CN110960559B - Food, oral cleaning and pharmaceutical composition for inhibiting lactic acid bacteria strain of oral pathogenic bacteria - Google Patents
Food, oral cleaning and pharmaceutical composition for inhibiting lactic acid bacteria strain of oral pathogenic bacteria Download PDFInfo
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- CN110960559B CN110960559B CN201811147290.7A CN201811147290A CN110960559B CN 110960559 B CN110960559 B CN 110960559B CN 201811147290 A CN201811147290 A CN 201811147290A CN 110960559 B CN110960559 B CN 110960559B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT 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
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- A—HUMAN NECESSITIES
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- 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
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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
- A61K8/99—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
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- 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
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- 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
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- 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
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- 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
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Abstract
The present invention relates to food, oral cleansing and pharmaceutical compositions for inhibiting lactic acid bacterial strains of oral pathogenic bacteria. The invention relates to application of Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain in tooth whitening and/or oral pathogenic bacteria inhibition. The present invention also relates to a lactic acid bacteria strain-containing food composition, pharmaceutical composition or oral cleaning composition comprising: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15077; and excipients, diluents and/or carriers. The lactobacillus strain has the function of inhibiting oral pathogenic bacteria, and can be in the form of food, oral cleaning or pharmaceutical composition.
Description
Technical Field
The present invention relates to a food composition, an oral cavity cleaning composition and a pharmaceutical composition, and particularly to a food composition, an oral cavity cleaning composition and a pharmaceutical composition comprising a lactic acid bacteria strain inhibiting oral pathogenic bacteria.
Background
Infant's primary teeth care will affect the permanent teeth in the future, and therefore, oral cleaning is very important from the infancy stage. If bacteria grow in the oral cavity, the bacteria are easy to attach to the surface of the gum, and the bacteria can be directly attached to the teeth of the baby after the teeth grow out to cause dental caries. The deciduous teeth are the basis for cutting teeth, and the deciduous teeth can maintain proper space and distance to provide for the growth of permanent teeth in the future. If the breast teeth are not well taken care of, the breast teeth are left to generate decayed teeth, the space can be seriously lost due to the decayed teeth, and bacteria and pustules are arranged around the permanent teeth when the permanent teeth develop in the future, so that the newly grown teeth are easy to form the decayed teeth, and even the incidence rate of adult and old periodontal diseases is influenced.
Consumption of Lactic Acid Bacteria (LAB) -containing products generally only has the effect of regulating the health of the intestinal tract. Although there are tens of thousands of strains of lactic acid bacteria in the natural world, only a few strains of lactic acid bacteria have been found to have the potential accessory traits of maintaining oral health and whitening teeth. The characteristics of the strains such as the capability of inhibiting oral pathogenic bacteria, the capability of adsorbing oral mucosa epidermal cells, whether the capability of inhibiting the pathogenic bacteria and generating hydrogen peroxide can be maintained in a tooth cleaning product and the like are important basis for exciting the strains to show functions in the oral cavity, controlling the micro-ecological balance in the oral cavity and screening strains with the functions of maintaining the oral health and whitening teeth.
The literature indicates that probiotics can compete with pathogenic bacteria for attached living areas and nutritional requirements, promote the pathogenic bacteria to generate aggregation characteristics, secrete antibacterial substances to enable the pathogenic bacteria to be weak or even kill the pathogenic bacteria, achieve the effects of reducing the number of the oral pathogenic bacteria and maintaining the oral health, and secrete hydrogen peroxide to remove oral odor and whiten teeth.
To date, only a few strains have been experimentally found to have the activity of maintaining oral health and whitening teeth. While the function of lactic acid bacteria on physical health lies in the specificity of strains (strains) rather than strains (species), such strains having a particular effect on human health are called functional probiotics (probiotic bacteria for the evaluation of probiotics in food; Report of joint FAO/WHO working group on drawing and identification peptides for the evaluation of probiotics in food; London Ontario, Canada April 30and May 1, 2002: 1-7).
In view of the above, the development of functional probiotics having an effect of inhibiting the activity of oral pathogenic bacteria is an object of the present efforts.
Disclosure of Invention
In some embodiments, the invention provides Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, which is deposited in the china common collection of microorganisms (deposit No. CGMCC No. 15077).
In some embodiments, the present invention provides a culture comprising said Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain. In some embodiments, the culture may also include suitable lactic acid bacteria media, including solid media and liquid media, such as MRS media. In some embodiments, the culture comprises a freeze-dried culture of Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for tooth whitening and/or inhibiting oral pathogens. In some embodiments, the present invention provides methods for tooth whitening and/or inhibiting oral pathogenic bacteria and/or treating diseases infected with oral pathogenic bacteria, the methods comprising administering to a subject a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain. In some embodiments, the present invention provides methods of treating diseases such as dental caries, tooth decay, periodontal diseases such as periodontitis, halitosis, and the like, comprising administering to a subject a Lactobacillus paracasei ET-22 strain. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain may be provided to a subject in the form of compositions such as food compositions, oral cleaning compositions, and pharmaceutical compositions. In some embodiments, the present invention provides methods of inhibiting oral pathogens, which may include Streptococcus mutans (Streptococcus mutans), Fusobacterium nucleatum (including subsp.
In some embodiments, the present invention provides a lactic acid bacteria strain-containing composition comprising: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15077; and excipients, diluents and/or carriers. In some embodiments, the composition is a composition suitable for use in the oral cavity, for example for tooth whitening and/or for inhibiting oral bacteria.
In some embodiments, the lactic acid bacterial strain is an active strain.
In some embodiments, the lactic acid bacterial strain is a inactivated strain.
In some embodiments, the composition may be in the form of any composition suitable for use, for example, a food (including beverage) composition, a pharmaceutical composition, or an oral cleaning composition. In some embodiments, the food composition, pharmaceutical composition or oral cleaning composition is a composition for the oral cavity, such as a food composition, pharmaceutical composition or oral cleaning composition for tooth whitening and/or for inhibiting oral bacteria. In some embodiments, the composition containing the inventive lactic acid bacterium strain has tooth whitening and/or oral pathogenic bacterium inhibiting effects, and is in the form of food, oral cleaning or pharmaceutical composition.
In some embodiments, the composition may be a food composition. In some embodiments, the excipient, diluent and/or carrier may be a food product.
In some embodiments, the food product may include a beverage product such as a food product, oil, meat product, dairy product, seafood product, can, sugary food product, cold food product, wine product, pet food product, and the like. In some embodiments, the food product may include beverages such as dairy drinks, tea, coffee, chewing gum and dentrifice, jerky for pets, or combinations thereof.
In some embodiments, the composition may be a pharmaceutical composition. In some embodiments, the composition may be formulated into an oral dosage form or a topical dosage form. In some embodiments, the composition may comprise a therapeutic and/or prophylactic composition. In some embodiments, the composition may be a nutritional composition. In some embodiments, the composition may be formulated as a dry or wet formulation. In some embodiments, the composition may be formulated as a gel, cream, spray, aerosol, ointment, emulsion, suspension, patch, buccal tablet, sublingual tablet, and the like.
In some embodiments, the composition may be an oral cleaning composition. In some embodiments, the excipient, diluent and/or carrier may be a toothpaste, dentifrice, mouthwash, breath freshening spray, fluoridating agent, denture cleanser, denture gel for pets, or depilatory cream for pets. In some embodiments, the excipient, diluent and/or carrier is a toothbrush, interdental brush, dental floss, oral swab, or pet bone. In some embodiments, furthermore, the composition may be a solid, powder, or slurry oral hygiene composition. In some embodiments, the composition may be a toothpaste, chewing gum, candy, liquid mouthwash such as mouthwashes, and the like.
In some embodiments, the compositions of the present invention may be used as probiotic and/or prebiotic compositions, pharmaceutical compositions for the treatment of oral infections, or functional foods. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain has activity in inhibiting oral pathogens.
In this context, probiotic bacteria may refer to the use of living microorganisms, which can be added to compositions such as food products (e.g. milk, cheese), pharmaceutical products (e.g. capsules, tablets, pills, powders, etc.) or oral hygiene products, etc., maintaining activity and exerting their physiological effect on the subject ingesting the composition containing said probiotic bacteria. Prebiotics may refer to substances added to compositions such as food products (e.g. milk, cheese), pharmaceutical products (e.g. capsules, tablets, pills, powders, etc.) or oral hygiene products, etc., which act on components of the microbiota, facilitating the establishment of bacteria beneficial to health and/or hindering the establishment of pathogenic bacteria. In some embodiments, the strains of the invention may be prepared in the form of probiotics or prebiotics.
In some embodiments, the present invention provides a food composition comprising a lactic acid bacterial strain comprising:
a lactic acid bacterium strain having an activity inhibiting effect against oral pathogenic bacteria, which is selected from at least one isolated lactic acid bacterium strain of the following group: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, preserved in China general microbiological culture Collection center (preservation number CGMCC No. 15077); and a physiologically acceptable excipient or diluent.
In some embodiments, the lactic acid bacterial strain is an active strain.
In some embodiments, the lactic acid bacterial strain is a inactivated strain.
In some embodiments, the excipient or diluent is a food product.
In some embodiments, the food product comprises a dairy drink, tea, coffee, chewing gum and dentrifice, jerky for pets, or a combination thereof.
In some embodiments, the present invention provides a pharmaceutical composition comprising a lactic acid bacterial strain comprising:
a lactic acid bacterial strain having an activity inhibiting effect against oral pathogenic bacteria, the lactic acid bacterial strain being selected from at least one isolated lactic acid bacterial strain of the group consisting of: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, preserved in China general microbiological culture Collection center (preservation number CGMCC No. 15077); and a pharmaceutically acceptable excipient or diluent.
In some embodiments, the lactic acid bacterial strain is an active strain.
In some embodiments, the lactic acid bacterial strain is a inactivated strain.
In some embodiments, the pharmaceutical composition is an oral dosage form or a topical dosage form.
In some embodiments, the present invention provides an oral cleaning composition comprising a lactic acid bacterial strain comprising:
a lactic acid bacterial strain having an activity inhibiting effect against oral pathogenic bacteria, the lactic acid bacterial strain being selected from at least one isolated lactic acid bacterial strain of the group consisting of: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is preserved in China general microbiological culture Collection center (preservation number CGMCC No. 15077).
In some embodiments, the lactic acid bacterial strain is an active strain.
In some embodiments, the lactic acid bacterial strain is a inactivated strain.
In some embodiments, the excipient or diluent of the oral cleaning composition is a toothpaste, dentifrice, mouthwash, breath freshening spray, fluorine-coated, denture cleanser, dentifrice for pets, or hair melting paste for pets.
In some embodiments, the carrier is a toothbrush, an interdental brush, dental floss, a dental floss stick, or a dental bone for pets.
In some embodiments, the present invention provides a food composition, an oral cleaning composition, and a pharmaceutical composition containing a lactic acid bacteria strain, which can inhibit the growth of oral pathogenic bacteria to maintain oral health.
The lactic acid bacteria strain-containing food composition according to an embodiment of the present invention comprises a lactic acid bacteria strain having an activity inhibiting effect against oral pathogenic bacteria, the lactic acid bacteria strain being selected from at least one isolated lactic acid bacteria strain selected from the group consisting of: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, preserved in China general microbiological culture Collection center (preservation number CGMCC No. 15077); and a physiologically acceptable excipient or diluent.
A pharmaceutical composition comprising a lactic acid bacterium strain according to another embodiment of the present invention comprises a lactic acid bacterium strain having an activity inhibiting effect against oral pathogenic bacteria, the lactic acid bacterium strain being selected from at least one isolated lactic acid bacterium strain selected from the group consisting of: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, preserved in China general microbiological culture Collection center (preservation number CGMCC No. 15077); and a physiologically acceptable excipient or diluent.
A further embodiment of the present invention provides an oral cleaning composition comprising a lactic acid bacterium strain selected from at least one isolated lactic acid bacterium strain selected from the group consisting of: lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, preserved in China general microbiological culture Collection center (preservation number CGMCC No. 15077); and a physiologically acceptable excipient or diluent.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for the preparation of a composition, such as a food composition, a pharmaceutical composition, or an oral cleaning composition.
In some embodiments, the present invention provides the use of a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for the preparation of a pharmaceutical composition for inhibiting oral pathogenic bacteria. In some embodiments, it has surprisingly been found that a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain has an excellent effect of inhibiting oral pathogens compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strains), which do not contain said strain. In some embodiments, Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain has superoxide generating activity compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strains), that does not contain the strain, and thus may be used, for example, for tooth whitening. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is capable of inhibiting or has an increased inhibitory activity (e.g., an increased inhibitory activity of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) of one or more of the following oral pathogens as compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strains), that does not contain the strain: streptococcus mutans (Streptococcus mutans), Fusobacterium nucleatum (Fusobacterium nucleatum) (including subsp. polysp. polytrichum), Actinobacillus actinomycetemcomitans (Agrobacterium actinomycetemcomitans), and Porphyromonas gingivalis (Porphyromonas gingivalis), among others. In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is capable of inhibiting all oral pathogens or has an increased inhibitory activity (e.g., increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) as compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strains), that does not contain the strain: streptococcus mutans (Streptococcus mutans), Fusobacterium nucleatum (Fusobacterium nucleatum) (including subsp. polymorpha), Actinobacillus actinomycetemcomitans (Agrobacterium actinomycetemcomatus), and Porphyromonas gingivalis (Porphyromonas gingivalis). In some embodiments, the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is capable of inhibiting or has an increased inhibitory activity (e.g., increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) of the following oral pathogens, in particular, as compared to a control or control strain, such as other Lactobacillus strains (e.g., Lactobacillus paracasei strains), that does not contain the strain: fusobacterium nucleatum (including subsp. polymorpha), Actinomyces actinomycetemcomitans (Actinomyces actinomycetemcomitans), and Porphyromonas gingivalis (Porphyromonas gingivalis). In some embodiments, the pharmaceutical compositions can be used to treat diseases such as dental caries, tooth decay, periodontal diseases such as periodontitis, halitosis, and the like. In some embodiments, a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain may prepare a composition and/or kit for tooth whitening and/or treating oral diseases. In some embodiments, the tooth whitening and/or oral disease treatment composition and/or kit may further include other suitable tooth whitening and/or oral disease treatment substances.
In some embodiments, the present invention provides a method of preparing a composition, such as a food composition, a pharmaceutical composition, or an oral cleaning composition, comprising adding a Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain to the composition.
The purpose, technical content, features and effects of the present invention will be more readily understood by the following detailed description of the embodiments taken in conjunction with the accompanying drawings.
Drawings
FIG. 1 shows the results of the hydrogen peroxide production test by the lactic acid bacterium strain of the present invention.
FIG. 2 shows the results of the test for inhibiting oral pathogens with the active lactic acid bacterial strain of the present invention.
FIG. 3 shows the results of the test for the inhibition of oral pathogens by the inactivated Lactobacillus strain of the invention.
[ biological Material Collection ]
The Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain of the invention is preserved in:
china center for type culture Collection/China Committee for culture Collection of microorganisms common microbiological center (CGMCC), wherein the preservation date is 18 days 12 months in 2017, and the preservation number is CGMCC No. 15077; the address of the depository is: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
Detailed Description
The following detailed description of the embodiments of the invention is provided in connection with the accompanying drawings. Aside from the details given herein, this invention is capable of broad application to other embodiments and that various other substitutions, modifications, and equivalents may be made in the embodiments without departing from the scope of the invention as defined by the appended claims. In the description of the specification, numerous specific details are set forth in order to provide a thorough understanding of the present invention; however, the present invention may be practiced without some or all of these specific details. In other instances, well-known steps or elements have not been described in detail so as not to unnecessarily obscure the present invention. The same or similar elements in the drawings will be denoted by the same or similar symbols. It is noted that the drawings are merely schematic and do not represent actual sizes or quantities of elements, and some details may not be fully drawn for brevity of the drawings.
The freeze-dried culture of the lactobacillus strain is preserved in China center for type culture Collection and China Committee for culture Collection of microorganisms. The details of the deposit are shown in table 1:
TABLE 1 deposited data of Lactobacillus strains
| Name of Strain | Classification | Deposit number | Date of storage |
| ET-22 | Lactobacillus paracasei | CGMCC 15077 | 12 month and 18 days 2017 |
The fermentation conditions of the strain are as follows:
MRS liquid medium: peptone, 10.0 g; 10.0g of beef extract; 5.0g of yeast extract powder; glucose, 20.0 g; dipotassium hydrogen phosphate, 5.0 g; diammonium hydrogen citrate, 2.0 g; sodium acetate, 5.0 g; magnesium sulfate heptahydrate, 0.5 g; 0.2g of manganese sulfate tetrahydrate; tween 80, 1.0 g; 15.0g of agar; 1000mL of distilled water. Adjusting the pH value to 6.2-6.4, and sterilizing for 15 minutes at 121 ℃.
L. paracasei ET-22 is a microaerophilic bacterium, grows well in a facultative anaerobic environment, produces lactic acid, has acid resistance, can resist an acid environment with a pH value of 2.5 and a 0.4% bile salt environment for 4 hours, is mesophilic, has a growth temperature range of 15-45 ℃, and has an optimal growth temperature of about 37 ℃.
Among the lactic acid bacteria deposited as listed in Table 1, Lactobacillus paracasei ET-22 strain was found to have the ability to inhibit oral pathogenic bacteria such as caries bacteria, gingival bacteria, oral odor bacteria, etc. In addition, the lactobacillus paracasei ET-22 strain can secrete hydrogen peroxide, so that the effect of whitening teeth can be achieved besides inhibiting oral pathogenic bacteria.
The present invention comprises a lactic acid bacterial strain having an activity inhibiting effect on oral pathogenic bacteria, the lactic acid bacterial strain being selected from at least one isolated lactic acid bacterial strain of the following group: lactobacillus paracasei ET-22 strain, which is preserved in China general microbiological culture Collection center (preservation number CGMCC No.15077), and a physiologically acceptable excipient, diluent or carrier, or a pharmaceutical composition consisting of a pharmaceutically acceptable excipient or diluent. Wherein the lactic acid bacteria strain may be an inactivated (inactivated) strain.
In an embodiment of the food composition, the physiologically acceptable excipient or diluent may be a food product, for example, the food product may include, but is not limited to, milk drinks, tea, coffee, chewing gum, and dentrifices (e.g., buccal tablets, chews, fondants, etc.), jerky for pets, or combinations thereof, wherein the milk drinks may include fermented milk, yogurt, cheese, or powdered milk. In the case of oral cleaning compositions, the excipient or diluent may be a toothpaste, dentifrice, mouthwash, breath freshening spray, fluorine-based application (e.g., to the teeth of young children), denture cleanser, denture gel for pets, or depilatory cream for pets, etc.; the carrier can be toothbrush, interdental brush, dental floss, oral cotton stick or dental bone for pet. The pharmaceutical composition may comprise an oral dosage form or an external dosage form. For example, oral dosage forms can be tablets, capsules, solutions, powders, and the like.
In the composition, the strain may be present in an effective amount. For example, in a food composition or a pharmaceutical composition, the number of lactic acid bacteria strains is 106CFU or more, e.g. 107CFU above, 108CFU above, 109CFU above, 1010Above CFU; preferably, the number of lactic acid bacteria is 1010Above CFU.
Some strains are known to be effective in inhibiting caries and periodontal bacteria, but the literature indicates that most studies are directed to testing the efficacy of individual strains in the oral cavity, and not all results of the tests prove that lactic acid bacteria are helpful for oral health, or have individual differences in the efficacy of strains (Anna Haukioja, European Journal of Dentistry 2010 (4): 348-355). For example, Vutotto C et al (International Journal of Oral Science 2014 (6): 189-194) analyzed the inhibitory activity of probiotics on pathogenic bacteria, and the test results were sometimes opposite even for different strains of probiotics. These results further emphasize that the inhibition of oral pathogenic bacteria and the whitening of teeth by probiotics are indeed strain-specific phenomena.
In fact, it is understood from the following test results of the present invention that most of the lactobacillus strains do not have the effects of inhibiting oral pathogenic bacteria and whitening teeth. At present, the literature reports of the research aiming at the function of probiotics related to the maintenance of oral health are gradually increased in recent times at home and abroad. In the early days, it was thought that lactic acid bacteria among probiotics may cause caries because of the coexistence of acid-producing characteristics with oral pathogenic bacteria and the possibility of causing enamel erosion. However, experiments show that lactic acid bacteria not only can inhibit caries bacteria and periodontal bacteria, but also can compete with the caries bacteria and periodontal bacteria for living space and nutrient sources of oral mucosa, and cause aggregation of oral pathogenic bacteria for easy elimination. However, these properties must be confirmed by experiments, and not all strains have the same properties and experimental results. It should be noted that the claimed strain of the present invention only comprises lactobacillus paracasei ET-22 strain deposited in the china typical culture collection center/china general microbiological culture collection center, and deposited in the china general microbiological culture collection center (collection number CGMCC No.15077), and does not broadly comprise all the above lactic acid bacteria of the same species.
Example 1: morphology and general Properties of lactic acid bacteria for maintaining oral health
The taxonomical characteristics of the strain were confirmed based on the results of 16S rDNA sequence analysis and analysis by the API bacterial identification system. The morphological and general characteristics of the above strains are detailed in table 2:
TABLE 2 morphological and general Property characteristics of the strains of lactic acid bacteria
Example 2: analyzing the hydrogen peroxide-producing characteristics of the strain to confirm the function for tooth whitening
The ability of the lactic acid bacterium strain of the present invention, lactobacillus paracasei ET-22 strain, to produce hydrogen peroxide was examined to thereby verify the tooth whitening ability of the lactic acid bacterium strain of the present invention. The experimental procedure was as follows:
1. preparing a probiotic hydrogen peroxide production screening plate.
2. Trimethylboron (Trimethylborane TMB)0.25mg/mL and Horseradish Peroxidase (Horseradish Peroxidase HR)0.01mg/mL were added to the plate.
3. The target strains were cultured in four-zone format on the plates.
4. After two days of culture, there were hydrogen peroxide-producing lactic acid bacteria strains that appeared blue around the colonies.
5. The hydrogen peroxide concentration of the lactobacillus thallus and the secondary metabolite is detected by using the hydrogen peroxide sensing test paper.
6. The lactic acid bacteria solution was cultured and centrifuged at 4500rpm for 5 minutes.
7. The cell fractions were dissolved in 4.9 ml of 100mM Piperazine-1, 4-diethylsulfonic acid (PIPERAzine-1, 4-bisethanesulfonic acid, PIPES).
8. After centrifugation, 10. mu.L (. mu.L) of the supernatant or the precipitated cell was dropped on a hydrogen peroxide test paper (Merck) and reacted for 10 seconds, and the color change was observed and the concentration was recorded by comparison with a color chart, after 5 hours of incubation at 37 ℃ and 220 rpm.
The results of the experiment are shown in FIG. 1. FIG. 1 shows the results of the ability of Lactobacillus paracasei ET-22 strain to secrete hydrogen peroxide on a hydrogen peroxide detection plate after the strain is cultured on the plate.
As is apparent from the test results of FIG. 1, the Lactobacillus paracasei ET-22 strain of the present invention exhibited good hydrogen peroxide secretion during the growth of the strain after two days of culture. It should be noted that lactic acid bacteria strains other than the same species of the present invention, such as Lactobacillus paracasei 9 strain (BCRC 16093), do not have the ability to secrete hydrogen peroxide, as shown in FIG. 1.
Example 3: inhibition ability of active lactic acid bacteria for maintaining oral health on oral pathogenic bacteria
If the lactic acid bacteria have the oral health care function, the resistance of the first oral pathogenic bacteria is high. Oral pathogenic bacteria include not only Streptococcus mutans (Streptococcus mutans), which is well known to the public, but also Fusobacterium nucleatum subsp. The symptoms caused by each strain are: streptococcus mutans causes dental caries; fusobacterium nucleatum polytype subspecies mainly cause problems of periodontal disease, halitosis, colon cancer and the like; actinomyces actinomycetemcomitans are the main bacterial species causing periodontitis, oral inflammation and pneumonia; porphyromonas gingivalis is the main cause of adult periodontitis and halitosis. In addition, for oral odor, besides porphyromonas gingivalis, which is a major species causing halitosis, fusobacterium nucleatum, actinobacillus syngenesis and porphyromonas gingivalis all produce sulfides, which are also one of the major species affecting oral odor.
Therefore, the invention carries out a pathogenic bacteria inhibition test to evaluate the capability of the active lactobacillus paracasei ET-22 strain of the invention to inhibit oral pathogenic bacteria, thereby achieving the effect of maintaining oral health. The following experimental procedure was used:
1. the active lactobacillus is coated on the two cm diameter part in the center of the MRS plate and cultured for two days.
2. Pouring into the upper part of the plate for culturing the oral pathogenic bacteria based on the grown lactic acid bacteria, and after solidification, coating the high-concentration oral pathogenic bacteria on the plate for culturing the pathogenic bacteria uniformly.
3. Culturing at 37 deg.C for 2-4 days.
4. The size of the inhibition diameter was measured as to whether the part of the center lower layer medium coated with lactic acid bacteria inhibited the growth of pathogenic bacteria.
The experimental results are shown in fig. 2, and fig. 2 is an analysis of the ability of the lactic acid bacteria strain of the present invention to inhibit pathogenic bacteria in the oral cavity.
As shown in FIG. 2, the ET-22 strain of Lactobacillus paracasei of the present invention showed the most significant effect of inhibiting the survival of pathogenic bacteria in the oral cavity, compared with another strain of Lactobacillus paracasei 9 strain.
Example 4: inhibition of pathogenic bacteria in the oral cavity by inactivated lactic acid bacteria for maintaining oral health
In addition to the function of the active lactic acid bacteria to inhibit oral pathogenic bacteria, it is not known whether the ability of the inactivated lactic acid bacteria to inhibit oral pathogenic bacteria is retained, and therefore, the present invention performs a pathogenic bacteria inhibition test to evaluate the ability of the inactivated lactobacillus paracasei ET-22 strain of the present invention to inhibit oral pathogenic bacteria, thereby achieving the effect of maintaining oral health. The following experimental procedure was used:
1. activate pathogenic bacteria in oral cavity.
2. The number of the bacteria is adjusted by thermally killed lactobacillus, and the bacteria and pathogenic bacteria are cultured together according to the proportion of billions of bacteria per milliliter.
3. Culturing at 37 deg.C for 2-4 days under anaerobic condition at 150 rpm.
4. The number of pathogenic bacteria was counted.
The experimental results are shown in fig. 3, fig. 3 is an analysis of the ability of the inactivated lactic acid bacterial strain of the present invention to inhibit oral pathogens; the histograms are expressed as Mean ± SD and compared with another strain of lactobacillus paracasei 9.
As shown in FIG. 3, the result of the inhibition of the survival of oral pathogenic bacteria by the inactivated Lactobacillus paracasei ET-22 strain of the present invention was that ET-22 was most significant as compared with the inhibitory effect of Lactobacillus paracasei 9 strain.
By combining the test results, the food composition and the medical composition containing the lactic acid bacteria strain can inhibit the growth of pathogenic bacteria in the oral cavity, and can be applied to reducing the decayed tooth, the periodontal disease, the halitosis and the like of the human body. Preferably, the lactic acid bacteria strain capable of secreting hydrogen peroxide also has an effect on whitening teeth. The invention finds out the lactic acid bacteria which have no side effect on human bodies and are beneficial to maintaining the health of the oral cavity as a new choice for maintaining the health of the oral cavity.
Example 5: intestinal flora regulating effect (sequencing results)
This example is intended to demonstrate the efficacy of the Lactobacillus paracasei of the present invention in terms of gut modulation, the principle and procedure of which are described in "test and evaluation of health food-criteria for functional assessment of gut flora". The results of 16S rDNA sequencing were used in this experiment.
Experimental samples: the bacterial powder sample (the number of viable bacteria is shown in the packaging specification) is provided by the company Limited responsibility of technical research institute of inner Mongolia dairy industry.
Experimental materials: 182 healthy adult BABL/c mice, 6 weeks old, weigh about 18-20 g.
Experimental procedure
1. Sample preparation
Live bacteria sample (ET-22): according to the specification of the sample, 1g of viable bacteria sample is weighed respectively, and the viable bacteria sample is suspended to 40ml by using PBS solution, namely the viable bacteria concentration is 2.5x109CFU/ml。
High dose group: calculated according to the gavage amount of 0.2ml/10g of the mice, the gavage amount of 20g of the mice is 0.4ml, and the gavage dose of 10 high-dose group mice is9CFU/20g。
The medium dose group: adding PBS into 5ml of the high-dose suspension respectively to reach a constant volume of 50ml, calculating according to the gavage amount of 0.2ml/10g of the mice, the gavage amount of 0.4ml of 20g of mice and the gavage amount of 10 of medium-dose group of mice8CFU/20g。
Low dose group: adding PBS into 5ml of the middle-dose group suspension respectively to reach a constant volume of 50ml, calculating according to the gavage amount of 0.2ml/10g of the mice, wherein the gavage amount of 20g of the mice is 0.4ml, and the gavage amount of the low-dose group mice is 107CFU/20g。
2. Experiment for regulating intestinal flora
(1) The BABL/c mice of 6 weeks old are raised in a clean animal room with temperature of 22 ℃, humidity of 10-60%, 12-hour illumination of alternating light and shade, and fed with standard feed and freely drinking water.
(2) Adaptive feeding was performed for 5 days, and 182 mice were randomly divided into 13 groups of 14 mice each, and the grouping was shown in table 3.
TABLE 3 Experimental groups for regulating intestinal flora
(3) Before beginning the gavage, the feces of each mouse were collected under sterile conditions, labeled, stored at-20 ℃ and tested for intestinal flora.
(4) Experimental groups were each administered with a intragastric dose of 0.2ml/10g, control groups were administered with PBS for 1-14 days, and experimental groups were each administered with a corresponding dose of test substance intragastric according to table 3. Mice were weighed once a week and gavage was adjusted according to body weight.
(5) Collecting feces of each mouse under sterile condition after 14 days, marking, storing at-20 deg.C, and detecting intestinal flora.
3 variation in intestinal microbial diversity in mice
The microbial diversity is based on an Illumina HiSeq sequencing platform, and a small fragment library is constructed for sequencing by using a double-ended sequencing (Paired-End) method. Species composition of the sample can be revealed by performing splicing filtration on Reads, OTUs (operational Taxonomic units) clustering, and performing species annotation and abundance analysis; and clustering the optimized sequences, dividing the OTUs, and obtaining species classification according to the sequence composition of the OTUs. And (4) performing taxonomic analysis on the samples at each classification level based on the OTU analysis result to obtain the community structure of each sample at the genus level.
The variety diversity of the species in a single sample is researched through Alpha diversity analysis, the Ace, Chaol, Shannon and Simpson indexes of each sample under the 97% similarity level are counted, and a sample dilution curve and a grade abundance curve are drawn; further performing Alpha Diversity analysis (Alpha Diversity), Beta Diversity analysis (Beta Diversity), and significant species difference analysis, among others, can mine the differences between samples. And (3) comparing the intestinal flora alpha diversity indexes: chaol and ACE refer to the measure of species abundance, i.e., number of species; the Shannon and Simpson indices are used to measure species diversity, and are influenced by species abundance and species uniformity in the sample population. The larger the homogeneity of each species in the population, the greater the diversity of the population, and the larger the Shannon index value and the smaller the Simpson index value, the higher the diversity of the species in the sample. At the genus level, ET-22 stem prognosis significantly increased the relative abundance in the mouse gut compared to the control group. The OTU, ACE and Chaol indexes of the low-dose group are obviously increased, and the ET-22 low dose is shown to be capable of obviously increasing the intestinal microbial diversity.
Yellow (underlined) indicates: statistically significant differences (p < 0.05) were observed compared to the control group.
At the level of pathopoiesia, vibrio desulfovis (Desulfovibrio) in the intestinal tracts of both the ET-22 low dose and medium dose groups of mice was significantly reduced compared to the control group; the number of Helicobacter pylori (Helicobacter) in the middle dose group and the high dose group is obviously reduced; the high-Escherichia Shigella (Escherichia-Shigella) was significantly reduced in both the low-dose and medium-dose groups. BL-99 can significantly increase the relative abundance of Lactobacillus (Lactobacillus) in the intestinal tract of mice, and BL-99 low dose group increases Lactobacillus (Lactobacillus) to the most significant extent.
Therefore, different doses of ET-22 are supplemented to regulate the balance of intestinal flora and inhibit the number of harmful bacteria, even pathogenic bacteria, so that the potential health effect is achieved.
Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, processing conditions, and so forth used in the specification (including the claims) are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. The term "at least" preceding a series of elements is to be understood as referring to each element in the series, unless otherwise indicated. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The appended claims are intended to cover such equivalents.
It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not meant to be limiting in any way. The true scope and spirit of the invention is indicated by the appended claims, and the specification and examples are exemplary only.
The above-mentioned embodiments are only for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to implement the same, so that the scope of the present invention should not be limited by the above-mentioned embodiments, i.e., all equivalent changes or modifications made in the spirit of the present invention should be covered in the scope of the present invention.
Claims (10)
1. The application of Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for tooth whitening, wherein the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15077.
2. A lactic acid bacteria strain-containing composition comprising:
lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15077; and an excipient, diluent and/or carrier, wherein the composition is a food composition, a pharmaceutical composition or an oral cleaning composition.
3. The lactic acid bacteria strain-containing composition according to claim 2, wherein the lactic acid bacteria strain is an active strain and/or a deactivated strain.
4. A composition comprising a lactic acid bacterial strain according to claim 2 or 3, wherein the composition is a composition for tooth whitening and/or inhibiting oral pathogens.
5. A lactic acid bacteria strain-containing composition according to claim 2 or 3 wherein the excipient, diluent and/or carrier is a food product.
6. The lactic acid bacteria strain-containing composition of claim 5, wherein the food product comprises dairy drinks, tea, coffee, chewing gum and dentrifice, jerky for pets, or combinations thereof.
7. The lactic acid bacterial strain-containing composition according to claim 2 or 3, which is in an oral dosage form or an external dosage form.
8. A composition comprising a lactic acid bacterial strain according to claim 2 or 3, wherein the excipient, diluent and/or carrier is a toothpaste, a dentifrice, a mouthwash, a breath freshening spray, a fluoride coating, a denture cleanser, a denture adhesive or cream for pets, a toothbrush, an interdental brush, a dental floss, a cotton-tipped oral stick or a bone for pets.
9. Use of Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for the preparation of a food composition, a pharmaceutical composition or an oral cleaning composition, wherein the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is deposited in the China general microbiological culture Collection center with the deposit number CGMCC No. 15077.
10. Use of Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain for preparing a pharmaceutical composition for inhibiting oral pathogenic bacteria, wherein the Lactobacillus paracasei (Lactobacillus paracasei) ET-22 strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 15077.
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| CN201811147290.7A CN110960559B (en) | 2018-09-30 | 2018-09-30 | Food, oral cleaning and pharmaceutical composition for inhibiting lactic acid bacteria strain of oral pathogenic bacteria |
| EP19865158.0A EP3822338A4 (en) | 2018-09-30 | 2019-09-23 | Lactobacillus paracasei et-22 and use thereof |
| US17/259,137 US11274275B2 (en) | 2018-09-30 | 2019-09-23 | Lactobacillus paracasei ET-22 and use thereof |
| PCT/CN2019/107274 WO2020063531A1 (en) | 2018-09-30 | 2019-09-23 | Lactobacillus paracasei et-22 and use thereof |
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| CN110892989B (en) * | 2019-06-27 | 2023-03-14 | 内蒙古伊利实业集团股份有限公司 | Lactobacillus paracasei ET-22 with function of relieving constipation |
| CN111642746B (en) * | 2020-05-26 | 2023-04-28 | 浙江亲水园生物科技有限公司 | Food, oral cleaning and pharmaceutical composition for inhibiting oral pathogenic bacteria |
| CN113349254B (en) * | 2020-11-26 | 2022-11-29 | 内蒙古伊利实业集团股份有限公司 | Antioxidant and blood pressure regulating lactobacillus paracasei ET-22 and application thereof |
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