Fermented dried orange peel metagen as well as preparation method and application thereof

Abstract

The invention provides fermented pericarpium citri reticulatae after-generation and a preparation method and application thereof, and relates to the technical field of oral health. The invention provides a metaplasm obtained by fermenting pericarpium citri reticulatae on the basis of saliva combined with lactobacillus, the metaplasm has the effect of treating periodontitis, and through the synergistic effect of various mechanisms of inhibiting growth of periodontal pathogenic bacteria such as porphyromonas gingivalis and the like and formation of biological membranes, reducing the level of inflammatory factors in serum, promoting bone mass increase and the like, the periodontitis treatment effect is achieved. The periodontitis symptom is improved from a plurality of links, the treatment effect is obviously better than that of a single-component or single-mechanism product, and a new treatment medicine component is provided for periodontitis and other oral inflammation diseases.

Description

Technical Field

[0001] This application relates to the field of oral health technology, and in particular to a post-biotic derived from fermented tangerine peel, its preparation method, and its application. Background Technology

[0002] Periodontitis is a multifactorial chronic inflammatory disease caused by dental plaque biofilm, leading to progressive destruction of periodontal supporting structures, including the periodontal ligament and alveolar bone. *Porphyromonas gingivalis* is a key pathogen in periodontitis; its virulence factors activate a macrophage-mediated inflammatory cascade, triggering excessive secretion of pro-inflammatory cytokines, resulting in collagen degradation and pathological bone resorption in periodontal tissues.

[0003] Traditional treatments, such as antibiotics, while effectively inhibiting the growth of pathogens like *Porphyromonas gingivalis*, can lead to dysbiosis of the normal oral flora with long-term use, resulting in drug resistance. They can also irritate the oral mucosa, potentially causing adverse reactions like oral ulcers. Furthermore, methods like periodontal scaling and root planing are local treatments. While they can remove some plaque and tartar, their effectiveness in addressing existing periodontal pockets and alveolar bone resorption is limited, and they are difficult to cure completely. There is a lack of systemic treatment options that can simultaneously regulate the body's immune response and inflammation levels.

[0004] In recent years, probiotics have shown promising applications in the treatment of periodontitis as a method for regulating the microbiota. Specific strains of Lactobacillus, such as Lactobacillus salivarius, have been proven to have positive effects on periodontal health. In addition, phytochemicals from medicinal plants, such as dried tangerine peel, offer another therapeutic dimension; however, the therapeutic potential of herbal compounds is often limited by bioavailability, and their therapeutic efficacy still needs improvement. Existing research mostly focuses on the application of single probiotics or their postbiotics, with limited research on the comprehensive application of probiotics combined with fermented postbiotics from traditional Chinese medicine. Summary of the Invention

[0005] The purpose of this invention is to provide a fermented tangerine peel post-biotic for treating oral inflammation and its application. By combining specific probiotics with fermented traditional Chinese medicine, a synergistic effect is achieved, resulting in a new functional pharmaceutical ingredient. Verification has shown that this fermented tangerine peel post-biotic can effectively improve periodontitis symptoms.

[0006] On the one hand, this application provides a method for preparing post-fermented tangerine peel, characterized in that the method includes: fermenting tangerine peel with *Ligilactobacillus salivarius*.

[0007] Preferably, the concentration of the dried tangerine peel is greater than 0.1% by mass; more preferably, greater than 1.25%; even more preferably, 1.25%-10%.

[0008] The concentration of the dried tangerine peel can be any value among 0.1%, 0.2%, 0.3%, 0.3125%, 0.4%, 0.5%, 0.6%, 0.625%, 0.7%, 0.8%, 0.9%, 1%, 1.25%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%.

[0009] Furthermore, the sialic acid-associated lactobacillus includes sialic acid-associated lactobacillus with accession number CICC 23175 and / or sialic acid-associated lactobacillus with accession number GDMCC No: 63646.

[0010] Lactobacillus salivarius (accession number GDMCC No: 63646) CCFM1327 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 14, 2023. Lactobacillus salivarius (accession number CICC 23175) was purchased from the China Industrial Microbial Culture Collection Center.

[0011] The activation or culture of lactobacillus in conjunction with sialic acid can be carried out using conventional methods for activating or culturing lactobacillus species.

[0012] In a preferred embodiment, the live bacteria content in the activated sialic acid-containing Lactobacillus bacterial solution is ≥1×10⁻⁶. 6 CFU / mL; more preferably, ≥1×10 9 CFU / mL.

[0013] In a preferred embodiment, the activation method includes the following steps:

[0014] Step 1: Pick a single colony from an MRS solid plate and inoculate it into MRS liquid medium. Incubate at 37°C for 20 hours to perform one activation and obtain a one-activated fermentation broth.

[0015] Step 2: Add 2% (w / w) of the first-activated fermentation broth to MRS liquid medium and incubate at 37°C for 20 hours for a second activation to obtain the fermentation broth. At this point, the viable cell count in the fermentation broth should reach ≥10⁻⁶. 6 CFU / mL.

[0016] Furthermore, the method includes the following steps:

[0017] Step 1: Ferment tangerine peel using saliva and lactobacillus to obtain fermentation broth;

[0018] Step 2: Centrifuge and filter the fermentation liquid to obtain post-fermented tangerine peel extract.

[0019] Preferably, the concentration of the dried tangerine peel is greater than 0.1% by mass; more preferably, greater than 1.25%; even more preferably, 1.25%-10%.

[0020] The concentration of the dried tangerine peel can be any value among 0.1%, 0.2%, 0.3%, 0.3125%, 0.4%, 0.5%, 0.6%, 0.625%, 0.7%, 0.8%, 0.9%, 1%, 1.25%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%.

[0021] Preferably, the dried tangerine peel is soluble in a solvent.

[0022] In a preferred embodiment, the solvent may be water and / or a culture medium.

[0023] Those skilled in the art can choose the solvent for the tangerine peel solution according to the actual situation; no specific requirements are made here.

[0024] Preferably, the tangerine peel solution is sterilized after preparation to prevent contaminating bacteria from affecting the subsequent fermentation process.

[0025] Furthermore, the sialic acid-associated lactobacillus includes sialic acid-associated lactobacillus with accession number CICC 23175 and / or sialic acid-associated lactobacillus with accession number GDMCC No: 63646.

[0026] Preferably, step one further includes the step of activating Lactobacillus salivans.

[0027] More preferably, the viable bacteria content in the bacterial solution after activation by saliva-based Lactobacillus is ≥1×10⁻⁶. 6 CFU / mL; more preferably, ≥1×10 9 CFU / mL.

[0028] Preferably, the inoculation amount is 1%-5%.

[0029] The inoculation amount can be any value among 1%, 2%, 3%, 4%, and 5%.

[0030] Preferably, the fermentation conditions include: fermentation at 37°C for 12-24 hours.

[0031] Fermentation time can be any value among 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, and 24h.

[0032] Preferably, the centrifugation is performed at 4℃-15℃, at 10000-20000g for 10-30 minutes.

[0033] More preferably, the centrifugation is performed at 4°C, 13000g for 10 minutes.

[0034] The centrifugation process only needs to remove the bacterial cells (including live or dead bacteria) by precipitation; no specific numerical requirements are specified for the conditions.

[0035] In a preferred embodiment, the filtration is performed using a 0.22 μm sterile filter membrane.

[0036] Those skilled in the art can use conventional filtration methods to remove any possible residual microorganisms or cell debris; the specific method is not required.

[0037] In a preferred embodiment, the method includes the following steps:

[0038] Step 1: Use Lactobacillus sialic acid-binding bacteria with accession number CICC 23175 and / or Lactobacillus sialic acid-binding bacteria with accession number GDMCC No: 63646 to ferment dried tangerine peel with a concentration greater than 0.1%, with an inoculum size of 1%-5%, and ferment at 37℃ for 12-24 hours to obtain fermentation broth;

[0039] Step 2: Centrifuge the fermentation liquid at 4℃-15℃, 10000-20000g for 10-30 minutes, and filter it through a sterile filter membrane to obtain the post-fermented tangerine peel saccharin.

[0040] On the other hand, this application also provides a post-biotic for fermented tangerine peel prepared by the method described above.

[0041] Preferably, the metabiotic is a metabolite of Lactobacillus salivarius, or any of the above-mentioned processed products.

[0042] On the other hand, this application also provides compositions that contain at least the fermented tangerine peel postbiotic in a physiologically acceptable medium.

[0043] This application may also add excipients, which may be appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0044] The compositions of this application can be prepared by a common method, wherein one or more physiologically acceptable diluents or carriers may be added, such as gels, pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0045] One aspect of this application relates to compositions, which are any compositions capable of achieving the effects described in this application. The compositions include, but are not limited to, the simultaneous or sequential use of the components. "Simultaneous use" includes using them together in the same formulation or separately in different formulations. "Sequential use" includes using them sequentially in different formulations, with no restriction on the order of sequential use.

[0046] On the other hand, this application also provides the use of the method or the post-biotic or the composition described herein in the preparation of products for relieving and / or treating oral inflammation.

[0047] Preferably, the oral inflammation includes periodontal inflammation and / or gingivitis.

[0048] On the other hand, this application also provides the use of the method or the post-genetic agent or the composition described herein in the preparation of products for relieving and / or treating periodontal inflammation and / or gingivitis.

[0049] Preferably, the application includes: inhibiting the growth of Porphyromonas gingivalis, inhibiting the formation of Porphyromonas gingivalis biofilm, anti-inflammatory effects, improving dental bone quality and / or improving bone volume fraction.

[0050] In a preferred embodiment, alveolar bone is used as a tooth sample for testing. Those skilled in the art will understand that the same or similar effects apply to other types of teeth, which will not be listed here.

[0051] On the other hand, this application also provides the use of the method or the metabiotic or the composition described herein in the preparation of products that inhibit the growth of Porphyromonas gingivalis and / or inhibit the formation of Porphyromonas gingivalis biofilms.

[0052] Preferably, the inhibition rate of Porphyromonas gingivalis biofilm formation can reach more than 80%, and more preferably, more than 90%.

[0053] On the other hand, this application also provides the use of the method, the post-biotic, or the composition described herein in the preparation of anti-inflammatory products.

[0054] Preferably, the application includes downregulating the levels of inflammatory cytokines to reduce the inflammatory response in periodontal tissues; more preferably, the inflammatory cytokines include tumor necrosis factor-α (TNF-α) and / or interleukin-8 (IL-8).

[0055] On the other hand, this application also provides the use of the method or the post-genetic agent or the composition described herein in the preparation of products that improve tooth bone quality and / or bone volume fraction.

[0056] The application promotes bone mass increase by increasing the BV / TV ratio, thereby protecting teeth, especially alveolar bone, and achieving a bone protection effect.

[0057] The present invention has the following beneficial effects:

[0058] This application proposes a post-biotic obtained from fermented dried tangerine peel using saliva and Lactobacillus. This post-biotic has therapeutic effects on periodontitis. It improves periodontitis symptoms from multiple aspects through a synergistic effect of multiple mechanisms, such as inhibiting the growth and biofilm formation of periodontal pathogens such as Porphyromonas gingivalis, reducing the level of inflammatory factors in serum, and promoting bone mass increase. Its therapeutic effect is significantly better than products with single ingredients or single mechanisms, providing a new therapeutic drug component for periodontitis and other oral inflammatory diseases.

[0059] Compared to traditional antibiotic treatments, treatment using the post-biotic prepared in this application not only effectively inhibits periodontal pathogens in a short period of time but also improves the oral microecology, maintaining stable therapeutic effects with long-term use and improving patient compliance and treatment outcomes. It avoids the problem of antibiotic-induced dysbiosis and drug resistance.

[0060] This application also provides a method for preparing postbiotics obtained by fermenting dried tangerine peel with *Lactobacillus salivarius*. This method is the first to discover a synergistic effect between *Lactobacillus salivarius* and dried tangerine peel. Furthermore, through fermentation technology, *Lactobacillus salivarius* can convert the active ingredients in dried tangerine peel into a form more easily absorbed by the human body, improving its bioavailability and enhancing the product's therapeutic effect. Simultaneously, it overcomes the drawback of the shelf life of live bacteria products being affected by environmental conditions, maximizing their positive effects. Moreover, this method is simple to operate, the process is green and safe, the product is stable, and it is suitable for industrial production and widespread application. Attached Figure Description

[0061] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0062] Figure 1 Statistical chart of serum TNF-α(A) and IL-8(B) levels in rats;

[0063] Figure 2 Comparison of the bone-to-tissue volume (BV / TV) ratio at the distal root of the mandibular first molar. Detailed Implementation

[0064] Technical terms:

[0065] Metabiotics: Metabiotics refer to preparations of non-living microorganisms or their components that are beneficial to the health of the host, including inactivated probiotics, probiotic metabolites, and probiotic cell components.

[0066] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0067] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0068] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0069] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0070] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0071] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0072] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0073] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0074] The culture media involved in the following examples are as follows:

[0075] MRS liquid culture medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, balance water, pH 6.2-6.4.

[0076] MRS solid culture medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, agar 20 g / L, balance water, pH 6.2-6.4.

[0077] BHI liquid culture medium: tryptone 10.0 g / L, bovine heart extract 17.5 g / L, sodium chloride 5.0 g / L, yeast extract 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate dodecahydrate 2.5 g / L, 0.5% vitamin K1, heme chloride 1 mL / L, sterile defibrinated sheep blood 50 mL / L, balance water, pH 7.2-7.4.

[0078] BHI solid medium: tryptone 10.0 g / L, bovine heart extract 17.5 g / L, sodium chloride 5.0 g / L, yeast extract 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate dodecahydrate 2.5 g / L, 0.5% vitamin K1, heme chloride 1 mL / L, sterile defibrinated sheep blood 50 mL / L, agar 20 g / L, balance water, pH 7.2-7.4.

[0079] *Porphyromonas gingivalis*: *Porphyromonas gingivalis* strain ATCC33277 was provided by the Guangdong Provincial Microbial Culture Collection Center. *Porphyromonas gingivalis* preserved in glycerol tubes was activated with BHI solid medium and cultured anaerobically at 37°C for 3 days. Subsequently, single colonies were selected and transferred to BHI liquid medium, cultured at 37°C for 48 hours, and then the bacteria were collected, centrifuged for 30 minutes, and resuspended to a concentration of 1×10⁻⁶. 5 CFU / mL is used for in vitro experiments, and 1×10 9 CFU / mL is used for in vivo studies.

[0080] Lactobacillus salivarius CCFM1327 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 14, 2023, with accession number GDMCC No: 63646.

[0081] Lactobacillus salivarius CICC 23175 was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 23175.

[0082] A strain of *Ligilactobacillus salivarius*, CCFM1327, taxonomically named *Ligilactobacillus salivarius*, was deposited on July 14, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No.: 63646), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. This strain has been described in Chinese patent application No. 2025105185708.

[0083] Example 1: Antibacterial effect of different concentrations of dried tangerine peel on Porphyromonas gingivalis

[0084] This embodiment investigates the specific antibacterial effect of dried tangerine peel on Porphyromonas gingivalis, using the following method.

[0085] Soak 20g of dried tangerine peel powder completely in 200mL of water for 30min, then boil the mixture for 1h, repeating twice. Collect the extract, filter with gauze, and concentrate to a final volume of 20mL. Centrifuge the solution at 13000 rpm for 10min, collect the supernatant, and sterilize it by passing it through a 0.22μm sterile filter membrane. Then, prepare the supernatant to concentrations of 10%, 5%, 2.5%, 1.25%, 0.625%, and 0.3125% (based on the mass of dried tangerine peel powder), and take 150μL of each concentration, adding them to separate Oxford cups (the Oxford cups are placed in a solution containing 1×10⁻⁶...). 5 The bacteria were incubated in BHI solid medium (CFU / mL) for *Porphyromonas gingivalis* ATCC33277, then diffused at 4°C for 6 h, followed by incubation at 37°C for 48 h. The formation of the antimicrobial zone around the Oxford cup in the medium was observed, and the diameter of the inhibition zone (DIZ) was measured. An equal volume of BHI liquid medium was used as a negative control, and an equal volume of 0.02% chlorhexidine was used as a positive control. The experimental results are shown in Table 1.

[0086] Table 1. Inhibitory effect of different concentrations of dried tangerine peel on the growth of Pseudomonas gingivalis.

[0087]

[0088] As shown in Table 1, lower concentrations (0.3125%-0.625%) of dried tangerine peel exhibited weaker antibacterial activity (DIZ < 8 mm). A critical threshold was observed at a concentration of 1.25%, reaching a clinically significant DIZ of 12.21 ± 1.46 mm. Doubling the concentration to 2.5% only slightly increased the DIZ to 12.54 ± 1.21 mm, and this increase was not statistically significant. Subsequently, at higher concentrations (5%-10%), the DIZ stabilized at 11.35-12.11 mm. This indicates that bacterial growth inhibition can be achieved at a concentration of 1.25%. Example 2: Inhibitory effect of saliva-fermented dried tangerine peel on *Porphyromonas gingivalis* biofilm.

[0089] Under the preferred conditions of Example 1, this example further uses saliva combined with Lactobacillus fermentation of dried tangerine peel to prepare postbiotics, and explores the inhibitory effect of fermented dried tangerine peel postbiotics on Porphyromonas gingivalis biofilm, the method is as follows.

[0090] Preparation method of post-fermentation biotic of *Lactobacillus salivarius* CCFM1327: *Lactobacillus salivarius* CCFM1327 stored in glycerol tubes was activated with MRS solid medium. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 20 h. This process was repeated for two generations to obtain the fermentation broth. The broth was pasteurized to inactivate the bacteria, and the absence of viable bacteria was confirmed by the plating method. The bacteria were lysed using a high-pressure homogenizer (1200 bar / 10 min), followed by centrifugation at 13000 g for 10 min. The supernatant was collected and filtered through a 0.22 μm sterile filter to obtain a cell-free supernatant, which is the post-fermentation biotic of *Lactobacillus salivarius* CCFM1327.

[0091] Preparation method of tangerine peel solution: Dissolve an appropriate amount of tangerine peel powder in MRS liquid culture medium to prepare the required concentration (1.25%) for the experiment, and then autoclave (121℃ / 15min). Then incubate at 37℃ for 20h. After incubation, centrifuge at 13000g for 10min and collect the supernatant to obtain the tangerine peel solution.

[0092] Preparation method of postbiotics from fermented dried tangerine peel by *Lactobacillus salivarius* CCFM1327: *Lactobacillus salivarius* CCFM1327 stored in glycerol tubes was activated with MRS solid medium. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 20 h. This process was repeated for two generations to obtain the fermentation broth. Simultaneously, an appropriate amount of dried tangerine peel powder was dissolved in MRS liquid medium to prepare the required concentration (1.25%), which was then autoclaved (121℃ / 15 min). 2% of the *Lactobacillus salivarius* CCFM1327 fermentation broth was inoculated into the tangerine peel solution and incubated at 37℃ for 20 h. After incubation, the fermentation broth was centrifuged at 13000g for 10 min at 4℃. The supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain a cell-free supernatant, which is the postbiotic from fermented dried tangerine peel by *Lactobacillus salivarius* CCFM1327.

[0093] Preparation method of post-biotic of fermented dried tangerine peel by *Lactobacillus salivarius* CICC 23175: The lyophilized powder of *Lactobacillus salivarius* CICC 23175 was dissolved and activated with MRS solid medium. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 20 h. This process was repeated for two generations to obtain the fermentation broth. Simultaneously, an appropriate amount of dried tangerine peel powder was dissolved in MRS liquid medium to prepare the required concentration (1.25%), which was then autoclaved (121℃ / 15 min). 2% of the *Lactobacillus salivarius* CICC 23175 fermentation broth was inoculated into the tangerine peel solution and incubated at 37℃ for 20 h. After incubation, the fermentation broth was centrifuged at 13000g for 10 min at 4℃. The supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain a cell-free supernatant, which is the post-biotic of fermented dried tangerine peel by *Lactobacillus salivarius* CICC 23175.

[0094] Quantitative analysis of biofilm formation was performed using crystal violet staining.

[0095] 80 μL of a concentration of 1×10 5 A CFU / mL suspension of *Porphyromonas gingivalis* was added to each well of a 96-well cell culture plate and incubated for 24 h. Subsequently, 80 μL of 0.02% chlorhexidine (CHX group), 1.25% dried tangerine peel solution (dried tangerine peel group), *Lactobacillus salivarius* CCFM1327 self-fermentation postbiotic (CCFM1327 postbiotic group), *Lactobacillus salivarius* CCFM1327 fermented dried tangerine peel postbiotic (CCFM1327 fermented dried tangerine peel group), and *Lactobacillus salivarius* CICC 23175 fermented dried tangerine peel postbiotic (CICC 23175 fermented dried tangerine peel group) were added, along with a control group (containing an equal volume of MRS liquid medium). Incubation continued for another 24 h without shaking to promote multilayer biofilm formation. The culture medium was then aspirated, the plates were washed with PBS, and the biofilms were stained with 0.01% crystal violet for 20 min. After removing the crystal violet, the plates were washed three times with PBS. The bound crystal violet was dissolved in 1.5 mL of 33% acetic acid and shaken at 150 rpm for 15 min. The absorbance was measured at 600 nm using a BioTek Synergy HT microplate reader. The inhibition rate of *Porphyromonas gingivalis* biofilm was calculated from the absorbance, and the experimental results are shown in Table 2.

[0096] The inhibition rate is calculated as follows: Inhibition rate (%) = (OD) NC -OD sample ) / OD NC *100%

[0097] Table 2 Inhibition rate of Porphyromonas gingivalis biofilm

[0098]

[0099] As shown in Table 2, although the anti-biofilm activity of 1.25% unfermented tangerine peel was negligible (inhibition rate <5%), the product after fermentation with Lactobacillus saliva-coated CCFM1327 showed a significantly enhanced effect, with a biofilm inhibition rate of 94.50%, which was significantly higher than that of the post-CCFM1327 group.

[0100] Example 3: Animal experiments related to the prevention and treatment of periodontitis using saliva-based fermented tangerine peel containing Lactobacillus CCFM1327.

[0101] In this embodiment, the following experiments were conducted using samples of self-fermented post-biotics, tangerine peel solution, *Lactobacillus salivarius* CCFM1327 fermented tangerine peel post-biotics, and *Lactobacillus salivarius* CICC 23175 fermented tangerine peel post-biotics prepared by the method in Example 2.

[0102] The animals used in the experiment were 5-week-old male Wistar rats, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were randomly divided into 7 groups: (1) Control group: healthy rats; (2) Model group: rats that successfully modeled the disease; (3) CHX group: treated with 0.02% chlorhexidine; (4) Tangerine peel group: treated with 1.25% tangerine peel solution; (5) CCFM1327 post-fermentation group: treated with post-fermentation of saliva combined with Lactobacillus CCFM1327; (6) CCFM1327 fermented tangerine peel group: treated with post-fermentation of saliva combined with Lactobacillus CCFM1327 fermented tangerine peel; (7) CICC 23175 fermented tangerine peel group: treated with post-fermentation of saliva combined with Lactobacillus CICC 23175 fermented tangerine peel.

[0103] Rats were housed at 22℃-24℃ with a 12-hour light / dark cycle and free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Jiangsu Provincial Institute of Parasitic Diseases (IACUC-JIPD-2024-107).

[0104] On the first day of the experiment, rats were anesthetized by isoflurane inhalation, and the left maxillary second molar of each rat was ligated with a 0.22 mm sterile orthodontic wire. The wire was inserted as deeply as possible into the gingival sulcus. After surgery, the rats were placed in a clean, warm environment to recover and were allowed to awaken. During the subsequent 7-day recovery period, the rats received antibiotic treatment for the first three days to reduce endogenous oral infection. From day 7 to day 20, 0.5 ml of *Porphyromonas gingivalis* solution was applied to the ligation site of the rat molars five times a week using a sterile syringe.

[0105] From day 21 to day 35, each group of rats received 0.5 mL of the designated therapeutic substance (CHX, post-fermentation glycogen, tangerine peel solution, post-fermentation glycogen from tangerine peel fermented by Lactobacillus saliva CCFM1327 or post-fermentation glycogen from tangerine peel fermented by Lactobacillus saliva CICC23175) 6 times a week via sterile syringe. After each treatment, the rats were fasted for 30 minutes.

[0106] (1) Effects on inflammation levels

[0107] Blood was collected from rats via the abdominal aorta, and serum was prepared by centrifugation at 3000g for 10 min at 4°C. The serum was then stored at -80°C for further analysis. Using the corresponding kits provided by Nanjing Senbeijia Biotechnology Co., Ltd., and following the manufacturer's protocol, the levels of tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) in the serum were quantitatively detected. The results are shown in Table 3. Figure 1 As shown.

[0108] Table 3. Serum TNF-α and IL-8 levels in rats (mean values)

[0109]

[0110]

[0111] Note: Different letters indicate significant differences between groups (P < 0.05), such as a and b; the same letters indicate no significant differences between groups (P > 0.05), such as a and a, b and b.

[0112] As shown in Table 3 and Figure 1 As shown, the levels of inflammatory cytokines IL-8 (P<0.001) and TNF-α (P<0.001) significantly increased after modeling. Compared with the model group, treatment with CCFM1327 fermented tangerine peel significantly reduced the levels of these two inflammatory cytokines (P<0.01), which was very close to the effect of CHX. Compared with other intervention groups, including the tangerine peel group, post-biotic group, and CICC23175 fermented tangerine peel group, the anti-inflammatory effect of CCFM1327 fermented tangerine peel group was significantly improved (P<0.05).

[0113] (2) Effects on the alveolar bone of rats

[0114] Rats were euthanized by exsanguination after deep anesthesia. The maxilla on the ligated side of the rat was dissected with a scalpel and then immersed in a 4% formaldehyde solution. The cleaned rat maxilla was then subjected to micro-computed tomography (Micro-CT) with the following parameters: voltage 90 kV, current 88 μA, pixel size 18 μm, and imaging rotation angle 180 degrees. The region of interest was normalized to the bifurcation of the second molar using CTAn software, and the ratio of bone volume fraction to total volume (BV / TV) was calculated. The results are shown in Table 4 and... Figure 2 As shown.

[0115] Table 4. Bone to tissue volume ratio (BV / TV) of the distal periapical region of the mandibular first molar (mean).

[0116]

[0117] Note: Different letter labels indicate significant differences between groups (P < 0.05), such as a and b, a and c, and b and c; the same letter labels indicate no significant differences between groups (P > 0.05), such as c and c, abc and a or b or c.

[0118] Bone volume fraction (BV / TV) refers to the proportion of bone tissue volume to the total volume of bone tissue and its internal pores (i.e., the total volume of the region of interest, ROI). It is a core parameter for quantitatively describing the microstructure of cancellous bone and reflects the amount of bone (the proportion of bone tissue). A higher value indicates denser trabeculae and generally more compact bone; conversely, a lower value may indicate bone loss.

[0119] As shown in Table 4 and Figure 2 As shown, the BV / TV ratio in the model group was significantly lower than that in the control group (P<0.01). After intervention, the BV / TV ratio in the CCFM1327 fermented tangerine peel group (P<0.05) significantly increased. Furthermore, compared with other intervention groups, such as the tangerine peel group, post-biotic group, and CICC23175 fermented tangerine peel group, the BV / TV ratio in the CCFM1327 fermented tangerine peel group was also significantly increased (P<0.05).

[0120] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing post-biotics from fermented tangerine peel, characterized in that, The method includes fermenting dried tangerine peel with *Ligilactobacillus salivarius*.

2. The preparation method according to claim 1, characterized in that, The aforementioned *Lactobacillus salivarius* includes *Lactobacillus salivarius* with accession number GDMCC No: 63646.

3. The method according to claim 1 or 2, characterized in that, The method includes the following steps: Step 1: Ferment tangerine peel using saliva and lactobacillus to obtain fermentation broth; Step 2: Centrifuge and filter the fermentation liquid to obtain post-fermented tangerine peel extract.

4. A post-biotic for fermented tangerine peel prepared by the method described in any one of claims 1-3.

5. A composition, characterized in that, It contains at least the post-biotic of fermented tangerine peel as described in claim 4 in a physiologically acceptable medium.

6. The use of the method as described in any one of claims 1-3, or the post-biotic as described in claim 4, or the composition as described in claim 5, in the preparation of products for relieving and / or treating oral inflammation.

7. The use of the method of any one of claims 1-3, or the post-biotic of claim 4, or the composition of claim 5, in the preparation of products for relieving and / or treating periodontal inflammation and / or gingivitis.

8. The use of the method of any one of claims 1-3, the metabiotic of claim 4, or the composition of claim 5 in the preparation of products that inhibit the growth of Porphyromonas gingivalis and / or inhibit the formation of Porphyromonas gingivalis biofilms.

9. The use of the method as described in any one of claims 1-3, or the post-biotic as described in claim 4, or the composition as described in claim 5, in the preparation of anti-inflammatory products.

10. The use of the method of any one of claims 1-3, or the metagener of claim 4, or the composition of claim 5, in the preparation of products that improve tooth bone quality and / or bone volume fraction.

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