Probiotics with decayed tooth resisting effect as well as metagen and application thereof

Abstract

The probiotic is characterized in that the probiotic is a lactobacillus pentosus B-1-35 strain, the preservation number of the lactobacillus pentosus B-1-35 strain is CGMCC (China General Microbiological Culture Collection Center) No.33870, the metagen is derived from fermentation liquor of the lactobacillus pentosus B-1-35, the preservation number of the metagen is CGMCC No.33870, the preservation number of the metagen is CGMCC No.33870, and the metagen is derived from the fermentation liquor of the lactobacillus pentosus B-1-35. The preparation method comprises the following steps: adding the lactobacillus pentosus B-1-35 into a fermentation culture medium according to the inoculum size of 2-4% in volume ratio, activating to the medium logarithm, and inoculating the activated bacterium liquid into the fermentation culture medium according to the inoculum size of 2-4% in volume ratio to obtain fermentation liquid; the method comprises the following steps: performing water bath inactivation and cooling on fermentation liquor, performing ultrasonication and centrifugation, taking supernate, filtering the supernate by using a nanofiltration membrane to remove part of water, adding nonfat dry milk powder accounting for 1-2% of the mass of the suspension into the nano-filtered suspension, and then concentrating to obtain metagen powder with a decayed tooth resisting effect, and further preparing the metagen powder into metagen buccal tablets. The toothpaste has the advantages of inhibiting caries pathogen activity and improving oral health.

Description

Technical Field

[0001] This invention relates to a probiotic and its postbiotic, and more particularly to a probiotic with anti-caries effect, its postbiotic, and its application. Background Technology

[0002] In today's fast-paced society, due to lifestyle habits such as diet, hygiene, and sleep patterns, the prevalence of dental caries is on the rise, reaching as high as 90% among adolescents. Early signs of dental caries include visible discolored plaque and tiny black spots or patches on the enamel surface. If cariogenic bacteria are not treated promptly, they will continue to erode the dentin and even the pulp, causing irreversible damage to tooth function and seriously affecting overall health. Dental plaque is essentially a complex biofilm formed by the accumulation of various oral microorganisms, saliva, and food debris. *Streptococcus mutans* is generally considered the primary cariogenic bacterium, while *Candida albicans* in an unbalanced oral environment can transform into virulent hyphae, promoting the virulence of *Streptococcus mutans*.

[0003] Currently, methods for preventing early tooth decay include fissure sealing, fluoride varnish, use of antibiotics or fluoride-containing medications, and ingestion of live bacteria preparations. However, antibiotic use can lead to antibiotic resistance in pathogenic bacteria; fluoride-containing medications can cause chronic or acute fluorosis due to overdose; live bacteria are sensitive to their environment, and their beneficial effects require colonization, with excessive intake potentially disrupting the balance of oral flora. In contrast, lactic acid bacteria postbiotics, as a food additive, are highly stable, easily absorbed by the body, and do not require consideration of bacterial activity and colonization, making them suitable for use in products that improve the oral microecology.

[0004] The screening of functional lactic acid bacteria postbiotic strains and the preparation of postbiotic formulations are currently the main research directions for lactic acid bacteria postbiotics. However, research on the control of caries-causing bacteria by lactic acid bacteria postbiotics is still limited, and the inhibitory effects and mechanisms of lactic acid bacteria postbiotics on caries-causing bacteria are unclear. Furthermore, there is a lack of corresponding postbiotic oral probiotic products on the market. Therefore, in order to effectively inhibit dental caries and maintain the dental health of residents, there is an urgent need to develop a postbiotic lozenge with anti-caries effects. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a probiotic with anti-caries effect that can effectively inhibit the growth of caries-causing bacteria, as well as its post-biotic and application.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: This invention provides a probiotic with anti-caries effects, wherein the probiotic is *Lactobacillus pentosus* (…). Lactiplantibacillus pentosus The strain B-1-35, with accession number CGMCC No. 33870, is used.

[0007] This invention also provides a metabiotic powder with anti-caries effect, wherein the metabiotic is derived from Lactobacillus pentosus (… Lactiplantibacillus pentosus B-1-35, wherein the pentosacchari lactobacillus ( Lactiplantibacillus pentosus The preservation number of strain B-1-35 is CGMCC No. 33870.

[0008] This invention also provides a method for preparing a post-caries powder with anti-caries effect, comprising the following steps: adding *Lactobacillus pentosus* B-1-35 to a fermentation medium at a volume ratio of 2-4% and activating it to mid-logarithmic phase; taking the activated bacterial solution and inoculating it into the fermentation medium at a volume ratio of 2-4%; allowing it to grow statically at 35-40 ℃ until OD≥1.7 and pH≤3.70, thus obtaining a fermentation broth; after inactivating and cooling the fermentation broth in a water bath, performing ultrasonic disruption and centrifugation to obtain the supernatant; filtering the supernatant through a nanofiltration membrane to remove some water; adding 1-2% of skim milk powder by weight of the nanofiltration suspension; and then concentrating to obtain a post-caries powder with anti-caries effect.

[0009] Furthermore, the ultrasonic cell disruptor used for ultrasonic disruption has the following operating parameters: frequency 20-25KHz, ultrasonic on for 3 seconds, ultrasonic off for 3 seconds, ultrasonic power 40%, and working time 10 minutes.

[0010] Furthermore, the fermentation medium is prepared by dissolving MRS solid medium in 1L of distilled water and sterilizing it at 121°C for 15 minutes. The formula of the MRS solid medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 80ml Tween, 0.5g magnesium sulfate, 0.25g manganese sulfate and 15g agar powder.

[0011] Furthermore, the concentration is specifically as follows: 1-2% of skim milk powder is added to the nanofiltration suspension, and the suspension is concentrated by vacuum centrifugation until the solid content is above 40%. The concentrated suspension is then spray-dried to obtain a post-caries powder with anti-caries effect.

[0012] Furthermore, the vacuum centrifugation conditions are as follows: temperature 50℃, rotation speed 2000 rpm, vacuum pressure -0.1Mpa, and the spray drying conditions are as follows: inlet air temperature 100℃, outlet air temperature 70℃, and feed rate 500 mL / h.

[0013] The present invention also provides the use of the post-natal powder prepared by the above method to prepare post-natal lozenges with anti-caries effects. The specific formula of the post-natal lozenges is as follows: 8-12 parts resistant starch, 4-6 parts cyclodextrin, 4-6 parts xylitol, 8-12 parts fructooligosaccharide, 4-6 parts isomaltulose, 14-18 parts post-natal powder, 45-51 parts microcrystalline cellulose, and 0.5-2 parts magnesium stearate.

[0014] The present invention also provides the application of the above-mentioned probiotics with anti-caries effects in the preparation of products that improve oral microecology.

[0015] The present invention also provides the use of the above-mentioned probiotics with anti-caries effects in the preparation of inhibitors of Streptococcus mutans and / or Candida albicans.

[0016] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a probiotic with anti-caries effect, its post-biotic, and its application, and the Pentosacchari lactobacillus obtained through screening ( Lactiplantibacillus pentosus The B-1-35 strain, after fermentation and inactivation, yields a metabiotic with anti-caries effects, slowing the proliferation rate of Streptococcus mutans and Candida albicans; inhibiting the adhesion and biofilm formation of Streptococcus mutans; and compared to live bacteria, the metabiotic does not require a special colonization environment and has less impact on the balance of oral flora. Using *Lactobacillus pentosus* (… Lactiplantibacillus pentosus The preparation of metagenin lozenges with anti-caries effects by fermentation of strain B-1-35 can significantly inhibit the acid production of Streptococcus mutans and Candida albicans, reduce the erosion of hydroxyapatite discs by mixed biofilms, and have a certain therapeutic and alleviating effect on plaque demineralization of tooth enamel. It has a significant protective effect on the oral cavity and fills the gap in the market for anti-caries metagenin lozenges.

[0017] The above-mentioned Lactobacillus pentosus ( Lactiplantibacillus pentosus B-1-35, with accession number CGMCCNo.33870, was deposited on March 19, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0018] Figure 1 Co-culture of Candida albicans and lactic acid bacteria fermentation broth; Figure 2 The inhibition rate of lactic acid bacteria postbiotics on cariogenic bacteria biofilms; where A is the inhibition rate of lactic acid bacteria postbiotics on Streptococcus mutans biofilms, and B is the inhibition rate of lactic acid bacteria postbiotics on Candida albicans biofilms. Figure 3 Results of strain identification; Figure 4 This is a postbiotic lozenge product containing Lactobacillus pentosus B-1-35. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0020] Specific Implementation Example 1: Screening of probiotics with anti-caries effects.

[0021] 1. Antibacterial test Preparation of test sample solution: Take the lactic acid bacteria stored in glycerol at -80℃ and thaw them in a water bath. Add them to MRS broth at a 3% v / v inoculation rate and activate them for 6 hours to the mid-log phase (OD=0.6-0.8). Take the activated bacterial solution and inoculate it into MRS broth at a 3% v / v inoculation rate. Incubate at 37℃ for 24 hours (OD=1.6-1.8) to obtain the lactic acid bacteria fermentation broth.

[0022] Two indicator bacteria, Streptococcus mutans strain BNCC 336931 and Candida albicans strain ATCC 10231, purchased from the bacterial culture center, were revived, activated, and passaged to phenotypic stability using BHI and YPD broths, respectively. The logarithmic-phase bacterial cultures were then stored in glycerol at -80°C for subsequent experiments.

[0023] Four sample solutions were obtained by different treatments of the lactic acid bacteria fermentation broth. The specific operations are as follows: 1) Heat-killed and ultrasonically-treated (HK+US) group: The lactic acid bacteria fermentation broth was inactivated by water bath at 90 ℃ for 10 min, and the cells were disrupted using an ultrasonic cell disruptor. The working parameters of the ultrasonic cell disruptor were: frequency 20-25KHz, ultrasonic on for 3 s, ultrasonic off for 3 s, ultrasonic power 40%, and working time 10 min. 1) Centrifuge the disrupted bacterial culture at 3000 rpm for 10 min, and filter the supernatant through a 0.22 μm PES membrane for sterilization; 2) Gentle heat-killed (GHK 70℃) group: Inactivate the lactic acid bacteria fermentation broth in a water bath at 70 ℃ for 1 h, centrifuge the bacterial culture at 3000 rpm for 10 min, and filter the supernatant through a 0.22 μm PES membrane for sterilization; 3) Cell-free supernatant (CFS) group: Centrifuge the lactic acid bacteria fermentation broth at 10000 rpm for 15 min, collect the supernatant, and filter the supernatant through a membrane for sterilization; 4) Fermentation broth group (FER): Vortex the cultured fermentation broth thoroughly, take a certain amount and store it at 4 ℃ for later use. Store the above (1-3) group samples at -20 ℃.

[0024] Oxford Cup Antibacterial Ring Oxford Cup Method: The *Streptococcus mutans* inhibition assay uses the Oxford cup-double-layer agar diffusion method. Prepare 10 mL of BHI agar for the bottom layer of the plate beforehand, dry it under UV light, and refrigerate it. Prepare 50% BHI soft agar, sterilize it, and allow it to cool to approximately 50-55 °C. Inoculate with *Streptococcus mutans* indicator culture and gently shake to mix (final concentration 10). 6 Pour the prepared lower agar (CFU / mL) into the agar. After the upper agar solidifies, place an Oxford cup and gently press it to ensure there are no gaps between the cup and the agar. Add 100 μL of each of the four sample solutions prepared above to the Oxford cup, pre-diffuse at 4 °C for 2-4 h, and incubate at 37 °C for 10 h. Observe and measure the diameter of the inhibition zone.

[0025] Based on the inhibition zone data in Table 1, three strains with good antibacterial effects were screened: B-1-35, G-2-36, and 51MY-3. The antibacterial effect of the CFS group was similar to that of the FER group, while the HK+US group and the GHK group were basically the same, slightly weaker than the first two groups.

[0026] Table 1. Inhibition zones of lactic acid bacteria strains

[0027] Note: Lowercase letters indicate differences between treatment groups within the same peer group, P < 0.05.

[0028] (2) Antibacterial test using the well plate method The activated Candida albicans culture was inoculated into YPD broth at 3% v / v and incubated overnight at 37 ℃ with shaking at 180 rpm. The OD of the culture medium was adjusted to achieve a bacterial concentration of 10. 7 CFU / ml. Equal volumes of 100 μL indicator bacterial suspension and three groups of lactic acid bacteria sample solutions (excluding the fermentation broth group) were added to each well of a 96-well plate. The control group was replaced with sterile MRS broth. The plates were incubated at 37 ℃ for 48 h. At 24 h and 48 h of incubation, the absorbance of each well was measured at 600 nm using a microplate reader, and the measurements were recorded. The inhibition rate was calculated using formula 1 as follows: Antibacterial rate (%) = (OD control - OD experiment) / OD control × 100 Formula 1 Based on the data in Table 2 regarding the inhibition rate of lactic acid bacteria metabiotics against Candida albicans using the 96-well method, three strains with good antibacterial effects were selected: B-1-35, G-2-36, and 51MY-3. Analysis of the data showed that the inhibition rate of Candida albicans decreased slightly with prolonged growth time. This is likely because Candida albicans formed a mature biofilm at the bottom and walls of the wells during the later stages of growth, reducing the effectiveness of the antibacterial substances. The CFS group showed significantly better antibacterial effects than other treatment groups at both 24 h and 48 h. The HK+US group was slightly better than the GHK inactivation group. This is likely because the heat treatment process destroyed some volatile and heat-sensitive substances in the lactic acid bacteria fermentation broth; the higher the heat inactivation temperature and the longer the time, the greater the damage to the active substances in the metabiotics.

[0029] Table 2 Growth inhibition rate using microplate method

[0030] Note: Uppercase letters indicate differences between strains in the same column, P < 0.01.

[0031] (3) Co-culture antibacterial experiment Mix 0.5 mL of lactic acid bacteria fermentation broth sample with 0.5 mL of Candida albicans culture (concentration 10). 7 The indicator bacterial culture (CFU / mL) was added to a 15 mL centrifuge tube containing 9 mL of YPD broth for co-culture. The control group consisted of 0.5 mL of indicator bacterial culture and 9.5 mL of YPD medium. The culture was carried out at 37 °C with shaking at 180 rpm for 48 h. Samples were taken at 24 h and 48 h of co-culture, serially diluted with sterile water, and the viable count of indicator bacteria at the two time points was calculated and recorded using the plate count method.

[0032] Depend on Figure 1 It was found that the fermentation broth of strain B-1-35 had a strong inhibitory effect on the growth of Candida albicans, reducing its activity by two orders of magnitude after 48 hours. The live lactic acid bacteria cells in the fermentation broth were able to interact with Candida albicans cells across species, significantly inhibiting its growth.

[0033] 2. Biomembrane inhibition ability Biofilm inhibition experiment: Equal volumes of 100 μL indicator bacterial solution (Streptococcus mutans concentration of 10) were used. 8 CFU / mL, Candida albicans was 10. 7The CFU / mL of the above-mentioned three groups of lactic acid bacteria test solutions (except for the fermentation broth group) were added to the wells of a 96-well plate. After incubation at 37°C for 24 h, the culture medium in the wells was discarded, and the plate was washed twice with PBS buffer, with continuous and vigorous shaking during washing to remove unattached bacteria. The PBS buffer was then discarded, and the indicator bacterial biofilm formed in the wells was fixed with 200 μL of methanol at room temperature for 10 min. After discarding the methanol, the plate was allowed to air dry at room temperature. The plate was then stained with 200 μL of 0.1% (w / v) crystal violet and shaken at 60 rpm at room temperature for 10 min. After staining, the plate was washed three times with deionized water to remove excess dye and allowed to air dry at room temperature. The crystal violet adhering to the biofilm was dissolved in 200 μL of 33% (v / v) glacial acetic acid, and the plate was then shaken on a shaker for 10 min to ensure complete dye release. The control group used sterile MRS broth instead of the lactic acid bacteria test solution, and the absorbance was measured at 590 nm using a microplate reader. The biofilm inhibition rate is calculated using the following formula 2: Biomembrane inhibition rate (%) = (OD control - OD experiment) / (OD control) × 100 Formula 2 Depend on Figure 2 China A Figure 2 As shown in Figure B, different strains of metagenic solutions exhibited varying degrees of inhibitory effects on biofilm formation in the two indicator bacteria. Specifically, the HK+US group of strain B-1-35 showed a biofilm inhibition rate of 45.7% against *Streptococcus mutans* and 65.9% against *Candida albicans*. Considering the combined biofilm inhibition rates against both indicator bacteria, the HK+US group demonstrated the best effect. Lactic acid produced by lactic acid bacteria has an inhibitory effect on *Candida*, reducing the formation of *Candida albicans* mycelium and early biofilm. Strain B-1-35 exhibits excellent fermentation performance, producing a large amount of lactic acid, and shows significant anti-biofilm effects against caries bacteria that have not adhered or formed biofilms.

[0034] Based on the antibacterial effect and the ability to inhibit biofilm formation, strain B-1-35, which had the strongest overall ability, was finally screened out and then identified as a strain.

[0035] 3. Strain identification 16S rDNA sequence alignment and identification: Using logarithmic-phase bacterial culture as a template, PCR amplification was performed using universal primers F (5'-AGAGTTTGATCCTGGCTCAG-3') and R (5'-GGTTACCTTGTTACGACTT-3'). The obtained PCR products were subjected to 2% agarose gel electrophoresis, and PCR products with a length of approximately 1500 bp were sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. The obtained spliced ​​sequences were aligned using NCBI, and a phylogenetic tree was constructed using MEGA 11.0 to analyze the phylogenetic relationships between strains. A phylogenetic tree was constructed using the neighbor-joining method, and the step-distance method was used for validation. The algorithm mode selected was p-distance, and *Lactobacillus plantarum* was used as the outermost strain.

[0036] Table 3 PCR reaction system

[0037] Note: PCR reaction conditions: 94 ℃ pre-denaturation for 10 min; 94 ℃ denaturation for 30 s, 50~60 ℃ annealing for 30 s, 72 ℃ extension for 1 min, 72 ℃ extension for 10 min, for a total of 30 cycles.

[0038] The 16S rDNA sequence of strain B-1-35 obtained from sequencing was compared with that in Genebank. The results showed that strain B-1-35 had the highest sequence similarity to several strains of *Lactiplantibacillus pentosus*. Figure 3 It can be seen that strain B-1-35 and CP115741.1:2563049-2564482 Lactiplantibacillus pentosus strain EP3 chromosome complete genome The bacteria clustered together, indicating a close phylogenetic relationship. Based on the morphological and physiological-biochemical characteristics of this strain, strain B-1-35 was ultimately identified as *Lactobacillus pentosaccharis*, and named *Lactobacillus pentosaccharis* (…). Lactiplantibacillus pentosus )B1-35.

[0039] In summary, the anti-biofilm efficacy of the metatrophic agents was initially screened using the Oxford cup method and co-culture method. It was found that all three metatrophic agent samples significantly inhibited the growth and biofilm formation of the two cariogenic bacteria. Considering both metatrophic agent performance and anti-cariogenic efficacy, strain B-1-35 was selected, and its 16S rDNA was sequenced to construct a phylogenetic tree. Based on the morphological and physiological-biochemical characteristics of this bacterium, it was finally identified and named *Lactobacillus pentosus* (…). Lactiplantibacillus pentosus B-1-35. Pentosacchariformis Lactobacillus B1-35 exhibits excellent fermentation performance and is a safe and controllable probiotic.

[0040] The above-mentioned Lactobacillus pentosus ( Lactiplantibacillus pentosusB-1-35, with accession number CGMCCNo.33870, was deposited on March 19, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] Specific Example 2: Application of a post-caries powder prepared using Lactobacillus pentosus B1-35 with anti-caries effects.

[0042] 1. Preparation of post-caries powder with anti-caries effect, including the following steps: The *Lactobacillus pentosus* B-1-35 glycerol-preserved bacterial suspension was activated at 3% v / v inoculum for 6 h to reach mid-logarithmic growth. The same inoculum was then statically cultured at 37 ℃ until OD ≥ 1.7 and the pH of the fermentation broth ≤ 3.70. The fermentation broth was inactivated in a 90 ℃ water bath for 10 min. After cooling, the broth was ultrasonically disrupted (ultrasonic cell disruptor operating parameters: frequency 20-25 kHz, ultrasonic on for 3 s, ultrasonic off for 3 s, ultrasonic power 40%, operating time 10 min). A two-step concentration method was used. First, the ultrasonically disrupted suspension was filtered through a 90% nanofiltration membrane desalination device to remove some water. 1.5% skim milk powder (heat protectant) was added to the nanofiltration suspension, and the mixture was vacuum centrifuged for 10 h at 50 ℃, 2000 rpm, and -0.1 MPa until the solids content was above 40%. The concentrated suspension was spray-dried (inlet air temperature 100 ℃, outlet air temperature 70 ℃, feed rate 500 mL / h) to obtain a brown powder. The post-biotic powder was further refined by a grinder and stored in an inverted glass bottle.

[0043] 2. Analysis of the components of post-natal vitamin powder (1) Determination of organic acids Experimental method: Accurately weigh an appropriate amount of post-biotic powder sample into a 2 mL EP tube, accurately add 500 μL of 30% methanol aqueous solution (containing 0.1% formic acid), vortex for 60 s, centrifuge at 12000 rpm for 10 min at 4 ℃, take 20 μL of supernatant, add 980 μL of 30% methanol aqueous solution (containing 0.1% formic acid), mix well, and then add to the test bottle. Chromatographic conditions: An ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters Corporation, USA) was used. The injection volume was 5 μL, the column temperature was 40℃, and the mobile phases were A-water (containing 0.1% formic acid) and B-methanol (containing 0.1% formic acid). The gradient elution conditions were: 0–6 min, 28% B; 6–9 min, 28–40% B; 9–10 min, 40–50% B; 10–11 min, 50% B; 11–13 min, 30% B; and the flow rate of mobile phase B was 0.25 mL / min.

[0044] (2) Determination of polypeptide content Accurately weigh 10 mg of the prepared post-biotic powder into a 10 mL volumetric flask, add a small amount of mobile phase, sonicate for 10 min to fully dissolve the sample, then dilute to the mark with mobile phase, filter through a 0.2 μm organic membrane, and determine according to GBT22492-20008.

[0045] (3) Determination of extracellular polysaccharide and lipoteichoic acid content The extracellular polysaccharide content of the post-biotic powder in this embodiment was determined using the anthrone-sulfuric acid method. Glucose was used as a standard curve, and the extracellular polysaccharide content was calculated based on the standard curve. Lipoteichoic acid was detected using a lipoteichoic acid ELISA kit (Jianglai Biotechnology).

[0046] (4) Results Analysis The organic acid content in the *Pediococcus pentosaceus* B-1-35 postbiotic powder is shown in Table 4. The total organic acid content in the postbiotic powder was 105 mg / g. The most abundant organic acids were: lactic acid (76352.132 μg / g), citric acid (18726.459 μg / g), pantothenic acid (4431.019 μg / g), succinic acid (2156.071 μg / g), malic acid (1621.088 μg / g), and phenyllactic acid (869.341 μg / g), consistent with the actual fermentation pH results of the strain. It also contained short-chain fatty acids such as acetic acid, propionic acid, hexanoic acid, and malonic acid. These organic acids and short-chain fatty acids are believed to have antibacterial and biofilm degradation effects on pathogenic bacteria. The polypeptide content in the *Pediococcus pentosaceus* SC13 postbiotic powder reached 3.2% (inoculum size 3%, fermentation time 24 h). The extracellular polysaccharide content in the postbiotic powder was determined to be 2165 mg / kg using the anthrone-sulfuric acid method, and the lipoteichoic acid content was determined to be 28.9 ng / kg using an ELISA kit, both of which were relatively abundant. Extracellular polysaccharides secreted by lactic acid bacteria possess anti-biofilm activity and probiotic properties that stimulate the growth of beneficial bacteria. Cell membrane components contained in lactic acid bacteria postbiotics, such as lipoteichoic acid, can interfere with the recognition between pathogens and host cells.

[0047] Table 4 Organic acid content in post-natal powder

[0048] Specific embodiment three: Application of the above-mentioned post-natal powder in preparing post-natal lozenges with anti-caries effects.

[0049] 1. Post-caries lozenges with anti-caries effects The post-biotic tablets are in lozenge form. The specific formula (based on a total of 100 parts) is: 10 parts resistant starch, 5 parts cyclodextrin, 5 parts xylitol, 10 parts fructooligosaccharides, 5 parts isomaltulose, 16 parts post-biotic powder, 44 parts microcrystalline cellulose, and 5 parts magnesium stearate. The direct compression method is used, where the above powders are thoroughly mixed in a granulator and then synthesized into lozenges using a fully automatic rotary tablet press.

[0050] 2. Lozenge index testing (1) Weight difference Take 20 tablets of the test sample, accurately weigh the total weight, and calculate the average tablet weight. Then accurately weigh each tablet separately. Compare the weight of each tablet with the average tablet weight (for tablets without content determination or for traditional Chinese medicine tablets with labeled tablet weight, the weight of each tablet should be compared with the labeled tablet weight). No more than 2 tablets should exceed the weight difference limit, and no tablet should exceed the limit by more than 1 time.

[0051] (2) Disintegration time limit The test shall be conducted in accordance with the Disintegration Time Limit Test Method (General Rule 0921) and shall comply with the requirements.

[0052] (3) Foaming amount Take 10 graduated test tubes with stoppers (inner diameter 1.5 cm; if the tablet diameter is large, the inner diameter can be changed to 2.0 cm), add 15 mL of water to each tube, place them in a water bath at 37 ± 1℃ for 5 minutes, and put one tablet of the test sample into each tube. Observe the volume of the maximum foaming within 20 minutes. The average foaming volume should not be less than 6 mL, and no more than 2 tablets should have a foaming volume of less than 4 mL.

[0053] (4) Dispersion uniformity According to the disintegration time test method (General Rule 0921), the inner diameter of the stainless steel wire mesh sieve is 710 μm, and the water temperature is 15-25 ℃. Take 6 test samples, and they should all disintegrate and pass through the sieve within 3 minutes. If a small number cannot pass through the sieve, but have softened into light and floated without a hard core, they meet the requirements.

[0054] (5) Microbial limits Microbial limit testing for non-sterile products: Microbial count method (General Rule 1105) and microbial limit standard for non-sterile drugs (General Rule 1107).

[0055] (6) Results Analysis like Figure 4As shown, the lozenges prepared by the direct compression method are light brown in color, round in shape, with a smooth surface and a certain gloss. Testing revealed that the weight difference of the lozenges, calculated based on a labeled weight of 300 mg, did not exceed the variation limit. Regarding disintegration time, all tested lozenges disintegrated completely within 30 minutes, meeting the disintegration performance requirements. In terms of foaming volume, the average foaming volume of the tested lozenges within 20 minutes was 6.5 ± 0.2 mL, with no lozenges less than 4 mL. Regarding dispersion uniformity, most of the tested lozenges disintegrated completely and passed through the sieve within 3 minutes. A small number did not pass through the sieve, but these softened into light, floating particles without a hard core, meeting the requirements. Using the plate count method, the results of the total aerobic bacteria count, mold count, and yeast count of the tested lozenges all met the requirements for this product. This lozenge is for oral administration and contains microbial preparations; Escherichia coli and Salmonella were not detected in the microbial limits. After testing for the aforementioned relevant indicators, the post-natal lozenges meet the requirements of the Pharmacopoeia General Rules for Tablets and can be used in actual production.

[0056] 3. Evaluation of the efficacy of the lozenge in vitro model (1) Establish an in vitro static mixed biofilm model Human saliva was collected using a non-stimulatory method, and the supernatant was obtained by centrifugation. Hydroxyapatite discs were soaked in the supernatant for a certain period to ensure they were fully coated with saliva components. The discs were then removed and placed vertically against the well walls of a 6-well plate. 2 mL of an equal volume of 10... 7 CFU / mL Streptococcus mutans and 10 6 The discs were submerged in a CFU / mL Candida albicans suspension. An appropriate amount of post-biotic tablets was added to a concentration of 5% (w / v) to allow complete disintegration and release of active substances. Broth without indicator bacteria served as a blank control, the absence of post-biotic tablets served as a double bacterial control, and the addition of excipients without post-biotic components served as another treatment group. All groups were incubated at 37 ℃ for 24 h, with three replicates per group. After incubation, the culture medium was collected, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. pH and calcium ion concentration were measured. Calcium ion concentration was determined using inductively coupled plasma mass spectrometry (ICP-MS) to reflect the amount of decalcification in the hydroxyapatite model of salivary tablets. The decalcification rate of the discs was calculated using the following formula 3, where the value represents the calcium ion concentration: Decalcification rate of discs (%) = (Treatment - Blank) / (Double control - Blank) × 100 Formula 3 (2) Results Analysis The presence of dental plaque biofilm matrix prevents the diffusion and dilution of cariogenic bacteria metabolites within the film. Acid-producing bacteria lower the local pH to a critical value, leading to gradual demineralization of the enamel surface. This process is accompanied by the loss of calcium ions from hydroxyapatite, the main component of enamel. Table 5 shows that the pH of the dual-bacterial culture medium treated with the post-biotic lozenges significantly increased, reducing the amount of demineralization of the hydroxyapatite discs by the mixed biofilm. The pH and calcium ion concentration of the dual-bacterial culture medium treated with the post-biotic excipients showed no significant difference compared to the dual-bacterial control group, indicating that the post-biotic components in the lozenges played a major role. The results indicate that the *Lactobacillus pentosus* B-1-35 post-biotic lozenges have a certain therapeutic and alleviating effect on plaque-induced demineralization of tooth enamel.

[0057] Table 5 Effects of post-natal vitamin tablets on the model

[0058] Note: Lowercase letters indicate differences between different groups within the same row, p<0.05.

[0059] 3. Toxicological experiments of lozenges An acute oral toxicology test was conducted, and the specific experimental protocol is as follows: (1) Grouping Forty healthy 8-week-old SPF mice, each weighing approximately 20 g, were divided into a post-biotic tablet group and a blank control group, with 20 mice in each group (10 males and 10 females).

[0060] (2) Administration All experimental mice were fasted for 4-6 hours before administration, but had free access to water. The Hou Sheng Yuan tablets were fully dissolved in water, weighed, and then administered orally via gavage using a syringe on an empty stomach. The gavage volume was 40 mL / kg body weight. Following the dose-limiting method, the Hou Sheng Yuan tablet suspension was administered as a single dose of 100 mg / kg body weight. The control group received physiological saline via gavage. After gavage, mice were allowed free access to food and water.

[0061] (3) Observation indicators Animal weights were weighed and recorded at the beginning of the experiment and at the end of the 7-day experiment. Daily diet, behavior, and mortality were also recorded for both groups of mice. After the gavage experiment was completed, mice were euthanized by cervical dislocation and dissected. The thymus and spleen were removed, weighed, and organ coefficients were determined.

[0062] (4) Results Analysis As shown in Table 6, within 7 days after a single oral administration of the Houshengyuan lozenge suspension, the mice exhibited normal activity and good growth and development, with no abnormal signs or deaths observed. There were no significant differences in body weight and organ indices between the lozenge group and the control group, suggesting that oral administration of the Houshengyuan lozenge has no acute toxicity in animals and demonstrates good safety.

[0063] Table 6 Observation Indicators

[0064] Note: p A value less than 0.05 indicates a significant difference from the control group, while ns indicates no significant difference from the control group.

[0065] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A probiotic with anti-caries effect, characterized in that: The probiotic mentioned is *Lactobacillus pentosus* (…). Lactiplantibacillus pentosus The strain B-1-35, with accession number CGMCC No. 33870, is used.

2. A postnatal vitamin powder with anti-caries effect, characterized in that: The metabiotic mentioned is derived from Lactobacillus pentosus ( Lactiplantibacillus pentosus B-1-35, wherein the pentosacchari lactobacillus ( Lactiplantibacillus pentosus The preservation number of strain B-1-35 is CGMCC No. 33870.

3. A method for preparing the post-caries powder with anti-caries effect as described in claim 2, characterized in that... The process includes the following steps: *Lactobacillus pentosus* B-1-35 is added to the fermentation medium at a volume ratio of 2-4% and activated to mid-log phase. The activated bacterial solution is then inoculated into the fermentation medium at a volume ratio of 2-4% and cultured at 35-40 ℃ until OD ≥ 1.7 and pH ≤ 3.70, yielding the fermentation broth. After inactivation and cooling of the fermentation broth via water bath, it is ultrasonically disrupted and centrifuged to obtain the supernatant. The supernatant is filtered through a nanofiltration membrane to remove some water. 1-2% (by weight) of skim milk powder is added to the nanofiltration suspension, and then the mixture is concentrated to obtain a post-caries powder with anti-caries effects.

4. The method for preparing a post-caries powder with anti-caries effect according to claim 3, characterized in that: The ultrasonic cell disruptor used for ultrasonic disruption has the following operating parameters: frequency 20-25 kHz, ultrasonic on for 3 seconds, ultrasonic off for 3 seconds, ultrasonic power 40%, and working time 10 minutes.

5. The method for preparing the post-caries powder with anti-caries effect according to claim 3, characterized in that: The fermentation medium is prepared by dissolving MRS solid medium in 1L of distilled water and sterilizing at 121℃ for 15 min. The formula of the MRS solid medium is 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 80ml Tween, 0.5g magnesium sulfate, 0.25g manganese sulfate and 15g agar powder.

6. The method for preparing a post-caries powder with anti-caries effect according to claim 3, characterized in that: The two-step concentration method is as follows: the suspension obtained by ultrasonic crushing is filtered with a nanofiltration membrane to remove some water, and 1-2% of the mass of the nanofiltration suspension is added to the suspension. The suspension is then concentrated by vacuum centrifugation until the solid content is above 40%. The concentrated suspension is then spray-dried to obtain a post-caries powder with anti-caries effect.

7. The method for preparing a post-caries powder with anti-caries effect according to claim 6, characterized in that: The vacuum centrifugation conditions are as follows: temperature 50℃, rotation speed 2000 rpm, vacuum pressure -0.1 MPa. The spray drying conditions are as follows: inlet air temperature 100℃, outlet air temperature 70℃, and feed rate 500 mL / h.

8. A postnatal powder prepared using the method described in any one of claims 3-7, used in the preparation of postnatal lozenges with anti-caries effects, characterized in that: The specific formula of the post-biotic tablets is as follows: 8-12 parts resistant starch, 4-6 parts cyclodextrin, 4-6 parts xylitol, 8-12 parts fructooligosaccharides, 4-6 parts isomaltulose, 14-18 parts post-biotic powder, 45-51 parts microcrystalline cellulose, and 0.5-2 parts magnesium stearate.

9. The application of the probiotic with anti-caries effect as described in claim 1 in the preparation of a product that improves oral microecology.

10. The use of the probiotic with anti-caries effect as described in claim 1 in the preparation of inhibitors of Streptococcus mutans and / or Candida albicans.

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