Subsp. paracasei CCFM1333 and application thereof

Abstract

This invention discloses a strain of *Lactobacillus paracasei* CCFM1333 with multi-target regulatory effects and its application in regulating oral microecology, belonging to the field of microbial technology. This invention yields a strain of *Lactobacillus paracasei* CCFM1333, whose fermented milk and inactivated fermented milk not only inhibit odor-causing bacteria in the oral cavity and regulate oral microecology, but also alleviate inflammation. Specifically, it can reduce the number of odor-causing bacteria in the oral cavity of rats with increased odor-causing bacteria; downregulate the levels of serum inflammatory factors IL1β, IL-6, and IL-17A; and reduce the relative abundance of pathogenic bacteria in the oral cavity of rats, effectively regulating the oral microecology of rats.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus paracasei CCFM1333 that can inhibit oral odor-causing bacteria and relieve inflammation, and its applications, belonging to the field of microbial technology. Background Technology

[0002] Halitosis is a common oral health problem that seriously affects patients' social interactions and mental health. Statistics show that 80%–90% of halitosis stems from oral flora imbalance. Among the many factors leading to oral flora imbalance, Fusobacterium nucleatum (…) Fusobacterium nucleatum , Fn) and Porphyromonas gingivalis ( Porphyromonas gingivalis The abnormal proliferation of Gram-negative anaerobic bacteria, represented by *P. galbium* (Fn), is a key characteristic. These bacteria are not only important pathogens of infectious diseases such as periodontitis, but also produce volatile sulfides by decomposing sulfur-containing amino acids, thus becoming a major microbial source of oral odor problems such as halitosis. *Fn* possesses strong adhesion and copolymerization capabilities, promoting oral biofilm formation and creating ecological niches for the colonization of other pathogenic bacteria. *P. galbium*, on the other hand, produces gingival proteases and lipopolysaccharides, damaging host tissues and exacerbating local inflammatory responses, further disrupting the oral microenvironment. Therefore, the regulation of these odor-causing / pathogenic bacteria is of great significance for maintaining the balance of the oral microecology and promoting overall oral health.

[0003] Currently, clinical intervention for the excessive proliferation of oral pathogens mainly relies on chemical antibacterial therapy. While these methods can temporarily suppress pathogens, the effects are not lasting and may disrupt the overall microbial balance due to non-specific killing. In recent years, intervention strategies centered on regulating the microecology have received considerable attention. Probiotics, with their mechanisms of competitively inhibiting pathogen colonization and regulating local immune responses, have demonstrated good potential for maintaining oral health.

[0004] Some studies have found that certain strains of *Lactobacillus* and *Bifidobacterium* can inhibit the growth of oral pathogens and improve oral health through mechanisms such as competitive colonization, acid production, bacteriocin production, copolymerization, and modulation of host immune responses. However, current research and applications mainly focus on the prevention and treatment of dental caries, periodontitis, or Candida infections. Studies specifically targeting oral odor-causing bacteria and systematically investigating their effects on the quantity of oral odor-causing bacteria, microecological balance, and systemic inflammation are still insufficient. Furthermore, the effects of probiotics are strain-specific; not all *Lactobacillus* strains possess equal efficacy. Therefore, screening and identifying probiotic strains with highly efficient and specific inhibitory capabilities against oral odor-causing bacteria is crucial.

[0005] Lactobacillus paracasei, as one of the common commensal bacteria in humans, has been extensively studied for its beneficial functions in the gastrointestinal tract. However, its potential in oral health, particularly in the precise regulation of odor-causing bacteria, has not been fully explored. Currently, there is a lack of systematic evidence regarding whether specific strains of Lactobacillus paracasei and their fermentation products can effectively inhibit multiple key odor-causing bacteria in vitro and in vivo, effectively reverse dysbiosis, and reduce systemic inflammation levels.

[0006] Based on this, the present invention aims to fill the above-mentioned technological gap and provide a new solution based on specific probiotics and their fermented milk to prevent and treat oral odor in a safer and more microecologically compliant manner. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides a strain of *Lactobacillus paracasei* CCFM1333 that can inhibit oral odor-causing bacteria and alleviate inflammation, and its application. The aim is to solve the technical problem of lacking *Lactobacillus paracasei* strains that can effectively inhibit multiple key odor-causing bacteria in vitro and in vivo, effectively reverse dysbiosis, and reduce systemic inflammation levels.

[0008] The first technical solution provided by this invention is a type of Lactobacillus paracasei ( Lacticaseibacillus paracasei (CCFM1333) The Lactobacillus paracasei is Lactobacillus paracasei GDMCC NO: 63789.

[0009] The *Lactobacillus paracasei* CCFM1333 strain was derived from dairy products in Xining, Qinghai. Sequencing analysis revealed its 16S rDNA sequence to be shown in SEQ ID NO.1. Nucleic acid sequence comparison was performed using NCBI, confirming the strain as *Lactobacillus paracasei* with 100% homology; it was named *Lactobacillus paracasei* (CCFM1333). Lacticaseibacillus paracasei (CCFM1333)

[0010] The *Lactobacillus paracasei* CCFM1333 colonies on MRS medium are white, raised, and have smooth edges.

[0011] The second technical solution provided by the present invention is a microbial preparation containing Lactobacillus paracasei CCFM1333 as described in the first technical solution.

[0012] In one embodiment, the number of *Lactobacillus paracasei* CCFM1333 cells in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0013] The third technical solution provided by the present invention is an epigenetic agent, wherein the epigenetic agent is prepared using the *Lactobacillus paracasei* CCFM1333 described in the first technical solution or the microbial preparation described in the second technical solution.

[0014] In one embodiment, the metabiotic is obtained by inactivating the fermentation broth of Lactobacillus paracasei CCFM1333 and then crushing it under high pressure.

[0015] The fourth technical solution provided by the present invention is a product, which contains Lactobacillus paracasei CCFM1333 as described in the first technical solution, a microbial preparation as described in the second technical solution, or a metabiotic as described in the third technical solution.

[0016] In one embodiment, the product includes food, medicine, or daily chemical products.

[0017] In one embodiment, the food includes yogurt, freeze-dried yogurt cubes, milk tablets, etc.

[0018] In one embodiment, the daily chemical product includes mouthwash, mouthwash pellets, toothpaste, dental powder, whitening strips, periodontal gel, dental gel, or tooth wax.

[0019] The fifth technical solution provided by this invention is the application of Lactobacillus paracasei CCFM1333 described in the first technical solution, the microbial preparation described in the second technical solution, or the metabiotic described in the third technical solution in the preparation of products that inhibit oral odor-causing bacteria and / or improve oral microecology.

[0020] In one embodiment, the odor-causing bacteria include Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn).

[0021] In one embodiment, the product has any one or more of the following uses: (a) Reduce the number of Porphyromonas gingivalis and Fusobacterium nucleatum in the oral cavity of individuals with halitosis; (b) Reduce the level of inflammatory factors in the serum of individuals with halitosis.

[0022] In one embodiment, the product includes food, medicine, or daily chemical products.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention yielded a strain of *Lactobacillus paracasei* CCFM1333, which exhibits inhibitory effects on halitosis-causing bacteria, specifically manifested in: (1) Lactobacillus paracasei CCFM1333 inhibits biofilm formation in Fusobacterium nucleatum and Porphyromonas gingivalis. The effect is reflected in the fact that the biofilm inhibition rates against Porphyromonas gingivalis and Fusobacterium nucleatum reached 95.59% and 94.83%, respectively.

[0024] (2) Fermented milk containing *Lactobacillus paracasei* CCFM1333 can inhibit biofilm formation of *Fusobacterium nucleatum* and *Porphyromonas gingivalis*. The effect is reflected in the fact that the biofilm inhibition rates against *Porphyromonas gingivalis* and *Fusobacterium nucleatum* reached 79.59% and 87.56%, respectively.

[0025] (3) Live *Lactobacillus paracasei* CCFM1333 and postbiotics can effectively inhibit the proliferation and growth of halitosis pathogens in rat oral cavity. The effects are reflected in: the number of *Fusobacterium nucleatum* in the rat oral cavity decreased from 8.58 and 8.71 at the first sampling to 7.45 and 7.58 lg (DNA copy number / μL) at the end of the experiment; and the number of *Porphyromonas gingivalis* decreased from 8.70 and 8.84 at the first sampling to 7.44 and 7.53 lg (DNA copy number / μL) at the end of the experiment.

[0026] (4) Live Lactobacillus paracasei CCFM1333 and postbiotics can effectively alleviate the inflammatory response in rats caused by pathogenic bacteria. The effect is reflected in the reduction of IL-6 levels in rat serum from 52.87 ng / L to 38.18 and 43.58 ng / L, respectively.

[0027] (5) Live *Lactobacillus paracasei* CCFM1333 and postbiotics can effectively reduce the relative abundance of odor-causing bacteria in the oral cavity of rats. The effects are reflected in: reducing the relative abundance of *Fusobacterium* from 0.59% to 0.20% and 0.06% in the oral cavity of rats; and reducing the relative abundance of *Porphyromonas* from 0.61% to 0.20% and 0.20%.

[0028] (6) Fermented milk containing *Lactobacillus paracasei* CCFM1333 and inactivated fermented milk can effectively inhibit the proliferation and growth of halitosis pathogens in the oral cavity of rats. The effects are reflected in: the number of *Fusobacterium nucleatum* in the oral cavity of rats decreased from 8.00 and 8.02 at the first sampling to 7.41 and 7.49 lg (DNA copy number / μL) at the end of the experiment, respectively; and the number of *Porphyromonas gingivalis* decreased from 8.46 and 8.26 at the first sampling to 7.37 and 7.23 lg (DNA copy number / μL) at the end of the experiment, respectively.

[0029] (7) Fermented milk containing *Lactobacillus paracasei* CCFM1333 and inactivated fermented milk can effectively alleviate the inflammatory response in rats caused by pathogenic bacteria. The effects are as follows: fermented milk reduced the serum IL-6, IL-1β, and IL-17A levels in rats from 52.4, 112.14, and 120.4 ng / L to 34.21, 83.09, and 66.12 ng / L, respectively; inactivated fermented milk reduced the serum IL-6, IL-1β, and IL-17A levels in rats to 34.75, 84.99, and 58.91 ng / L, respectively.

[0030] (8) Fermented milk containing *Lactobacillus paracasei* CCFM1333 and inactivated fermented milk can effectively reduce the relative abundance of odor-causing bacteria in the oral cavity of rats. The effect is reflected in the reduction of the relative abundance of *Fusobacterium* in the oral cavity of rats from 0.56% to 0.16% and 0.08%, respectively.

[0031] Therefore, this Lactobacillus paracasei CCFM133 has a very broad application prospect in the preparation of food products that alleviate halitosis (such as fermented milk).

[0032] Preservation of biological materials A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1333, taxonomically named Lacticaseibacillus paracasei It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 12, 2023, with accession number GDMCC No: 63789, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0033] Figure 1 The effect of live Lactobacillus paracasei CCFM1333 and postbiotics on the number of odor-causing bacteria in the oral cavity of rats is shown in the figure.

[0034] Figure 2 The figure shows the effects of live Lactobacillus paracasei CCFM1333 and postbiotics on serum inflammatory factors in rats.

[0035] Figure 3 The diagram shows the effects of live Lactobacillus paracasei CCFM1333 and postbiotics on the oral microbiota of rats.

[0036] Figure 4 The figure shows the effect of live Lactobacillus paracasei CCFM1333 and postbiotics on odor-causing bacteria in the oral cavity of rats.

[0037] Figure 5 The graph shows the effect of Lactobacillus paracasei CCFM1333 fermented milk and inactivated fermented milk on the number of odor-causing bacteria in the oral cavity of rats.

[0038] Figure 6Effect diagram of fermented milk and inactivated fermented milk of Lactobacillus paracasei CCFM1333 on rat serum inflammatory factors.

[0039] Figure 7 Effect diagram of fermented milk and inactivated fermented milk of Lactobacillus paracasei CCFM1333 on rat oral flora.

[0040] Figure 8 Effect diagram of fermented milk and inactivated fermented milk of Lactobacillus paracasei CCFM1333 on odor-producing bacteria in rat oral cavity. Detailed implementation manners

[0041] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0042] Porphyromonas gingivalis involved in the following embodiments was purchased from the Guangdong Provincial Microbial Culture Collection Center, with the product number ATCC33277 (GDM1.851); Fusobacterium nucleatum involved was purchased from Beina Chuanglian Biotechnology Co., Ltd., with the product number ATCC25586 (BNCC337407). The SPF grade Wistar rats involved in the following embodiments were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license number SCXK(Beijing)2012 0001). The ELISA kits involved in the following embodiments were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.; other reagents involved in the following embodiments were purchased from Sinopharm Chemical Reagent Co., Ltd. The media involved in the following embodiments are as follows: MRS solid medium ( / L): peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 20 g / L.

[0043] MRS liquid medium ( / L): peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L.

[0044] BHI solid culture medium ( / L): tryptone 10.0g, beef heart extract 17.5g, sodium chloride 5.0g, yeast extract 5.0g, glucose 2.0g, disodium hydrogen phosphate dodecahydrate 2.5g, 0.5% vitamin K1 heme chloride 1mL / L, agar 20 g / L, 50mL / L sterile defibrinated sheep blood, pH 7.2~7.4.

[0045] BHI liquid culture medium ( / L): tryptone 10.0g, beef heart extract 17.5g, sodium chloride 5.0g, yeast extract 5.0g, glucose 2.0g, disodium hydrogen phosphate dodecahydrate 2.5g, 0.5% vitamin K1 heme chloride 1mL / L, pH 7.2~7.4.

[0046] The preparation method of the bacterial suspension involved in the following examples is as follows: Porphyromonas gingivalis bacterial suspension: Single colonies were obtained by anaerobic culture at 37°C for more than 48 hours on BHI solid medium; single colonies were picked and inoculated into BHI liquid medium and anaerobically cultured at 37°C for 48 hours to obtain culture medium 1; culture medium 1 was inoculated into BHI liquid medium at an inoculation rate of 4% of the total volume of BHI medium, and anaerobically cultured at 37°C for 48 hours, followed by anaerobic culture at 37°C for 24 hours, and then cultured at 8000× g Collect the bacterial sludge by centrifugation for 10 minutes, and adjust the concentration to 1×10⁻⁶ with sterile PBS. 9 CFU / mL.

[0047] Fusobacterium nucleatum suspension: Single colonies were obtained by anaerobic culture at 37°C for more than 24 hours on BHI solid medium; single colonies were picked and inoculated into BHI liquid medium and anaerobically cultured at 37°C for 48 hours to obtain culture medium 1; culture medium 1 was inoculated into BHI liquid medium at an inoculation rate of 4% of the total volume of BHI medium, and anaerobically cultured at 37°C for 24 hours, followed by inoculation at 8000× g Collect the bacterial sludge by centrifugation for 10 minutes, and adjust the concentration to 1×10⁻⁶ with sterile PBS. 8 CFU / mL.

[0048] Live *Lactobacillus paracasei*: Single colonies were obtained by culturing on MRS solid medium at 37°C for 20 h. A single colony was picked and inoculated into MRS liquid medium, and cultured at 37°C for 20 h to obtain culture medium 1. Culture medium 1 was inoculated into MRS medium at an inoculation rate of 2% of the total volume, and cultured at 37°C for 20 h. The cells were then collected, washed twice with PBS, and the concentration was adjusted to 1×10⁻⁶ PBS. 9 CFU / mL.

[0049] The preparation method of the postgenetic agent involved in the following embodiments is as follows: Postbiotics of *Lactobacillus paracasei*: After culturing *Lactobacillus paracasei* in MRS liquid medium for 20 h as described above, the bacterial concentration was adjusted to 1 × 10⁻⁶ using MRS medium. 9 CFU / mL, the fermentation broth containing the bacterial cells was pasteurized, and the completely inactivated *Lactobacillus paracasei* culture was then homogenized using a high-pressure homogenizer at 5000× g Centrifuge for 10 minutes, and the supernatant obtained after centrifugation is the postgenetic agent.

[0050] The preparation method of long-term fermented milk involved in the following examples is as follows: Add 7% glucose to raw milk, pasteurize, cool to room temperature, inoculate with 2% Lactobacillus paracasei CCFM1333, and incubate at 37℃ for 16 hours. At this time, the pH is measured to be 4.45, and the fermented milk preparation is complete.

[0051] The preparation method of inactivated fermented milk involved in the following examples is as follows: The prepared fermented milk is pasteurized to obtain inactivated fermented milk.

[0052] The preparation method of the fermented milk supernatant involved in the following examples is as follows: The prepared inactivated fermented milk is then subjected to 8000× g Centrifuge for 10 min, and pass the resulting supernatant through a 0.22 μm sterile filter membrane to obtain the fermented milk supernatant.

[0053] Example 1: Screening and identification of Lactobacillus paracasei CCFM1333 The inhibitory effects of 26 *Lactobacillus paracasei* strains on *Pg* and *Fn* were evaluated using the Oxford cup method, and the results are shown in Table 1. *Lactobacillus paracasei* strain CCFM1333 showed the best inhibitory effect on *Pg*, with an inhibition zone diameter of 13.60 ± 0.37 mm. The inhibition zones of the other 25 *Lactobacillus paracasei* strains on *Pg* were all less than 10 mm in diameter, indicating that CCFM1333 had an inhibitory effect on *Pg*. The inhibitory effects of the 26 *Lactobacillus paracasei* strains on *Fn* were evaluated using the same method, and the results showed that none of the 26 strains inhibited the growth of *Fn*.

[0054] Table 1. Inhibition of Pg and Fn by Lactobacillus paracasei

[0055] The identification sequence of Lactobacillus paracasei CCFM1333 is shown in SEQ ID NO.1.

[0056] Example 2: Biofilm inhibition rate of Lactobacillus paracasei CCFM1333 against Porphyromonas gingivalis and Fusobacterium nucleatum. The inhibitory effect of *Lactobacillus paracasei* on biofilm formation of *Porphyromonas gingivalis* and *Fusobacterium nucleatum* was determined using the crystal violet method. 100 μL of *Porphyromonas gingivalis* or *Fusobacterium nucleatum* suspension was added to each well of a 96-well cell culture plate, followed by 100 μL of *Lactobacillus paracasei* post-gene in each well. The plates were then anaerobically incubated at 37°C for 48 h. A negative control was an equal volume of blank MRS liquid medium, and a positive control was an equal volume of 0.02% chlorhexidine. Each sample was repeated three times. After incubation, the biofilm was washed twice with PBS, fixed with 99% methanol for 15 min, dried at room temperature for 10 min, and then allowed to air dry at room temperature. 100 μL of 0.1% crystal violet solution was added to each well for staining for 10 min. After staining, the plates were washed with PBS until no residual color remained. 100 μL of 95% ethanol was added to dissolve the biofilm, and the plates were shaken for 10 min. The absorbance was read at OD600. Biofilm inhibition rate = (OD negative control - OD sample) / OD negative control × 100%.

[0057] The biofilm inhibition rates of *Lactobacillus paracasei* CCFM1333 against *Porphyromonas gingivalis* and *Fusobacterium nucleatum* are shown in Tables 1 and 2, respectively. *Lactobacillus paracasei* CCFM1333 achieved a biofilm inhibition rate of 95.59% against *Porphyromonas gingivalis*, comparable to chlorhexidine and significantly superior to the other 25 *Lactobacillus paracasei* strains. *Lactobacillus paracasei* CCFM1333 also achieved a biofilm inhibition rate of 94.83% against *Fusobacterium nucleatum*, significantly better than chlorhexidine and the other 25 *Lactobacillus paracasei* strains.

[0058] Table 2 Biofilm inhibition rate of Lactobacillus paracasei against Pg

[0059] Table 3 Biofilm inhibition rate of Lactobacillus paracasei against Fn

[0060] Example 3: Biofilm inhibition rate of Lactobacillus paracasei CCFM1333 fermented milk against Porphyromonas gingivalis and Fusobacterium nucleatum. The specific detection method is the same as in Example 2. The crystal violet method was used to determine the biofilm inhibition rate of Pg and Fn in the supernatant of fermented milk of Lactobacillus paracasei CCFM1333. The negative control was raw milk and the positive control was chlorhexidine.

[0061] The biofilm inhibition rates of *Lactobacillus paracasei* CCFM1333 fermented milk against Pg and Fn are shown in Tables 4 and 5, respectively. The biofilm inhibition rate of *Lactobacillus paracasei* CCFM1333 fermented milk against Pg reached 79.59%, slightly lower than that of *Lactobacillus paracasei* CCFM1333; the biofilm inhibition rate of *Lactobacillus paracasei* CCFM1333 fermented milk against Fn reached 87.56%, significantly better than chlorhexidine but slightly lower than that of *Lactobacillus paracasei* CCFM1333.

[0062] Table 4. Biofilm inhibition rate of Pg by fermented milk containing *Lactobacillus paracasei* CCFM1333

[0063] Table 5. Biofilm inhibition rate of Fn by Lactobacillus paracasei CCFM1333 fermented milk.

[0064] Example 4: The effect of Lactobacillus paracasei CCFM1333 on improving the oral microecology of a rat model with increased specific odor-causing bacteria. 1. Effect of Lactobacillus paracasei CCFM1333 on the number of odor-causing bacteria in the oral cavity of rats (1) Experimental methods The rat feeding and diet involved in the examples are as follows: Thirty male Wistar rats were randomly divided into five groups (n=6 per group): control group, model group, chlorhexidine group, CCFM1333 live bacteria group, and CCFM1333 postbiotic group. Before the experiment, all rats were placed in a constant temperature environment of 22–24°C with alternating 12-hour light and 12-hour dark cycles for one week to acclimatize. The experiment lasted for five weeks, a total of 35 days.

[0065] The modeling method involved in the embodiments is as follows: Three days after the rats' acclimatization period, they were fed distilled water containing 125 μg / mL ampicillin sodium daily to eliminate the pre-existing oral flora. On the first day of the experiment (D1), the rats' oral cavity was infected with Pg and Fn, specifically as follows: Pg and Fn were injected into the rats' oral cavity using a sterile syringe at a concentration of 10 μg / mL. 9 Pg bacterial suspensions in the CFU / mL range and concentrations of 10 8 0.50 mL of Fn bacterial suspension (CFU / mL) was slowly rinsed into the oral cavity of rats, who were then fasted and deprived of water for half an hour. The infection frequency was once every two days for a total of 2 weeks.

[0066] Two weeks after Pg and Fn infection (D14), rats were treated with preventative measures, the methods of which were as follows: After the pathogenic bacteria infection ended, the chlorhexidine group, the CCFM1333 live bacteria group, and the CCFM1333 post-biotic group were treated daily with chlorhexidine at a concentration of 0.02%, 10%, and 10%, respectively. 9 CCFM1333 bacterial suspension on the order of CFU / mL and equivalent to a concentration of 10 9 After slowly rinsing the oral cavity of rats with CCFM1333 at the CFU / mL level, and withholding food and water for half an hour, the treatment was carried out 6 times a week for 2 weeks.

[0067] After the pathogenic bacteria infection was completed (D14), one week after probiotic intervention (D21), and two weeks after probiotic intervention (D28), saliva samples were collected from rats by swabbing their mouths for 10 seconds using disposable sterile swabs. The collected sterile swabs were stored at -80°C for further analysis. DNA was extracted from *Porphyromonas gingivalis* ATCC33277, *Fusobacterium nucleatum* ATCC25586, and rat saliva samples. DNA concentration and quality were detected using an ultra-micro nanophotometer. The real-time quantitative PCR (RT-qPCR) reaction system (10 μL) settings are shown in Table 5. The reaction program was set as follows: 95°C, 30 s; 40 cycles (95°C, 30 s; 60°C, 30 s), and the melting curve was set to increase by 0.5°C per second from 65°C to 95°C.

[0068] Table 5 RT-qPCR reaction system

[0069] RT-qPCR was used to quantify *Porphyromonas gingivalis* ATCC33277 and *Fusobacterium nucleatum* ATCC25586, and a standard curve was constructed. DNA copy number was calculated according to formula (1). Primers for *Porphyromonas gingivalis* and *Fusobacterium nucleatum* are shown in Table 6.

[0070] (1) Table 6 Specific Primers

[0071] (2) Experimental results During the experiment, oral saliva samples were collected three times, and Fn and Pg were counted. The experimental results are as follows: Figure 1 As shown in the figure. No Fn or Pg were detected in the blank group during the three counts. After the odor-causing bacteria colonized the oral cavity (D14), the levels of Fn and Pg in the oral cavity of rats in all groups reached above 8.00 lg CFU / mL. In the model group, the levels of both bacteria remained consistently high (>8.00 lg CFU / mL) throughout the experiment, indicating that Fn and Pg successfully colonized the rat oral cavity.

[0072] One week after intervention with chlorhexidine, live bacteria, and postbiotics (D21), the Fn count significantly decreased from 8.42, 8.58, and 8.71 to 7.35 ( ). p <0.01), 7.34 ( p <0.01), 7.51 lg (DNA copy number / μL) p <0.01); the amount of Pg decreased from 8.55, 8.70, and 8.84 to 7.64 ( p <0.05), 8.07 ( p >0.05), 8.06 ( p <0.05). Two weeks after intervention (D28), the number of Fn was 7.53 ( p <0.01), 7.45 ( p <0.01), 7.58 ( p <0.01), with no significant rebound observed; the amount of Pg decreased to 7.64 ( p <0.01), 7.44 ( p <0.01), 7.53 ( p The concentration of Fn and Pg decreased further (<0.01), indicating that the live bacteria and postbiotics had a significant inhibitory effect on Fn and Pg, and the inhibitory effect was comparable to that of chlorhexidine, maintaining stability.

[0073] 2. Effects of Lactobacillus paracasei CCFM1333 on serum inflammatory factors in rats (1) Experimental methods The specific implementation method is the same as step 1. After the experiment, the rats were anesthetized with isoflurane, and blood was collected from the abdominal aorta at 3000× g Rat serum was separated by centrifugation for 10 min, and the IL-6 content in the serum was measured using an ELISA kit.

[0074] (2) Experimental results Quantitative analysis of serum inflammatory factor IL-6 was performed on each group. The results showed that ( Figure 2 Compared with the control group, the IL-6 level in the model group increased from 36.17 to 52.87 ng / L. p <0.05 indicates activation of systemic inflammatory response. Compared with the model group, the IL-6 levels in the live bacteria group and the post-biotic group decreased to 38.18 ( p <0.05) and 43.58 ( p >0.05). This indicates that live Lactobacillus paracasei CCFM1333 can improve inflammation.

[0075] 3. Effects of Lactobacillus paracasei CCFM1333 on oral flora in rats (1) Experimental methods The specific implementation method is the same as in step 1. After the experiment, oral swabs were collected from rats, and DNA was extracted, strictly following the instructions. Using the above DNA as a template, specific primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) were used to amplify the hypervariable region of the bacterial 16S rRNA gene V3-V4, with an amplification length of 465 bp. After the amplification reaction, the target fragment was separated by 1.5% agarose gel electrophoresis, and the specific band was recovered using a gel purification kit. Based on the DNA concentration determination results, the purified products were mixed in equal mass concentrations to prepare a mixed library with a total mass of 200 ng and a final volume of 50 μL. After constructing the library according to the Turseq DNA LT sample preparation kit instructions, the DNA fragment size was determined, and then the library concentration was accurately determined using the KAPA kit instructions. The constructed library was diluted, denatured, and finally added to the reagent kit cartridge for sequencing.

[0076] (2) Experimental results To clarify the impact of *Lactobacillus paracasei* CCFM1333 on the composition of the oral microbiota in rats, taxonomic analysis of the sequencing sequences was performed based on the Silva database, annotating a total of 14 phyla and 266 genera. Figure 3 At the phylum level, the dominant bacterial groups are, in descending order, Proteobacteria (…). Proteobacteria Firmicutes ( Firmicutes ) and Actinobacteria ( Actinobacteria At the genus level, the genus *Rodentia* has the highest relative abundance. Rodentibacter ), followed by Streptococcus spp. ( Streptococcus ), genus *Agglutinosa* ( Aggregatibacter ), genus Rocheria ( Rothia ) and Corynebacterium spp. Corynebacterium Further analysis revealed that *Fusobacterium* spp. were present in the oral cavity of the model group rats. Fusobacterium The relative abundance (0.59%) was significantly higher than that of the control group (0.21%). p <0.05%. Chlorhexidine (0.15%) p <0.01%, live bacteria (0.20%) p <0.01) and post-natal (0.06%, p After intervention with <0.01), the abundance of this genus was significantly reduced. ​ Similarly, *Porphyromonas* spp. ( ​ The percentage of cases was also significantly higher in the model group (0.61% vs. 0.06% in the control group). p<0.01%, in chlorhexidine (0.19%, p<0.05), live bacteria (0.20%, p<0.01), p <0.01) and post-natal (0.20%, p <0.01) Significantly decreased after intervention ( ​ The above results indicate that both live *Lactobacillus paracasei* CCFM1333 and postbiotics can effectively inhibit *Fusobacterium* and *Porphyromonas* in the oral cavity of rats.

[0077] Example 5: The effect of Lactobacillus paracasei CCFM1333 fermented milk and inactivated fermented milk on improving the oral microecology of a rat model with increased specificity of odor-causing bacteria. 1. Effects of Lactobacillus paracasei CCFM1333 fermented milk and inactivated fermented milk on the number of odor-causing bacteria in the oral cavity of rats. (1) Experimental methods Thirty-six male Wistar rats were randomly divided into six groups (n=6 per group): control group, model group, chlorhexidine group, raw milk group, CCFM1333 fermented milk group, and inactivated CCFM1333 fermented milk group. The remaining experimental procedures were the same as step 1 in Example 3.

[0078] (2) Experimental results During the experiment, oral saliva samples were collected three times, and Fn and Pg were counted. The experimental results are as follows: ​ As shown in the figure. No Fn and Pg were detected in the blank group during the three counts. After the odor-causing bacteria colonized the oral cavity (D14), the levels of Fn and Pg in the oral cavity of rats in all groups reached above 8.00 lg CFU / mL. The levels of both bacteria in the model group and the raw milk group remained consistently high (>8.00 lg CFU / mL) throughout the experiment, indicating that Fn and Pg successfully colonized the rat oral cavity.

[0079] One week after intervention with chlorhexidine, fermented milk, and inactivated fermented milk (D21), the Fn count significantly decreased from 8.43, 8.00, and 8.02 to 7.44, respectively. p <0.01), 7.60 ( p <0.05), 7.35 lg (DNA copy number / μL) p <0.01); the amount of Pg decreased from 8.59, 8.46, and 8.26 to 7.91 ( p <0.01), 7.75 ( p <0.05), 7.58 ( p >0.05). Two weeks after intervention (D28), the number of Fn was 7.62 ( p <0.01), 7.41 ( p <0.01), 7.49 ( p<0.01), with no significant rebound observed; the amount of Pg decreased to 7.58 ( p <0.01), 7.37 ( p <0.05), 7.23 ( p The concentration of Fn and Pg in fermented milk was <0.05, indicating a further decrease. This suggests that the inhibitory effects of fermented milk and inactivated fermented milk on Fn and Pg are comparable to those of chlorhexidine and can remain stable.

[0080] 2. Effects of Lactobacillus paracasei CCFM1333 fermented milk and inactivated fermented milk on serum inflammatory factors in rats. (1) Experimental methods Same as step 2 in Example 4.

[0081] (2) Experimental results Quantitative analysis was performed on serum inflammatory factors IL-6, IL-1β, and IL-17A in each group. Results showed ( ​ Compared with the control group, the levels of IL-6, IL-1β, and IL-17A in the model group increased from 35.57, 88.67, and 87.89 to 52.4 ng / L, respectively. p <0.05), 112.14 ( p >0.05) and 120.4 ( p >0.05), indicating activation of systemic inflammatory response. Compared with the model group, the IL-6 level in the fermented milk group and the inactivated fermented milk group decreased to 34.21 ( p <0.01) and 34.75 ( p <0.01), IL-1β levels decreased to 83.09 ( p >0.05) and 84.99 ( p <0.05), IL-17A levels decreased to 66.12 ( p <0.05) and 58.91 ( p <0.05). This indicates that both Lactobacillus paracasei CCFM1333 fermented milk and inactivated fermented milk can improve inflammation.

[0082] 3. Effects of Lactobacillus paracasei CCFM1333 fermented milk and inactivated fermented milk on oral flora in rats (1) Experimental methods Same as step 3 in Example 4.

[0083] (2) Experimental results To clarify the effects of fermented milk and inactivated fermented milk on the composition of oral microbiota in rats, taxonomic analysis was performed on the sequencing sequences based on the Silva database, annotating a total of 14 phyla and 266 genera. ​ At the phylum level, the dominant bacterial groups are, in descending order, Proteobacteria (…). ​Firmicutes ( ​ ) and Actinobacteria ( ​ At the genus level, the genus *Rodentia* has the highest relative abundance. ​ ), followed by Streptococcus spp. ( ​ ), genus *Agglutinosa* ( ​ ), genus Rocheria ( ​ ) and Corynebacterium spp. ​ Further analysis revealed that *Fusobacterium* spp. were present in the oral cavity of the model group rats. ​ The relative abundance (0.56%) was significantly higher than that of the control group (0.29%). p <0.05%. Chlorhexidine (0.16%) p <0.01%, fermented milk (0.14%) p <0.01) and inactivated fermented milk (0.08%) p After intervention with <0.01), the abundance of this genus was significantly reduced. ​ The above results indicate that both fermented milk and inactivated fermented milk can effectively inhibit Fusobacterium spp. in the oral cavity of rats.

[0084] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A type of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1333, characterized in that, The Lactobacillus paracasei is Lactobacillus paracasei GDMCC NO: 63789.

2. A microbial preparation containing Lactobacillus paracasei CCFM1333 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, In the microbial preparation, the bacterial count of *Lactobacillus paracasei* CCFM1333 is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. An epigenetic agent, characterized in that, The epigenetic agent is a postgenetic agent prepared using Lactobacillus paracasei CCFM1333 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 3.

5. The epigenetic agent according to claim 4, characterized in that, The metabiotic is obtained by inactivating the fermentation broth of Lactobacillus paracasei CCFM1333 and then crushing it under high pressure.

6. A product characterized in that, The product contains Lactobacillus paracasei CCFM1333 as described in claim 1, the microbial preparation as described in any one of claims 2-3, or the metabiotic as described in any one of claims 4-5.

7. The product according to claim 6, characterized in that, The products include food, medicine, or daily chemical products.

8. The application of *Lactobacillus paracasei* CCFM1333 according to claim 1, the microbial preparation according to any one of claims 2-3, or the metabiotic according to any one of claims 4-5 in the preparation of products that inhibit oral odor-causing bacteria and / or improve oral microecology is characterized in that... The products include pharmaceuticals or daily chemical products.

9. The application according to claim 8, characterized in that, The odor-causing bacteria include Porphyromonas gingivalis and Fusobacterium nucleatum.

10. The application according to claim 8, characterized in that, The product has one or more of the following uses: (a) Reduce the number of Porphyromonas gingivalis and Fusobacterium nucleatum in the oral cavity of individuals with halitosis; (b) Reduce the level of inflammatory factors in the serum of individuals with halitosis.

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