered saline (DPBS, pH 7.2). The binding of Alexa 488-labeled LPS and LTA to cells was then analyzed by flow cytometry (FACS calibur, BD Biosciences, Sparks, MD, USA). Data were collected at 10,000 cell counts, and Alexa Fluor was detected in the FL-1 channel. TM The combination of 488-labeled LPS and LTA was analyzed using CellQuest software (BD biosciences).

[0120] like Figure 12As shown, treatment with *Porphyromonas gingivalis* LPS, *T. forsythia* LPS, and *F. alocis* LTA significantly increased the number of fluorescent cells (i.e., cells with LPS and LTA adhering to them), while co-culturing with LTA derived from the *S. salivarius* type strain (Ss-type LTA) or LTA of the *S. salivarius* G7 (accession number: KCCM13161P) of the present invention resulted in a significant decrease in the number of fluorescent cells. In particular, compared to the *S. salivarius* type strain, treatment with LTA of the *S. salivarius* G7 (accession number: KCCM13161P) of the present invention significantly reduced the number of cells with LPS and LTA adhering to them.

[0121] 4.2 Analysis of adhesion inhibition by CD14 and LBP

[0122] For *Porphyromonas gingivalis* LPS, *T. forsythia* LPS, and *F. alocis* LTA, in order to induce inflammatory cytokines via TLR4 and TLR2, respectively, LPS and LTA must first bind to CD14 and LBP on the oral cell membrane. Therefore, it was confirmed that *S. salivarius* LTA can competitively inhibit the adhesion of periodontal bacteria's LPS and LTA to CD14 and LBP.

[0123] Porphyromonas gingivalis LPS, T. forsythia LPS, and F. alocis LTA, all coated with fluorescent substances, were cultured with CD14 or LBP protein in the presence of different concentrations of LTA derived from the Salivarius type strain (Ss type LTA) or the LTA of Salivarius G7 (accession number: KCCM13161P) of this invention. The degree of adhesion was measured, and the results are presented below. Figure 13 middle.

[0124] Anti-LBP monoclonal antibody (Anti-LBP mAb) (MAB870, R&D systems, Minneapolis, MN, USA) and anti-CD14 antibody (AF982, R&D systems) were dissolved in DPBS at a concentration of 1 μg / ml. The antibody (50 ng / well) was aliquoted into EIA plates (Corning Co., Corning, NY, USA) and coated at 4°C for 12 hours. The antibody-coated EIA plates were washed five times with phosphate-buffered saline (PBST) containing 0.1% Tween 20, and then aliquoted into DPBST containing 1% bovine serum albumin (BSA) and inactivated by gentle shaking for 2 hours. Subsequently, recombinant human LBP (rhLBP, R&D system) and human CD14 (rhCD14, R&D system) were dissolved in DPBS at a concentration of 2 μg / ml and added to the wells of an EIA plate coated with the antibodies. After incubating the plate at room temperature for 4 hours, it was washed five times with PBST and then treated individually with biotin-labeled LPS or LTA, or co-treated with *S. salivarius* LTA, in the prepared wells and incubated at room temperature for 2 hours. To measure the biotin-labeled LPS and LTA binding to rhLBP and rhCD14, 50 μl of HRP-labeled streptavidin (1 μg / ml) was reacted in PBST containing 2% BSA for 1 hour. After washing five times with PBST, 3,3',5,5'-tetramethylbenzidine (TMB) solution was added to the wells, and the mixture was incubated at room temperature for 30 minutes. The enzyme reaction was then terminated with 1N sulfuric acid. The absorbance was measured at 450 nm using a microplate reader (Biotek, Winooski, VT, USA).

[0125] like Figure 13As shown, with increasing concentrations of LTA derived from the *S. salivarius* type strain (Ss type LTA) or the *S. salivarius* G7 (accession number: KCCM13161P) of the present invention, the binding of *Porphyromonas gingivalis* LPS, *T. forsythia* LPS, and *F. alocis* LTA to CD14 or LBP decreased. In particular, compared to the *S. salivarius* type strain, the *S. salivarius* G7 (accession number: KCCM13161P) LTA of the present invention significantly inhibited the binding of LPS or LTA to CD14 or LBP.

[0126] <Example 5: Evaluation of the ability to suppress volatile sulfur compounds>

[0127] To confirm whether the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of this invention has a halitosis-inhibiting effect, an experiment was conducted using a culture medium containing *Streptococcus gingivalis*, which generates volatile sulfur compounds. 1 ml of *Porphyromonas gingivalis* (1 × 10⁻⁶) was used. 8 (300 cells / ml) were inoculated into 10 ml of fresh culture medium and cultured for 48 hours. Then, 300 μl of the bacterial culture was transferred to a clean 50 ml centrifuge tube. Next, 300 μl or 600 μl of the culture medium of *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention was added to the centrifuge tube containing *Streptococcus gingivalis*, or 10 μl of the culture medium of *Streptococcus salivarius* G7 (accession number: KCCM13161P). 7 Or 10 8 Add the live cells themselves, then add culture medium until the total volume reaches 5 ml. Afterward, shake the centrifuge tube vertically for 1 minute and incubate at room temperature for 5 minutes. Then, obtain 1 ml of air directly above the surface of the solution using a 1 ml syringe, and analyze the result using an oral-chromosome gas chromatograph. TM The concentrations of hydrogen sulfide (H2S) and methanethiol (CH3SH) were determined using Nissha FIS, Inc., Osaka, Japan, and the results are presented in [the table / incomplete]. Figure 14 and Figure 15 middle.

[0128] like Figure 14As shown, the culture medium of Streptococcus salivarius G7 (accession number: KCCM13161P) of the present invention reduced the concentration of halitosis-inducing volatile sulfur compounds (i.e. hydrogen sulfide (H2S) and methanethiol (CH3SH)) of Porphyromonas gingivalis in a concentration-dependent manner.

[0129] And, as Figure 15 As shown, the *Streptococcus salivarius* G7 strain (accession number: KCCM13161P) of the present invention also reduced the concentrations of hydrogen sulfide (H2S) and methanethiol (CH3SH) in a concentration-dependent manner.

[0130] The above results confirm that the *Streptococcus salivarius* G7 (accession number: KCCM13161P) of the present invention can effectively inhibit halitosis caused by *Porphyromonas gingivalis*.

[0131] <Example 6: Evaluation of Biofilm Formation Inhibition>

[0132] 6.1 Inhibition of biofilm formation

[0133] The efficacy of the *Streptococcus saliva* G7 (accession number: KCCM13161P) of this invention in inhibiting biofilm formation was evaluated. Saliva was collected from 10 adults without periodontitis, mixed, and aliquoted into two tubes. First, to prepare a saliva-coated plate, an equal volume of phosphate-buffered saline was added to one tube and centrifuged at 6000 × g for 10 minutes at 4°C. The supernatant was then transferred to a clean tube. An equal volume of BHI medium was added to the saliva mixture in the other tube and centrifuged at 2000 × g for 10 minutes. The supernatant was then transferred to a clean tube, thus preparing the salivary bacteria.

[0134] The saliva used for coating was dispensed into 200 μl portions onto 8-well glass plates and dried in an oven at 40 °C. This process was repeated 10 times. The saliva-coated plates were then sterilized in an ultraviolet (UV) sterilizer. Subsequently, to form a cariogenic biofilm, 100 μl of *Streptococcus mutans* (S. mutans) (1 × 10⁻⁶) was added to 10 ml of a saliva bacterial suspension containing 2% sucrose. 8Cells / ml) were added to 8-well glass plates and 1 ml of 12-polystyrene plates, respectively, and cultured under aerobic conditions. Subsequently, for the control group, the medium was changed daily using BHI medium containing 2% sucrose for 7 days; for the experimental group, the medium was changed for 7 days using the following medium, wherein the medium used for replacement was BHI medium containing 2% sucrose inoculated with the *Streptococcus salivarius* G7 (accession number: KCCM13161P) strain of the present invention at two concentrations, so that each medium contained 10 cells / ml. 7 and 10 8 One strain of bacteria was identified. Subsequently, to analyze the amount of biofilm, 8-well plates were stained using a Bacterial Live / Dead Staining Kit (Invitrogen), and 3D observation was performed using a confocal laser microscope, as shown in the figure. Figure 16 middle.

[0135] like Figure 16 As shown, it was confirmed that saliva-containing strains and Streptococcus mutans (S. mutans) formed a biofilm on a plate coated with saliva. When the saliva-containing Streptococcus G7 (S. salivarius G7; accession number: KCCM13161P) of the present invention was added, the apoptosis of the biofilm-forming strains increased, thereby loosening the biofilm.

[0136] 6.2 Determination of the number of bacteria in biofilm

[0137] The total bacterial counts (Oral streptococci, Streptococcus mutans, and Streptococcus salivarius G7; accession number: KCCM13161P) in the formed biofilm were determined. 1 ml of DPBS was added to a 12-well plate to physically detach the cultured biofilm. After centrifuging at 5000×g for 1 minute to obtain a bacterial pellet, total DNA was extracted from the bacterial pellet using a G-spin Genomic DNA extraction kit (iNtRON Biotechnology, Gyeonggi, Korea). Bacterial quantification was then performed using a real-time quantitative gene amplification system.

[0138] Real-time gene analysis was performed using standard quantitative DNA and samples. After mixing TB green premix (Takara Co., Tokyo, Japan), ROX II dye, primers, and DNA, the mixture was preheated at 94°C for 5 minutes, and then repeated 40 times with the following steps: 10 seconds at 95°C (denaturation stage), 10 seconds at 60°C (annealing stage), and 33 seconds at 72°C (extension stage). To analyze non-specific binding, a melting curve was used. A standard curve was plotted using the standard quantitative DNA for Ct values ​​and bacterial count. Based on this standard curve, the bacterial count was calculated using the Ct values ​​of the sample DNA, and the results are presented below. Figure 17 The primers used at this time are shown in Table 4.

[0139] [Table 4]

[0140]

[0141] like Figure 17 As shown, the amount of Streptococcus salivarius G7 (accession number: KCCM13161P) in the biofilm increased with the increase of treatment amount, but Streptococcus mutans, a cariogenic bacterium, decreased significantly with the increase of the treated Streptococcus salivarius G7 (KCCM13161P) of the present invention.

[0142] [Collection Number]

[0143] Name of depository: Korean Center for Microbial Collection (KCCM)

[0144] Accession number: KCCM13161P

[0145] Deposit date: April 21, 2022

[0146] [Sequence List]

[0147] Attached is an electronic file of the sequence list.

Claims

1. The use of a Streptococcus salivarius strain G7 with accession number KCCM13161P in the preparation of a drug with antibacterial activity, characterized in that, The antibacterial activity refers to its antibacterial activity against one or more strains selected from Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus.

2. The use of one or more of the following: Streptococcus salivarius strain G7 with accession number KCCM13161P, its concentrate, its dried product, and its culture, in the preparation of compositions for the prevention or improvement of diseases, characterized in that... The disease is caused by one or more of the following bacteria: Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus.

3. The use of a probiotic composition comprising Streptococcus salivarius strain G7 or a culture thereof with accession number KCCM13161P in the preparation of probiotics with antibacterial activity, characterized in that, The antibacterial activity refers to its antibacterial activity against one or more strains selected from Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus.

4. The use according to claim 3, characterized in that, The probiotic composition is selected from one or more dosage forms chosen from the group consisting of tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, chewing gums, and candies.

5. The use of one or more of the following: a Streptococcus salivarius strain G7 with accession number KCCM13161P, its concentrate, its dried product, and its culture, in the preparation of a pharmaceutical composition for the prevention or treatment of a disease, characterized in that... The disease is caused by one or more of the following bacteria: Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus.

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