WO2022016266A1 - Probiotique pour la santé buccale - Google Patents

Probiotique pour la santé buccale Download PDF

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WO2022016266A1
WO2022016266A1 PCT/CA2021/051001 CA2021051001W WO2022016266A1 WO 2022016266 A1 WO2022016266 A1 WO 2022016266A1 CA 2021051001 W CA2021051001 W CA 2021051001W WO 2022016266 A1 WO2022016266 A1 WO 2022016266A1
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salivarius
strain
salivarius strain
oral
disease
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Ted JIN
Mizue NAITO
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13400719 Canada Inc
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention generally relates to the field of probiotics, and more particularly relates to a novel probiotic that promotes oral health.
  • the oral cavity houses one of the most diverse microbiotas in the human body. It provides several unique niches for bacterial colonization, such as the saliva, the tongue, the cheek, other mucosal surfaces, as well as the non-shedding surfaces of teeth. There are close to 800 unique oral bacterial species identified on the Human Oral Microbiome Database, with more species expected to be added with further sampling and identification. As with microbiota of other sites of the human body, a balanced oral microbiota is essential to the health of the human host. Alterations to this balance, or a dysbiotic microbial community, can not only lead to various diseases in the mouth, such as dental caries, periodontal disease, and halitosis, but can also cause disease systemically.
  • Dental caries is the most prevalent oral disease worldwide, estimated to have affected 2.4 billion people in 2016, while periodontal disease is the 11th most prevalent disease globally. Halitosis is a common problem in many individuals, and has a worldwide prevalence of 22% to 50%. Dental caries is caused by a combination of host factors and excessive dietary sugar intake, which leads to fermentation and production of acidic metabolites and extracellular polymeric substances by acidogenic bacteria residing in the supragingival plaque. Continued exposure to fermentable carbohydrates creates a positive feedback loop, where localized regions of low pH within the plaque biofilms produced by the acidogenic bacteria continues to select for acidogenic and aciduric bacteria.
  • Streptococcus mutans is the oral bacteria that is most associated with causing dental caries. Targeting S. mutans has been shown to be a successful method to reduce the incidence of dental caries.
  • Periodontal disease is an inflammatory disease induced by the subgingival microbiota, leading to the destruction of the supporting structures of the tooth. Unlike most dysbiotic diseases, periodontal disease is associated with increased microbial diversity in the subgingival crevice. Pathogenic species associated with periodontal disease include Porphyromonas gingivalis, Tannerella forsythia , Fusobacterium nucleatum and anaerobic species enriched in virulence factors that are able to survive in the highly inflammatory environment of the diseased gum. When periodontal disease is severe, not only can it lead to tooth loss, but also to various systemic diseases such as atherosclerosis, adverse pregnancy outcomes, and rheumatoid arthritis.
  • VSCs volatile sulphur compounds
  • Comparisons of tongue coatings of halitosis patients against healthy controls have revealed several VSC-producing bacterial species that are commonly found in halitosis patients.
  • the genera Prevotella and Leptotrichia are considered to be highly associated with halitosis, as well as other taxa such as Peptostreptococcus stomatis, Capnocytophaga gingivalis and Ta. forsythia.
  • probiotics Due to the recent awareness on the importance of maintaining a healthy microbiota, probiotics have become an exciting method for preventing or treating diseases associated with dysbiosis. Probiotics are defined by the Food and Agriculture Organization/World Health Organization (F AO/WHO) as " live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host" . While the majority of probiotics developed are targeted to gut health, oral health probiotics are emerging as an important means to maintain the oral microbiota and prevent oral diseases.
  • F AO/WHO Food and Agriculture Organization/World Health Organization
  • a novel probiotic has now been developed that promotes oral health.
  • the probiotic is a novel strain of Streptococcus salivarius.
  • a novel strain of Streptococcus salivarius which encodes for the bacteriocin, subtilosin.
  • a method of preventing disease in a mammal, or restoring oral health in mammal comprising administering a therapeutically effective amount of an S. salivarius probiotic that produces subtilosin to the oral cavity of the mammal.
  • an S. salivarius strain which exhibits anti microbial activity that is protease-resistant.
  • Figure 1 graphically illustrates the results of a two-species growth competition assay performed against S. salivarius DB-B5 and S. mutans UA159, and against S. salivarius ATCC 13419 and S. mutans UA159;
  • Figure 2 illustrates the phylogenetic reconstruction of S. salivarius DB-B5, including A)
  • 16S rRNA Bayesian and Maximum Likelihood phylogenetic tree and B) a multi-gene (dnaG, firr, infC, nusA, pgk, rplA, rpoB, rpsC, smpB, tsf) Bayesian and Maximum Likelihood phylogenetic tree.
  • Bayesian posterior probabilities greater than 0.95 are shown at nodes, and branches with bootstrap support over 70% are thickened;
  • Figure 3 provides the amino acid sequences of the coding regions within the subtilosin bacteriocin locus of the S. salivarius DB-B5 strain.
  • Figure 4 illustrates the full DNA sequence of the subtilosin bacteriocin locus of the S. salivarius DB-B5 strain. Coding regions are underlined, italic font indicates complement strand, bold font indicates forward strand and font with wave underline indicates overlapping start/stop regions.
  • a novel Streptococcus salivarius strain is provided which is useful to prevent and/or treat disease in a mammal.
  • the present S. salivarius strain herein referred to as S. salivarius DB-B5
  • disease is used herein to encompass disease within the oral cavity such as dental caries, periodontal disease (e.g. gingivitis and periodontitis), halitosis, dysbiosis within the oral cavity which can lead to systemic disease, as well as disease that occurs in other parts of the body, such as cancer or gastrointestinal disease by pathogenic microorganisms that originate in the oral cavity.
  • periodontal disease e.g. gingivitis and periodontitis
  • halitosis e.g., dysbiosis within the oral cavity which can lead to systemic disease, as well as disease that occurs in other parts of the body, such as cancer or gastrointestinal disease by pathogenic microorganisms that originate in the oral cavity.
  • the present S. salivarius strain advantageously inhibits oral pathogens linked to disease.
  • oral pathogen refers to pathogenic microorganisms that exist in the oral cavity, or which originate in the oral cavity but may localize in other parts of the body to result in disease beyond the oral cavity.
  • oral pathogens inhibited by the present S. salivarius strain include, but are not limited to, strains of the Prevotella genus such as Pr. shahii, strains of the Porphyromonas genus such as Po. gingivalis, strains of the Fusobacterium genus such as F. nucleatum , strains of the Leptotrichia genus such as L. wadei, and strains of the Peptostreptococcus genus such as Pe.
  • an S. salivarius strain to inhibit pathogens may be determined using known assays such as an agar overlay assay in which a target pathogenic strain in liquid agar is added to an agar plate comprising homogeneous growth of the present S. salivarius strain. Detection of zones of inhibition, i.e. clear zones in which there is no growth of the target pathogen, indicates that the present S. salivarius strain inhibits the target pathogenic strain. The greater the zone (or width) of inhibition, the more virulent the inhibition.
  • the anti -pathogenic properties of the present S. salivarius strain are due, at least in part, to its bacteriocin expression profile.
  • the present S. salivarius strain uniquely produces subtilosin, a bacteriocin which S. salivarius strains do not characteristically produce.
  • the subtilosin locus of an S. salivarius strain according to an embodiment of the invention is shown in Figure 4, and the proteins it encodes are shown in Figure 3. Exemplary proteins encoded by this locus include an Ml 3 family metallopeptidase, ABC transporter and ABC transporter ATP binding protein, radical S-adenosyl-L- methionine proteins, a helix-turn-helix domain-containing protein and phosphoglycerate mutase.
  • Anti- pathogenic properties of the present S. salivarius strain are protease-resistant, at least in part, for example, these properties are resistant to proteases, such as at least one of pronase, subtilisin, or Proteinase K.
  • the present strain may encode other anti-pathogenic compounds, such as a thiazolyl peptide bacteriocin, not typically produced by S. salivarius , and a bacteriocin-like peptide (blp) bacteriocin.
  • the bacteriocin loci may be found in the chromosomal DNA of the organism, or in extrachromosomal structures such as plasmids or episomes.
  • the unique bacteriocin expression profile of the present S. salivarius strain provides the strain with inhibitory properties that allow it to target a broad range of pathogenic microorganisms.
  • the present S. salivarius strain has a genome consisting of one circular chromosome (2, 143,863 bp) with a GC content of 40.2%, one megaplasmid referred to as pIKMIN-B501 (138,497 bp) with a GC content of 35.6%, one small plasmid referred to as pIKMIN-B503 (3,225 bp) with a GC content of 39.6%, and one linear phage-like episome referred to as pIKMIN-B502 (57,714 bp) with a GC content of 39.1%.
  • the strain possesses three separate loci encoding bacteriocins, a thiazolyl peptide (thiopeptide) locus on the megaplasmid, and subtilosin and blpU bacteriocin loci on the chromosome.
  • the genome sequences were deposited at the National Centre for Biotechnology Information under GenBank accession numbers CP054153, CP054154, CP054155 and CP054156, the contents of which are incorporated herein by reference.
  • the present S. salivarius strain also advantageously exhibits significant growth dominance over the pathogen, S. mutans.
  • S. mutans When mixed and grown together under suitable growth conditions, e.g. grown on BHI agar and incubated at 37 °C under 5% CO2, the present S. salivarius strain outgrows S. mutans by at least about 10-90%, preferably by at least about 20%, 30% or 40%, for example, by about 40-80%.
  • the present S. salivarius strain is hydrophobic in nature, and thus, able to colonize human cell surfaces, enabling the strain to maintain a strong presence within the oral cavity on administration and thereby limit pathogen proliferation.
  • the present S. salivarius strain exhibits a hydrophobicity of at least about 85%, preferably at least 90%-95%.
  • other strains of S. salivarius such as strain Ml 8
  • Hydrophobicity may be determined using assays established in the art, for example, the Microbial Adhesion to Hydrocarbon (MATH) assay which is based on the determination of microbial hydrophobicity by differential partitioning at an aqueous- hydrocarbon interface.
  • MATH Microbial Adhesion to Hydrocarbon
  • the assay includes mixing of the bacterial cell suspension with a hydrocarbon, preferably an aliphatic hydrocarbon such as dodecane, hexadecane, octane or p-xylene, for a predetermined period to allow optimal interaction of the bacteria with the hydrocarbon phase. Partitioning of the bacterial suspension between the aqueous and hydrocarbon phases is determined, and expressed as the percentage of cells adsorbed by the hydrocarbon phase.
  • surface hydrophobicity is based on the difference in absorbance of cells in suspension at 595 nm initially and following incubation with a hydrocarbon.
  • hydrophobicity is determined using varying concentrations of ammonium sulfate (the greater the degree of aggregation at lower concentrations, the greater the hydrophobicity).
  • the present S. salivarius strain is physiologically acceptable for use as a probiotic for administration to the oral cavity.
  • the present strain is susceptible to antibiotics such as Vancomycin (glycopeptides), Gentamicin (aminoglycosides), Streptomycin (aminoglycosides), Clindamycin (lincosamides), Erythromycin (macrolides), Ampicillin and Penicillin (penicillins), tetracycline and chloramphenicol (phenicols).
  • the present strain has been determined to be a non-producer of toxic biogenic amines, such as histamine, tyramine, cadaverine, tryptamine, serotonin, putrescine and spermine/spermidine.
  • the present strain does not encode virulent factors based on genome analysis that would render its use in mammals to be detrimental.
  • the present S. salivarius strain ferments carbohydrates.
  • Exemplary carbohydrates fermented by the S. salivarius strain include, but are not limited to, D-galactose, D-glucose, fructose, mannose, N-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, raffmose, gentiobiose, salicin, cellobiose, maltose, melibiose, saccharose, trehalose and inulin.
  • the present S. salivarius strain is useful to prevent and/or treat disease in a mammal.
  • mammal is used herein to refer to both human and non-human mammals, including domesticated animals such as, but not limited to, cats, dogs and other pets, horses, livestock, etc.
  • the method includes administering a therapeutically effective amount of the S. salivarius strain to a mammal to treat or prevent disease in the mammal.
  • therapeutically effective refers to an amount of probiotic which is effective to reduce or minimize at least one factor associated with a target disease such as oral disease, including reducing the presence or activity of pathogenic microorganisms linked to dental caries such as S.
  • mutans pathogenic species associated with periodontal disease such as Porphyromonas gingivalis , and Fusobacterium nucleatum
  • VSC volatile sulphur compound
  • Prevotella e.g. Pr. shahii and Pr. intermedia
  • Leptotrichia e.g. L. wadei
  • Peptostreptococcus stomatis e.g. Capnocytophaga gingivalis
  • pathogenic species that may spread to cause disease elsewhere in the body, e.g. Fusobacterium nucleatum.
  • a therapeutically effective amount of the present S. salivarius strain is about 500 million to 20 billion CFUs per day.
  • the present S. salivarius strain may be formulated for administration in the form of a powder, tablet, capsule, suspension, and the like.
  • probiotic cells such as the present S. salivarius cells, are dried to enhance viability/shelf-life, either by freeze-drying or spray-drying, using known techniques.
  • the cells may be admixed with protective agents, such as, sweet whey concentrate, concentrated milk, lactose, saccharose, trehalose, galactose, starch, sorbitol, casein, beta-lactoglobulin, alpha- lactalbumin, soy, serum albumin, glutenin, prolamin, lysine, cysteine, glycine, or vitamins, to enhance survival following spray-drying.
  • protective agents such as, sweet whey concentrate, concentrated milk, lactose, saccharose, trehalose, galactose, starch, sorbitol, casein, beta-lactoglobulin, alpha- lactalbumin, soy, serum albumin, glutenin, prolamin, lysine, cysteine, glycine, or vitamins, to enhance survival following spray-drying.
  • protective agents such as, sweet whey concentrate, concentrated milk, lactose, saccharose, trehalose, galactose, star
  • Suitable excipients are those which do not adversely affect S. salivarius growth and/or colonization.
  • common excipients include, but are not limited to, microcrystalline cellulose (as binder/diluent), rice maltodextrin (as binder/diluent), mannitol or dextrin (filler), silicon dioxide (gliding/anti-caking agent), magnesium stearate (as lubricant), and hydroxy propyl methylcellulose (as suspending/viscosity agent).
  • the dried cells may be administered in dry form, or may be suspended in a liquid, e.g. an aqueous-based liquid, for oral administration, e.g. as an oral rinse, which may be ingested or discarded following rinsing of the oral cavity for a sufficient period of time, e.g. 30 seconds to 1 or 2 minutes.
  • the S. salivarius strain may also be formulated for administration in foods or beverages such as milk-based products, e.g. milk, yogurt, ice cream, puddings, whipped toppings or cheese products, drinks, fruit juices, soy or cereal-based products, confectionery products such as candy, gummies, chocolate, cookies, pastries and other baked goods, frosting and the like, and various other products.
  • milk-based products e.g. milk, yogurt, ice cream, puddings, whipped toppings or cheese products
  • drinks fruit juices, soy or cereal-based products
  • confectionery products such as candy, gummies, chocolate, cookies, pastries and other baked goods, frosting and the like
  • Chewing gums, lozenges, tooth paste or gel, aqueous gel and oral strips may also be prepared comprising the S. salivarius strain.
  • the present formulations may additionally include ingredients to enhance taste, including flavors, e.g. fruit or mint flavor, and/or sweetener, preferably non-fermentable sweetener such as aspartame, sucralose, stevia (steviol glycosides), and monk fruit extract, as well as carbohydrates that are metabolized via different pathways such as xylitol, erythritol, glycerol, and allulose.
  • flavors e.g. fruit or mint flavor
  • sweetener preferably non-fermentable sweetener such as aspartame, sucralose, stevia (steviol glycosides), and monk fruit extract
  • carbohydrates that are metabolized via different pathways such as xylitol, erythritol, glycerol, and allulose.
  • the present strain may be combined with one or more additional probiotics, including those that also treat or prevent oral disease, and those which treat other ailments.
  • additional probiotics include other S. salivarius strains, e.g. K12 and Ml 8 strains, other strains of Streptococcus species such as S. thermophilus , strains of Lactobacillus such as L. reuteri, L. brevis, L. helveticus, L. plantarum, L. paracasei, L. casei, L. fermentum, L. rhamnosus, and L. acidophilus, strains of Bifidobacterium such as B. bifidum, B. longum, B. lactis, B. infantis , and B. breve, and strains of Bacillus such as B. subtilis and B. coagulans.
  • S. salivarius formulations may be supplemented with one or more additional therapeutic agents or dietary supplements which do not adversely affect S. salivarius growth and/or colonization.
  • additional therapeutic agents include, but are not limited to, fluoride, vitamins (e.g. vitamin D, vitamin E), minerals, prebiotics, agents that prevent cariogenic activity such as calcium phosphate salts, antioxidants and mixtures thereof.
  • the formulation may include a preservative, which does not adversely affect S. salivarius growth and/or colonization, such as essential oils (orange oil, grape oil, clove oil, etc.), benzoates, sorbates, and the like.
  • a preservative which does not adversely affect S. salivarius growth and/or colonization, such as essential oils (orange oil, grape oil, clove oil, etc.), benzoates, sorbates, and the like.
  • Antimicrobial Activity Against Oral Pathogens were assessed for their ability to inhibit oral bacteria associated with various oral diseases, using the bacterial inhibition overlay assay as described in Chew et al. (2015) J Appl Microbiol 118:1180-1190. https://doi.org/10.llll/jam.12772.
  • the target bacteria used were: Po. gingivalis ATCC 33277, Pr. shahii DSM 15611, F. nucleatum DSM 15643, L. wadei DSM 19758 and Pe. stomatis DSM 17678.
  • DNA extraction was performed using DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions for Gram-Positive Bacteria.
  • qPCR was used to quantify the percent composition of the 2-species mixture, using the primers Ssaliv-qF and Ssaliv-qR for S. salivarius ATCC 13419, rpoB2-qF and rpoB2-qR for S. salivarius DB-B5, and Smut4-qF and Smut4-qR for S. mutans UA159 (Table 1).
  • PCR products were purified with QIAquick PCR purification kit (Qiagen) and sequenced using Sanger sequencing (The Centre for Applied Genomics, The Hospital for Sick Children) with the 8F and 1492R primers (Table 1). The resulting sequences were aligned and analyzed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the NCBI database.
  • 16S rRNA Phylogenetic Analysis - 16S rRNA phylogeny reconstruction was performed as previously described (Naito et al. (2015). Proc Natl Acad Sci USA 112:7791-7796. https://doi.org/10.1073/pnas.1501676112).
  • DNA was extracted from S. salivarius DB-B5 using DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's guide for Gram-Positive Bacteria. The DNA was used as a template for the amplification of 16S rRNA using the universal primers 8F and 1492R (Table 1).
  • Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific) was used following the manufacturer's instructions, with an initial denaturation time of 30 s at 98 °C, 30 cycles consisting of 10 s at 98 °C, 15 s at 55 °C, and 45 s at 72 °C, followed by a final extension of 7 min at 72 °C.
  • the PCR product was purified with QIAquick PCR purification kit (Qiagen) and sequenced using Sanger sequencing (The Centre for Applied Genomics, The Hospital for Sick Children) with the 8F and 1492R primers (Table 1).
  • Multi-gene Phylogenetic Analysis The multi-gene phylogenetic reconstruction was performed as previously described, with slight modifications (Naito et al. 2015). The amino acid sequences of the following genes, based on the Genomic Encyclopedia of Bacteria and Archaea were selected and concatenated: dnaG, frr, infC, nusA, pgk, rplA, rpoB, rpsC, smpB, tsf. The S. salivarius DB-B5 genes were obtained through Illumina sequencing of the full genome. Genes from all other bacterial species were obtained through IMG (https://img.jgi.doe.gov/).
  • Carbohydrate Fermentation Profile To determine the carbohydrate metabolic capability of S. salivarius DB-B5, the API 50CH (bioMerieux) system was used, following the manufacturer's instructions. The API strip containing S. salivarius DB-B5 was incubated aerobically at 37 °C, and the fermentation profile was measured at both 24 h and 48 h.
  • Antibiotic Susceptibility Test Antibiotic susceptibility was measured using ETEST strips
  • CARD Genome Mining for Antibiotic Resistance Genes using CARD -
  • the genome of S. salivarius DB-B5 was screened for genes involved in antibiotic resistance using the Comprehensive Antibiotic Resistance Database (CARD; https://card.incinaster.ca/) (as described by Jia et al. (2017) Nucleic Acids Res 45:D566-D573).
  • CARD is an online bioinformatic database of antibiotic resistance determinants organized through the Antibiotic Resistance Ontology (ARO).
  • ARO Antibiotic Resistance Ontology
  • ORF The protein sequences of all predicted open reading frames (ORF) of S. salivarius DB-B5 was used as input for the Resistance Gene Identifier (RGI) tool on the CARD website.
  • VFDB VFDB
  • the VFDB core database was downloaded and a local reciprocal blastp analysis was performed against the protein sequences of all predicted ORFs of S. salivarius DB-B5 using the BLAST+ software (Camacho et al. (2009). BMC Bioinformatics 10:421). Greater than 50% identity match and E-values of less than 10 5 were used as cut-off values.
  • Table 2 shows the inhibition properties of the prime candidate probiotic, named S. salivarius DB-B5, and the common lab strain S. salivarius ATCC 13419 against the above pathogens.
  • S. salivarius DB-B5 showed inhibitory properties against all of the pathogens, in differing amounts to the lab strain.
  • nucleatum (periodontal, halitosis) 4 4
  • salivarius DB-B5 was able to ferment 17 of the 49 carbohydrates tested. The comparison with other S. salivarius strains indicates that there are differences between the strains. The fermentation profile was found to be stable under numerous lab propagations, as well as fermentation and freeze-drying processes (data not shown).
  • S. salivarius DB-B5 The cell surface hydrophobicity of S. salivarius DB-B5 was also assessed, to determine the likelihood of the strain to be able to colonize human cell surfaces, such as the oral environment. Using the MATH assay, S. salivarius DB-B5's measured hydrophobicity was 98.70%. The high cell surface hydrophobicity indicates that the candidate probiotic is able to attach and colonize the human oral environment well. [0056] Safety Evaluation of S. salivarius DB-B5 - S. salivarius DB-B5 was assessed for its antibiotic susceptibility using Etest strips (bioMerieux). A diverse group of antibiotic classes were tested, and S.
  • salivarius DB-B5 was found to be susceptible to all antibiotics tested, according to both the European (EFSA) and the U.S.'s (CLSI) breakpoints for Streptococcus species (Table 4).
  • EFSA European Food Safety Authority
  • CCSI Clinical and Laboratory Standards Institute
  • MIC Minimum inhibitory concentration
  • S. salivarius DB-B5 were run on the RGI tool from the CARD bioinformatic database, to detect potential antibiotic resistance genes. None of the ORFs from S. salivarius DB-B5 had "Perfect” (100% identical to the reference) or "Strict” (match bitscore above the curated BLASTP bitscore cutoff) hits against the database. RGI predicted 140 ORFs as "Loose” hits, which generally indicates distant homologs which may or may not have a role in antibiotic resistance.
  • VFDB protein sequences of all predicted ORFs of S. salivarius DB-B5 were also analyzed for potential virulence genes using VFDB.
  • the 15 VFDB-positive genes were compared to their actual predicted roles based on NCBI Blastp (nr database).
  • NCBI Blastp nr database
  • the genes' presence in 7 commercially available probiotics were also assessed.
  • the screened probiotics' genomes are Bifidobacterium longum 35624, Lactobacillus helveticus R0052, Lactobacillus reuteri SD2112/ATCC 55730, Lactobacillus rhamnosus GG, L. rhamnosus R0011, S. salivarius K12, and S. salivarius Ml 8.
  • UTP-glucose-1- UDP-glucose Elyaluronic acid capsule is a virulence factor
  • the protein may act
  • ACP dehydratase (100%) ACP dehydratase 42 (6 bacteria. Not related to virulence in [Streptococcus sp.] [LPS - B. (50.8%) /7) Streptococcus.
  • Probiotics have the ability to prevent disease while maintaining or restoring a healthy microbiota, and thus have become an attractive method as a preventative treatment in oral diseases.
  • a major beneficial action of probiotics is their ability to protect the host against pathogen colonization.
  • This study has indicated that at least two independent methods of protecting against pathogens exists in S. salivarius DB-B5: the secretion of an antimicrobial molecule that actively kills oral pathogens, including Po. gingivalis, Pr. shahii , F. nucleatum, L. wadei, and Pe. stomatis, and a mechanism that allows S. salivarius DB-B5 to outcompete S. mutans and inhibit its growth when grown together.
  • S. salivarius DB-B5 showed an extremely high cell surface hydrophobicity of 98.70%.
  • a high cell surface hydrophobicity is used as a proxy for the bacteria's ability to be able to colonize the host, and thus, is a desirable trait in probiotics.
  • probiotics for oral health the probiotics must adhere to various surfaces inside the mouth. Depending on the mode of delivery, the oral probiotics will have less time to colonize the oral environment, compared to the time gut probiotics have to colonize the gastrointestinal tract. Thus, possessing a high cell surface hydrophobicity is a desirable characteristic in oral probiotics.
  • S. salivarius DB-B5 Extensive in vitro and in silico safety analysis of S. salivarius DB-B5 was conducted to ensure its suitability for human consumption. Generally, S. salivarius is considered a safe species since it is a normal commensal of the oral microbiota, acquired immediately after birth.
  • Antibiotic resistance and the horizontal transfer of resistance genes is one of the most important crises facing the medical community today.
  • the susceptibility of probiotics to antibiotics is essential if it is to be widely used by consumers.
  • S. salivarius DB-B5 was shown to be susceptible to the various antibiotics tested, which spanned a wide-range of antibiotic classes. The breakpoints to indicate susceptibility was based on the European Food Safety Authority (EFSA), as well as the Clinical and Laboratory Standards Institute (CLSI) documents.
  • EFSA European Food Safety Authority
  • CLSI Clinical and Laboratory Standards Institute
  • VFDB-positive genes were analyzed for their potential roles as virulence factors. All of the genes analyzed were common genes found in most bacteria, such as heat shock proteins, or genes involved in the biosynthesis of surface structures. Some of the genes flagged by VFDB are part of gene clusters involved in the biosynthesis of virulence factors only in specific taxa. For instance, a gene identified as UDP -glucose pyrophosphorylase by VFDB is involved in hyaluronic acid capsule biosynthesis in Group A Streptococcus such as Streptococcus pyogenes.
  • Lactobacillus rhamnosus L8020 was used to ferment a yogurt product, which showed significant decrease in oral pathogen levels, compared to a placebo yogurt [1] A combination of L.
  • rhamnosus GG and Bifidobacterium lactis BB-12 normally used as gut probiotics, was delivered in lozenge format to healthy participants, and was shown to be able to reduce plaque index, gingival index, and decrease levels of gingival pathogens [2]
  • Several clinical trials using Lactobacillus salivarius WB21 -containing tablets have been performed, and the probiotic was shown to decrease periodontal conditions, periodontal pathogens, halitosis, and halitosis-associated pathogens [3-5]
  • S. salivarius ll has been shown to reduce halitosis levels when used in conjunction with chlorhexidine, while S.
  • salivarius M18 usage was shown to reduce cariogram outcome in at-risk children [6,7]
  • the above trials indicate that oral probiotics are able to prevent and treat a wide-range of oral diseases, using a variety of delivery formats.
  • the results of this study thus, indicates that the present oral probiotic strain that can tackle three prevalent oral diseases, e.g. dental caries, periodontal disease, and halitosis.
  • S. salivarius DB-B5 was grown on BHI media (Hardy Diagnostics) at 37 °C under 5%
  • Unicycler (v0.4.8.0) was used to perform a de novo hybrid assembly with 1,722,228 Illumina paired-end reads and 629,563 PacBio long reads, with a genome coverage of -lOOOx (9).
  • the complete genome consists of one circular chromosome (2,143,863 bp) with a GC content of 40.2%, one megaplasmid named pIKMIN-B501 (138,497 bp) with a GC content of 35.6%, one small plasmid named pIKMIN-B503 (3,225 bp) with a GC content of 39.6%, and one linear phage like episome named pIKMIN-B502 (57,714 bp) with a GC content of 39.1%.
  • the genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (10).
  • the genome contains a total of 2,041 protein coding genes, 18 complete rRNA genes, 4 non-coding RNA genes (ncRNA), and 68 tRNA genes.
  • a search of the genome of S. salivarius DB-B5 was conducted to identify bacteriocins using BAGEL4 (http://bagel4.molgenrug.nl/), a web-based genome mining tool for bacteriocins and RiPPs (ribosomally synthesized and post-translationally modified peptides) (11).
  • BAGEL4 http://bagel4.molgenrug.nl/
  • RiPPs ribosomally synthesized and post-translationally modified peptides
  • the S. salivarius DB-B5 strain was deposited at The International Depository Authority of Canada (IDAC) under accession no. 160720-1 (depositor reference, DOSEBIOSYSTEMS- SS DBB5), on July 16, 2020, in accordance with the Budapest Treaty.
  • Proteinase K were added at a lmg/mL concentration and various halitosis-causing bacterial cells were incubated in the protease-containing extract for 2 hours at 37 °C. A control with no protease was also incubated with microbial cells. To all samples was added 10 ul of lOx TRIS, CaCl buffer. Protease only controls were also included (protease, 10 ul of lOx Tris, and 100 ul of phosphate buffer), as well as a heat-inactivated sample (100 ul of DB-B5 extract, boiled at 100 °C for 30 minutes).
  • the active antimicrobial compound against these halitosis-causing bacteria is protease-resistant.
  • S. salivarius DB-B5 was tested for the ability to produce BAs using both an in silico method to detect the presence of genes required for BA production, as well as an in vitro plating method to detect the presence of the BA. In silico results indicated that the genes involved in BA production were not present in the S. salivarius DB-B5 genome as shown in Table 7. Table 7.
  • ATCC 31616 were streaked on decarboxylase media containing different amino acid precursors. S. salivarius growth did not cause a pH color change, whereas E. coli growth caused the decarboxylase media to change to a distinct purple color, indicating the rise of pH to more alkaline conditions due to the presence of biogenic amines.
  • the DB-B5 water was swirled around in the mouth for 15 seconds prior to ingestion. Participants took DB-B5 water twice daily, for 4 weeks.
  • Hydrogen sulfide and methyl mercaptan levels were measured at baseline and 4 weeks using an OralChroma device.
  • Hydrogen sulfide is a volatile sulfur compound that is associated with halitosis-causing bacteria residing on the dorsum of the tongue, and accounts for about 90% of the cause of halitosis.
  • Methyl mercaptan is a volatile sulfur compound that is associated with periodontal disease- causing bacteria.
  • DB-B5 was able to reduce the levels of hydrogen sulfide in the mouth of healthy participants by about 50%, as well as reduced levels of methyl mercaptan as shown in Table 8. Table 8.
  • 'Positive numbers indicates a lower value at week 4 compared to baseline.
  • Negative numbers indicates a higher value at week 4 compared to baseline.

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Abstract

La présente invention concerne une nouvelle souche de Streptococcus salivarius qui présente une activité antimicrobienne, et est, par conséquent, utile pour traiter une maladie. L'activité antimicrobienne de la présente souche de S. salivarius, au moins en partie, est résistante aux protéases. La souche de S. salivarius produit des bactériocines, y compris la subtilosine. La souche de S. salivarius inhibe avantageusement des pathogènes buccaux et, par conséquent, est utile pour traiter ou prévenir une maladie buccale telle que des caries dentaires, une maladie parodontale (par exemple la gingivite et la parodontite), l'halitose et la dysbiose à l'intérieur de la cavité buccale.
PCT/CA2021/051001 2020-07-23 2021-07-20 Probiotique pour la santé buccale Ceased WO2022016266A1 (fr)

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WO2026075575A1 (fr) 2024-10-03 2026-04-09 Instituto Superior Técnico Bioencres électroconductrices, leur procédé d'obtention et leur utilisation, procédé d'obtention d'un produit alimentaire et ledit produit alimentaire

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CN113957006A (zh) * 2021-09-27 2022-01-21 微康益生菌(苏州)股份有限公司 一种植物乳杆菌n13及其在预防或治疗龋齿和牙周炎上的应用
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CN114621895A (zh) * 2022-03-14 2022-06-14 江南大学 一种能够抑制具核梭菌并改善口气的唾液乳杆菌
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CN116574634A (zh) * 2023-03-13 2023-08-11 广东悦创生物科技有限公司 一株唾液链球菌嗜热亚种jf2及其在制备抗炎和解脂食品药品中的应用
CN116574634B (zh) * 2023-03-13 2023-11-03 广东悦创生物科技有限公司 一株唾液链球菌嗜热亚种jf2及其在制备抗炎和解脂食品药品中的应用
CN118685290A (zh) * 2023-03-23 2024-09-24 株式会社绿色商店 新型唾液链球菌菌株以及包含其口腔用组合物
EP4435091A1 (fr) * 2023-03-23 2024-09-25 Greenstore Inc. Nouvelle souche de streptococcus salivarius ayant une activité antibactérienne, antifongique, anti-inflammatoire et inhibant les caries dentaires et composition orale la comprenant
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CN119842518A (zh) * 2024-12-12 2025-04-18 江南大学 一株缓解骨吸收与牙周组织损伤的唾液链球菌ccfm1399及其后生元
CN119842518B (zh) * 2024-12-12 2025-12-30 江南大学 一株缓解骨吸收与牙周组织损伤的唾液链球菌ccfm1399及其后生元

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