WO2023008879A1 - 식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 균주 포자를 포함하는 조성물의 구강 건강 개선을 위한 병용 사용 - Google Patents

식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 균주 포자를 포함하는 조성물의 구강 건강 개선을 위한 병용 사용 Download PDF

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WO2023008879A1
WO2023008879A1 PCT/KR2022/010973 KR2022010973W WO2023008879A1 WO 2023008879 A1 WO2023008879 A1 WO 2023008879A1 KR 2022010973 W KR2022010973 W KR 2022010973W WO 2023008879 A1 WO2023008879 A1 WO 2023008879A1
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Prior art keywords
composition
sod
oral
strain
spores
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PCT/KR2022/010973
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English (en)
French (fr)
Korean (ko)
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반재구
김의중
박영찬
Original Assignee
주식회사 제노포커스
주식회사 바이옴로직
주식회사 벡스퍼트
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Publication of WO2023008879A1 publication Critical patent/WO2023008879A1/ko

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms
    • 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
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/312—Foods, ingredients or supplements having a functional effect on health having an effect on dental health
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/591—Mixtures of compounds not provided for by any of the codes A61K2800/592 - A61K2800/596

Definitions

  • the present invention relates to a composition
  • a composition comprising an extract of Aucuba japonica and superoxide dismutase and/or spores of a Bacillus sp. strain and its use for promoting oral health, maintaining oral hygiene, or preventing or treating oral diseases. .
  • Oral diseases most often begin with poor oral hygiene, leading to tartar, tooth decay, gingivitis, etc., and can be accompanied by severe bad breath.
  • the main causes of bad breath also called halitosis, are periodontal diseases such as gingivitis and periodontitis, odor-causing bacteria on the tongue, certain foods and alcoholic beverages, and smoking. If the food in the mouth is not properly removed, a soft deposit on the tooth surface called plaque is formed, and the plaque becomes tartar over time.
  • Tartar is a mineral in saliva and gingival crevicular fluid within plaque that is calcified and firmly adhered to the tooth surface. Tartar cannot be removed by brushing but can be removed only by scaling.
  • Periodontitis not only causes excruciating pain, but also requires special attention as bacteria can infect the heart or kidneys.
  • antibacterial agents include antibiotics.
  • antibiotics can cause the appearance of resistant bacteria in the oral cavity along with systemic side effects on our body, long-term use is difficult, so there is a disadvantage in that they must be used as a short-term treatment.
  • antibacterial agents used in mouth fresheners include Sangquinarine, Listerine, and Chlorhexidine. It has the disadvantage of being expensive, and although Listerine has alcohol as its main component and has some bacteriostatic action, it only shows a temporary effect in the oral cavity and may cause harm to tissues when used for a long time.
  • the object of the present invention is to solve the problems of the prior art described above.
  • Another object of the present invention is to improve oral health or manage oral hygiene by using a pine extract, superoxide dismutase and/or Bacillus sp. strain spores together.
  • Another object of the present invention is to prevent or treat oral diseases by using a pine extract, superoxide dismutase and/or spores of a Bacillus sp. strain together.
  • an oral composition comprising at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores.
  • an oral product comprising the composition is provided.
  • compositions for preventing or treating oral diseases comprising the composition.
  • a veterinary composition comprising the composition is provided.
  • a food composition comprising the composition is provided.
  • a feed composition comprising the composition is provided.
  • pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores are administered to the subject at least one selected from the group consisting of, or the subject.
  • SOD superoxide dismutase
  • Bacillus sp. strain spores are administered to the subject at least one selected from the group consisting of, or the subject.
  • composition according to the present invention can be usefully used for preventing or treating oral diseases such as bad breath, calculus, plaque, dental caries, gingivitis or periodontitis without causing side effects, as well as reducing pain caused by such oral diseases. can be usefully used for
  • Figure 1 shows the results of measuring the degree of edema and redness of the gums when a beagle dog with gingivitis according to Experimental Example 1.2 was treated with a cedar extract.
  • Figure 2 shows the results of measuring the degree of alveolar bone destruction when the cedar extract was administered to rats induced with periodontitis according to Experimental Example 1.3.
  • Figure 3 shows the criteria for converting the calculus index into a score.
  • Figure 4 shows the criteria for converting the gingivitis index into a score.
  • Figure 6 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 2.4. * indicates P ⁇ 0.05 versus week 0.
  • FIG. 7 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 2.4.
  • FIG. 10 is a graph showing the results of the halitosis index observed after administration of a test substance according to Experimental Example 3.4.
  • 11 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 3.4. * indicates P ⁇ 0.05 versus week 0, ** indicates P ⁇ 0.01 versus week 0.
  • Figure 12 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 3.4. * indicates P ⁇ 0.05 versus week 0.
  • FIG. 13 is a graph showing the results of the gingivitis index observed after administration of the test substance according to Experimental Example 3.4. ** indicates P ⁇ 0.01 versus week 0.
  • 16 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 4.4. * indicates P ⁇ 0.05 versus week 0.
  • 17 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 4.4. * indicates P ⁇ 0.05 versus week 0, ** indicates P ⁇ 0.01 versus week 0.
  • Figure 19 shows the overall procedure for preparing a recombinant production strain expressing SodA2 (BSBA310).
  • rrnB T1T2 represents a transcription terminator
  • rep(pBR322) represents the replicon from pBR322 that works in E. coli
  • rep(pUB110) is a replicon from pUB110 that works in B. subtilis
  • KanR stands for kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase)
  • the BJ27 promoter represents a strong promoter for B. subtilis.
  • subject is used interchangeably with “patient”, and is a mammal in need of oral hygiene or prevention or treatment of oral disease, such as a primate (eg, human, monkey, chimpanzee, etc.), companion It may be an animal (eg dog, cat, etc.), a domestic animal (eg cow, pig, horse, sheep, goat, etc.) or a laboratory animal (eg rat, mouse, guinea pig, etc.).
  • primate eg, human, monkey, chimpanzee, etc.
  • companion It may be an animal (eg dog, cat, etc.), a domestic animal (eg cow, pig, horse, sheep, goat, etc.) or a laboratory animal (eg rat, mouse, guinea pig, etc.).
  • treatment generally means obtaining a desired pharmacological and/or physiological effect.
  • This effect has a therapeutic effect in terms of partially or completely curing diseases and/or other unwanted or undesirable conditions (such as halitosis, tartar, plaque, dental caries, gingivitis, periodontitis, pain, etc.).
  • Desirable therapeutic effects include preventing occurrence or recurrence of the disease, amelioration of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or amelioration.
  • Prognosis includes, but is not limited to.
  • treatment may refer to medical intervention for a pre-existing disease or disorder.
  • prevention means obtaining a desired prophylactic pharmacological and/or physiological effect in terms of partially or completely preventing a disease or symptom thereof.
  • administration means providing an active ingredient to achieve a prophylactic or therapeutic purpose to a subject.
  • the present invention partially applies superoxide dismutase (SOD) or Bacillus sp. strain spores in combination with cedar extract to the oral cavity of a subject, or superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract. It is based on the finding that the spores, when applied to the oral cavity of a subject, are effective in promoting oral hygiene or preventing or treating oral diseases. Accordingly, according to one aspect of the present invention, there is provided an oral composition comprising (i) a cedar extract and (ii) superoxide dismutase (SOD), Bacillus sp. strain spores, or SOD and Bacillus sp.
  • SOD superoxide dismutase
  • SOD superoxide dismutase
  • Bacillus sp. strain spores or SOD and Bacillus sp.
  • the composition may include either superoxide dismutase or spores of a Bacillus sp. strain in combination with a pine extract.
  • the composition may include superoxide dismutase and spores of a Bacillus sp. strain in combination with a pine extract.
  • Aucuba japonica is a kind of evergreen plant of the dicotyledonous umbel dogwood family, and is mainly distributed in the southern part of Korea, especially in the Ulleungdo region. In other words, it is also referred to as Doyeop Coral, Cheongakpan, or Blue Tree.
  • extract refers to a liquid component obtained by immersing a desired material in various solvents and then extracting at room temperature, low temperature or elevated temperature for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It means the result of solid content, etc.
  • it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.
  • the pine tree extract may be an extract extracted from the whole of the aerial part of a pine tree, a part thereof, or a material derived therefrom.
  • the pine extract is preferably a hot water extract, but is not limited thereto.
  • the part of the tree may be a stem, leaf, flower, petal or seed of the tree.
  • the pine extract is preferably extracted from leaves, stems or mixtures thereof of pine trees, but is not limited thereto.
  • Whole, part of, or material derived therefrom used for extraction may be ground or shredded or suitably dried.
  • the extraction solvent is not particularly limited, and for example, water or an organic solvent may be used, and the organic solvent includes methanol, ethanol, propanol, isopropanol, alcohol having 1 to 4 carbon atoms including butanol, acetone, ether, and benzene. , Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination, but are not limited thereto.
  • any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used, preferably hot water extraction.
  • one or more of the above extraction methods may be used in combination.
  • the desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method.
  • any known method may be used.
  • the pine extract may be a hot water extract of pine tree, and 3 to 20 times the weight of the medicinal material is added, preferably 5 to 15 times the water, and the temperature is 80 to 120 °C, preferably 85 to 110 °C, more Preferably, it may be extracted at a temperature of 90 to 105 ° C. for 1 to 24 hours, preferably for 2 to 12 hours, and more preferably for 3 to 8 hours, but the hot water extraction method is not limited thereto.
  • the extract may be subjected to additional processes such as filtration, concentration, and drying as necessary after the extraction.
  • additional processes such as filtration, concentration, and drying as necessary after the extraction.
  • the method and conditions of the additional process are not particularly limited, and may be performed by a method known in the art or under commonly performed conditions.
  • the final concentration of the pine tree extract may be 100 to 50,000 mg/kg, preferably 5,000 to 25,000 mg/kg, but is not limited thereto.
  • Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of superoxide (O 2 ⁇ - ) radicals into ordinary molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ).
  • SOD plays an important role in reducing oxidative stress by scavenging reactive oxygen species.
  • SODs are widely distributed in prokaryotic and eukaryotic cells and are classified into four classes according to the different types of metal centers (copper/zinc, nickel, manganese and iron).
  • Manganese-containing SOD [Mn-SOD] is widely present in the chloroplasts, mitochondria, and cytoplasm of many bacterial or eukaryotic cells.
  • SOD may be used interchangeably with (poly)peptide having superoxide dismutase activity.
  • SOD may include a polypeptide having superoxide dismutase activity or a fragment thereof, or a fusion product containing the same.
  • the SOD can be manganese bound (Mn-SOD).
  • the SOD may be deamidated Mn-SOD.
  • the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 2 (SodA).
  • SOD may be one in which amino acid residues at positions 74 and 137 of SEQ ID NO: 2 are substituted with Asp.
  • the SOD may include or consist of the amino acid sequence represented by SEQ ID NO: 4 (SodA2).
  • the SOD or polypeptide having SOD activity of the present invention also includes an amino acid sequence showing substantial identity to the above amino acid sequence.
  • the substantial identity is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more when the aligned sequences are analyzed using an algorithm commonly used in the art. Means an amino acid sequence that exhibits identity.
  • the SOD is a modified or engineered polypeptide having SOD enzymatic activity that does not affect or affect various aspects (in vivo, in vitro or ex vivo stability, uniformity, and/or morphological alteration). may include one or more mutations, such as deletions, insertions or substitutions of one or more amino acids.
  • the polypeptide may be added with a heterologous material (eg, a tag known in the art, including a HIS tag, an HA tag, a myc tag, a GFC and/or an Fc domain of an antibody) for purification, detection, in vivo delivery, or increased stability. can be included with a heterologous material (eg, a tag known in the art, including a HIS tag, an HA tag, a myc tag, a GFC and/or an Fc domain of an antibody) for purification, detection, in vivo delivery, or increased stability. can be included with a heterologous material (eg, a tag known in the art, including
  • the SODs of the present invention may be derived from a variety of sources including natural, mutant or recombinant microorganisms.
  • SOD may be derived from bacteria.
  • the SOD may be derived from a bacterium that is generally regarded as safe (GRAS) for use in drugs or foods, such as a Bacillus sp. strain or variant thereof.
  • GRAS regarded as safe
  • SOD may be obtained from a Bacillus amyloliquesfaciens strain (eg, GF423 strain or GF424 strain) or a culture supernatant thereof.
  • the GF423 strain and the GF424 strain were deposited with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and March 13, 2017, respectively (accession numbers KCTC 13222 BP and accession numbers KCTC 13227 BP, respectively).
  • the Bacillus amyloliquefaciens GF424 strain is a strain obtained by mutating the Bacillus amyloliquefaciens GF423 strain by UV irradiation in order to improve sod gene expression.
  • the SOD enzyme (SodA) derived from the Bacillus amyloliquefaciens GF423 or GF424 strain is Mn-SOD and has the amino acid sequence of SEQ ID NO: 2 (its nucleotide sequence is shown in SEQ ID NO: 1). Additionally, the SOD may be a recombinant polypeptide. For example, based on SEQ ID NO: 2, amino acid residues at positions 74 and 137 may be deamidated SOD in which Asp is substituted (SodA2), which has the amino acid sequence of SEQ ID NO: 4 (its nucleotide sequence is SEQ ID NO: marked 3). The sequences of SEQ ID NOs: 1 to 4 are as shown in Table 1.
  • SOD may be derived from other recombinant strains (eg, Bacillus Subtilis species strains) containing the SOD expression gene of the Bacillus amyloliquefaciens strain.
  • the recombinant strain may be prepared by recombinant technology using a conventional protein production strain known in the art.
  • the Bacillus subtilis strain (the parent strain of the recombinant strain) may be KCTC 3135, and the KCTC 3135 strain may be distributed from the Korea Research Institute of Bioscience and Biotechnology (KCTC).
  • KCTC Korea Research Institute of Bioscience and Biotechnology
  • one or more of the genes shown in Table 2 may be removed from the recombinant strain to facilitate post-processing.
  • the recombinant strain may be prepared through the process shown in FIG. 19 .
  • the recombinant strain may include the expression vector shown in FIG. 20 .
  • sodA and sodA2 represent genes encoding SOD.
  • SOD derived from the strain is an enzyme secreted outside the cell, when manufacturing SOD using the strain, it is possible to mass-produce SOD that is safe for individuals without going through an expensive purification process (eg, column purification). Because of this, efficient production is possible.
  • the SOD of the present invention can be obtained by culturing the natural, mutant or recombinant microorganism in various culture media.
  • SOD included in the oral composition may be isolated from the culture supernatant of Bacillus amyloliquefaciens strain GF423 or strain GF424.
  • a culture solution may be obtained by culturing the Bacillus amyloliquefaciens strain in various types of media.
  • the strain is grown at about 25° C. to about 42° C. for about 1 day to about 4 days using a complex medium (pH 6.0 to 7.0).
  • LB medium Luria-Bertani (LB) medium, International Streptomyces Project (ISP) medium, nutrient agar (NA) medium, brain heart infusion agar (BHI) medium, SDA (sabouraud dextrose agar) medium, PDA (potato dextrose agar) medium, NB (nutrient broth) medium, and the like.
  • ISP International Streptomyces Project
  • NA nutrient agar
  • BHI brain heart infusion agar
  • SDA sabouraud dextrose agar
  • PDA potato dextrose agar
  • NB nutrient broth
  • SODs can also be sourced from other natural, mutant or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).
  • the SODs of the present invention may be isolated or purified from cultures of natural, variant or recombinant strains. wherein the isolated or purified SOD or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it was derived.
  • the purified product may be a pure product separated from a strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like, or a culture concentrate obtained through ultrafiltration, concentration, or the like.
  • substantially free of cellular material includes preparations of the protein in which the protein is isolated from cellular components of the cell from which the protein is isolated or recombinantly produced.
  • the phrase “substantially free of cellular material” means less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 10% by dry weight of unwanted proteins. Preferably it includes preparations of less than about 5% protein.
  • the SOD may be preferably purified by the following purification method, but is not limited thereto.
  • the culture supernatant is collected by centrifuging the culture solution obtained by previously culturing the Bacillus amyloliquefaciens strain.
  • the supernatant fraction is pretreated by solid phase extraction, then isolated and purified by chromatography.
  • SOD can be purified using a variety of methods of chromatography. Preferably, hydrophobic interaction chromatography is used.
  • the SOD may be included in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof.
  • strain lysate means obtained by mechanically or chemically disrupting the strain by culturing it, and may include all of the additional processes such as extraction, dilution, concentration, and purification therefrom.
  • strain culture may refer to a culture medium itself or a supernatant thereof obtained by culturing a strain.
  • strain culture concentrate refers to a culture concentrate obtained by ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like from a strain culture, or obtained through ultrafiltration, concentration, or the like.
  • strain culture extract refers to an extract obtained from the culture medium or a concentrate thereof, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof, or a fraction thereof.
  • the dried form may include a freeze-dried form.
  • the SOD may include cellular material from the cell or tissue source from which it was derived, such as extracellular vesicles.
  • the cellular material containing SOD can be separated from the culture solution by filtration, concentration, etc. by culturing various sources including natural, mutant or recombinant hosts using conventional techniques known in the art as described above. can
  • the spores of the Bacillus species strain may be obtained by culturing the Bacillus species strain in a suitable medium and isolating the spores produced after inducing sporulation.
  • spores of Bacillus sp. strains may be sourced from GRAS bacteria generally considered safe for use in drugs or foods.
  • Bacillus species strain spores are known to be resistant to proteases and low pH (Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28:214-220.
  • spores of strains of Bacillus species are GRAS probiotics approved in several countries.
  • the spores of the Bacillus species strain may be derived from a Bacillus amyloliquefaciens strain (eg, GF423 or GF424 strain). Sporulation can be induced using conventional techniques known in the art.
  • spores of a strain obtained by inducing sporulation in a medium such as DSM, NB, SYP, LB2, etc. during main culture after pre-cultivation of the strain various sources including natural, mutant, or recombinant hosts are cultivated to produce feeder cells After removal, it can be separated through filtration, concentration, or centrifugation.
  • pine extract and the SOD enzyme and / or Bacillus sp. strain spores may be included in the composition in a form such that they can be administered sequentially, in reverse order or simultaneously with each other.
  • the oral composition may include pine extract, SOD, and Bacillus sp. strain spores, or may be included in a form in which they may be used in combination.
  • prevention of bad breath, calculus, dental plaque, dental caries or periodontal disease (eg, gingivitis or periodontitis) when a mixture of pine tree extract, SOD, and Bacillus species strain spores is applied to the oral cavity. , it was confirmed that it was effective for improving or treating oral health, oral hygiene management, or preventing or treating oral diseases. In addition, it was confirmed that the mixture was effective in reducing pain due to oral diseases.
  • the pine extract, SOD and spores of the Bacillus sp. strain may be mixed in an appropriate ratio to exert the effect.
  • the pine tree extract has an amount corresponding to a dose of 0.1 to 10,000 mg/kg, preferably 1 to 1,000 mg/kg, more preferably 1 to 100 mg/kg, and still more preferably 5 to 50 mg/kg per day.
  • SOD and Bacillus sp. strain spores are mixed in a ratio such that the SOD titer is 5,000 to 20,000 U per g dry weight and the spores are 0.1 x 10 ⁇ 10 to 1 x 10 ⁇ 11 cfu per g dry weight.
  • the final composition may be, for example, a mixture of 0.5% to 5% of Japanese oak extract and 0.5% to 12% of SOD and/or spores of a Bacillus species strain.
  • the final composition may preferably be a mixture of 2% of cedar extract and 2.5% of SOD and/or spores of a Bacillus species strain.
  • the composition can be used for oral health promotion or oral hygiene management.
  • the composition can be used for preventing or treating oral diseases.
  • the composition can be used to prevent, ameliorate, or treat one or more diseases selected from halitosis, tartar, plaque, dental caries, periodontal disease, and stomatitis (eg, chronic stomatitis).
  • diseases selected from halitosis, tartar, plaque, dental caries, periodontal disease, and stomatitis (eg, chronic stomatitis).
  • the composition can be used to reduce oral pain.
  • the composition may have various oral forms known in the art, including a liquid form, a solid form, a gel form, a powder form, a paste form, or a form impregnated or applied to a carrier.
  • an oral product containing the composition for oral cavity includes at least one selected from the group consisting of (i) pine extract and (ii) SOD and Bacillus sp. strain spores as described above.
  • the product may be one or more selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral patch, and mouthwash, but is not limited thereto.
  • the oral product of the present invention in addition to containing the pine extract and SOD and/or Bacillus sp. strain spores as active ingredients, may contain additional ingredients known in the art required for its formulation.
  • the oral product when the oral product is toothpaste, it may further include an abrasive, a moisturizing agent, a foaming agent, a sweetener, a whitening agent, or a flavoring agent.
  • the oral product when the oral product is an oral gel, it may further include a thickener such as a medium chain fatty acid for viscosity or adhesiveness of the gel.
  • a mouthwash it may further include a carrier such as non-toxic alcohol.
  • a pharmaceutical composition or a veterinary composition comprising a composition comprising at least one selected from the group consisting of pine extract as described above, superoxide dismutase (SOD) and spores of Bacillus sp. is provided.
  • the pharmaceutical composition of the present invention may be used interchangeably with the term veterinary composition when applied to animals other than humans.
  • the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
  • SOD superoxide dismutase
  • Bacillus sp. strain spores in combination with cedar extract.
  • the composition can be used for preventing or treating oral diseases.
  • the spherical disease may be one or more diseases selected from halitosis, calculus, dental plaque, dental caries, periodontal disease, and chronic stomatitis, but is not limited thereto.
  • the composition may be for preventing or treating oral pain.
  • the oral pain may be due to various oral diseases or undesirable conditions.
  • oral pain may be due to plaque, plaque, dental caries, periodontal disease or chronic stomatitis.
  • the pharmaceutical or veterinary composition of the present invention may further include one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents.
  • the pharmaceutically acceptable carrier, excipient and/or diluent may be one commonly used in the art.
  • Carriers, excipients or diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Mineral oils such as cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and silicon dioxide, etc. It may include, but is not limited thereto.
  • additives such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Additives for the formulation may be selected from those commonly used in the pharmaceutical field.
  • the pharmaceutical or veterinary composition of the present invention may be formulated in a preferred form depending on the method of use, and particularly known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated by adopting a method. Specific examples of such formulations include tablets, pills, powders, granules, syrups, solutions, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, limonade, aerosols, and extracts. There are extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, and patches.
  • compositions of the present invention can be prepared using a suitable method known in the art or using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA). can be formulated accordingly.
  • the pharmaceutical or veterinary composition of the present invention may be administered orally or parenterally depending on the desired method, and in the case of parenteral administration, nasal spray, external skin application or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection Injection or intrathoracic injection is preferred.
  • the pharmaceutical or veterinary composition of the present invention may be preferably applied through intraoral injection methods such as oral spray, oral application, and intraoral injection.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the complex composition, for example, starch, calcium carbonate, sucrose, lactose, It may be prepared by mixing gelatin or the like.
  • lubricants such as magnesium stearate and talc may also be used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents.
  • SOD may be coated with a coating such as shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof.
  • a coating such as shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof.
  • the coating agent is not limited thereto.
  • Forms for parenteral administration include toothpastes, mouthwashes, topical formulations (eg, gels, creams, ointments, dressing solutions, sprays, and other coating agents).
  • topical formulations eg, gels, creams, ointments, dressing solutions, sprays, and other coating agents.
  • the active ingredient of the present invention may be impregnated with a carrier such as gauze made of natural or synthetic fibers.
  • the gel, cream or ointment it may be suitable for direct application on or around affected areas including teeth and gums or carious areas.
  • the spray it can be prepared by a conventional spray preparation method, and can be filled and packaged in a compression container or other spray container, and sprayed and applied to the oral disease area from time to time to prevent or treat oral diseases.
  • the dressing solution it can be prepared by a conventional dressing solution manufacturing method, and oral diseases can be prevented or treated by dressing an oral disease site or dressing another bacterial infection site.
  • the pharmaceutical or veterinary composition of the present invention is administered in a pharmaceutically or veterinarily effective amount.
  • pharmaceutically effective amount or “veterinarily effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical or veterinary treatment, and the effective dosage level is the level of the patient or animal. Weight, gender, age, health condition, severity, activity of drug, sensitivity to drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field. can
  • the pharmaceutical or veterinary composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously.
  • the pharmaceutical composition may be administered single or multiple times as needed. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this amount can be easily determined by a person skilled in the art.
  • composition comprising at least one selected from the group consisting of (i) cedar extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above
  • SOD superoxide dismutase
  • the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
  • SOD superoxide dismutase
  • Bacillus sp. strain spores in combination with cedar extract.
  • Such food compositions include medical or nutraceutical food compositions.
  • medical food or “nutraceutical food” refers to food made from raw materials or ingredients that have the potential to have beneficial functions in the human body, which maintain or improve health by maintaining normal functions or activating the physiological functions of the human body. Refers to food, as defined by, but not limited to, the Ministry of Food and Drug Safety, and does not exclude any conventional health food in that sense.
  • the food includes, but is not limited to, various foods, food additives, beverages (eg, functional drinks, natural fruit juice and vegetable drinks), gum, tea, vitamin complexes, health functional foods, and other functional foods. .
  • beverages eg, functional drinks, natural fruit juice and vegetable drinks
  • gum e.g., tea
  • vitamin complexes e.g., vitamin complexes, health functional foods, and other functional foods.
  • the food may be prepared by a conventional method known in the art.
  • the medical food, nutraceutical food, or health functional food further includes at least one of a carrier, diluent, excipient, and additive in addition to the SOD for the purpose of improving oral hygiene or preventing or improving oral disease, tablets, It may be formulated into one selected from the group consisting of pills, powders, granules, powders, capsules and liquid formulations.
  • Specific examples of the carrier, excipient, diluent, and additive are well known in the art, and a person skilled in the art can prepare them by combining appropriate components according to the formulation.
  • the content of the extract of the present invention, superoxide dismutase and/or Bacillus species strain spores as active ingredients in the above-described formulation can be appropriately adjusted depending on the type and purpose of use, patient condition, type and severity of symptoms, etc. And, based on the weight of the solid content, it may be 0.001 to 99.9% by weight, preferably 0.01 to 50% by weight, but is not limited thereto.
  • the dosage of the food of the present invention may vary depending on the patient's age, weight, sex, dosage form, state of health and degree of disease, and divided administration once a day to several times at regular time intervals according to the judgment of a doctor or pharmacist You may.
  • the daily dosage may be 10 to 1,000 mg/kg based on the active ingredient content.
  • the above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dose of the health functional food of the present invention is less than the above dose, a significant effect may not be obtained, and if it exceeds more than that, it is uneconomical and may cause undesirable side effects because it is out of the range of the usual dose. there is.
  • a composition comprising at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above
  • a feed composition is provided.
  • the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
  • the feed composition of the present invention may be prepared in any formulation commonly used in the art.
  • the feed composition of the present invention may include auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes, and other probiotic-type microorganism preparations; grains such as ground or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugar and dairy products, such as various powdered milk and whey powder; main components such as lipids, for example, animal fats and vegetable fats optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventives may be further included.
  • the feed composition of the present invention may be in the form of a powder or liquid formulation, and may include a feed additive excipient (calcium carbonate, horse powder, zeolite, jade powder or rice bran, etc.).
  • a feed additive excipient calcium carbonate, horse powder, zeolite, jade powder or rice bran, etc.
  • administering to a subject at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above
  • a method for improving oral hygiene comprising a.
  • prevention of oral diseases comprising the step of administering to the subject at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above or method of treatment is provided.
  • the method may include administering SOD and Bacillus sp. strain spores to the subject in combination with a cedar extract.
  • the oral disease may include one or more selected from the group consisting of oral pain, halitosis, plaque, calculus, dental caries, periodontal disease, and chronic stomatitis.
  • the pine extract, SOD and Bacillus sp. strain spores when administered in combination, may be administered simultaneously as a single composition or as separate compositions containing each or any two components.
  • the pine extract, SOD and Bacillus sp. strain spores may be administered sequentially or in reverse order as separate compositions containing either or any two components.
  • the pine extract, SOD and Bacillus sp. strain spores may be administered by the same route or by different routes depending on the dosage form. The composition, dosage form, route of administration, etc. are as described above for the composition.
  • the leaves and stems of the cedar tree were collected from the forest in Jisepo-ri, Irun-myeon, Geoje-si, Gyeongsangnam-do in August 2020, and the evidence specimen is stored in the sample storage room of Chonbuk National University.
  • 900 L of distilled water was added to 100 kg of pine tree, shaken at 100 ° C. for 3 hours, concentrated under reduced pressure, and then dried to obtain a hot water extract powder of pine tree.
  • Bacillus amyloliqueurfaciens strains used in this example are the Bacillus amyloliqueurpaciens GF423 strain and the Bacillus amyloliqueurpaciens GF424 strain, and these strains were published by the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and 2017, respectively. It was deposited with accession number KCTC 13222 BP and accession number KCTC 13227BP as of March 13, 2018.
  • the Bacillus amyloliquefaciens GF424 strain is a strain obtained by inducing mutation by irradiating UV to the Bacillus amyloliquefaciens GF423 strain in order to improve the expression of the sodA gene.
  • Example 2.1 Bacillus amyloliquefaciens Cultivation of GF423 or GF424 strains
  • LB agar medium Lia-Bertani (LB) agar; tryptophan 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L
  • MnSO 4 manganese sulfate
  • the cell culture medium obtained in Example 2.1 was centrifuged at 4° C. and 3,578 x g for 20 minutes to obtain a supernatant, and then concentrated 10 times using ultrafiltration (UF, MWCO 10,000). The concentrated solution was filtered through a sterilization filter and lyophilized. The activity of SOD was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide radicals by 50%. The activity of the dried SOD enzyme was 30 ⁇ 6 U/mg.
  • the medium used in this example was SYP or DSM.
  • SYP medium contains 1.5% soy tone, 0.5% yeast extract, 0.5% K 2 HPO 4 , 0.1% MnSO 4 , 0.1% MgSO 4 , 10 mM FeSO 4 , 0.04% (NH 4 ) 2 SO 4 , 0.04% % (NH 4 ) 2 PO 4 , 0.1% CaCl 2 and 2% glucose.
  • the DSM medium contains Bacto nutrient broth 8 g/L, KCl 1 g/L, MgSO 4 0.25 g/L, Ca(NO 3 ) 2 0.16415 g/L, MnCl 2 0.9521 mg/L and FeSO 4 0.152 mg/L. .
  • MnSO 4 , MgSO 4 , FeSO 4 , (NH 4 ) 2 SO 4 , (NH 4 ) 2 PO 4 and CaCl 2 were dissolved in ddH 2 O and added before use.
  • a single colony of Bacillus amyloliquefaciens strain GF423 or GF424 was inoculated into 1 mL of LB in a 14 mL tube and incubated at 37° C. and 200 rpm for 12 hours.
  • 1 mL of the culture medium was transferred to 50 mL of LB medium in a 500 mL flask, and cultured at 37° C. and 200 rpm for 12 hours.
  • 20 mL of culture medium was transferred to 1 L of SYP or DSM in a 2.5 L baffled flask.
  • the inoculated culture was cultured at 37° C. and 200 rpm for 24 to 120 hours.
  • lysozyme (0.5 g/L) was added to the culture broth and incubated for 1 hour at 37° C. and 200 rpm to remove remaining feeder cells. Crude spores were harvested by centrifugation at 6000 rpm for 10 minutes. The harvested crude spores were purified by washing twice with water, then with 0.02% SDS, again with water twice, and then suspended in a PBS solution. The spore suspension was stored at -20 °C. The number of spores was determined by plating the diluted spore solution on LB agar plates and then counting colonies.
  • a test using beagle dogs or rats was performed to determine whether the pine tree extract exhibited oral health promotion and oral hygiene management effects.
  • Test materials were prepared as follows. An oral ointment containing 2% of the pine extract obtained in Example 1 (test group) and an oral ointment not containing the pine extract (control group, vehicle only) were prepared with the compositions shown in Table 3 below.
  • Test group contains 2% cedar extract
  • Control cedar extract 200 mg - Mucoadhesive (Sodium polyacrylate, PAA-Na) 300mg 300mg
  • Flavor enhancer Meltol 100 mg 100 mg Preservative (Methyl paraben) 18mg 18mg Preservative (Poly paraben) 2 mg 2 mg
  • the test substance of Experimental Example 1.1 was applied and administered to the gums (external gingival region of both maxillary molars) twice daily for 4 weeks.
  • the dosage was administered at 1.0 g/day by 0.5 g per time.
  • the content of the active ingredient contained in 1.0 g/day was 20 mg/day.
  • an oral examination was performed and the gingivitis index was evaluated according to the criteria shown in Table 4 below.
  • the gingivitis index deteriorated over time in the beagle dog administered with the control (vehicle) not containing the pine extract, whereas the oral ointment containing the pine extract was administered.
  • the gingivitis index was reduced by about 15%.
  • Anesthesia was induced by intraperitoneal injection of a mixture of zoletin and ketamine at a ratio of 1:2 (1.5 ml/kg) to the rats after the acclimatization process was completed.
  • periodontitis was induced by ligating the cervical region of the lower left second molar with a silk suture (0.5 mm).
  • a mandibular right second molar that was not ligated with a suture was set as a control group.
  • the day of inducing periodontitis was set as day 0, and the rats were sacrificed 2 weeks after the day of inducing periodontitis.
  • pine extract test substance in Experimental Example 1.1
  • DW vehicle
  • the rats were sacrificed and the mandibles were removed. Thereafter, all soft tissues were removed by treatment with 1M NaOH for about 1 hour, and the degree of alveolar bone loss was quantified by measuring the length from the cemento-enamel junction to the crest of the alveolar bone.
  • pine extract was useful for improving oral diseases such as gingivitis and destruction of the alveolar bone.
  • a test using beagle dogs was performed to determine whether a mixture of superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spores exhibits oral health promotion and oral hygiene management effects.
  • SOD superoxide dismutase
  • Bacillus amyloliquefaciens strain spores exhibits oral health promotion and oral hygiene management effects.
  • Superoxide dismutase (SOD) and Bacillus amyloliqueurfaciens strain spores were purified from Bacillus amyloliqueurfaciens GF424 in Example 2 and superoxide dismutase (SOD) prepared in Example 3. It was prepared by mixing the spores of the amiloliquefiaciens GF423 strain (2.5% mixture, SOD 10,000 U/g).
  • the mixture is adjusted to a desired viscosity by dissolving a medium-chain fatty acid such as lauric acid for maintaining the viscosity / adhesion of oral gel at room temperature, mixing tasty ingredients such as betaine, oligosaccharide, and egg yolk, and finally It was obtained by homogeneously mixing SOD and 2.5% of spores (SOD titer: 12,510 U/g, spores: 1.2 x 10 ⁇ 10 CFU/g). This mixture (SOD + spores) was used in a tube in the form of oral gel, which is a gel type (20 g/tube, 250 U/g gel).
  • a breeding room equipped with environmental conditions of temperature 23 ⁇ 3 °C, relative humidity 30 ⁇ 10 %, ventilation frequency 10 to 15 times / hour, lighting 12 hours, illumination intensity 150 to 200 Lux was used, and during the period of purification and testing, stainless steel was used. They were raised individually in cages, one per cage. Feed and drinking water were provided autonomously.
  • Test groups G1 (“G1_Vehicle”) and G2 (“G2_BASOD”) were constructed as follows.
  • test substance of Experimental Example 2.1 was applied to the gums (external gingival region of both maxillary molars) and administered every day for 4 weeks.
  • the dose was 0.25 g (SOD 62.5 U)/head twice a day.
  • Example 2 In order to confirm that the purified superoxide dismutase (SOD) in Example 2 exhibits oral health promotion and oral hygiene management effects, a test using a cat was performed.
  • SOD superoxide dismutase
  • Test materials used were prepared in the same manner as in Experimental Example 2.1, except for the spores of the Bacillus amyloliquefaciens GF423 strain.
  • test group G1 the breed is mixed, KSH (Korean short hair), Exotic, or Siamese, gender is neutered male (NM) or neutered female (SF), and age is 2 to 10 was three
  • test group G2 the breed was T.A. (Turkish Angora) or KSH (Korean short hair), gender was neutered male (NM) or neutered female (SF), and the age was 1 to 11 years.
  • test substance of Experimental Example 3.1 was applied and administered to the gingival region of the right maxillary molar every day for 4 weeks.
  • the dosage was 0.3 g (SOD 75 U)/head twice a day.
  • Feed intake of the test cats was evaluated at week 0 (before the start of administration), week 2, and week 4 (see table below).
  • the halitosis index did not change until the end of the test, but in the SOD group, it was confirmed that the halitosis index continued to decrease (FIG. 10).
  • the plaque index significantly increased during the test period, whereas in the SOD group, the plaque index decreased with statistical significance from the 2nd week to the end of the test (FIG. 11).
  • a test using cats was performed to determine whether a mixture of pine extract, superoxide dismutase (SOD), and spores of Bacillus amyloliquefaciens strain showed oral health promotion and oral hygiene management effects.
  • SOD superoxide dismutase
  • Test materials were prepared as follows. An oral gel containing a mixture of pine extract powder obtained in Example 1, SOD prepared in Example 2, and spores prepared in Example 3 was prepared with the composition shown in the table below.
  • the mixture dissolves pine extract powder in purified water at 60 ° C. for 1 hour, dissolves carboxymethylcellulose and sodium polyacrylate for maintaining the viscosity / adhesion of oral gel at room temperature, and contains vitamins, oligosaccharides, and egg yolk powder. After mixing such tasty ingredients, it was prepared by adding superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spore mixture at room temperature. Finally, the oral gel containing the above 2% pine tree extract and 2.5% SOD and spore mixture (SOD titer: 250 U/g, spores: 2 x 10 ⁇ 8 CFU/g) was put into a tube and used.
  • SOD superoxide dismutase
  • spore mixture 2 x 10 ⁇ 8 CFU/g
  • Test groups G1 (“G1_Vehicle”) and G2 (“G2_Vehicle+BASOD”) were constructed as follows.
  • test group G1 the breed is mixed, KSH (Korean short hair), Exotic, or Siamese, gender is neutered male (NM) or neutered female (SF), and age is 2 to 10
  • test substance of Experimental Example 4.1 was applied and administered to the gingival region of the right maxillary molar every day for 4 weeks.
  • the dose was administered twice a day at 0.3 g/head once.
  • Feed intake of the test cats was evaluated at week 0 (before the start of administration), week 2, and week 4 (see table below).
  • test group G1 activity was significantly improved from the 2nd week, and in test group G2, activity gradually increased, but there was no statistical significance (data not shown).
  • test group G1 there was little change in palpation pain until the end of the test, but in test group G2, the palpation pain index significantly decreased with statistical significance from 2 weeks after administration (FIG. 14).
  • test group G1 the halitosis index did not change until the end of the test, but in test group G2, the halitosis index decreased by about 50% compared to week 0 at the 2nd week of administration, and the bad breath was statistically significantly improved until the 4th week (FIG. 15) .
  • test group G1 the plaque index significantly increased during the test period, whereas in test group G2, the plaque index gradually decreased until the end of the test, although there was no statistical significance (FIG. 16).
  • test group G1 there was no significant change in the gingivitis index, but in test group G2, the gingivitis index decreased statistically significantly from week 2 to week 4 (FIG. 18).
  • a mixture of pine extract and SOD and spores as test substances was applied to the cat's gums twice daily at a dose of 0.3 g/head once. It was confirmed that the index, plaque index, calculus index, and gingivitis index all decreased. In particular, symptoms of palpable pain, halitosis, calculus, and gingivitis were improved statistically significantly compared to 0 weeks. In addition, it was confirmed that the test substance was not harmful because no special adverse reaction was observed in the entire test process.
  • the mixture of test substances, pine extract and SOD, and spores is not only useful for improving oral diseases such as bad breath, plaque, tartar and gingivitis, but also reduces pain caused by such oral diseases. It was also found to be useful.

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PCT/KR2022/010973 2021-07-27 2022-07-26 식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 균주 포자를 포함하는 조성물의 구강 건강 개선을 위한 병용 사용 WO2023008879A1 (ko)

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