WO2020063531A1 - 副干酪乳杆菌et-22及其用途 - Google Patents

副干酪乳杆菌et-22及其用途 Download PDF

Info

Publication number
WO2020063531A1
WO2020063531A1 PCT/CN2019/107274 CN2019107274W WO2020063531A1 WO 2020063531 A1 WO2020063531 A1 WO 2020063531A1 CN 2019107274 W CN2019107274 W CN 2019107274W WO 2020063531 A1 WO2020063531 A1 WO 2020063531A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
strain
subject
oral
cfu
Prior art date
Application number
PCT/CN2019/107274
Other languages
English (en)
French (fr)
Inventor
刘伟贤
洪维鍊
孙婷
司徒文佑
赵雯
Original Assignee
内蒙古伊利实业集团股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201811147290.7A external-priority patent/CN110960559B/zh
Priority claimed from CN201811147289.4A external-priority patent/CN110964653B/zh
Priority claimed from CN201811164616.7A external-priority patent/CN110964658B/zh
Application filed by 内蒙古伊利实业集团股份有限公司 filed Critical 内蒙古伊利实业集团股份有限公司
Priority to US17/259,137 priority Critical patent/US11274275B2/en
Priority to EP19865158.0A priority patent/EP3822338A4/en
Publication of WO2020063531A1 publication Critical patent/WO2020063531A1/zh

Links

Images

Classifications

    • 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
    • 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, e.g. protozoa; 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/381—Microorganisms
    • 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, e.g. protozoa; 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
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/165—Paracasei
    • 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
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • 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
    • C12R2001/225—Lactobacillus

Definitions

  • the invention relates to the technical field of microorganisms, in particular to Lactobacillus paracasei and uses thereof, for example, to promote the growth of bifidobacteria and lactic acid bacteria, to promote humoral immunity and NK cell killing activity, and to inhibit oral pathogenic bacteria.
  • Probiotics refer to living microorganisms that can be added to a composition, maintain activity, and exert a physiological effect on a subject ingesting a composition containing the probiotics.
  • the World Health Organization's definition of probiotic products is that food contains a sufficient number of live microorganisms, and through various processes of food processing, and after entering the human intestine, it can still maintain an appropriate number of live bacteria and bacterial activity. Therefore, the strain should be able to maintain a relatively stable number of viable bacteria after the production of bacterial powder, production and processing of products, and gastric acid and bile salt stress in the human gastrointestinal tract.
  • the balance of intestinal flora is closely related to human health.
  • the intestinal flora includes beneficial bacteria, neutral bacteria and harmful bacteria.
  • Beneficial bacteria in the healthy human intestine occupy a dominant position, and they constantly interact with harmful bacteria to maintain human health. If the beneficial bacteria in the intestine are reduced due to various factors, harmful bacteria will multiply, and the microecological balance in the intestine will be broken, causing various clinical symptoms such as enteritis and diarrhea.
  • a large number of antibiotics are usually used clinically. Although they can effectively kill harmful bacteria, they also kill beneficial bacteria at the same time, causing imbalance of intestinal flora and causing diseases.
  • oral probiotic supplementation is a direct and effective way to regulate the intestinal flora.
  • Oral supplementation of probiotic preparations or products containing probiotics can directly or indirectly regulate the composition of the intestinal flora, activate the host's endogenous microbiota or immune system activity to achieve probiotic effects.
  • Immune is an important physiological function of the human body. It involves non-specific immunity and specific immunity. Non-specific immunity does not require prior contact with the antigen. Once the pathogen enters the body, it will quickly clear the pathogen. Both phagocytic cells and natural killer cells have clearance and killing effects and are important members of the non-specific immune system. Specific immunity has immune cells (T lymphocytes and B lymphocytes) that specifically recognize foreign bodies and have immune memory capacity. The most important information transmitter between the immune systems is cytokines. Each cytokine has different stimulating functions for different cell types. They bind to specific receptors on the cell surface and induce cell growth and functional activity.
  • the human body relies on this function to recognize "self” and “non-self” components, thereby destroying and repelling the antigenic substances entering the human body or the damaged cells and tumor cells generated by the human body to maintain human health, resist or prevent microbes or send Infection by organisms or invasion of other unwanted organisms.
  • Baby teeth care for young children will affect the permanent teeth of the future, so from the beginning of the baby, oral cleanliness is very important. If there is bacteria in the oral cavity, the bacteria will easily adhere to the gum surface, and after the baby teeth grow, they will directly attach to the baby's baby teeth and cause dental caries.
  • the deciduous teeth are the foundation of all teeth. The deciduous teeth can maintain the proper space and distance to provide future permanent tooth growth. If the deciduous teeth are not taken care of, they will cause caries, which will lead to serious loss of space. In the future, when the permanent teeth develop, the permanent teeth will be surrounded by bacteria and pustules. As a result, the newly-grown teeth are more likely to form caries, and even affect Incidence of periodontal disease in adulthood and old age.
  • lactic acid bacteria Generally, the products containing lactic acid bacteria (LAB) only have the effect of regulating intestinal health. Although tens of thousands of lactic acid bacteria strains exist in nature, only a small number of lactic acid bacteria strains have been found to have potential auxiliary characteristics for maintaining oral health and whitening teeth. The ability of these strains to inhibit oral pathogenic bacteria, the ability to adsorb oral epithelial cells, and whether they can still maintain the ability to inhibit pathogenic bacteria and produce hydrogen peroxide in dentifrice products, are to stimulate the strains to exhibit functions in the oral cavity and control the oral cavity. The micro-ecological balance and screening have important basis for maintaining oral health and whitening dental functional strains.
  • probiotics can compete with pathogenic bacteria for their attached living areas and nutritional requirements, promote the aggregation characteristics of pathogenic bacteria, and secrete bacteriostatic substances to make pathogenic bacteria weak or even kill pathogenic bacteria, in order to reduce the number of oral pathogenic bacteria and maintain oral health. , And the purpose of secreting hydrogen peroxide to remove oral odor and whiten teeth.
  • the ET-22 strain (deposited as CGMCC No. 15077) alone has a variety of beneficial activities, including, for example, the ability to significantly promote the growth of intestinal bifidobacteria and lactic acid bacteria, which can tolerate in vitro simulated gastrointestinal fluid Stress environment; significantly enhance humoral immunity, activate NK cell activity, including, for example, increasing the number of antibody-producing cells, half of the hemolytic value HC 50 , activating NK cell activity, can improve the problem of low immune capacity caused by irregular diet and increased work pressure ; Inhibits oral pathogenic bacteria and has a peroxide-generating activity, and can be used for, for example, tooth whitening.
  • the strain has no oral acute toxicity, is not resistant to antibiotics, and is safe for food processing.
  • the present invention provides Lactobacillus paracasei having beneficial activities and uses thereof.
  • the invention relates to, but is not limited to, the following.
  • Lactobacillus paracasei ET-22 strain which was deposited in the CGMCC of the General Microbiology Center of the China Microbial Species Collection Management Committee.
  • a composition comprising the Lactobacillus paracasei ET-22 strain according to item 1, and an excipient, a diluent, and / or a carrier.
  • composition of item 2 wherein the composition is a food composition, a pharmaceutical composition, or an oral cleansing composition.
  • composition of item 2 wherein the ET-22 strain is an active strain and / or a deactivated strain.
  • composition of item 3 wherein the composition is a food composition
  • excipients, diluents, and / or carriers include dairy beverages, tea, coffee, chewing gum and dentifrice, and meat for pets Dry or a combination of the above.
  • composition of item 3 wherein the composition is an oral cleaning composition
  • the excipients, diluents, and / or carriers are toothpaste, dentifrice, mouthwash, fresh breath spray, fluoride agent, Denture cleaners, dentifrices for pets or pet hair creams, toothbrushes, interdental brushes, dental floss, oral cotton swabs or pet dental bones.
  • composition according to item 3 wherein the composition is a pharmaceutical composition, the composition: 1) is made into an oral dosage form; 2) includes a therapeutic and / or preventive composition; 3) includes a nutritional composition 4) made into a dry or aerosol; or 5) made into a gel, cream, spray, aerosol, ointment, emulsion, suspension, patch, buccal tablet or sublingual tablet.
  • composition according to item 2 wherein the composition 1) is used for tooth whitening and / or inhibition of oral pathogens; 2) is used to adjust the subject flora balance; 3) is used to promote bifidobacteria and / Or the growth of lactic acid bacteria; and / or 4) for enhancing the immunity of a subject, such as enhancing the humoral and / or cellular immunity of the subject, such as increasing the number of antibody-producing cells, increasing the half-hemolysis value HC 50, and / or Or increase NK cell activity.
  • composition of item 2 wherein the composition is administered to a subject, such as a mammalian subject, by a gastrointestinal route.
  • composition according to item 2 wherein the content of the ET-22 strain in the composition is 10 6 CFU or more, such as 10 7 CFU or more, 10 8 CFU or more, 10 9 CFU or more, 10 10 CFU or more, or 10 11 CFU or more.
  • a method for treating a subject comprising administering an effective amount of the Lactobacillus paracasei ET-22 strain according to item 1 to a subject in need thereof, thereby 1) for use in teeth Whitening and / or inhibiting oral pathogens; 2) for regulating the subject's flora balance; 3) for promoting the growth of bifidobacteria and / or lactic acid bacteria; and / or 4) for enhancing the immunity of the subject, for example enhancing humoral immunity of the subject and / or cellular immunity, such as increasing the number of antibody-producing cells, total hemolytic improving and / or increasing NK cell activity HC 50.
  • the invention provides a culture comprising the Lactobacillus paracasei ET-22 strain.
  • the culture may further include a suitable lactic acid bacteria culture medium.
  • the lactic acid bacteria medium includes a solid medium and a liquid medium, such as an MRS medium.
  • the culture comprises a freeze-dried culture of Lactobacillus paracasei ET-22 strain.
  • the present invention provides a method for regulating a subject's flora balance, the method comprising administering a Lactobacillus paracasei ET-22 strain to the subject to regulate it Flora balance.
  • the invention provides a method for enhancing the immunity of a subject, the method comprising administering a Lactobacillus paracasei ET-22 strain to the subject to enhance its immunity force.
  • the present invention provides a method for increasing the number of antibody-producing cells in a subject, increasing the half-hemolytic value HC 50 and / or increasing the activity of NK cells, said method comprising lactobacillus paracasei ET
  • the -22 strain is administered to the subject.
  • the subject's humoral and / or cellular immunity is enhanced by the methods of the invention.
  • the method increases the number of antibody-producing cells in a subject.
  • the present invention provides a method for whitening teeth and / or inhibiting oral pathogenic bacteria and / or treating diseases caused by oral pathogenic bacteria, the method comprising administering Lactobacillus paracasei ET-22 strain to Subject.
  • the present invention provides a method for treating diseases such as dental caries, tooth decay, periodontal diseases such as periodontitis, halitosis, etc., the method comprising administering a Lactobacillus paracasei ET-22 strain to a test subject By.
  • the Lactobacillus paracasei ET-22 strain can be provided to a subject in the form of a composition such as a food composition, an oral cleaning composition, and a pharmaceutical composition.
  • the present invention provides a method of inhibiting and / or treating a disease caused by oral pathogenic bacteria.
  • the oral pathogenic bacteria may include Streptococcus mutans, Fusobacterium nunucleatum (including polytypes) Subsp. Polymorphum), actinomycetecomitans and porphyromonas gingivalis.
  • the strain is an active strain. In some embodiments, the strain can be administered to the subject by a gastrointestinal route. In some embodiments, the strain can be administered to the subject orally. In some embodiments, the strain is administered to a subject by other means suitable for intestinal absorption, such as by a means such as a feeding tube that is not passed through the mouth, throat, and esophagus.
  • the subject may be a mammal.
  • the subject can be a mammal, such as human, monkey, horse, cow, dog, cat, mouse, rat, pig, and the like.
  • the methods and compositions of the invention promote the growth of bifidobacteria and lactic acid bacteria.
  • the use of the methods and compositions of the present invention does not affect the growth of enteric bacteria such as Enterobacter.
  • the invention provides a method of promoting the growth of bifidobacteria and / or lactic acid bacteria, the method comprising administering a Lactobacillus paracasei ET-22 strain to the subject to promote Growth of bifidobacteria and / or lactic acid bacteria.
  • the use of the methods and compositions of the present invention does not affect the growth of enteric bacteria such as Enterobacter.
  • the composition of the present invention may be in the form of any composition suitable for use, for example, it may be a food (including beverage) composition, a pharmaceutical composition, or an oral cleaning composition.
  • the food composition, pharmaceutical composition, or oral cleansing composition is a composition for the oral cavity, such as a food composition, a pharmaceutical composition for dental whitening and / or for inhibiting oral bacteria, or Oral cleaning composition.
  • the composition containing the lactic acid bacteria strain of the present invention has the effects of tooth whitening and / or inhibition of oral pathogenic bacteria, and is present in the form of a food, oral cleansing or pharmaceutical composition.
  • the composition may include suitable excipients, diluents, and / or carriers, such as physiologically or pharmaceutically acceptable excipients, diluents, and / or carriers.
  • the composition may be a food composition.
  • the excipient, diluent, and / or carrier can be a food product.
  • the food may include beverages such as food products, oils, meat products, dairy products, aquatic products, canned foods, sugary foods, cold foods, alcoholic beverages, pet foods, and the like.
  • the food may include beverages such as dairy drinks, tea, coffee, chewing gum and dentifrice, jerky for pets, or a combination of the above.
  • the composition may be a pharmaceutical composition.
  • the composition can be formulated as an oral or topical dosage form.
  • the composition may include a therapeutic and / or prophylactic composition.
  • the composition may be a nutritional composition.
  • the composition can be formulated as a dry or aerosol.
  • the composition can be made in the form of a gel, cream, spray, aerosol, ointment, emulsion, suspension, patch, buccal tablet, sublingual tablet, and the like.
  • the composition may be an oral cleaning composition.
  • the excipient, diluent, and / or carrier can be toothpaste, dentifrice, mouthwash, breath freshening spray, fluoride application agent, denture cleaner, dentifrice for pets, or pet hair cream .
  • the excipient, diluent, and / or carrier is a toothbrush, interdental brush, dental floss, oral cotton swab, or dentifrice for pets.
  • the composition may be a solid, powder, or slurry oral hygiene composition.
  • the composition may be a toothpaste, chewing gum, candy, a liquid mouthwash such as a mouthwash, and the like.
  • the composition of the present invention can be used as a probiotic and / or prebiotic composition, a pharmaceutical composition for treating an oral infection, or a functional food.
  • the Lactobacillus paracasei ET-22 strain has an activity to inhibit oral pathogens.
  • probiotics can refer to the use of live microorganisms that can be added to compositions such as foods (such as milk, cheese), pharmaceuticals (such as capsules, tablets, pills, powders, etc.) or oral hygiene products, etc. It remains active and exerts its physiological effects on a subject ingesting a composition containing the probiotic.
  • Prebiotics can refer to substances that are added to a composition such as food (such as milk, cheese), pharmaceuticals (such as capsules, tablets, pills, powders, etc.) or oral hygiene products, etc.
  • the components work to help build healthy bacteria and / or hinder the creation of pathogenic bacteria.
  • the strains of the invention can be prepared in the form of probiotics or prebiotics.
  • the pharmaceutical composition comprises an oral dosage form or a topical dosage form.
  • the excipient or diluent of the oral cleaning composition is toothpaste, dentifrice, mouthwash, fresh breath spray, fluoride coating agent, denture cleaner, pet dentifrice, or pet hair cream .
  • the carrier is a toothbrush, an interdental brush, dental floss, an oral cotton swab, or a dentifrice for pets.
  • the present invention provides a lactic acid bacteria-containing food composition, an oral cleaning composition, and a pharmaceutical composition, which can inhibit the growth of oral pathogenic bacteria to maintain oral health.
  • the strain may be present in an effective amount.
  • the content of the lactic acid bacteria strain in the composition may be 10 6 CFU or more, such as 10 7 CFU or more, 10 8 CFU or more, 10 9 CFU or more, 10 10 CFU or more, 10 11 CFU or more; preferably, the number of the strains is 10 9 CFU or more.
  • the effects of probiotics to promote the growth of bifidobacteria and / or lactic acid bacteria and / or to regulate flora balance have been found to be strain-specific.
  • Lactobacillus paracasei ET-22 strains have been found to have superior promotion of bifidobacteria and / or control strains such as other Lactobacillus strains (such as common Lactobacillus paracasei strains) that do not contain the strain and / Or the effect of lactic acid bacteria growth and / or regulating the flora balance.
  • the Lactobacillus paracasei ET-22 strain has an improved promoting double compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain). Bifidobacterium and / or Lactobacillus growth and / or role in regulating flora balance.
  • Lactobacillus paracasei ET-22 strain is capable of promoting Bifidobacterium compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain).
  • lactic acid bacteria and / or increase the subject's flora balance e.g., increase by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.
  • promoting the growth of bifidobacteria and / or lactic acid bacteria and / or regulating the balance of the subject's flora can be measured by methods known in the art.
  • promoting the growth of bifidobacteria and / or lactic acid bacteria and / or adjusting the balance of the subject's flora can be performed, for example, by comparing changes between themselves and between groups of bifidobacteria, lactic acid bacteria, enterococci, and enterobacteria. get on.
  • the Lactobacillus paracasei ET-22 strain can be used to prepare compositions and / or kits for promoting the growth of bifidobacteria and / or lactic acid bacteria and / or regulating the balance of the subject's flora. .
  • the composition and / or kit for promoting the growth of bifidobacteria and / or lactic acid bacteria and / or regulating the balance of the subject's flora may further include other suitable bifidobacteria and / or lactic acid bacteria A substance that regulates the growth and / or balance of the subject's flora.
  • the Lactobacillus paracasei ET-22 strain has been found to have the ability to inhibit oral pathogenic bacteria, such as dental caries, gingival bacteria, and oral odor bacteria.
  • Lactobacillus paracasei ET-22 strain can secrete hydrogen peroxide, in addition to inhibiting oral pathogenic bacteria, and can also whiten teeth.
  • the Lactobacillus paracasei ET-22 strain has superior immunomodulation compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains, such as a common Lactobacillus paracasei strain effect.
  • the Lactobacillus paracasei ET-22 strain has an improved subject compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain). Effects of humoral and / or cellular immunity.
  • the Lactobacillus paracasei ET-22 strain is capable of increasing the subject as compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain). Humoral and / or cellular immunity (e.g.
  • improving a subject's humoral and / or cellular immunity can be measured by methods known in the art, such as by antibody-producing cell experiments and / or half-hemolytic value HC 50 determinations, and NK cell activity assays, etc. .
  • Lactobacillus paracasei ET-22 strain can be prepared into a composition and / or kit for enhancing the immunity of a subject.
  • the subject's immunity-enhancing composition and / or kit may further include other suitable immunity-enhancing substances.
  • Lactobacillus paracasei ET-22 strains have been found to be superior to control or control strains that do not contain the strain, such as other Lactobacillus strains (such as common Lactobacillus paracasei strains). Inhibition of oral pathogens.
  • the Lactobacillus paracasei ET-22 strain is capable of producing peroxide compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain). Active and thus can be used, for example, for tooth whitening.
  • the Lactobacillus paracasei ET-22 strain is capable of inhibiting the following when compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain)
  • a control or control strain that does not contain the strain such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain)
  • One or more oral pathogenic bacteria or have increased inhibitory activity e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times) , 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more
  • inhibitory activity e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times
  • Streptococcus mutans e.g., 10%, 20%
  • the Lactobacillus paracasei ET-22 strain is capable of inhibiting all oral pathogenic bacteria compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain). Or have increased inhibitory activity (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times , 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more) inhibitory activities: Streptococcus mutans, Fusobacterium nucleus (including subtype subsp.
  • the Lactobacillus paracasei ET-22 strain is particularly capable of inhibiting the following when compared to a control or control strain that does not contain the strain, such as other Lactobacillus strains (such as a common Lactobacillus paracasei strain) Oral pathogens may have increased inhibitory activity (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more) inhibitory activities: Fusobacterium nucleus (including polytype subsp.
  • the pharmaceutical composition can be used to treat diseases such as dental caries, tooth decay, periodontal diseases such as periodontitis, halitosis, and the like.
  • Lactobacillus paracasei ET-22 strains can be prepared in compositions and / or kits for tooth whitening and / or treatment of oral diseases.
  • the composition and / or kit for tooth whitening and / or treatment of oral diseases may also include other suitable teeth whitening and / or treatment materials for oral diseases.
  • the present invention provides a method for preparing a composition, such as a food composition, a pharmaceutical composition, or an oral cleansing composition, the method comprising adding Lactobacillus paracasei ET-22 strain to the composition .
  • Figure 1 Results of evaluation of the tolerance of Lactobacillus paracasei ET-22ET-22 to simulated intestinal environmental tolerance, gastric acid and bile salt environment.
  • Figure 4 Comparative results of Lactobacillus paracasei ET-22 and Lactobacillus casei LC-01 regulating changes in intestinal colonies.
  • Lactobacillus paracasei ET-22 strain of the present invention is deposited in:
  • CGMCC China Type Culture Collection Center / China General Microbial Collection Center (CGMCC), Deposit Date: December 18, 2017, Deposit Number CGMCC No. 15077; The address of this deposit is: Beichen, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences.
  • Lactobacillus paracasei strain K56 is deposited with:
  • CGMCC China Type Culture Collection / China General Microbial Collection Center (CGMCC), Deposit Date: December 29, 2017, CGMCC No. 15139;
  • the address of the deposit unit is: Beichen, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences.
  • the freeze-dried culture of the lactic acid bacteria strain according to the present invention has been deposited in the China Type Culture Collection Center and the General Microbial Center of the China Microbial Culture Collection Management Committee. Details of the deposit are shown in the table below:
  • the fermentation conditions of this strain are:
  • MRS liquid medium peptone, 10.0g; beef extract, 10.0g; yeast extract, 5.0g; glucose, 20.0g; dipotassium hydrogen phosphate, 5.0g; diammonium hydrogen citrate, 2.0g; sodium acetate, 5.0g ; Magnesium sulfate heptahydrate, 0.5 g; Manganese sulfate tetrahydrate, 0.2 g; Tween 80, 1.0 g; 15.0 g of agar; 1000 mL of distilled water. Adjust the pH to between 6.2 and 6.4 and sterilize at 121 ° C for 15 minutes.
  • L.paracasei ET-22 is a microaerobic bacterium, which grows better in a facultative anaerobic environment, produces lactic acid, has acid resistance, can withstand an acidic environment with a pH of 2.5, and a 0.4% bile salt environment for 4 hours, and is mesophilic
  • the growth temperature range of bacteria is 15 ⁇ 45 °C, and the optimum growth temperature is about 37 °C.
  • Example 1 Artificial gastric juice, intestinal fluid tolerance, artificial bile tolerance
  • the coverslip was clamped with a pair of forceps, and after being sterilized by an alcohol lamp, it was put into a 6-well plate for later use.
  • the Caco-2 cells were removed from the storage tube, the cells were adjusted, and then transferred to a 6-well plate for cultivation. After the plate was full, the experiment was performed. During the test, remove the liquid from the dish and wash it twice with PBS buffer.
  • This example is intended to confirm the effect of Lactobacillus paracasei of the present invention on intestinal regulation.
  • mice Thirty-six healthy SPF-grade BABL / c mice were taken and weighed 18-22 g (provided by Beijing Huafukang Biotechnology Co., Ltd.). After 3 days of adaptive breeding, they were randomly divided into 3 groups, 12 in each group, namely the blank control group and the sample group. To each group of animals, sterile water (gavage volume 0.2mL / 10g) in which Bifidobacterium lactis BL-99 bacterial powder was dissolved was administered orally, and the same volume of sterile water was administered to the blank control group. Once a day, feed or gavage continuously for 14 days.
  • Gavage measurement 1.3 ⁇ 10 7 CFU / ml (according to the human requirement is 2 ⁇ 10 9 CFU / day, the conversion factor for human and mouse is 0.0026). Take sterilized centrifuge tubes, number them, collect mouse feces under aseptic conditions after adaptive feeding, and 2-3 capsules each, about 100mg, transfer to aseptic operation room at low temperature for bacterial detection. At the end of the experiment, mouse feces were collected again. After the mice were numbered with picric acid, they were weighed on the 8th and 14th days after the administration of the test substance, and the amount of intragastric administration was calculated. The mice were weighed once at the end of the experiment. Colony count: Prepare a selective medium according to the strain to be identified, see the table below for the strain to be tested and the corresponding medium, sterilize, shake well, and cool to 45 ° -50 ° C and pour the plate for later use.
  • mice The collected feces of the mice were placed in a sterilized tube filled with 0.5 mL of physiological saline to prepare a bacterial suspension, and shaken for 1 min before use.
  • Another 0.1mL micropipette tip was taken, and according to this method, a 10-fold gradient dilution was performed until 10 -7 g / ml. According to the number of live bacteria of the bacterial species to be identified, select two consecutive appropriate dilutions.
  • Each dilution uses a 10 ⁇ L micropipette to pipette 10 ⁇ L of the bacteria suspension, and the surface is coated on a selective agar plate. The culture conditions are shown. The method of colony counting is referred to the "GB 4789.2-2010 National Food Safety Standard Food Microbiological Determination of Total Colony Determination”.
  • Lactobacillus paracasei ET-22 has the effect of regulating the intestinal flora in this study ( Figure 3).
  • ET-22 is a Lactobacillus genus, but it can significantly increase the bifidobacterium genus by more than an order of magnitude, and the increase is higher than LC-01, showing that it has a very strong ability to regulate intestinal bacteria.
  • Example 3 is intended to confirm the effect of the Lactobacillus paracasei on the intestinal regulation of the present invention.
  • the principle and procedure refer to "Technical Specifications for Inspection and Evaluation of Health Foods-Criteria for Adjusting the Function of the Intestinal Microbiota".
  • the difference from Example 3 is that 16S rDNA sequencing results were used in this experiment.
  • Bacterial powder samples (see packaging specifications for the number of live bacteria) were provided by Inner Mongolia Dairy Technology Research Institute Co., Ltd.
  • mice 182 healthy 6-week-old adult BABL / c mice weighing about 18-20 g.
  • Live bacteria sample (ET-22): According to the sample specifications, each 1g live bacteria sample was weighed and suspended to 40ml with PBS solution, that is, the live bacteria concentration was 2.5x10 9 CFU / ml.
  • High dose group calculated in accordance with 0.2ml / 10g mouse gavaged volume, mice fed an amount of 20g 0.4ml gavage dose of high dose group mice 10 9 CFU / 20g.
  • Medium-dose group 5ml high-dose suspension was added to PBS to make up to 50ml. Calculated according to 0.2ml / 10g of mice, 20g mice were given 0.4ml, and medium-dose mice were given 10 8 CFU / 20g.
  • Low-dose group 5ml medium-dose group suspensions were added to PBS to make up to 50ml, calculated according to 0.2ml / 10g of mice, 20g mice were given 0.4ml, and low-dose mice were given 10 7 CFU / 20g.
  • mice were randomly divided into 13 groups with 14 in each group.
  • the grouping conditions are shown in Table 9.
  • each test substance was administered according to an intragastric amount of 0.2ml / 10g, and the control group was administered PBS for 1-14 days, and the experimental group was administered an appropriate dosage of the test substance according to Table 9.
  • Mice were weighed once a week and the amount of gavage was adjusted according to body weight.
  • Microbial diversity is based on the Illumina HiSeq sequencing platform and uses a paired-end sequencing method to construct a small fragment library for sequencing.
  • OTUs Orthogonal Taxonomic Units
  • species composition of the sample can be revealed; the optimized sequence is clustered, the OTU is divided, and its species are obtained based on the OTU sequence composition classification.
  • the taxonomic analysis of the samples at various classification levels was performed to obtain the community structure of each sample at the genus level.
  • the species diversity within a single sample was studied through Alpha diversity analysis.
  • the Ace, Chao1, Shannon, and Simpson indices of each sample at the 97% similarity level were calculated, and the sample dilution curve and grade abundance curve were drawn; further alpha diversity was performed.
  • Sex analysis (Alpha Diversity), beta diversity analysis (Beta Diversity) and significant species difference analysis, etc. can excavate the differences between samples.
  • Comparison of intestinal flora alpha diversity index: Chao1 and ACE refer to the measurement of species abundance, that is, the number of species; Shannon and Simpson indices are used to measure species diversity, which is affected by species abundance and species uniformity in the sample community. Under the same species abundance, the greater the uniformity of each species in the community, the greater the diversity of the community is considered.
  • ET-22 significantly increased the relative abundance in the intestines of mice after intervention with ET-22.
  • the OTU, ACE, and Chao1 indexes of the low-dose group increased significantly, indicating that low-dose ET-22 can significantly increase the intestinal microbial diversity.
  • H. pylori The number of Helicobacter decreased significantly; the Escherichia-Shigella genus in the low and medium dose groups decreased significantly.
  • BL-99 can significantly increase the relative abundance of Lactobacillus in the intestine of mice, and the low-dose BL-99 group has the most significant increase in Lactobacillus.
  • SRBC sheep red blood cells
  • PBS suspension was used to adjust the concentration of live bacteria samples (approximately 10 7 -10 9 CFU / day) according to different gavage amounts before the animals were suspended. Each mouse was injected intraperitoneally for 28 days. SRBC 0.2mL was immunized. Mice immunized with SRBC for 4 days were sacrificed, and the spleen was removed to prepare a cell suspension of 5 ⁇ 10 6 cells / mL. After dissolving the agarose, mix it with an equal amount of double Hank's solution, aliquot small test tubes, 0.5mL per tube, and add 20% (V / V, configured with physiological saline) to the tube to accumulate 50 ⁇ L of SRBC.
  • Spleen cells 200 ⁇ L of suspension mix quickly, pour onto a six-well plate with a thin layer of agarose brushed, solidify with agar, put it in a carbon dioxide incubator and continue incubation for 1h, then add the complement diluted with SA buffer (1:10) , Continue incubation for 2h, count the number of hemolytic plaques.
  • the number of antibody-producing cells is shown in Figure 5. Compared with the control group, the number of L-paracasei ET-22 and L.paracasei K56 (CGMCC 15139) promoted antibody-producing cells was significantly different from the control group (p ⁇ 0.05). .
  • each mouse was injected intraperitoneally with 0.2 mL of SRBC for immunization. After 4 days, the eyeballs were removed and blood was collected in a 1.5 mL centrifuge tube, which was left at 4 ° C for about 1 hour to allow the serum to be analyzed, and the serum was collected by centrifugation at 2000 r / min for 10 minutes. The serum was diluted 100-fold with SA buffer. Add the diluted serum to a 96-well plate, 100 ⁇ L per well, and then add 10% (v / v) SRBC 50 ⁇ L, 100 ⁇ L complement (diluted 1: 8 with SA solution), and incubate in a 37 ° C thermostatic water bath for 30 min.
  • the amount of hemolysin is expressed as a half hemolysis value (HC 50 ) and is calculated according to the following formula:
  • the half hemolytic value HC 50 of the L.paracasei ET-22 group has a significant difference (p ⁇ 0.05), and is higher than that of L.paracasei K56.
  • the animals were continuously sampled for 28 days.
  • the target cell YAC-1 was subcultured 24 hours before the experiment.
  • the cells were washed twice with Hank's solution before use, and the cell concentration was adjusted to 1 ⁇ 10 with RPMI 1640 complete culture solution containing 10% calf serum. 5 cells / mL (target cells). Sudden death of mouse cervical dislocation, spleen was taken aseptically, made into a skin cell suspension, washed twice with Hank's solution, centrifuged at 1000 rpm for 10 min, and resuspended in 2 mL of RPMI 1640 complete medium containing 10% calf serum.
  • Trypan blue live cell staining count (the number of live cells should be above 95%), adjust the cell concentration to 1 ⁇ 10 7 cells / mL (effect cells), and make the effect target ratio to 100: 1.
  • the target cells will release 100 ⁇ L each of the target cells and the culture medium, and the target cells will release 100 ⁇ L each of the target cells and 1% NP40.
  • Three parallel holes Incubate for 4 h at 37 ° C in a 5% CO 2 incubator. Centrifuge the 96-well culture plate at 1500 rpm for 5 minutes.
  • L.paracasei ET-22 antibody-producing cell test and the half hemolysis value HC 50 are positive, the sample is determined to be L.paracasei ET-22 humoral immunopositive; if the NK cell activity test is positive, the sample L.paracasei ET-22 showed positive NK cell activity. In summary, it is believed that L.paracasei ET-22 has the effect of enhancing immunity.
  • Example 6 Analysis of hydrogen peroxide-producing properties of strains to confirm the function for tooth whitening
  • the ability of the Lactobacillus paracasei ET-22 strain of the present invention to produce hydrogen peroxide was examined to verify the whitening ability of the Lactobacillus strain of the present invention.
  • the experimental steps are as follows:
  • TMB trimethylboron
  • HRP horseradish peroxidase
  • PPES piperazine-1,4-diethanesulfonic acid
  • FIG. 8 is a result showing the ability of Lactobacillus paracasei ET-22 strain to secrete hydrogen peroxide on a plate after being cultured on a hydrogen peroxide detection plate.
  • Example 7 Inhibition of oral pathogenic bacteria by active lactic acid bacteria to maintain oral health
  • Lactic acid bacteria need to play a role in oral health care, first of all whether the resistance of oral pathogenic bacteria is strong.
  • Oral pathogens are not just Streptococcus mutans, which are more commonly known to the public, but also include Fusobacterium nucleus subsp. Polymorphum, Aggregatibacter actinomycetecomitans, and Porphyromonas gingivalis Porphyromonas gingivalis and so on.
  • Symptoms caused by each strain are: dental caries caused by Streptococcus mutans; polymorphous subtypes of Fusobacterium nucleus mainly cause problems such as periodontal disease, halitosis and colon cancer; actinomycete actinomycetes cause periodontitis, oral cavity The main species of inflammation and pneumonia; Porphyromonas gingivalis is the main cause of adult periodontitis and halitosis.
  • P. gingivalis which is the main species causing halitosis
  • polymorphisms of Fusobacterium nucleus, actinomycetes, and P. gingivalis all produce sulfides. It is also one of the main bacteria affecting oral odor.
  • the present invention performs a pathogen suppression test to evaluate the ability of the active Lactobacillus paracasei ET-22 strain of the present invention to inhibit oral pathogenic bacteria, thereby achieving the effect of maintaining oral health.
  • a pathogen suppression test to evaluate the ability of the active Lactobacillus paracasei ET-22 strain of the present invention to inhibit oral pathogenic bacteria, thereby achieving the effect of maintaining oral health.
  • FIG. 9 is an analysis of the ability of the lactic acid bacteria strain of the present invention to inhibit oral pathogenic bacteria.
  • Figure 9 shows the results of the inhibition of oral pathogenic bacteria by the Lactobacillus paracasei ET-22 strain of the present invention. Compared with another strain of Lactobacillus paracasei 9, the inhibitory effect of ET-22 is most significant.
  • Example 8 Inhibition of oral pathogenic bacteria by deactivated lactic acid bacteria to maintain oral health
  • the present invention performs a pathogen suppression test to evaluate the inactive Lactobacillus paracasei ET of the present invention.
  • FIG. 10 is an analysis of the ability of the inactivated lactic acid bacteria strain of the present invention to inhibit oral pathogenic bacteria; the histograms are expressed as Mean ⁇ SD and compared with another strain of Lactobacillus paracasei 9.
  • the inactivated Lactobacillus paracasei ET-22 strain of the present invention inhibits the survival of oral pathogenic bacteria. Compared with the inhibitory effect of Lactobacillus paracasei 9 strain, ET-22 is the most significant.
  • the food composition and pharmaceutical composition containing the lactic acid bacteria strain of the present invention can inhibit the growth of oral pathogenic bacteria, and can be used to reduce human dental caries, periodontal disease, and bad breath.
  • a strain of lactic acid bacteria capable of secreting hydrogen peroxide is also effective for whitening teeth.
  • the present invention finds lactic acid bacteria that have no side effects on the human body and is beneficial to maintaining oral health as a new option for maintaining oral health.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nutrition Science (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Birds (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Pediatric Medicine (AREA)
  • Physiology (AREA)
  • Animal Husbandry (AREA)
  • Immunology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种副干酪乳杆菌(Lactobacillus paracasei)ET-22菌株、含有该菌株的组合物及其用途，该菌株的保藏编号为CGMCC No.15077。

Description

副干酪乳杆菌ET-22及其用途 技术领域
本发明涉及微生物技术领域，尤其是涉及副干酪乳杆菌(Lactobacillus paracasei)及其用途，例如用于促进双歧杆菌和乳酸菌的生长，促进体液免疫和NK细胞杀伤活性，以及抑制口腔病原菌等。
背景技术
1.益生菌与益生菌产品
益生菌指活的微生物，其能够添加到组合物中，保持活性并且对摄取含有所述益生菌的组合物的对象发挥生理学作用。世界卫生组织对益生菌产品的定义为食品中含有充足数量的活的微生物，经过食品加工的各个过程，以及进入人体肠道以后，仍能保持适当的活菌数量和菌活性。因此菌株在菌粉的制作、产品的生产加工以及经人体胃肠道的胃酸、胆盐胁迫后，菌株应能够保持较为稳定的活菌数量。
2.益生菌与肠道菌群失调
肠道菌群的平衡与人体健康息息相关。肠道菌群包括有益菌、中性菌和有害菌。健康的人体肠道中有益菌占据优势地位，并且与有害菌不断相互作用，维护人体健康。如果由于各种因素影响使肠道中有益菌减少，有害菌便会大量繁殖，肠内微生态平衡被打破，引起肠炎、腹泻等多种临床症状。同时，临床上通常使用大量的抗生素，虽然能有效的杀害有害菌，但同时杀灭有益菌，造成肠道菌群失调，引起疾病。
对于肠道菌群失调者，经口补充益生菌是调节肠道菌群直接有效的办法。经口补充益生菌制剂或含益生菌产品可以直接或间接调节肠道菌群组成，激活宿主内源性微生物群或者免疫系统活性来实现益生作用。
3.益生菌与免疫功能
免疫是人体的一种重要的生理功能，涉及非特异性免疫和特异性免疫，非特异性免疫不需要预先接触抗原，一旦病原体进入了机体，则会快速清除病原体。吞噬细胞和自然杀伤细胞都具有清除和杀伤作 用，是非特异性免疫系统中的重要成员。特异性免疫具有特异地识别外来异物并具有免疫记忆能力的免疫细胞(T淋巴细胞和B淋巴细胞)。免疫系统之间最首要的信息传导子就是细胞因子，每种细胞因子对不同的细胞类型具有不同的刺激功能，它们通过结合细胞表面的特异性受体而诱导细胞的生长发育和功能活性。人体依靠这种功能识别“自己”和“非己”成分，从而破坏和排斥进入人体的抗原物质或人体本身所产生的损伤细胞和肿瘤细胞等，以维持人体的健康，抵抗或防止微生物或寄生物的感染或其它所不希望的生物的侵入。
增强人体免疫力是儿童、青少年、成年人与老年人多年龄群体及各个社会阶层共同的需求。研究同时指出，多种人体疾病如感冒、肠炎、关节炎症等均与免疫能力低下相关。益生菌具有多种功能，如调节肠道菌群失调、增强肠道免疫功能，抑制过敏反应。其中对益生菌免疫调节作用的研究表明益生菌可以同时促进特异性和非特异性免疫，对人体健康起到促进作用。因此是否能筛选得到具有提高免疫力特性的益生菌是本发明希望解决的问题。
4.益生菌与口腔卫生
幼儿的乳齿保健将会影响到未来的恒久齿，因此，从婴儿时期开始，口腔清洁即十分重要。若口腔有细菌滋生，细菌容易附着于牙龈表面，待乳齿长出后会直接附着于婴儿的乳齿上造成龋齿。乳齿是一切牙齿的基础，乳齿可维系适当的空间及距离以提供未来恒久齿的生长。若乳齿没有照顾好，放任乳齿产生龋齿，龋齿会导致空间严重丧失，且将来恒久齿在发育时，恒久齿的周围都是细菌与脓包，导致新长出来的牙齿也比较容易形成龋齿，甚至影响成年及老年后的牙周病发生率。
一般食用含乳酸菌(LAB)的产品仅具有调整肠道健康的效果。虽然有数以万计的乳酸菌菌株存在自然界，但目前仅发现少数乳酸菌菌株可能具有维护口腔健康及美白牙齿的潜在性附属特质。这些菌株所具有的抑制口腔病原菌的能力、吸附口腔黏膜表皮细胞的能力、在洁牙产品中是否尚能维持抑制病原菌及产生过氧化氢的能力等特性，是激发菌株在口腔展现功能，控制口腔中的微生态平衡、筛选有维护口腔健康及美白牙齿功能性菌株时的重要依据。
文献指出益生菌可以与病原菌竞争贴附的生存区域及营养需求，促进病原菌产生聚集特性，并分泌抑菌物质使得病原菌较虚弱或甚至 杀死病原菌，来达到减少口腔病原菌数量并维持口腔健康的功效，以及分泌过氧化氢去除口腔气味及美白牙齿的目的。
时至今日，仅有少数菌株经实验过程发现具备有维持口腔健康及美白牙齿的活性效果的乳酸菌菌株被确认。而乳酸菌对身体健康的功能在于菌株(strain)的特异性而非菌种(species)，此种对于人的身体健康有特殊功效的菌株称为功能性益生菌(Guidelines for the evaluation of probiotics in food；Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food；London Ontario，Canada April 30 and May 1，2002：1-7)。
综上所述，开发具有有益活性的功能性益生菌是目前需努力的目标。
发明内容
发明人研究发现ET-22菌株(保藏编号为CGMCC No.15077)单菌即具有多种有益活性，包括例如：显著促进肠道双歧杆菌和乳酸菌增长的能力，可以耐受体外模拟胃肠液胁迫环境；显著增强体液免疫，激活NK细胞活性，包括例如提高抗体生成细胞数，半数溶血值HC 50，激活NK细胞活性，可以改善由于饮食不规律和工作压力增加而引发的免疫能力低下等问题；抑制口腔病原菌，具备产生过氧化物活性，由此可以用于例如牙齿美白。实验表明该菌株无口服急性毒性，无抗生素耐受，安全可以用于食品加工。
因此，本发明提供了具有有益活性的副干酪乳杆菌及其用途。
在一些实施方案中，本发明涉及下述各项，但不限于此。
1.副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株，其保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏日期为2017年12月18日，保藏编号为CGMCC No.15077。
2.一种组合物，其包含项目1所述的副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株，和赋形剂、稀释剂和/或载体。
3.项目2所述的组合物，其中所述组合物为食品组合物、医药组合物或口腔清洁组合物。
4.项目2所述的组合物，其中所述ET-22菌株为具有活性的菌株和/或去活性的菌株。
5.项目3所述的组合物，其中所述组合物为食品组合物，所述 赋形剂、稀释剂和/或载体包括乳制饮品、茶、咖啡、口香糖及洁牙糖、宠物用肉干或以上的组合。
6.项目3所述的组合物，其中所述组合物为口腔清洁组合物，所述赋形剂、稀释剂和/或载体为牙膏、洁牙粉、漱口水、口气清新喷雾、涂氟剂、假牙清洁剂、宠物用洁牙胶或宠物用化毛膏、牙刷、牙间刷、牙线、口腔棉棒或宠物用洁牙骨。
7.项目3所述的组合物，其中所述组合物为医药组合物，所述组合物：1)制成口服剂型；2)包括治疗和/或预防用组合物；3)包括营养组合物；4)制成干剂或湿剂；或5)制成凝胶、乳膏、喷雾剂、气溶胶、软膏、乳剂、混悬剂、贴片、口含片剂或舌下片剂形式。
8.项目2所述的组合物，其中所述组合物1)用于牙齿美白和/或抑制口腔病原菌；2)用于调节受试者菌群平衡；3)用于促进双歧杆菌和/或乳酸菌的生长；和/或4)用于增强受试者免疫力，例如增强所述受试者的体液免疫和/或细胞免疫，例如提高抗体生成细胞数、提高半数溶血值HC 50和/或提高NK细胞活性。
9.项目2所述的组合物，其中所述组合物通过胃肠道途径施用给受试者，例如哺乳动物受试者。
10.项目2所述的组合物，其中所述组合物中ET-22菌株的含量为10 6CFU以上，例如10 7CFU以上，10 8CFU以上，10 9CFU以上，10 10CFU以上，或10 11CFU以上。
11.一种治疗受试者的方法，其包括将有效量的项目1所述的副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给有需要的受试者，由此1)用于牙齿美白和/或抑制口腔病原菌；2)用于调节受试者菌群平衡；3)用于促进双歧杆菌和/或乳酸菌的生长；和/或4)用于增强受试者免疫力，例如增强所述受试者的体液免疫和/或细胞免疫，例如提高抗体生成细胞数、提高半数溶血值HC 50和/或提高NK细胞活性。
12.项目11所述的方法，其中所述ET-22菌株为项目2所述的组合物的形式。
13.项目11所述的方法，其中所述ET-22菌株为具有活性的菌株和/或去活性的菌株。
14.项目11所述的方法，其中所述ET-22菌株通过胃肠道途径施用给受试者，例如哺乳动物受试者。
15.项目11所述的方法，其中所述ET-22菌株以10 6CFU以上，例如10 7CFU以上，10 8CFU以上，10 9CFU以上，10 10CFU以上，或10 11CFU以上的量施用给所述受试者。
在一些实施方案中，本发明提供一种培养物，其包括所述的副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株。在一些实施方案中，所述培养物还可以包括适当的乳酸菌培养基。在一些实施方案中，所述乳酸菌培养基包括固体培养基和液体培养基，例如MRS培养基。在一些实施方案中，所述培养物包括副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株的冷冻干燥培养物。
在一些实施方案中，本发明提供一种用于调节受试者菌群平衡的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给所述受试者以调节其菌群平衡。
在一些实施方案中，本发明提供一种用于增强受试者免疫力的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给所述受试者以增强其免疫力。在一些实施方案中，本发明提供一种提高受试者抗体生产细胞数、提高半数溶血值HC 50和/或提高NK细胞活性的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给所述受试者。在一些实施方案中，通过本发明的方法，所述受试者的体液免疫和/或细胞免疫得到增强。在一些实施方案中，所述方法提高受试者的抗体生成细胞数。
在一些实施方案中，本发明提供用于牙齿美白和/或抑制口腔病原菌和/或治疗口腔病原菌感染的疾病的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给受试者。在一些实施方案中，本发明提供治疗例如龋齿、蛀牙、牙周病如牙周炎、口臭等疾病的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给受试者。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株可以以组合物如食品组合物、口腔清洁组合物以及医药组合物的形式提供给受试者。在一些实施方案中，本发明提供抑制口腔病原菌和/或治疗口腔病原菌感染的疾病的方法，所述口腔病原菌可以包括变形链球菌(Streptococcus mutans)，具核梭杆菌(Fusobacterium nucleatum)(包括多型亚种subsp.polymorphum)、放线共生放线杆菌(Aggregatibacter  actinomycetemcomitans)和牙龈卟啉单孢菌(Porphyromonas gingivalis)等。
在一些实施方案中，所述菌株为具有活性的菌株。在一些实施方案中，所述菌株可以通过胃肠道途径施用给所述受试者。在一些实施方案中，所述菌株可以通过口服施用给所述受试者。在一些实施方案中，所述菌株通过其它适合肠道吸收的方式施用给受试者，例如通过不经过口腔、咽喉和食道的方式如饲管施用给受试者。
在一些实施方案中，所述受试者可以为哺乳动物。在一些实施方案中，所述受试者可以为哺乳动物，例如人、猴、马、牛、犬、猫、小鼠、大鼠和猪等。
在一些实施方案中，本发明的方法和组合物促进双歧杆菌和乳酸菌的生长。
在一些实施方案中，本发明的方法和组合物使用后肠杆菌等肠道细菌的生长不受影响。
在一些实施方案中，本发明提供一种促进双歧杆菌和/或乳酸菌的生长的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给所述受试者以促进双歧杆菌和/或乳酸菌的生长。
在一些实施方案中，本发明的方法和组合物使用后肠杆菌等肠道细菌的生长不受影响。
在一些实施方案中，本发明的组合物可以为适合使用的任何组合物形式，例如可以为食品(包括饮料)组合物、医药组合物或口腔清洁组合物。在一些实施方案中，所述食品组合物、医药组合物或口腔清洁组合物为用于口腔的组合物，例如用于牙齿美白和/或用于抑制口腔细菌的食品组合物、医药组合物或口腔清洁组合物。在一些实施方案中，含有本发明乳酸菌菌株的组合物具有牙齿美白和/或抑制口腔病原菌的作用，且以食品、口腔清洁或医药组合物的形式存在。
在一些实施方案中，所述组合物可以包含适当的赋形剂、稀释剂和/或载体，例如生理上或药学上可接受的赋形剂、稀释剂和/或载体。
在一些实施方案中，所述组合物可以为食品组合物。在一些实施方案中，该赋形剂、稀释剂和/或载体可以为食品。在一些实施方案中，该食品可以包括饮品如粮食制品、油、肉制品、乳制品、水产品、罐头、含糖食品、冷食、酒品、宠物食品等。在一些实施方案中，所 述食品可以包括饮品如乳制饮品、茶、咖啡、口香糖及洁牙糖、宠物用肉干或以上的组合。
在一些实施方案中，所述组合物可以为医药组合物。在一些实施方案中，所述组合物可以制成口服剂型或外用剂型。在一些实施方案中，所述组合物可以包括治疗和/或预防用组合物。在一些实施方案中，所述组合物可以是营养组合物。在一些实施方案中，所述组合物可以制成干剂或湿剂。在一些实施方案中，所述组合物可以制成凝胶、乳膏、喷雾剂、气溶胶、软膏、乳剂、混悬剂、贴片、口含片剂、舌下片剂等形式。
在一些实施方案中，所述组合物可以为口腔清洁组合物。在一些实施方案中，该赋形剂、稀释剂和/或载体可以为牙膏、洁牙粉、漱口水、口气清新喷雾、涂氟剂、假牙清洁剂、宠物用洁牙胶或宠物用化毛膏。在一些实施方案中，该赋形剂、稀释剂和/或载体为牙刷、牙间刷、牙线、口腔棉棒或宠物用洁牙骨。在一些实施方案中，此外，所述组合物可以为固体、粉状、或浆状口腔卫生组合物。在一些实施方案中，所述组合物可以为牙膏，口香糖，糖果、液体漱口液如漱口剂等。
在一些实施方案中，本发明的组合物可以用作益生菌和/或益生元组合物、用于治疗口腔感染的药物组合物、或功能食品。在一些实施方案中，该副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株具有抑制口腔病原菌的活性。在一些实施方案中，益生菌可以指使用活的微生物，其能够添加到组合物如食品(如牛奶、乳酪)、药品(如胶囊、片剂、丸剂、粉剂等)或口腔卫生用品等中，保持活性并且对摄取含有所述益生菌的组合物的对象发挥其生理学作用。益生元可以指这样的物质，所述物质被添加到组合物如食品(如牛奶、乳酪)、药品(如胶囊、片剂、丸剂、粉剂等)或口腔卫生用品等中，其对微生物群的组分发挥作用，有利于建立对健康有利的细菌和/或阻碍致病细菌建立。在一些实施方案中，本发明的菌株可以制备成益生菌或益生元的形式。
在一些实施方案中，该医药组合物包括口服剂型或外用剂型。
在一些实施方案中，该口腔清洁组合物的赋形剂或稀释剂为牙膏、洁牙粉、漱口水、口气清新喷雾、涂氟剂、假牙清洁剂、宠物用洁牙胶或宠物用化毛膏。
在一些实施方案中，该载体为牙刷、牙间刷、牙线、口腔棉棒或宠物用洁牙骨。
在一些实施方案中，本发明提供一种含乳酸菌菌株的食品组合物、口腔清洁组合物以及医药组合物，其可抑制口腔病原菌的生长，以维持口腔健康。
在本发明的组合物中，所述菌株可以以有效量存在。例如组合物中乳酸菌菌株的含量可以为10 6CFU以上，例如10 7CFU以上，10 8CFU以上，10 9CFU以上，10 10CFU以上，10 11CFU以上；优选所述菌株的数量为10 9CFU以上。
在一些实施方案中，已经发现益生菌促进双歧杆菌和/或乳酸菌的生长和/或调节菌群平衡的作用是菌株特异性的。已经发现副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比具有优异的促进双歧杆菌和/或乳酸菌的生长和/或调节菌群平衡的作用。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比具备提高的促进双歧杆菌和/或乳酸菌的生长和/或调节菌群平衡的作用。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比能够促进双歧杆菌和/或乳酸菌的生长和/或提高受试者的菌群平衡(例如提高10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或更多倍)。在一些实施方案中，促进双歧杆菌和/或乳酸菌的生长和/或调节受试者菌群平衡可以通过本领域已知的方法测量。在一些实施方案中，促进双歧杆菌和/或乳酸菌的生长和/或调节受试者菌群平衡可以通过例如比较实验前后自身及组间双歧杆菌、乳酸菌、肠球菌、肠杆菌的变化情况进行。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株可以制备用于促进双歧杆菌和/或乳酸菌的生长和/或调节受试者菌群平衡的组合物和/或试剂盒。在一些实施方案中，所述促进双歧杆菌和/或乳酸菌的生长和/或调节受试者菌群平衡的组合物和/或试剂盒还可以包括其它适当的促进双歧杆菌和/或乳酸菌的生长和/或调节受试者菌群平衡的物质。
在一些实施方案中，已经发现副干酪乳酸杆菌ET-22菌株被发现具有抑制口腔病原菌的能力，例如龋齿菌、牙龈菌及口腔气味菌等。此外，副干酪乳酸杆菌ET-22菌株可以分泌过氧化氢，除了抑制口腔病原菌之外，还可以达到美白牙齿的功效。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比具有优异的免疫调节作用。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比具备提高受试者体液和/或细胞免疫力的作用。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比能够提高受试者体液和/或细胞免疫力(例如提高10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或更多倍)。在一些实施方案中，提高受试者体液和/或细胞免疫力可以通过本领域已知的方法测量，例如通过抗体生成细胞实验和/或半数溶血值HC 50测定，以及NK细胞活性实验等测定。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株可以制备用于增强受试者免疫力的组合物和/或试剂盒。在一些实施方案中，所述增强受试者免疫力的组合物和/或试剂盒还可以包括其它适当的增强免疫力的物质。
在一些实施方案中，已经发现副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比具有优异的抑制口腔病原菌的作用。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比具备产生过氧化物活性，由此可以用于例如牙齿美白。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比能够抑制下述一种或多种口腔病原菌或具有提高的抑制活性(例如提高10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或更多倍)的抑制活 性：变形链球菌(Streptococcus mutans)，具核梭杆菌(Fusobacterium nucleatum)(包括多型亚种subsp.polymorphum)、放线共生放线杆菌(Aggregatibacter actinomycetemcomitans)和牙龈卟啉单孢菌(Porphyromonas gingivalis)等。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比能够抑制全部口腔病原菌或具有提高的抑制活性(例如提高10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或更多倍)的抑制活性：变形链球菌(Streptococcus mutans)，具核梭杆菌(Fusobacterium nucleatum)(包括多型亚种subsp.polymorphum)、放线共生放线杆菌(Aggregatibacter actinomycetemcomitans)和牙龈卟啉单孢菌(Porphyromonas gingivalis)。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株与不含所述菌株的对照或对照菌株如其它乳杆菌菌株(如普通副干酪乳酸杆菌菌株)相比尤其能够抑制下述口腔病原菌或具有提高的抑制活性(例如提高10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或更多倍)的抑制活性：具核梭杆菌(Fusobacterium nucleatum)(包括多型亚种subsp.polymorphum)、放线共生放线杆菌(Aggregatibacter actinomycetemcomitans)和牙龈卟啉单孢菌(Porphyromonas gingivalis)。在一些实施方案中，所述医药组合物可以用于治疗例如龋齿、蛀牙、牙周病如牙周炎、口臭等疾病。在一些实施方案中，副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株可以制备用于牙齿美白和/或治疗口腔疾病的组合物和/或试剂盒。在一些实施方案中，所述牙齿美白和/或治疗口腔疾病的组合物和/或试剂盒还可以包括其它适当的牙齿美白和/或治疗口腔疾病的物质。
在一些实施方案中，本发明提供制备组合物如食品组合物、医药组合物或口腔清洁组合物的方法，所述方法包括将副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株加入到组合物中。
以下藉由具体实施例配合所附的图式详加说明，当更容易了解本发明的目的、技术内容、特点及其所达成的功效。
附图说明
图1.副干酪乳杆菌ET-22ET-22模拟肠道环境耐受性，胃酸、胆盐环境耐受能力评价结果。
图2.副干酪乳杆菌ET-22肠道黏附性评价结果。
图3.副干酪乳杆菌ET-22调节肠道菌群结果。
图4.副干酪乳杆菌ET-22与干酪乳杆菌LC-01调节肠道菌落变化的比较结果。
图5.L.paracasei ET-22激活抗体生成细胞数。
图6.L.paracasei ET-22血清溶血素半数溶血值(HC 50)。
图7.L.paracasei ET-22激活NK杀伤细胞活力。
图8.副干酪乳杆菌ET-22产过氧化氢的试验结果。
图9.副干酪乳杆菌ET-22抑制口腔病原菌的试验结果。
图10.副干酪乳杆菌ET-22去活性乳酸菌菌株抑制口腔病原菌的试验结果。
【生物材料保藏】
本发明的副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株保藏于：
中国典型培养物保藏中心/中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏日期为2017年12月18日、保藏编号CGMCC No.15077；该保藏单位的地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
副干酪乳酸杆菌(Lactobacillus paracasei)K56菌株保藏于：
中国典型培养物保藏中心/中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏日期为2017年12月29日、保藏编号CGMCC No.15139；该保藏单位的地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
具体实施方式
除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。
本发明所述的乳酸菌菌株的冷冻干燥培养物已保藏于中国典型培养物保藏中心以及中国微生物菌种保藏管理委员会普通微生物中心。保藏的详细资料如下表所示：
表1 乳酸菌菌株的保藏资料
菌株名 分类 保藏编号 保藏日期
ET-22 副干酪乳酸杆菌 CGMCC 15077 2017年12月18日
乳酸菌菌株的形态学以及一般性质
根据16S rDNA序列分析以及API细菌鉴定系统分析结果来确认菌株在分类学上的特征。上述菌株在形态学及一般性质上的特征详细列于下表：
表2 乳酸菌菌株的形态学及一般性质特征
Figure PCTCN2019107274-appb-000001
本菌株的发酵条件为：
MRS液体培养基：蛋白胨，10.0g；牛肉膏，10.0g；酵母浸粉，5.0g；葡萄糖，20.0g；磷酸氢二钾，5.0g；柠檬酸氢二铵，2.0g；乙酸钠，5.0g；七水合硫酸镁，0.5g；四水合硫酸锰，0.2g；吐温80，1.0g；琼脂15.0g；蒸馏水1000mL。调整pH至6.2～6.4之间，121℃灭菌15分钟。
L.paracasei ET-22为微好氧菌，兼性厌氧环境生长较佳，产乳酸，具有耐酸性，可耐受pH值2.5的酸性环境及耐0.4％的胆盐环境4小时，嗜中温菌，生长温度范围在15～45℃，最适生长温度在37℃左右。
实施例1：人工胃液、肠液耐受性、人工胆汁耐受性
L.paracasei ET-22活化三次后分装8支，取一支进行菌落计数，计算初始活菌数。其余七只，以4000rpm离心10min，除去上清液后置于pH2.5培养基中培养，37℃，培养1～3小时，每小时取一管混合 液，以4000rpm离心10min，除上清液后，用5mL RO water清洗两次后计算活菌数。另外4支3小时候以4000rpm离心10min，去除上清液(pH 2.5 MRS broth)后，以含1.5％牛胆汁MRS broth将4管充分混匀，在平均分配至4管，37℃培养1～4小时。每小时取1管混合液，以4000rpm离心10min，去除上清液后，用5mL RO Water清洗两次后计算存活菌数。统计不同时间测得活菌数的变化，进行比较分析，菌株的存活率见图1。
结果显示，总菌数经由酸性培养基及胆盐环境连续处理，L.paracasei ET-22经模拟消化环境连续7小时之胃酸、胆碱处理仍维持5次方菌数，证明L.paracasei ET-22是可以通过人体消化系统环境的严格考验。
实施例2.肠道细胞吸附效果
将载玻片以镊子夹取盖玻片，酒精灯杀菌后，放入6孔盘中备用。将Caco-2细胞从保藏管中取出，调整细胞后，移至6孔盘中培养，待满盘后，再进行实验。试验时，移除盘中液体，以PBS缓冲液清洗两次后，每孔加入1.5mL菌液(1×10 9CFU/mL)及1.5mL细胞培养液(含有10％PBS及1％penicilin-streptomycin)混匀，于恒温培养箱中培养(37℃，5％CO 2)4小时后，无菌PBS清洗两次，甲醇固定细胞，革兰氏染色，显微镜下计算活菌数。
通过黏附实验对菌株对Caco-2细胞的黏附性进行检测，结果如图2，L.paracasei ET-22对Caco-2细胞具有较强的黏附性。
实施例3：肠道菌群调节效果
本实施例意图证实本发明的副干酪乳杆菌在肠道调节方面的效果。
取36只健康SPF级BABL/c小鼠，体重18-22g(由北京华阜康生物科技有限责任公司提供)。适应性饲养3天后，将其随机分为3组，每组12只，即空白对照组，样品组。向每组动物分别灌胃溶解了乳双歧杆菌BL-99菌粉的无菌水(灌胃体积0.2mL/10g)，空白对照组灌胃相同体积的无菌水。每天1次，连续饲喂或灌胃14天。灌胃计量：1.3×10 7CFU/ml(按人体需要量为2×10 9CFU/天，人体和小鼠换算系数0.0026进行换算)。取灭菌离心管，编号，适应性喂养后 于无菌条件下采集小鼠粪便，每只2-3粒，约100mg，低温条件转移至无菌操作间进行菌群的检测。实验结束时，再次采集小鼠粪便。使用苦味酸对小鼠进行分组编号后，分别于给予受试物第8天、第14天称重，计算小鼠灌胃量，实验结束时称重1次。菌落计数：根据待鉴定菌种配制选择性培养基，待测菌种及相应培养基见下表，灭菌，摇匀，冷至45℃-50℃倾注平板，备用。
表3 待测菌种及对应选择性培养基
Figure PCTCN2019107274-appb-000002
将采集的小鼠粪便，置于装有0.5mL生理盐水的灭菌管中，配制成菌悬液，使用前震荡1min。使用0.1mL微量移液器吸取0.1mL菌悬液，缓慢注于0.9mL灭菌生理盐水中，震荡或反复吹打使其混匀，制成1∶10的菌悬液。另取0.1mL微量移液器吸头，依此方法，进行10倍梯度稀释，直至10 -7g/ml。根据待鉴定菌种的活菌数，选择两个连续的适宜稀释度，每个稀释度使用10μL微量移液器吸取10μL菌悬液，在选择性琼脂平板上进行表面涂布，按下表所示培养条件进行培养。菌落计数方法参考《GB 4789.2-2010食品安全国家标准食品微生物学检验菌落总数测定》进行。
表4 肠道菌群检验用培养基及鉴定方法
Figure PCTCN2019107274-appb-000003
采用SPSS17.0进行数据统计。比较实验前后自身及组间双歧杆菌、乳酸菌、肠杆菌的变化情况。
实验期间动物的体重变化结果如下表5所示。在实验期内，动物表征正常，给予受试物后未出现任何不良反应，实验周期内，两组动物体重并未出现显著差异。从表6-8可以看出，副干酪乳杆菌ET-22能显著促进双歧杆菌和乳杆菌的生长，对于肠杆菌无显著影响。通过自身组别干预前后的比较来看，ET-22的增幅明显高于业内知名菌株 干酪乳杆菌LC-01(DSM 19465)，具有优异的效果。
因此，本研究中副干酪乳杆菌ET-22具有调节肠道菌群的功效(图3)。
表5 动物的体重变化
Figure PCTCN2019107274-appb-000004
表6 受试前后动物肠道双歧杆菌的变化(LgCFU/g)
Figure PCTCN2019107274-appb-000005
结果显示，ET-22组别自身干预后的增幅比起业内知名菌株LC-01的增幅显著提高：ET-22增加了1.24个数量级，LC-01只增加了0.76个数量级(图4)。ET-22为乳酸杆菌属，却能够显著增加双歧杆菌属超过一个数量级，且增加的量高于LC-01，显示其增加肠道好菌的调节能力极强。
表7 受试前后动物肠道乳杆菌的变化(LgCFU/g)
Figure PCTCN2019107274-appb-000006
结果显示ET-22组别自身干预后的增幅比起业内知名菌株LC-01的增幅高：ET-22增加了0.77个数量级，LC-01增加了0.61个数量级(图4)。
表8 受试前后动物肠道肠杆菌的变化(LgCFU/g)
Figure PCTCN2019107274-appb-000007
Figure PCTCN2019107274-appb-000008
实施例4：肠道菌群调节效果(测序结果)
本实施例意图证实本发明的副干酪乳杆菌在肠道调节方面的效果，其原理和步骤参见“保健食品检验与评价技术规范-调节肠道菌群功能判定标准”。与实施例3不同之处在于本实验采用16S rDNA测序结果。
实验样品：菌粉样品(活菌数见包装规格)由内蒙古乳业技术研究院有限责任公司提供。
实验材料：6周龄健康成年BABL/c小鼠182只，体重约18-20g。
实验步骤
1.样品准备
活菌样品(ET-22)：依据样品规格，各称取1g活菌样品，分别使用PBS溶液悬浮至40ml，即活菌浓度均为2.5x10 9CFU/ml。
高剂量组：按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，高剂量组小鼠灌胃剂量为10 9CFU/20g。
中剂量组：分别取5ml高剂量悬浮液加入PBS定容至50ml，按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，中剂量组小鼠灌胃剂量为10 8CFU/20g。
低剂量组：分别取5ml中剂量组悬浮液加入PBS定容至50ml，按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，低剂量组小鼠灌胃剂量为10 7CFU/20g。
2.调节肠道菌群实验
(1)6周龄BABL/c小鼠饲养于清洁级动物房，温度22℃，湿度10-60％，12小时明暗交替照明，标准饲料喂养，自由饮水。
(2)适应性喂养5天，182只小鼠随机分为13组，每组14只，分组情况见表9。
表9 调节肠道菌群实验分组
Figure PCTCN2019107274-appb-000009
Figure PCTCN2019107274-appb-000010
(3)开始灌胃前，无菌条件下采集每只小鼠粪便，标记，-20℃保存，检测肠道菌群。
(4))实验按照0.2ml/10g灌胃量给予各受试物，对照组1-14天给予PBS，实验组分别依据表9灌胃给予相应剂量的受试物。小鼠每周称重一次，根据体重调整灌胃量。
(5)14天后无菌条件下采集每只小鼠粪便，标记，-20℃保存，检测肠道菌群。
3.小鼠肠道微生物多样性变化
微生物多样性是基于Illumina HiSeq测序平台，利用双末端测序(Paired-End)的方法，构建小片段文库进行测序。通过对Reads拼接过滤，OTUs(Operational Taxonomic Units)聚类，并进行物种注释及丰度分析，可以揭示样品的物种构成；将优化序列进行聚类，划分OTU，并根据OTU的序列组成得到其物种分类。基于OTU分析结果，对样品在各个分类水平上进行分类学分析，获得各样品在菌属水平上的群落结构。
通过Alpha多样性分析研究单个样品内部的物种多样性，统计了各样品在97％相似度水平下的Ace、Chao1、Shannon及Simpson指数，绘制了样品稀释曲线及等级丰度曲线；进一步进行α多样性分析(Alpha Diversity)、β多样性分析(Beta Diversity)和显著物种差异分析等等，可以挖掘样品之间的差异。肠道菌群α多样性指数比较：Chao1和ACE指衡量物种丰度即物种数量的多少；Shannon和Simpson指数用于衡量物种多样性，受样品群落中物种丰度和物种均匀度的影响。相同物种丰度情况下，群落中各物种具有越大的均匀度，则认为群落具有越大的多样性，Shannon指数值越大，Simpson指数值越小，说明样品的物种多样性越高。在属的水平与对照组相比，ET-22干预后可显著增加小鼠肠道中的相对丰度。其中低剂量组的OTU、ACE、Chao1指数显著上升，显示ET-22低剂量最能够明显增加肠道微生物多样性。
Figure PCTCN2019107274-appb-000011
下划线标示：与对照组相比有统计上显著差异(p＜0.05)。
在致病菌属的水平，控制组相比，ET-22低剂量与中剂量两组小鼠肠道中的脱硫弧菌(Desulfovibrio)均显著下降；中剂量与高剂量两组的幽门螺杆菌(Helicobacter)数量显著下降；低剂量与中剂量三组的高埃希氏菌-志贺氏菌属(Escherichia-Shigella)均显著下降。BL-99可显著增加小鼠肠道中的乳酸杆菌属(Lactobacillus)相对丰度，且BL-99低剂量组对乳酸杆菌(Lactobacillus)的增加程度最为显著。
因此，补充不同剂量的ET-22可以调节肠道菌群平衡，抑制有害菌甚至是致病菌数量，起到潜在的健康之功效。
实施例5：免疫调节活性分析
5.1体液免疫实验
5.1.1抗体生成细胞检测实验
经过绵羊红细胞(SRBC)免疫的小鼠脾细胞悬液与一定量的SRBC混合，在补体参与下，使分泌抗体的脾细胞周围的SRBC溶解，形成肉眼可见的空斑，溶血空斑数可反映抗体生成细胞数。
使用PBS悬浮于每日动物灌胃前依据不同灌胃量分别调整活菌样品浓度(用量为约10 7-10 9CFU/天)，动物连续给样28天后，经腹腔对每只小鼠注射SRBC 0.2mL进行免疫。将SRBC免疫4天后的小鼠处死，取脾，制成5×10 6个细胞/mL的细胞悬液。将琼脂糖加热溶解后，与等量双倍的Hank’s液混合，分装小试管，每管0.5mL，再向管内加20％(V/V，用生理盐水配置)压积SRBC 50μL，脾细胞悬液200μL，迅速混匀，倾倒至已刷琼脂糖薄层的六孔板上，带琼脂凝固后，放入二氧化碳培养箱中继续孵育1h，然后加入SA缓冲液稀释的补体(1∶10)，继续孵育2h，计数溶血空斑数。
抗体生成细胞数结果如图5所示，样品组与对照组相比，L.paracasei ET-22、L.paracasei K56(CGMCC 15139)促进抗体生成细胞数与对照组具有显著差异(p＜0.05)。
5.1.2血清溶血素半数溶血值(HC 50)的测定
动物连续给样28天后，经腹腔对每只小鼠注射SRBC 0.2mL进行免疫。4天后，摘除眼球取血于1.5mL离心管内，4℃放置约1h，使血清充分析出，2000r/min离心10min收集血清。取血清用SA缓冲液稀释100 倍。将稀释后的血清加入96孔板，每孔100μL，再依次加入10％(v/v)SRBC 50μL，补体100μL(用SA溶液按1∶8稀释)，置37℃恒温水浴中保温30min，1500r/min离心10min。然后样品孔和空白对照孔各取上清液50μL，加入另一个96孔培养板内，加文奇氏试剂150μL，同时设半数溶血孔，加入10％(v/v)SRBC 12.5μL，再加文奇氏试剂至200μL，用震荡期充分混匀，放置10min后，于540nm处用全自动酶标仪测定各孔光密度值。
溶血素的量以半数溶血值(HC 50)表示，按以下公式计算：
Figure PCTCN2019107274-appb-000012
从图6可以看出，与对照组相比，L.paracasei ET-22组半数溶血值HC 50具有显著性差异(p＜0.05)，且高于L.paracasei K56。
5.2 NK细胞活性试验
动物连续给样28天，开始实验前24h将靶细胞YAC-1进行传代培养，用前以Hank’s液洗2次，用含10％小牛血清的RPMI 1640完全培养液调整细胞浓度为1×10 5个/mL(靶细胞)。小鼠颈椎脱臼猝死，无菌取脾，制成皮细胞悬液，用Hank’s液洗2次，1000rpm离心10min，再用2mL含10％含小牛血清的RPMI 1640完全培养基重悬，用台盼蓝活细胞染色计数(活细胞数应在95％以上)，调整细胞浓度为1×10 7个/mL(效应细胞)，使效靶比为100∶1。取靶细胞和效应细胞各100μL，加入U型96孔培养板中，靶细胞自然释放孔加靶细胞和培养液各100μL，靶细胞最大释放孔加靶细胞和1％NP40各100μL，上述均设三个平行孔。37℃，5％CO 2培养箱中培养4h，将96孔培养板以1500rpm离心5min，每孔吸取上清100μL置平底96孔培养板中，同时加入LDH基质液100μL，反应3min，然后每孔加入1mol/L的HCL溶液30μL终止反应，在酶标仪490nm处测OD值，NK活性按下式计算：
Figure PCTCN2019107274-appb-000013
由图7可知，L.paracasei ET-22激活NK细胞活性的能力高于L.paracaset K56，且与对照组相比具有显著差异(p＜0.05)。
由实验结果可知，L.paracasei ET-22抗体生成细胞实验和半数溶血值HC 50结果阳性，则判定样品L.paracasei ET-22体液免疫阳性；NK细胞 活性实验结果阳性，可判定样品L.paracasei ET-22的NK细胞活性实验阳性。综上，可认为L.paracasei ET-22具有增强免疫力的功效。
L.paracasei ET-22抑制口腔病原菌
目前已知部份菌株对于抑制龋齿菌及牙周菌是有功效的，但文献指出，多数的研究都是针对个别菌株在口腔内的功效进行试验，且不是所有的试验结果皆证明乳酸菌对于口腔健康是有帮助的，还是有菌株功效上的个体差异(Anna Haukioja，European Journal of Dentistry 2010(4)：348-355)。举例而言，Vuotto C等(International Journal of Oral Science 2014(6)：189-194)针对益生菌对于病原菌的抑制能力分析，提到任何一株益生菌都不会是相同的，即便是同种不同株的益生菌，试验的结果有时都会是相反的。这样的结果更加强调益生菌对于口腔病原菌的抑制及美白牙齿等功效确实是有菌株特异性的现象。
事实上，由本发明的下列试验结果可知，多数乳酸杆菌菌株并不具有抑制口腔病原菌及美白牙齿的作用。国内外目前针对益生菌在维护口腔健康相关功能研究的文献报导在近期才有陆续增加的趋势。早期认为益生菌之中的乳酸菌各属，由于产酸的特性有可能与口腔病原菌共存之外，还有可能促成腐蚀珐琅质，而造成龋齿。但实际上，由实验显示乳酸菌不但可以抑制龋齿菌及牙周菌之外，还能与之竞争口腔黏膜的生存空间及营养来源，并且造成口腔病原菌的凝集而易排除。但这些特性都必须透过实验才能确认，并非所有的菌株皆有相同的特性及实验结果。
实施例6：分析菌株产生过氧化氢的特性，以确认对于牙齿美白的功能
检视本发明的乳酸菌菌株-副干酪乳酸杆菌ET-22菌株产生过氧化氢的能力，藉此来验证本发明的乳酸菌菌株的美白牙齿的能力。实验步骤如下：
1.制备益生菌产过氧化氢筛选平板。
2.在平板内加入三甲基硼(Trimethyl borane TMB)0.25mg/mL与辣根过氧化物酶(Horseradish Peroxidase HRP)0.01mg/mL。
3.在平板上以画四区方式培养目标菌株。
4.培养两天后，有产生过氧化氢的乳酸菌菌株，会在菌落周围呈现蓝 色。
5.利用过氧化氢感测试纸，侦测乳酸菌菌体及二次代谢物含过氧化氢的浓度。
6.培养乳酸菌菌液并以4500rpm离心5分钟。
7.取菌体的部分溶于4.9毫升100mM的哌嗪-1，4-二乙磺酸(Piperazine-1，4-bisethanesulfonicacid，PIPES)。
8.以37℃220rpm培养5小时，将菌体离心后取10微升(μL)上清液或沉降的菌体滴于过氧化氢检测试纸(Merck)反应10秒钟，观察颜色变化并与比色卡比对记录浓度。
实验结果如图8所示。图8为副干酪乳酸杆菌ET-22菌株在过氧化氢检测平板上培养后，在平板上显示分泌过氧化氢能力的结果。
由图8的试验结果可知，培养两天后，在菌株的生长过程中，本发明的副干酪乳酸杆菌ET-22菌株有良好的过氧化氢分泌情形。另须说明的是，非本发明的相同菌种的乳酸菌菌株，例如副干酪乳酸杆菌9菌株(BCRC16093)，则没有分泌过氧化氢的能力，如图8所示。
实施例7：维持口腔健康的活性乳酸菌对于口腔病原菌的抑制能力
乳酸菌要发挥口腔保健功能，首重口腔病原菌的抵抗能力是否强大。口腔病原菌不仅仅只是大众比较熟知的变形链球菌(Streptococcus mutans)，还包含具核梭杆菌多型亚种(Fusobacterium nucleatum subsp.polymorphum)、放线共生放线杆菌(Aggregatibacter actinomycetemcomitans)、牙龈卟啉单孢菌(Porphyromonas gingivalis)等。各菌株所引起的症状分别是：变形链球菌造成龋齿；具核梭杆菌多型亚种主要造成牙周病、口臭及结肠癌等问题；放线共生放线杆菌则是引起牙周炎、口腔发炎及肺炎的主要菌种；牙龈卟啉单孢菌则是成人型牙周炎致病菌及口臭的主因。此外，对于口腔气味，除了牙龈卟啉单孢菌是引起口臭的主要菌种外，具核梭杆菌多型亚种、放线共生放线杆菌以及牙龈卟啉单孢菌皆会产生硫化物，也是影响口腔气味的主要菌种之一。
因此，本发明进行病原菌抑制试验，评估本发明的活性副干酪乳酸杆菌ET-22菌株抑制口腔病原菌的能力，进而达成维持口腔健康的效果。使用如下的实验步骤：
1.将活性乳酸菌涂布于MRS平板中央两公分直径处，培养两天。
2.倒入口腔病原菌之培养基于已生长乳酸菌之平板上方，待凝固后，将高浓度口腔致病菌涂布于病原菌培养基平板上，涂布均匀。
3.培养在37℃环境中2-4天。
4.中央下层培养基有涂布乳酸菌的部分是否有抑制病原菌生长，测量抑制直径大小。
实验结果如图9所示，图9为本发明的乳酸菌菌株抑制口腔病原菌的能力分析。
如图9所示为本发明的副干酪乳酸杆菌ET-22菌株抑制口腔病原菌生存的结果，与另一株副干酪乳杆菌9比较，ET-22的抑制效果最为显著。
实施例8：维持口腔健康的去活性乳酸菌对于口腔病原菌的抑制能力
除了活性乳酸菌抑制口腔病原菌的功能外，去活性之乳酸菌对于抑制口腔病原菌的能力是否尚保有其功能由为未未知，因此，本发明进行病原菌抑制试验，评估本发明的去活性副干酪乳酸杆菌ET-22菌株抑制口腔病原菌的能力，进而达成维持口腔健康的效果。使用如下的实验步骤：
1.活化口腔病原菌。
2.将热致死乳酸菌调整菌数，依每毫升十亿菌体的比例与病原菌共同培养。
3.以厌氧方式、150rpm转速培养在37℃环境中2-4天。
4.计数病原菌的数量。
实验结果如图10所示，图10为本发明的去活性乳酸菌菌株抑制口腔病原菌的能力分析；柱状图皆以Mean±SD表示之，并与另一株副干酪乳杆菌9比较。
如图10所示为本发明的去活性副干酪乳酸杆菌ET-22菌株抑制口腔病原菌生存的结果，与副干酪乳杆菌9菌株的抑制效果相比，ET-22最为显著。
综合以上试验结果，本发明的含乳酸菌菌株的食品组合物以及医药组合物可抑制口腔病原菌的生长，可作为降低人体龋齿、牙周病及口气异味等应用。较佳者，可分泌过氧化氢的乳酸菌菌株对于牙齿的美白亦有作用能力。本发明找出对人体无副作用且有益于维持口腔健康乳酸菌作为维持口腔健康的新选择。
除非另有说明，本说明书(包括权利要求书)中使用的所有表示成分、细胞培养、处理条件等的量的数字应当理解为在所有条件下受到术语“约”的修饰。因此，除非另有相反的说明，数值参数为近似值，并且可以根据通过本发明试图获得的期望特性而变化。除非另有说明，一系列元素之前的术语“至少”应当理解为指该系列中的每个元素。本领域技术人员会认识到或者能够利用不超过常规实验确定本文所述的本发明的具体实施方案的许多等同物。所附权利要求书意图涵盖这类等同物。
本领域技术人员会清楚，可以进行本发明的许多修改和变化而不背离其精神和范围。本文所述的具体实施方案仅通过实例的方式提供，并不意味着以任何方式限制。本发明的真正范围和精神通过所附权利要求书示出，说明书和实施例仅是示例性的。
以上所述的实施例仅是为说明本发明的技术思想及特点，其目的在使本领域技术人员能够了解本发明的内容并据以实施，当不能以之限定本发明的专利范围，即大凡依本发明所揭示的精神所作的均等变化或修饰，仍应涵盖在本发明的专利范围内。

Claims (15)

  1. 副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株，其保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏日期为2017年12月18日，保藏编号为CGMCC No.15077。
  2. 一种组合物，其包含权利要求1所述的副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株，和赋形剂、稀释剂和/或载体。
  3. 权利要求2所述的组合物，其中所述组合物包括食品组合物、医药组合物和口腔清洁组合物。
  4. 权利要求2所述的组合物，其中所述ET-22菌株为具有活性的菌株和/或去活性的菌株。
  5. 权利要求3所述的组合物，其中所述组合物为食品组合物，所述赋形剂、稀释剂和/或载体包括乳制饮品、茶、咖啡、口香糖及洁牙糖、宠物用肉干或以上的组合。
  6. 权利要求3所述的组合物，其中所述组合物为口腔清洁组合物，所述赋形剂、稀释剂和/或载体为牙膏、洁牙粉、潄口水、口气清新喷雾、涂氟剂、假牙清洁剂、宠物用洁牙胶或宠物用化毛膏、牙刷、牙间刷、牙线、口腔棉棒或宠物用洁牙骨。
  7. 权利要求3所述的组合物，其中所述组合物为医药组合物，所述组合物：1)制成口服剂型；2)包括治疗和/或预防用组合物；3)包括营养组合物；4)制成干剂或湿剂；或5)制成凝胶、乳膏、喷雾剂、气溶胶、软膏、乳剂、混悬剂、贴片、口含片剂或舌下片剂形式。
  8. 权利要求2所述的组合物，其中所述组合物1)用于牙齿美白和/或抑制口腔病原菌；2)用于调节受试者菌群平衡；3)用于促进双歧杆菌和/或乳酸菌的生长；和/或4)用于增强受试者免疫力，例如增强所述受试者的体液免疫和/或细胞免疫，例如提高抗体生成细胞数、提高半数溶血值HC 50和/或提高NK细胞活性。
  9. 权利要求2所述的组合物，其中所述组合物通过胃肠道途径施用给受试者，例如哺乳动物受试者。
  10. 权利要求2所述的组合物，其中所述组合物中ET-22菌株的含量为10 6CFU以上，例如10 7CFU以上，10 8CFU以上，10 9CFU以上，10 10CFU以上，或10 11CFU以上。
  11. 一种治疗受试者的方法，其包括将有效量的权利要求1所述的副干酪乳酸杆菌(Lactobacillus paracasei)ET-22菌株施用给有需要的受试者，由此1)用于牙齿美白和/或抑制口腔病原菌；2)用于调节受试者菌群平衡；3)用于促进双歧杆菌和/或乳酸菌的生长；和/或4)用于增强受试者免疫力，例如增强所述受试者的体液免疫和/或细胞免疫，例如提高抗体生成细胞数、提高半数溶血值HC 50和/或提高NK细胞活性。
  12. 权利要求11所述的方法，其中所述ET-22菌株为权利要求2所述的组合物的形式。
  13. 权利要求11所述的方法，其中所述ET-22菌株为具有活性的菌株和/或去活性的菌株。
  14. 权利要求11所述的方法，其中所述ET-22菌株通过胃肠道途径施用给受试者，例如哺乳动物受试者。
  15. 权利要求11所述的方法，其中所述ET-22菌株以10 6CFU以上，例如10 7CFU以上，10 8CFU以上，10 9CFU以上，10 10CFU以上，或10 11CFU以上的量施用给所述受试者。
PCT/CN2019/107274 2018-09-30 2019-09-23 副干酪乳杆菌et-22及其用途 WO2020063531A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/259,137 US11274275B2 (en) 2018-09-30 2019-09-23 Lactobacillus paracasei ET-22 and use thereof
EP19865158.0A EP3822338A4 (en) 2018-09-30 2019-09-23 LACTOBACILLUS PARACASEI ET-22 AND USE OF IT

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201811164616.7 2018-09-30
CN201811147290.7A CN110960559B (zh) 2018-09-30 2018-09-30 用于抑制口腔病原菌的乳酸菌菌株的食品、口腔清洁以及医药组合物
CN201811147289.4 2018-09-30
CN201811147289.4A CN110964653B (zh) 2018-09-30 2018-09-30 一种可调节肠道菌群平衡的副干酪乳杆菌et-22
CN201811147290.7 2018-09-30
CN201811164616.7A CN110964658B (zh) 2018-09-30 2018-09-30 一种具有免疫调节功能的副干酪乳杆菌et-22

Publications (1)

Publication Number Publication Date
WO2020063531A1 true WO2020063531A1 (zh) 2020-04-02

Family

ID=69949305

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/107274 WO2020063531A1 (zh) 2018-09-30 2019-09-23 副干酪乳杆菌et-22及其用途

Country Status (3)

Country Link
US (1) US11274275B2 (zh)
EP (1) EP3822338A4 (zh)
WO (1) WO2020063531A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3811955A4 (en) * 2018-09-30 2021-06-16 Inner Mongolia Yili Industrial Group Co., Ltd. NEW USE OF LACTOBACILLUS PARACASEI K56 THAT IS ABLE TO REGULATE THE REST OF THE GASTROINTESTINAL FLORA
NL2035207A (en) * 2022-11-22 2023-07-27 Chongqing Tianyou Dairy Co Ltd Application of lactobacillus paracasei in preparation for reducing serum cholesterol levels

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110959676B (zh) * 2018-09-30 2022-11-25 内蒙古伊利实业集团股份有限公司 一种含乳双歧杆菌的发酵乳制品及其应用
CN117586926B (zh) * 2024-01-18 2024-04-16 东北农业大学 副干酪乳杆菌lp-116、lp-116后生元组合物及其制备方法与应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102021127A (zh) * 2010-07-28 2011-04-20 江南大学 一株副干酪乳杆菌及其应用
CN103070287A (zh) * 2013-02-26 2013-05-01 湖南省微生物研究所 副干酪乳杆菌发酵饲料及其制备方法、发酵设备和用途
CN108148784A (zh) * 2018-01-31 2018-06-12 石河子大学 一株副干酪乳杆菌菌株及其在制备果蔬汁发酵饮料中的应用
CN108338361A (zh) * 2017-01-24 2018-07-31 丰华生物科技股份有限公司 用于抑制口腔病原菌的乳酸菌菌株的食品、口腔清洁以及医药组合物
CN108603165A (zh) * 2016-01-19 2018-09-28 西姆莱斯有限公司 用于改变口腔生物膜之组成的益生菌

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1034787A1 (en) 1999-03-11 2000-09-13 Société des Produits Nestlé S.A. Lactobacillus strains preventing diarrhea caused by pathogenic bacteria
EP1634948A1 (en) 2004-09-10 2006-03-15 Basf Aktiengesellschaft Means and methods for preventing and/or treating caries
EP1972208A1 (en) 2007-03-16 2008-09-24 Kirin Holdings Kabushiki Kaisha Composition for improving intestinal microflora
AU2009257440B2 (en) * 2008-06-13 2014-09-25 ProBiora Health, LLC Use of hydrogen peroxide-producing bacteria for tooth whitening
WO2011007584A1 (ja) 2009-07-16 2011-01-20 国立大学法人広島大学 口腔内疾患の予防、改善又は治療剤
EP2420580A1 (en) * 2010-08-18 2012-02-22 AB-Biotics, S.A. Probiotic composition for oral health
US8980289B2 (en) 2010-08-31 2015-03-17 Niigata University Intestine immunomodulator
WO2012071380A1 (en) * 2010-11-24 2012-05-31 Oragenics, Inc. Use of bacteria to treat and prevent respiratory infections
JP5904028B2 (ja) 2012-06-19 2016-04-13 ライオン株式会社 乳酸菌及びその培養由来物、ならびにこれらを含有する組成物
AU2014288647B8 (en) 2013-07-12 2015-10-29 Morinaga Milk Industry Co., Ltd. Novel Lactic Acid Bacterium, Drug, Food or Drink, and Feed which Contain the Novel Lactic Acid Bacterium
WO2015159124A1 (en) 2014-04-15 2015-10-22 Compagnie Gervais Danone Use of lactobacillus paracasei for promoting intestinal clearance of opportunistic pathogens after antibiotic dysbiosis
CN104523762B (zh) 2014-12-10 2019-01-22 中国农业大学 具有抗过敏作用的副干酪乳杆菌的应用和功能食品组合物及其制备方法
CN104894021B (zh) 2015-06-03 2018-02-09 宜兰食品工业股份有限公司 一种副干酪乳杆菌及其应用
EP3117822A1 (en) 2015-07-17 2017-01-18 AB-Biotics, S.A. Self-film-forming composition for oral care
TWI592487B (zh) * 2016-10-05 2017-07-21 益福生醫股份有限公司 新穎副乾酪乳桿菌副乾酪亞種ｋ５６
EA202090769A1 (ru) * 2018-05-04 2020-06-11 Борис Славинович ФАРБЕР Пищевая, косметическая и фармацевтическая композиция с иммуномодулирующим и протективным антивирусным эффектом
US11559479B2 (en) * 2018-07-16 2023-01-24 ProBiora Health, LLC Beverage additive
US20210000890A1 (en) * 2019-07-02 2021-01-07 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization Compositions and methods for dental care

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102021127A (zh) * 2010-07-28 2011-04-20 江南大学 一株副干酪乳杆菌及其应用
CN103070287A (zh) * 2013-02-26 2013-05-01 湖南省微生物研究所 副干酪乳杆菌发酵饲料及其制备方法、发酵设备和用途
CN108603165A (zh) * 2016-01-19 2018-09-28 西姆莱斯有限公司 用于改变口腔生物膜之组成的益生菌
CN108338361A (zh) * 2017-01-24 2018-07-31 丰华生物科技股份有限公司 用于抑制口腔病原菌的乳酸菌菌株的食品、口腔清洁以及医药组合物
CN108148784A (zh) * 2018-01-31 2018-06-12 石河子大学 一株副干酪乳杆菌菌株及其在制备果蔬汁发酵饮料中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANNA HAUKIOJA, EUROPEAN JOURNAL OF DENTISTRY, no. 4, 2010, pages 348 - 355
See also references of EP3822338A4
VUOTTO C ET AL., INTERNATIONAL JOURNAL OF ORAL SCIENCE, no. 6, 2014, pages 189 - 194

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3811955A4 (en) * 2018-09-30 2021-06-16 Inner Mongolia Yili Industrial Group Co., Ltd. NEW USE OF LACTOBACILLUS PARACASEI K56 THAT IS ABLE TO REGULATE THE REST OF THE GASTROINTESTINAL FLORA
US11666613B2 (en) 2018-09-30 2023-06-06 Inner Mongolia Yili Industrial Group Co., Ltd. Use of Lactobacillus paracasei subsp. paracasei K56 capable of regulating gastrointestinal flora balance
NL2035207A (en) * 2022-11-22 2023-07-27 Chongqing Tianyou Dairy Co Ltd Application of lactobacillus paracasei in preparation for reducing serum cholesterol levels

Also Published As

Publication number Publication date
EP3822338A1 (en) 2021-05-19
EP3822338A4 (en) 2021-08-18
US20210253995A1 (en) 2021-08-19
US11274275B2 (en) 2022-03-15

Similar Documents

Publication Publication Date Title
TWI639389B (zh) 用於抑制口腔病原菌之乳酸菌菌株之食品、口腔清潔以及醫藥組合物
WO2020063531A1 (zh) 副干酪乳杆菌et-22及其用途
RU2446814C2 (ru) Микроорганизмы молока млекопитающего, их содержащие композиции и их применение для лечения мастита
Teughels et al. Probiotics and oral healthcare
JP4455333B2 (ja) プロバイオティック細菌：Ｌａｃｔｏｂａｃｉｌｌｕｓｆｅｒｍｅｎｔｕｍ
Bizzini et al. Probiotics and oral health
Jain et al. Probiotics and their efficacy in improving oral health: a review
CN110964653B (zh) 一种可调节肠道菌群平衡的副干酪乳杆菌et-22
CN110964658B (zh) 一种具有免疫调节功能的副干酪乳杆菌et-22
JP2016513631A (ja) ラクトバチルス・プランタルムｎｃｃ２９３６調製物及び口腔健康
EP2220210B1 (en) Strains of lactobacillus plantarum as probiotics with immunomodulatory specific effect
CN110960559B (zh) 用于抑制口腔病原菌的乳酸菌菌株的食品、口腔清洁以及医药组合物
JP2008502606A (ja) 乳酸菌及び下痢の予防におけるそれらの使用
CN107530382B (zh) 防龋剂和防龋组合物
Pande et al. Prospectus of probiotics in modern age diseases
JP5204977B2 (ja) 歯周病原因菌の除菌用微生物及び当該微生物を含有する歯周病予防又は治療のための医薬組成物、食品又は食品添加物
Gomes et al. Action of probiotics on oral pathogens: Efficacy and controversies
Pavithra et al. Probiotics–A Miracle in Periodontal Therapy
Sonawane et al. Probiotics: The Nano Soldiers for Periodontium
CN118146972A (zh) 一种改善口腔健康的瑞士乳杆菌及益生菌组合物
Shankar et al. Dr. Ambar Khan
TW202233217A (zh) 提升口腔免疫球蛋白a含量及抑制口腔病原菌之組成物及其用途
JP2021511822A (ja) 新規ラクトバチルス・サリバリウス株を含む組成物、及び中耳炎と上気道感染症の予防法／治療法
Sudhir An Assessment of the Effect of Short Term Consumption of Probiotic Curd on Salivary P H And Streptococcus Mutans Counts
GS Comparative Evaluation of the Efficacy of the Probiotic, Chlorhexidineand Fluoride Mouthwash Against Salivary Streptococcus Mutans-An in Vivo Study

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19865158

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 19865158.0

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE