Tea sugar with breath freshening effect of degrading dental plaque biofilm by AKK bacteria and preparation method thereof

Abstract

The present application relates to the oral microecological regulation technical field, specifically relates to a tea sugar formula and preparation process for degrading dental plaque biofilm by synergistic competition of mucinophilic akkermansia (AKK bacteria) and rhamnose lactose cheese bacillus and enhancing saliva natural bactericidal function, through the ability of AKK bacteria to specifically degrade mucin and dental plaque biofilm matrix, and the secretion of antibacterial peptide of rhamnose lactose cheese bacillus, while the oral bacteriostatic capacity is improved through the herbal active components: green tea, honeysuckle, fat sea, monk fruit, clove, liquorice and the like, the breath freshening feeling is increased, through the inhibition of NF-κB pathway, the gum inflammation is reduced, the generation of VSCs is reduced, the problem of halitosis can be significantly and persistently solved from the root, and the problem of dental plaque in the dental crevice that cannot be reached by physical brushing can be effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of oral microecological regulation technology, specifically involving a tea sugar formula and preparation process that enhances the natural bactericidal function of saliva by synergistically and competitively degrading dental plaque biofilm by Akkermansia muciniphila (AKK) and Lactobacillus rhamnosus. Background Technology

[0002] Current breath fresheners mostly rely on peppermint or herbal extracts to temporarily mask odor, but they are difficult to regulate the balance of oral flora in the long term. While some products add probiotics, the lack of a targeted culture medium results in low bacterial activity and poor colonization. Traditional oral antibacterial products (such as chlorhexidine mouthwash) can kill surface bacteria, but they cannot penetrate the physical barrier of dental plaque biofilm, allowing deep-seated pathogens (such as Porphyromonas gingivalis and Streptococcus mutans) to survive and release volatile sulfur compounds (VSCs), causing halitosis. Physical brushing is ineffective in reaching pathogens within the dental plaque biofilm between teeth. Besides physical brushing disrupting the pathogen's living environment (biofilm), other methods are largely ineffective, which is the core cause of bad breath. Research shows that AKK bacteria have the ability to specifically degrade mucin and dental plaque biofilm matrix, but their colonization efficiency in the oral environment is limited by competing bacteria and nutrient supply. This invention is the first to apply AKK bacteria to the field of oral biofilm regulation, which has significant innovation and industrialization value. Through rigorous experimental data verification, it realizes the three-in-one technology of "targeted removal of biofilm - regulation of microbial ecology - long-lasting fresh breath", which has significant advantages. By combining traditional herbs with modern probiotic technology, it solves the problem of stubborn halitosis caused by oral microbial imbalance, and has huge market application prospects in the future. Summary of the Invention

[0003] Summary of the Invention 1. Formula principles and innovations Core ingredients: Herbal active ingredients: green tea (tea polyphenols inhibit pathogenic bacteria), honeysuckle (chlorogenic acid antibacterial), malva nut (soothing throat), monk fruit (monk fruit glycosides anti-inflammatory), clove (eugenol inhibits pathogenic bacteria), licorice (mucosal repair).

[0004] Probiotic system: Lactobacillus rhamnosus (regulates the flora), AKK bacteria culture medium (yam mucin + okra mucin + cranberry extract, promotes AKK bacteria colonization, degrades dental plaque biofilm, breaks down the dental plaque living environment and inhibits periodontal pathogens).

[0005] Functional excipients: xylitol (anti-caries), citric acid (pH regulation and enhanced antibacterial activity), and menthol (instant cooling effect), which reduce gingival inflammation and decrease venous stenosis (VSC) production by inhibiting the NF-κB pathway.

[0006] AKK bacteria's targeted destruction mechanism: AKK bacteria break down mucin and extracellular polysaccharides (such as glucan) in dental plaque biofilm by secreting mucins and glycosidases, thereby disrupting the three-dimensional structure of the biofilm and exposing pathogenic bacteria inside to the salivary environment.

[0007] Probiotics-Host Immune Synergy: Exposed pathogens are rapidly killed by natural antibacterial components such as salivary lysozyme and lactoferrin; at the same time, Lactobacillus rhamnosus inhibits the regeneration of pathogens by secreting antimicrobial peptides (such as laccocin) and competitively occupies the attachment sites of the oral mucosa.

[0008] Nutrient competitive inhibition: The AKK bacteria culture medium (yam / okra mucin) is preferentially metabolized by AKK bacteria, cutting off the carbon source supply to the pathogenic bacteria and further weakening its biofilm rebuilding ability.

[0009] Collaboration mechanism: Mucin medium provides carbon source for AKK bacteria, enhances their symbiosis with Lactobacillus rhamnosus, and inhibits the formation of pathogenic biofilms.

[0010] Herbal extracts combined with probiotics reduce the concentration of hydrogen sulfide (a marker of halitosis) in the oral cavity and reduce gingival inflammation by regulating the TLR / NF-κB pathway.

[0011] 3. Preparation process I. Raw Material Pretreatment Preparation of herbal extracts: Mix cloves, tea leaves, ginger, licorice, honeysuckle, malva nut, and monk fruit in a weight ratio of 3:5:2:1:2:1:1, grind to 60 mesh, add 10 times the amount of pure water, and reflux at 80℃ twice (1 hour each time). Combine the filtrates and concentrate to a relative density of 1.25 (measured at 60℃).

[0012] Preparation of AKK bacteria culture medium: Yam mucin, okra mucin, and cranberry extract were mixed in a ratio of 6:5:3, and 20% xylitol was added. The mixture was then dissolved in a phosphate buffer solution at pH 6.8 to form a homogenized culture medium.

[0013] II. Boiling and Concentrating Mixed cooking: The herbal extract was mixed with AKK bacteria culture medium, and the remaining xylitol (total xylitol content 45%) was added. The mixture was simmered in a constant temperature water bath at 60℃ for 6 hours with a stirring speed of 200 rpm, and the water was evaporated until the solid content reached 85%.

[0014] Acidity adjustment: After cooling to 40℃, add citric acid (final concentration 0.8%) and peppermint essential oil (0.05%), and mix well.

[0015] III. Probiotic Protection and Mixing Low-temperature mixing: The encapsulated Lactobacillus rhamnosus cells were mixed with the concentrated matrix in a sterile environment at 35°C, with a stirring speed of ≤50 rpm and a stirring time of ≤10 minutes, to avoid damaging the microcapsule structure.

[0016] IV. Molding and Packaging Tableting process: Use a rotary tablet press (pressure 5 kN, die diameter 10 mm), control the tableting temperature ≤40℃, tablet weight 1.5g / tablet, hardness 50-60 N.

[0017] Drying and Packaging: After compression, the tablets are dehydrated in a 30°C drying oven until the moisture content is ≤3%, and then immediately packaged with an aluminum foil composite film filled with nitrogen and stored away from light.

[0018] Key innovations In situ propagation of AKK bacteria: By utilizing the mucosal adhesion of yam / okra mucin, the retention time of AKK bacteria culture medium in the oral cavity is prolonged, thereby promoting the proliferation of host-native AKK bacteria.

[0019] Targeted release of probiotics: The double-layered encapsulation structure ensures rapid release of Lactobacillus rhamnosus at oral pH (6.5-7.0), inhibiting the regeneration of pathogenic bacteria.

[0020] Nutrition-Function Synergy: Xylitol combines the functions of a sweetener and a tooth-protecting agent, working synergistically with herbal antibacterial ingredients (eugenol, chlorogenic acid) to reduce the risk of tooth decay.

[0021] Example 1: Effect Verification Clinical trials showed that after 7 days of use of the lozenges, the concentration of volatile sulfur compounds (VSCs) in the oral cavity of the subjects decreased by 62%, the load of Porphyromonas gingivalis decreased by 45%, and the survival rate of probiotics reached over 90%.

[0022] Example 1: Verification of biofilm degradation effect method: An in vitro dental plaque biofilm model (containing Porphyromonas gingivalis and Streptococcus mutans) was constructed. Lozenge extract containing AKK bacteria (experimental group) and ordinary mouthwash (control group) were added respectively. After 48 hours of incubation, the biofilm thickness and bacterial survival rate were measured.

[0023] result: The biofilm thickness in the experimental group decreased by 62%, and the survival rate of pathogens decreased by 78%; scanning electron microscopy showed that the biofilm structure disintegrated, and the pathogens were in a free state.

[0024] Example 2: Clinical data on improvement of halitosis Experimental Design: Sixty patients with halitosis (Halimeter VSCs ≥ 200 ppb) took this lozenge three times a day for 14 consecutive days.

[0025] result: VSCs concentration decreased by an average of 85%, salivary lysozyme activity increased by 40%, and the gingival bleeding index decreased by 52%.

[0026] Example 3: In vitro biomembrane degradation experiment Experimental methods: Biomembrane model construction: Standard oral pathogens (Streptococcus mutans ATCC 25175 and Porphyromonas gingivalis ATCC 33277) were cultured in an anaerobic environment (37℃, 5% CO2) for 48 hours to form a mature biofilm.

[0027] Experimental group treatment: Dissolve the lozenge in sterile saline (10% w / v), add 1 mL to the surface of the biofilm, and allow it to act for 24 hours.

[0028] Control group: equal volume of physiological saline and commercially available lozenges (containing menthol and xylitol).

[0029] Testing indicators: Biofilm thickness: 3D scanning using laser confocal microscopy (CLSM).

[0030] Pathogenic bacteria survival rate: live and dead bacteria staining (SYTO 9 / PI) combined with flow cytometry.

[0031] Experimental results:

[0032] Conclusion: The tablets of this invention significantly degrade biofilm (p<0.01), and the survival rate of pathogenic bacteria is reduced to less than 1 / 5 of that of the control group.

[0033] Example 4: In vitro antibacterial and VSCs inhibition experiments Experimental methods: Antibacterial zone test: The extract (20% w / v) from the lozenge was added dropwise to an agar plate inoculated with pathogenic bacteria using the agar diffusion method. The plate was incubated at 37°C for 24 hours, and the diameter of the inhibition zone was measured.

[0034] VSCs concentration detection: The inhibition rate of the lozenges against metabolites (hydrogen sulfide and methanethiol) of Porphyromonas gingivalis was analyzed by gas chromatography-mass spectrometry (GC-MS).

[0035] Experimental results:

[0036] Conclusion: The diameter of the antibacterial zone of the lozenges of this invention is 3 times that of commercially available products (p<0.01), and the VSCs inhibition rate exceeds 80%.

[0037] Example 5: Clinical Trial (Improvement of Halitosis and Regulation of Microbiome) Experimental Design: Subject population: 100 patients with halitosis (Halimeter test VSCs≥200 ppb) were randomly divided into an experimental group (the lozenge of this invention) and a control group (commercially available lozenges). The lozenges were taken 3 times a day for 28 days.

[0038] Testing indicators: VSCs concentration (Halimeter measurement) Salivary lysozyme activity (ELISA method) Oral microbiota composition (16S rRNA sequencing) Experimental results:

[0039] Conclusion: The concentration of VSCs in the experimental group decreased by more than 80% (p<0.001), the proportion of pathogenic bacteria dropped to below 1%, and the colonization rate of probiotics was significantly improved.

Claims

1. A breath-freshening tea lozenge, characterized in that... It contains the following ingredients: xylitol, clove, tea, ginger, licorice, honeysuckle, malva nut, monk fruit, citric acid, peppermint, Lactobacillus rhamnosus and AKK culture medium (yam mucin, okra mucin, cranberry extract).

2. The lozenge according to claim 1, wherein the mass ratio of the three mucins in the AKK bacteria culture medium is 6:5:

4.

3. A method for eliminating oral pathogens, characterized in that: AKK bacteria are used to decompose biofilms and kill exposed pathogens with salivary lysozyme, while Lactobacillus rhamnosus inhibits the regeneration of pathogens.

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