CN117264806A - Wettman's bacterium coagulans and application thereof in inhibiting oral pathogenic bacteria - Google Patents

Wettman's bacterium coagulans and application thereof in inhibiting oral pathogenic bacteria Download PDF

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CN117264806A
CN117264806A CN202310904907.XA CN202310904907A CN117264806A CN 117264806 A CN117264806 A CN 117264806A CN 202310904907 A CN202310904907 A CN 202310904907A CN 117264806 A CN117264806 A CN 117264806A
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oral
bacteria
microbial agent
pathogenic bacteria
condensation
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陈其亮
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Wuxi Aikepai Biotechnology Co ltd
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Wuxi Aikepai Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • 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
    • 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
    • C12R2001/07—Bacillus

Abstract

The invention discloses a condensation Wittman's bacterium and application thereof in inhibiting oral pathogenic bacteria, and belongs to the technical field of microorganisms. The invention obtains the condensation Wittman's bacterium which can inhibit a plurality of potential oral pathogenic bacteria, has low acid production and no volatile sulfide production, and can release, colonize and exert the effect of probiotics in the oral cavity. The Wittman's bacterium coagulans can be used for developing microbial agents or products for inhibiting oral pathogenic bacteria, such as health products, medicines or oral care products, so as to achieve the purposes of preventing oral diseases or improving oral health.

Description

Wettman's bacterium coagulans and application thereof in inhibiting oral pathogenic bacteria
Technical Field
The invention relates to a strain of condensation Wittman's bacteria and application thereof in inhibiting oral pathogenic bacteria, belonging to the technical field of microorganisms.
Background
Oral health is an important component of human health, and the World Health Organization (WHO) determines dental health as one of ten major criteria for human health. Oral health directly or indirectly affects general health. The oral cavity contains a plurality of microorganisms which balance each other and maintain health, once pathogenic microorganisms are propagated in a large quantity, various oral diseases can be caused, even if some microorganisms exist in the oral cavity for a long time, certain systemic diseases such as coronary heart disease, diabetes mellitus and the like can be caused or aggravated, the systemic health is endangered, and the quality of life is influenced.
Common oral problems include caries, periodontal disease, inflammation, halitosis, etc.; caries is mainly affected by bacteria, which are not only caused by a certain kind of bacteria, but many bacteria present on the tooth surface are associated with caries. The major pathogenic bacteria in the oral cavity currently considered by researchers include: fusobacterium nucleatum, porphyromonas gingivalis, actinobacillus actinomyces, streptococcus mutans, helicobacter pylori, staphylococcus aureus and the like. Wherein, the fusobacterium nucleatum and the actinobacillus concomitantly present the risk of causing periodontal disease; in particular, fusobacterium nucleatum can promote cancer cell proliferation, tumor immune escape, recurrence, chemotherapy resistance and the like. Porphyromonas gingivalis is one of periodontal pathogens, and can cause infectious endocarditis, even coronary heart disease, atherosclerosis and other common diseases. The streptococcus mutans has the largest proportion in the natural flora of the oral cavity and is one of the main components of dental plaque; streptococcus mutans adsorbs onto teeth layer by layer, forming a so-called "biofilm", i.e. plaque; they consume sugars, produce acids, erode the enamel layer of the tooth, create cavities, and eventually cause caries; caries is a bacterial infectious disease that occurs in dental hard tissues, with the highest incidence among oral diseases. Helicobacter pylori is a major pathogenic bacterium in chronic active gastritis, but at the same time it is also widely found in the oral mucosa, saliva and plaque, and is also associated with the severity of periodontal disease and recurrent oral ulceration. Many microorganisms can cause inflammation of the oral mucosa by invading the oral mucosa, and bacteria causing infection of the oral mucosa are mainly gram-positive cocci including staphylococcus aureus and the like.
Therefore, development of a method for inhibiting potential pathogenic bacteria capable of controlling oral flora is of great importance to human health. The acquisition of probiotics capable of inhibiting potential pathogenic bacteria of oral flora is an important method for solving the problem, and the substances can inhibit the growth and reproduction of harmful microorganisms and have the effects of preventing and protecting dental caries, periodontitis and the like caused by oral dysbacteriosis.
It has been reported that probiotics capable of inhibiting potential pathogenic bacteria of the oral cavity flora have been developed, such as lactococcus lactis, streptococcus thermophilus, lactobacillus plantarum, lactobacillus paracasei, lactobacillus rhamnosus, mannheimia coagulans, and the like. Some of these reports for M.weizmann condensation are as follows:
CN 110195029A discloses a highly effective antagonistic streptococcus mutans coagulating, a preparation method of the bacterial powder, chewing gum containing the bacterial powder and application thereof, and reported is CCTCC NO: the M2018889 condensed Weizhman bacteria can be used for bacteria, and the culture solution has obvious antibacterial effect on the cariogenic streptococcus mutans, has antibacterial potency up to more than 8000U/ml, and can survive in the high-temperature processing process of the plate-type chewing gum; however, it has only inhibitory effect on Streptococcus mutans, and cannot be used for inhibition of other oral pathogenic bacteria, and has limited effect.
CN 115607577A discloses a probiotic agent with the effect of relieving stomatitis, a preparation method and application thereof, and the probiotic agent is composed of specific lactobacillus salivarius of CGMCC No.19487, lactobacillus salivarius of CGMCC No.16922 and lactobacillus acidophilus of CGMCC No.1.12735, and the probiotic agent is matched with an auxiliary agent to inhibit the growth of pathogenic microorganisms, promote the restoration of oral mucosa and relieve stomatitis; however, the combination microbial agent needs to be applied to various strains and auxiliary agents, has complex formula, and has no mention of specific pathogenic microorganisms.
CN 114432227A discloses an oral care solution containing probiotics, which is found to inhibit the growth of helicobacter pylori, streptococcus mutans, porphyromonas gingivalis and staphylococcus aureus by developing an oral care solution containing Weizhman's coagulation BC 99; however, in this scheme, on the one hand, the strains that can be inhibited are limited, and the strains have no effect on Fusobacterium nucleatum and actinobacillus actinomycetemcomitans that are at risk of causing periodontal disease, and on the other hand, when the inhibition experiments of Streptococcus mutans and Porphyromonas gingivalis are performed, the concentrations of Streptococcus mutans and Porphyromonas gingivalis are very low, the time for achieving the inhibition effect is relatively long, and the antibacterial efficiency is to be improved.
In summary, it is necessary to further develop probiotics capable of efficiently inhibiting various potential pathogenic bacteria of oral flora, and improving the functional potential of probiotics in aspects of oral health maintenance, prevention and treatment of oral diseases, and the like.
Disclosure of Invention
In order to solve at least one of the problems, the invention screens and obtains the condensation Wittman bacterium which can effectively inhibit a plurality of potential pathogenic bacteria of oral flora. The invention carries out full screening and functional evaluation on the bacterial strain, and fully considers possible adverse effects (such as acid production, odor-producing compounds and the like) of probiotics.
The invention relates to a condensation Wittman's bacteria, which is preserved in China Center for Type Culture Collection (CCTCC) in the year 2023, month 6 and 29, wherein the preservation number is CCTCC NO: m20231138.
The inventive condensation of Wittman's bacteria has the following characteristics:
(1) Can inhibit a variety of potential oral pathogens including, but not limited to, fusobacterium nucleatum, porphyromonas gingivalis, actinomyces companion, streptococcus mutans, helicobacter pylori, staphylococcus aureus;
(2) High inhibition of pathogenic bacteria, including: can inhibit high-concentration pathogenic bacteria and achieve excellent (3) bacteriostasis in a short time;
(4) Low acid production;
(5) No volatile sulfide such as hydrogen sulfide, methyl mercaptan, dimethyl disulfide, dimethyl trisulfide and the like is generated.
The second object of the present invention is to provide a microbial agent comprising the inventive Wettman coagulans.
The microbial inoculum takes the condensation Wittman as a main microorganism.
In one embodiment, the viable count of the coagulated Wittman's bacteria in the microbial inoculum is 10 5 ～10 8 CFU/mL。
In one embodiment, the concentration of the condensed Wittman bacteria in the microbial inoculum is in the range of 10 5 ～10 8 CFU/mL。
In one embodiment, the microbial inoculum is prepared by preparing a seed solution of Wettman coagulans and then expanding the seed solution.
In one embodiment, the microbial agent is a liquid agent or a solid agent.
In one embodiment, the microbial inoculum also contains any strain of any species that can be used in food or health products or medicines, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis, lactobacillus, and the like.
A third object of the present invention is to provide a preparation for inhibiting oral pathogenic bacteria, which comprises the viable bacteria of the inventive M.Weizmanni coagulans.
In one embodiment of the invention, the oral pathogenic bacteria include, but are not limited to: fusobacterium nucleatum, porphyromonas gingivalis, actinobacillus actinomyces, streptococcus mutans, helicobacter pylori, and Staphylococcus aureus.
In one embodiment, the oral care implement is any one of a dental gel, a dentifrice, a toothpaste, a mouthwash, an oral spray, a chewing gum, a dental floss, a tooth cleaning solution, and a tooth cleaning foam.
In one embodiment, the article is a health product, a pharmaceutical product, or an oral care product.
In one embodiment, the product may be a probiotic solid beverage, a probiotic tablet candy, a probiotic formula, a functional health food, a dietary supplement, a health food, etc., further comprising a pharmaceutically acceptable carrier or additive. When the pathogenic bacteria inhibiting product is a health food, the formulation of the health food is powder, granules, tablets, capsules, suspending agents, emulsion, syrup or extractum.
In one embodiment, the article of manufacture further comprises a physiologically acceptable carrier and/or excipient and/or diluent. Optionally, the acceptable carrier may comprise one or more of the following: emulsifying agents (emulsifiers), suspending agents (suspending agents), disintegrating agents (decomponents), disintegrating agents (disintegrating agent), dispersing agents (dispersing agents), binding agents (binding agents), excipients (excipients), stabilizers (stabilizing agent), chelating agents (chelating agents), diluents (diluents), gelling agents (gelling agents), preservatives (preservatives), wetting agents (wetting agents), lubricants (lubricants), absorption retarders (absorption delaying agent), liposomes (lipomes), and the like. The choice and amount of these carriers will be readily apparent to those skilled in the art.
In some embodiments, the acceptable carrier may further comprise any one of the following solvents: water, normal saline (PBS), phosphate buffered saline (phosphate buffered saline, PBS), aqueous alcohol-containing solutions (aqueous solution containing alcohol), and any other suitable solvent.
In some embodiments, the oral care implement may further comprise an acceptable adjuvant (acceptable adjuvant) that is widely used in the art of oral care implement manufacturing. For example, an acceptable adjuvant may comprise one or more of the following: solvents, gelling agents, actives, preservatives, antioxidants, sequestering agents, chelating agents, surfactants, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and the quantity of the reagents can be properly adjusted according to actual requirements. The oral care implement may be manufactured in a form suitable for oral use, such as mouthwash, toothpaste, spray, patch, or the like, using techniques well known to those skilled in the art.
A fourth object of the present invention is to provide the use of the coagulated wiltmann bacterium for preparing a medicine or an oral care product for preventing oral diseases or improving oral health.
The invention has the beneficial effects that:
the invention obtains the condensation Wittman bacterium which can inhibit a plurality of potential oral pathogenic bacteria, has low acid production and does not produce volatile sulfide. Can be released and planted in the oral cavity to exert the effect of probiotics.
The Wittman's bacterium coagulans can be used for developing microbial agents or products for inhibiting oral pathogenic bacteria, such as health products, medicines or oral care products, so as to achieve the purposes of preventing oral diseases or improving oral health.
Preservation of biological materials
Welch's disease, VC77, taxonomic designation Weizmannia coagulans VC, was deposited at China center for type culture Collection, accession number CCTCC NO: m20231138, the preservation address is university of Wuhan in Wuhan, china.
Drawings
Fig. 1: co-culture time (h) when the inhibition rate of the Weizhman bacteria VC77 on pathogenic bacteria is more than 99%.
Detailed Description
In order to describe the possible application scenarios, technical principles, practical embodiments, and the like of the present application in detail, the following description is made with reference to the specific embodiments and the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples and are not intended to limit the scope of protection of the present application.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearances of the phrase "in various places in the specification are not necessarily all referring to the same embodiment, nor are they particularly limited to independence or relevance from other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in the embodiments may be combined in any manner to form a corresponding implementable technical solution.
Unless defined otherwise, technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains; the use of related terms herein is for the description of specific embodiments only and is not intended to limit the present application.
In the description of the present application, the term "and/or" is a representation for describing a logical relationship between objects, which means that there may be three relationships, e.g., a and/or B, representing: there are three cases, a, B, and both a and B. In addition, the character "/" herein generally indicates that the front-to-back associated object is an "or" logical relationship.
Without further limitation, the use of the terms "comprising," "including," "having," or other like terms in this application is intended to cover a non-exclusive inclusion, such that a process, method, or article of manufacture that comprises a list of elements does not include additional elements but may include other elements not expressly listed or inherent to such process, method, or article of manufacture.
The present invention relates to:
isolation medium: yeast extract 10.0g/L; 10.0g/L of beef extract; peptone 10.0g/L; sodium bicarbonate 1.5g/L; 1.5g/L of monopotassium phosphate; 1.0g/L of magnesium sulfate heptahydrate; manganese sulfate 0.1g/L; 1.0g/L of calcium chloride; deionized water 1L.
MRS medium (fermentation medium): 10.0g/L peptone, 10.0g/L beef powder, 5.0g/L yeast powder, 20.0g/L glucose, 0.1g/L magnesium sulfate, 5.0g/L sodium acetate, 2.0g/L ammonium citrate, 2.0g/L dipotassium hydrogen phosphate, 0.05g/L manganese sulfate, 801.0g/L Tween (solid medium is added with 20.0g/L agar powder).
GAM liquid medium (peptone 5g, soybean peptone 3g, peptone 5g, digested serum powder 10g, yeast extract 2.5g, cysteine hydrochloride 0.3g, sodium thioglycolate 0.3g, L-arginine 1g, vitamin k 15mg, heme 10mg, potassium dihydrogen phosphate 2.5g, sodium chloride 3.0g, water 1L, pH 7.3).
Trypticase Soytone Broth (TSB): in g/L, tryptone 17.0, sodium chloride 5.0, soybean papain hydrolysate 3.0, dipotassium hydrogen phosphate 2.5, glucose 2.5, pH value (25 ℃) 7.3+/-0.2.
Brain Heart Infusion (BHI) liquid medium: according to g/L, peptone 10.0, dehydrated calf brain extract 12.5, dehydrated calf heart extract 5.0, sodium chloride 5.0, glucose 2.0, disodium hydrogen phosphate 2.5, pH value (25 ℃) 7.4+ -0.2.
Commercial strains: fusobacterium nucleatum ATCC 25586; porphyromonas gingivalis ATCC 33277; actinobacillus ATCC 43717; streptococcus mutans ATCC 25175; helicobacter pylori ATCC 43504; staphylococcus aureus ATCC 25923. All are commercial standard strains and can be purchased.
Self-screening pathogenic bacteria for strain primary screening and performance detection: the separated dental calculus, saliva, feces and the like are taken as samples, and the fusobacterium nucleatum M1, the porphyromonas gingivalis M2, the actinobacillus concomitantly actinobacillus M3, the streptococcus mutans M4, the helicobacter pylori M5 and the staphylococcus aureus M6 are obtained through screening.
Example 1: isolation, screening and identification of strains
(1) Separating and purifying strains: firstly, adding 15mL of sterile water into a sample (crushed pickled pickle), shaking vigorously for 20min, and then performing water bath at 80 ℃ for 20min; then 100uL of diluent is coated on an agar separation culture medium flat plate by adopting a concentration gradient dilution method (the formula of the culture medium is 10.0g/L of yeast extract, 10.0g/L of beef extract, 10.0g/L of peptone, 1.5g/L of sodium bicarbonate, 1.5g/L of monopotassium phosphate, 1.0g/L of magnesium sulfate heptahydrate, 0.1g/L of manganese sulfate, 1.0g/L of calcium chloride and 1L of deionized water), 30 strains with large colonies and quick growth are selected and are cultivated for 48 hours at 45 ℃, and then the strains are streaked, separated and purified and preserved in glycerol pipes.
(2) Bacterial strain primary screening: qualitatively detecting the inhibition of bacteria liquid to oral pathogenic bacteria by using a filter paper method; the method specifically comprises the following steps:
preparing bacterial liquid: inoculating the bacterial liquid preserved by the glycerol pipe into an MRS culture medium, and culturing for 24 hours at a constant temperature of 37 ℃ and 120rpm under shaking to obtain 30 strains of single bacterial liquid for later use;
preparing an indicator bacteria plate: respectively activating Fusobacterium nucleatum, porphyromonas gingivalis, actinobacillus, streptococcus mutans, helicobacter pylori and staphylococcus aureus, and culturing; diluting the cultured bacterial liquid to OD600 = 1 by using sterile physiological saline to obtain bacterial suspension; adding 100ul of fungus suspension into solid culture medium cooled to 45-50 ℃, shaking rapidly, adding into a culture dish, spreading evenly, and solidifying to obtain an indicator fungus plate for later use.
Bacterial strain primary screening: sucking 20mL of the bacterial liquid on an ultra-static workbench into a sterile culture dish, placing sterile filter paper sheets into the culture dish by using sterile forceps, soaking for 10 minutes, clamping the bacterial liquid-containing paper sheets by using the sterile forceps, wiping off redundant bacterial liquid on the edges of the inner culture dish, pasting on the surface of a culture medium of an indicator bacteria plate, lightly pressing to enable the paper sheets to be in close contact with the culture dish, enabling the filter paper sheets to be 15mm away from the edge of the culture dish, uniformly placing 3 filter paper sheets on each culture dish, placing a constant temperature oven at 37 ℃, observing whether a bacteriostasis ring exists around the filter paper sheets containing the bacterial liquid after culturing for 24 hours, measuring the size of the bacteriostasis ring, and taking an average value for comparison and screening. The 6 strains with better comprehensive performance are obtained by primary screening, and are VC1, VC12, VC13, VC24, VC61 and VC77.
(3) Identification of Strain ITS:
6 strains with better comprehensive performance obtained by primary screening are centrifuged, cell sediment is obtained after fermentation broth is centrifuged, genome is extracted, the species is identified by determining the ITS sequence of a conserved region, and the primer sequences for PCR are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', the sequence is shown as SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', the sequence is shown as SEQ ID NO: 2).
Determining ITS gene sequence, and comparing with Genbank database to determine species; among them, the 2 strains VC12 and VC77 are Wettman coagulans. Wherein, the Weizhman's bacterium coagulating VC77 is preserved in China Center for Type Culture Collection (CCTCC) at the year 2023, 6 and 29, and the preservation number is CCTCC NO: m20231138, the preservation address is university of Wuhan in Wuhan, china.
Example 2: physiological and biochemical characteristics of Welch's disease-causing bacteria VC77
(1) Acid production characteristics
The strains VC12 and VC77 of Welch's disease were inoculated into 200mL MRS medium at an inoculum size of 1%, and after overnight culture, the acidity of the bacterial solution was measured. And meanwhile, the bacterial count of the bacterial liquid is measured by a plate dilution counting method. The acidity calculation formula is as follows: x=c×v×100/(m×0.1).
X = acidity of the broth in milliliters per 100g (mL/100 g) of 0.1mol/L sodium hydroxide consumed by 100g of sample. V = volume of sodium hydroxide standard solution consumed at the time of dripping in milliliters. C=molar concentration of sodium hydroxide standard solution in moles per liter. M = mass of sample in grams. Finally, the acid production capacity was evaluated by the following formula: acid production capacity = acidity/10 8 The higher the bacterial count, the stronger the acid producing ability. The results are shown in the following table.
TABLE 1 results of acid production capability experiments
(2) Capability of producing volatile sulfides
Bacteria were inoculated into 10mL of a modified GAM liquid medium (peptone 5g, soybean peptone 3g, peptone 5g, digested serum powder 10g, yeast extract 2.5g, cysteine hydrochloride 0.3g, sodium thioglycolate 0.3g, L-arginine 1g, vitamin k 15mg, heme 10mg, potassium dihydrogen phosphate 2.5g, sodium chloride 3.0g, water 1L, pH 7.3) containing 1% D- (+) -glucose and 0.5mM DL-methionine at an inoculum size, anaerobic culture was performed for 24 hours, and then, after the pH was reduced to 1 or lower by adding 0.16mL of 6M HCl to the culture medium, metabolism of the bacterial cells was stopped, and a proper amount of bacterial cells were collected in a headspace gas bottle and analyzed for volatile components (hydrogen sulfide, methyl mercaptan, dimethyl disulfide). As a result, as shown in Table 2, the bacteria did not produce volatile sulfides such as hydrogen sulfide, methyl mercaptan, dimethyl disulfide, and dimethyl trisulfide.
TABLE 2 volatile sulfide test results
Strain Hydrogen sulfide Methyl mercaptan Dimethyl disulfide Dimethyl trisulfide
VC77 N.D. N.D. N.D. N.D.
VC12 N.D. 20.35 36.82 7.49
Wherein, n.d.: non detected (not detected)
Example 3: inhibition of pathogenic bacteria by Welch's bacteria
Pathogenic bacteria (including Fusobacterium nucleatum, porphyromonas gingivalis, actinobacillus actinomyces, streptococcus mutans, helicobacter pylori, and Staphylococcus aureus) are cultured in corresponding culture medium and the concentration of the bacterial cells is measured by plate counting method in advance. The pathogenic bacteria are mixed with a certain initial concentration (1 x 10 3 ～1.2*10 3 CFU/mL or 1 x 10 6 CFU/mL), inoculated into a culture suitable for the growth of the corresponding pathogenic bacteria corresponding to Table 1 belowSimultaneously adding the respective concentrated Weizhmann bacteria VC77 to the corresponding initial concentration (1X 10 3 ～1.2*10 3 CFU/mL or 1 x 10 6 CFU/mL) and a blank (without addition of w.weintilman coagulans) were set for co-cultivation. And counting the number of the viable bacteria by adopting a plate pouring method at different time points, and calculating the viable bacteria concentration of the bacteria. Wherein, the oral potential pathogenic bacteria are respectively co-cultured for 0.5h, 1h, 2h, 4h, 6h, 12h, 18h, 24h, 30h, 36h and 48h, and viable bacteria count is carried out. Based on the counted number of viable bacteria, the inhibition ratio (%) was obtained by the following calculation method, and the time point when the inhibition ratio was 99% or more was obtained. Wherein, inhibition ratio (%) = (number of viable bacteria in blank group-number of viable bacteria in experimental group)/number of viable bacteria in blank group ×100%. The results are shown in FIG. 1.
TABLE 3 Table 3
Example 4: microbial agent containing Wittman coagulans VC77
1. Preparation of liquid microbial inoculum containing Wittman coagulans VC77
Inoculating the single colonies of the Welch mannfield bacteria to MRS seed culture medium, culturing at 37deg.C for 36h, transferring to fermentation culture medium, and culturing at 37deg.C for 40-56 h at 200r/min to obtain strain with a number of 1.0X10 8 -5.0×10 9 CFU/mL fermentation broth.
2. Preparation of solid microbial inoculum containing Welch's mannhei VC77
Centrifuging the bacterial liquid of the condensation Wittman's bacteria, mixing the spores and dry starch in a weight ratio of 1:2, drying for 20-24 hours at 40-50 ℃, crushing by a crusher, sieving, adding the ingredients of corncob powder or maltodextrin, mixing to prepare a solid microbial inoculum, wherein the spore content is up to 1 multiplied by 10 9 cdu/g.
Example 5: application of Welch's disease-causing bacteria VC77 in preparation of oral care products
An oral care product comprising the inventive frozen Wittman VC77. The oral care product can be any one of dental gel, dentifrice, toothpaste, mouthwash, mouth spray, chewing gum, dental floss, tooth cleaning liquid and tooth cleaning foam.
Optionally, the oral care implement may further comprise an acceptable adjuvant that is widely used in the art of oral care implement manufacture. For example, an acceptable adjuvant may comprise one or more of the following: solvents, gelling agents, actives, preservatives, antioxidants, masking agents, chelating agents, surfactants, coloring agents, thickeners, fillers, fragrances, and odor absorbers. The selection and the quantity of the reagents can be properly adjusted according to actual requirements.
Optionally, the oral care implement is an oral care solution. The mixture ratio is as follows: 1.5g of sorbitol, 1.0g of xylitol, 0.05g of dipotassium glycyrrhizinate, 0.28g of mint extract, 1.2g of propylene glycol, 0.1g of potassium sorbate, 0.01g of sodium fluoride, 0.02g of condensation of Wilmannomnia, and the balance of purified water; wherein the viable count of the Welch's disease is 5.10 7 The pH of the nursing liquid is regulated to be 6 by citric acid. Optionally, the oral care solution is for use with an oral atomizer; the oral care solution can pass through the atomizing sheet with the aperture more than or equal to 5 mu m, and the pores are not blocked in the use process.
It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present invention and their spirit, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the following claims.

Claims (10)

1. A strain of wegener's condensation, wherein the strain of wegener's condensation has been deposited at the China center for type culture collection, with a deposit number of cctccc NO: m20231138.
2. A microbial agent comprising the frozen Weizhman's bacterium according to claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent comprises the condensation of the bacteria of the genus westernum of claim 1 as a main microorganism.
4. The microbial agent according to claim 2, wherein the viable count of the condensed Wittiman bacteria in the microbial agent is 10 5 ～10 8 CFU/mL。
5. The microbial agent of claim 2, wherein the microbial agent is a liquid microbial agent or a solid microbial agent.
6. A product for inhibiting oral pathogenic bacteria comprising the viable bacteria of the species westernum coagulans of claim 1.
7. The article of manufacture of claim 6, wherein the oral pathogenic bacteria include, but are not limited to: fusobacterium nucleatum, porphyromonas gingivalis, actinobacillus actinomyces, streptococcus mutans, helicobacter pylori, and Staphylococcus aureus.
8. The article of claim 6, wherein the article is a health product, a pharmaceutical product, or an oral care product.
9. The article of claim 8 wherein the oral care implement is any one of a dental gel, a dentifrice, a toothpaste, a mouthwash, an oral spray, a chewing gum, a dental floss, a dental cleaning solution, a dental cleaning foam.
10. Use of the condensation of mansoni, as defined in claim 1, for the manufacture of a medicament for preventing oral disease or improving oral health, an oral care product.
CN202310904907.XA 2023-07-21 2023-07-21 Wettman's bacterium coagulans and application thereof in inhibiting oral pathogenic bacteria Pending CN117264806A (en)

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