Application of soybean saponin Bb in the preparation of anti-caries products

Abstract

This invention relates to the application of soybean saponin Bb in the preparation of anti-caries products, belonging to the field of biotechnology. This invention provides the application of soybean saponin Bb in the preparation of anti-caries products. In vitro experiments revealed that soybean saponin Bb inhibits the formation of cariogenic bacterial biofilms and acid production, reduces the degree of enamel demineralization, regulates the structure and metabolic function of dental plaque microbial communities, thereby improving the oral microecology. Simultaneously, soybean saponin Bb exhibits good biocompatibility and anti-caries potential in animals, suggesting that soybean saponin Bb has the potential to be developed into a novel natural anti-caries product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of soybean saponin Bb in the preparation of anti-caries products. Background Technology

[0002] Legumes are rich in bioactive compounds and are a low-cost, nutrient-rich food source for human diets and animal feed. Plant proteins, represented by legume protein, have advantages over animal proteins in terms of environmental sustainability, production cost, nutritional and health benefits, and ethical considerations, showing better application prospects. Soybeans are one of my country's major food crops, with advantages such as high yield, affordable price, and well-developed extraction processes for health-related components, attracting much attention from researchers both domestically and internationally. Previous studies have confirmed that soybean-derived compounds, including isoflavones, saponins, flavonoids, peptides, proteins, lipids, vitamins, and minerals, all have immunomodulatory effects. Among them, soyasaponins (Ss) are an important secondary metabolite of soybeans with various biological functions, including antibacterial, antifungal, antiviral, antioxidant, and immunomodulatory effects. Ss can inhibit various pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and exert disease prevention and control effects by regulating the host microbiome. However, the preventive and therapeutic effects of soyasaponins on the dental caries microecology and their mechanisms are not clear. Therefore, it is urgent to explore the potential of soyasaponins in preventing and treating dental caries and to provide new directions for the development of natural anti-caries drugs. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of soybean saponin Bb in the preparation of anti-caries products.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides the application of soybean saponin Bb in the preparation of anti-caries products.

[0006] Secondly, this invention provides the application of soybean saponin Bb in the preparation of anti-cariogenic bacteria products.

[0007] This invention, through in vitro experiments, found that soybean saponin Bb can inhibit the formation of cariogenic bacterial biofilms and acid production, reduce the degree of enamel demineralization, regulate the structure and metabolic function of dental plaque microbial communities, and thus improve the oral microecology. At the same time, soybean saponin Bb has good biocompatibility and anti-caries potential in animals, suggesting that soybean saponin Bb has the potential to be developed into a new type of natural anti-caries product.

[0008] As a preferred embodiment of the application described in this invention, the cariogenic bacteria include, but are not limited to, at least one of Streptococcus mutans, Lactobacillus, and Actinomycetes.

[0009] In a preferred embodiment of the application described in this invention, the lactobacillus includes Lactobacillus casei.

[0010] As a preferred embodiment of the application described in this invention, the anti-caries products and / or anti-cariogenic bacteria products include, but are not limited to, at least one of pharmaceuticals and daily chemical products.

[0011] As a preferred embodiment of the application described in this invention, the anti-caries products and / or anti-cariogenic bacteria products include, but are not limited to, oral care products. This invention has experimentally demonstrated that soybean saponin Bb has excellent anti-caries efficacy and can be added to oral care products such as mouthwashes, sustained-release gels, and toothpaste to exert its anti-caries effect.

[0012] As a preferred embodiment of the application described in this invention, the dosage form of the anti-caries product and / or anti-cariogenic bacteria product includes, but is not limited to, at least one of solid dosage form, liquid dosage form, semi-solid dosage form and gaseous dosage form.

[0013] In a preferred embodiment of the application described in this invention, when the anti-caries product and / or anti-cariogenic bacteria product is in liquid form, the content of soybean saponin Bb in the anti-caries product and / or anti-cariogenic bacteria product is 100-200 mg / L. When the concentration of soybean saponin Bb is 100 mg / L, it not only effectively prevents caries but also has good biocompatibility.

[0014] As a preferred embodiment of the application described in this invention, the anti-caries product and / or anti-cariogenic bacteria product further contains excipients.

[0015] As a preferred embodiment of the application described in this invention, the excipients include, but are not limited to, at least one of fillers, binders, wetting agents, disintegrants, lubricants, flow aids, preservatives, flavoring agents, colorants, solvents, and cosolvents.

[0016] As a preferred embodiment of the application described in this invention, the filler includes, but is not limited to, at least one of starch, dextrin, glucose, mannitol, hydroxybenzoic acid cellulose, and microcrystalline cellulose.

[0017] As a preferred embodiment of the application described in this invention, the adhesive and wetting agent include, but are not limited to, at least one of water, gelatin, starch paste, polyethylene glycol, and ethanol.

[0018] As a preferred embodiment of the application described in this invention, the disintegrant includes, but is not limited to, at least one of crospovidone, sodium carboxymethyl starch, and crospovidone carboxymethyl cellulose.

[0019] As a preferred embodiment of the application described in this invention, the lubricant and retention aid include, but are not limited to, at least one of gum arabic, polyethylene glycol, magnesium stearate, micronized silica gel, and talc.

[0020] As a preferred embodiment of the application described in this invention, the preservative includes, but is not limited to, at least one of domiphen, benzoic acid, sodium benzoate, propylene glycol, ethyl acetate, and sorbic acid.

[0021] In a preferred embodiment of the application described in this invention, the flavoring agent and coloring agent include, but are not limited to, at least one of sucrose, sorbitol, mannitol, orange peel oil, carmine, and malic acid.

[0022] As a preferred embodiment of the application described in this invention, the solvent and co-solvent include, but are not limited to, at least one of water, ethanol, glycerol and polysorbate 80.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention, through in vitro experiments, found that soybean saponin Bb can inhibit the formation of cariogenic bacterial biofilms and acid production, reduce the degree of enamel demineralization, regulate the structure and metabolic function of dental plaque microbial communities, and thus improve the oral microecology. At the same time, soybean saponin Bb has good biocompatibility and anti-caries potential in animals, suggesting that soybean saponin Bb has the potential to be developed into a new type of natural anti-caries product. Attached Figure Description

[0025] Figure 1 This figure illustrates the effect of different concentrations of Ss-Bb on the formation of Sm-Lc dual-species biofilm in Example 1 of this invention. Figure A shows the biofilm morphology after crystal violet staining, and Figure B shows the statistical results of biofilm formation in each group. Figures marked with " "" indicates that there is a statistically significant difference between this group and the PBS group. p <0.0001);

[0026] Figure 2 This figure illustrates the effect of different concentrations of Ss-Bb on 24-hour mature Sm-Lc dual-species biofilm in Example 1 of this invention. Figure A shows the biofilm morphology after crystal violet staining, and Figure B shows the statistical results of biofilm formation in each group. Figures marked with " "" indicates that there is a statistically significant difference between this group and the PBS group. p <0.0001);

[0027] Figure 3 This figure shows the demineralization depth and mineral loss of enamel blocks after different treatments in Example 1 of the present invention. A represents microscopic CT images of enamel blocks from different treatment groups, B represents the statistical results of enamel demineralization depth from different treatment groups, and C represents the statistical results of enamel mineral loss from different treatment groups. The figures are marked with "". "" indicates that there is a statistically significant difference between this group and the Sm-Lc group. p <0.05), " "" indicates that there is a statistically significant difference between this group and the Sm-Lc group. p <0.0001);

[0028] Figure 4 The figures show the cell viability of human oral epithelial cells (HOECs) treated with different concentrations of Ss-Bb in Example 2 of this invention. A represents the cell viability measurement results after 1 day of co-culture, B represents the cell viability measurement results after 3 days of co-culture, C represents the cell viability measurement results after 5 days of co-culture, and D represents the cell viability measurement results after 7 days of co-culture. The figures are marked with "". "Indicates a statistically significant difference compared to the Control group" p <0.05), " "Indicates a statistically significant difference compared to the Control group" p <0.01), " "Indicates a statistically significant difference compared to the Control group" p <0.001), " "Indicates a statistically significant difference compared to the Control group" p <0.0001);

[0029] Figure 5 This invention illustrates the effect of Ss-Bb on plaque biofilm formation and acid production in caries-active and caries-free individuals in Example 3. Figure A shows the biofilm formation amount, and Figure B shows the acid production concentration. The figure is marked with "...". "" indicates a statistically significant difference between the two groups. p <0.05), " "" indicates a statistically significant difference between the two groups. p <0.01), " "" indicates a statistically significant difference between the two groups. p <0.0001);

[0030] Figure 6 This is a diagram showing the relative abundance distribution of microorganisms in different treatment groups in Example 3 of the present invention. Others in the diagram represent species whose relative abundance ranks after 20th.

[0031] Figure 7 This is a KEGG functional abundance clustering heatmap for different treatment groups in Embodiment 3 of the present invention, where A represents level 1 functions, B represents level 2 functions, and C represents level 3 functions.

[0032] Figure 8This document presents micro-CT images of rat molars from different treatment groups in Example 4 of this invention, along with analyses of their enamel volume and density. A represents representative micro-CT images of rat molars from each group (yellow arrows indicate caries lesions), B shows the statistical results of enamel volume in rat molars from each group, and C shows the statistical results of enamel density in rat molars from each group. Images marked with "..." are included. "" indicates a statistically significant difference between the two groups. p <0.05), " "" indicates a statistically significant difference between the two groups. p <0.01), " "" indicates a statistically significant difference between the two groups. p <0.001), " "" indicates a statistically significant difference between the two groups. p <0.0001);

[0033] Figure 9 HE staining results of the heart, liver, spleen, lungs, and kidneys of rats in the Ss-Bb group and Con group in Example 5 of this invention. Detailed Implementation

[0034] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0035] Unless otherwise specified, all other materials and reagents used in the examples, comparative examples, and effect examples are commercially available.

[0036] The soybean saponin Bb described in this invention can be obtained from various commercial channels. In the following examples, soybean saponin Bb was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a purity of 98%, catalog number S873374, and CAS number 51330-27-9. Soybean saponin Bb was mixed with anhydrous ethanol to prepare a 5 mg / mL stock solution for later use.

[0037] Streptococcus mutans ( Streptococcus mutans Sm) was purchased from the American Type Culture Collection (ATCC), catalog number ATCC 25175; Lactobacillus casei ( Lactobacillus casei The Lc was purchased from the American Type Culture Collection (ATCC), catalog number ATCC 393.

[0038] Sm and Lc strains were stored in BHI liquid medium containing 30 v / v% glycerol at -80°C. Before use, the frozen Sm and Lc strains were thawed at room temperature. Using a disposable sterile inoculation loop, one loopful of bacterial culture was streaked onto BHI solid medium (Sm) and MRS solid medium (Lc), respectively. After incubation at 37°C and 6% oxygen for 24 h, single colonies of Sm and Lc were picked and inoculated into 5 mL of BHI liquid medium. The culture was then incubated at 37°C and 6% oxygen for 24 h, and the bacterial concentration was adjusted to 5 × 10⁻⁶. 5 CFU / mL was mixed in equal amounts to prepare Sm-Lc mixed bacterial culture.

[0039] BHI liquid medium: Dissolve 37 g of BHI medium powder in 1 L of pure water, autoclave, and store at 4°C.

[0040] BHI solid medium: Dissolve 37 g of BHI medium powder and 15 g / L agar powder in 1 L of pure water. After complete dissolution, autoclave the solution to prepare plates.

[0041] Neutral BHIS: Dissolve 37 g of BHI medium powder in 1 L of pure water, autoclave, add sterile sucrose solution to prepare BHI liquid medium containing 0.2 wt% sucrose, and store at 4°C.

[0042] Acidic BHIS: Dissolve 37 g of BHI medium powder in 1 L of pure water, add glacial acetic acid to adjust the pH to 5.5, autoclave, add sterile sucrose solution to prepare BHI liquid medium containing 0.2 wt% sucrose, and store at 4°C.

[0043] BHI culture medium powder was purchased from Guangdong Huankai Microbial Technology Co., Ltd., product number 028360.

[0044] MRS liquid medium: Dissolve MRS medium powder in pure water according to the instructions, autoclave, and store at 4°C. For MRS solid medium, add 15 g / L agar powder before autoclaving, dissolve completely, autoclave, and prepare plates.

[0045] MRS culture medium powder was purchased from Guangdong Huankai Microbial Technology Co., Ltd., product number 027312.

[0046] The CCK-8 test kit was purchased from GLPBIO, catalog number GK10001.

[0047] Metagenomic sequencing of bacterial community structure and functional metabolism in plaque biofilms was commissioned to Beijing Novogene Technology Co., Ltd.

[0048] Example 1

[0049] To investigate the anti-cariogenic efficacy of soy saponin Bb (Ss-Bb), soy saponin Bb was used to treat cariogenic bacteria Sm and Lc. The specific method is as follows:

[0050] 1. Effect of Ss-Bb on biofilm formation in Sm-Lc mixed bacterial culture.

[0051] Take a 96-well plate and add 100 µL of Sm-Lc mixed bacterial solution and 100 µL of Ss-Bb solution (500 mg / L, 400 mg / L, 300 mg / L, 200 mg / L, 150 mg / L, 100 mg / L, 50 mg / L) to each well. The final concentrations of Ss-Bb solution are 250 mg / L, 200 mg / L, 150 mg / L, 100 mg / L, 75 mg / L, 50 mg / L, and 25 mg / L, respectively. The final concentration of Sm-Lc mixed bacterial solution is 5 × 10⁻⁶. 5 CFU / ml. The positive control group received an equal volume of 100 µL of 1wt% chlorhexidine solution and 100 µL of Sm-Lc mixed bacterial culture. The negative control group received 100 µL of PBS and 100 µL of Sm-Lc mixed bacterial culture. The blank control group received 200 µL of neutral BHIS containing neither Ss-Bb nor Sm-Lc bacterial culture. Each treatment was performed in triplicate, with identical volumes for each treatment and each parallel well.

[0052] The cells were cultured at 37℃ and 6% oxygen for 24 h. The culture medium was removed, and the cells were washed three times with PBS. The cells were fixed with methanol for 15 min, dried for 15 min after methanol removal, and stained with 0.1 wt% crystal violet solution for 5 min. The crystal violet solution was removed, and the cells were washed twice with PBS. 200 μL of sodium deoxycholate was added to each well and mixed thoroughly. The optical density was measured at 600 nm to assess the biofilm mass in each group. The minimum biofilm inhibition concentration (MBIC) and the drug concentration at which 50% biofilm inhibition was achieved were calculated. 50 ), the results are shown Figure 1 .

[0053] Meanwhile, following the above grouping, the Sm-Lc mixed bacterial culture was first mixed with neutral BHI liquid medium and cultured for 24 h. Then, Ss-Bb stock solution was added according to the final Ss-Bb concentration. The minimum biofilm reduction concentration (MBRC) was determined by crystal violet staining. The results are shown in […]. Figure 2 .

[0054] MBIC is defined as the lowest concentration among (OD treatment group - OD blank group) / (OD negative control - OD blank group) × 100% ≤ 10%.

[0055] MBRC means the lowest concentration of (OD treatment group - OD blank group) / (OD negative control - OD blank group) × 100% ≤ 10%.

[0056] like Figure 1-2 As shown, the biomass of Ss-Bb and Sm-Lc biofilms co-cultured at concentrations ≥75 mg / L for 24 h was significantly lower than that of the negative control group. p <0.0001, Figure 1 (A and 1B), their MBIC is 100 mg / L, MBIC 50 The concentration was 75 mg / L; after treatment with Ss-Bb at a concentration ≥100 mg / L for 24 h, the biomass of the Sm-Lc dual-strain biofilm was significantly lower than that of the negative control group. p <0.0001, Figure 2 (A and 2B), with an MBRC of 200 mg / L. These results indicate that Ss-Bb can effectively inhibit the formation of biofilms by cariogenic bacteria at concentrations ≥75 mg / L, thereby achieving a caries-preventing effect.

[0057] 2. Effect of Ss-Bb on Sm-Lc biofilm-induced enamel demineralization.

[0058] Bovine incisors without fractures, cracks, or caries, and with fully developed roots, were selected under a stereomicroscope (×20). 4 mm × 4 mm × 3 mm tooth blocks were prepared from the buccal surface of the teeth using a diamond saw blade under running water. The surfaces were then smoothed and polished using 400–2000 grit silicon carbide sandpaper. The blocks were ultrasonically cleaned at 40 Hz for 10 min to remove debris and smears, and then allowed to air dry. All surfaces of the enamel blocks (except the experimental surfaces) were coated with a double layer of acid-resistant nail polish, and sterilized to obtain the enamel blocks.

[0059] The experiment was divided into four groups: an Ss-Bb treatment group (containing 200 mg / L, 100 mg / L, and 75 mg / L Ss-Bb solution, respectively), a positive control (0.5 wt% CHX), a caries model group (containing Sm-Lc mixed bacterial solution without Ss-Bb solution), and a blank control group (containing BHIS without Ss-Bb solution and bacterial solution). The experiment consisted of two parts:

[0060] Part A: In a 24-well sterile culture plate, add Sm-Lc mixed bacterial solution, Ss-Bb drug solution, CHX solution, etc., according to the above settings, with a total volume of 2 mL per well. After anaerobic culture at 37℃ for 24 h, remove the culture supernatant, wash twice with sterile PBS, and add 2 mL of neutral BHIS to each well for 8 h. Remove the culture supernatant, wash twice with sterile PBS, and add 2 mL of acidic BHIS to each well for 16 h. Remove the culture supernatant, wash twice with sterile PBS, and continue to culture the biofilm under pH cycling conditions (8 h neutral, 16 h acidic) for 14 days.

[0061] Part B: The effect of Ss-Bb on the demineralization of dental blocks after acting on mature Sm-Lc dual-strain biofilm. In 24-well sterile culture plates, 2 mL of Sm-Lc mixed bacterial solution was added to the soybean saponin treatment group and the positive control group, while 2 mL of neutral BHIS without drug solution or bacterial solution was added to the blank control group. After anaerobic incubation at 37℃ for 24 h, the culture supernatant was removed, and the plates were washed twice with sterile PBS. 2 mL of neutral BHIS containing Ss-Bb solution was added to each well of the soybean saponin treatment group, 2 mL of neutral BHIS containing 0.5 wt% CHX was added to each well of the positive control group, and 2 mL of neutral BHIS without drug solution or bacterial solution was added to each well of the blank control group. The plates were incubated for 24 h. Aspirate the culture supernatant, wash twice with sterile PBS, and add 2 mL of neutral BHIS for 8 h each time; aspirate the culture supernatant, wash twice with sterile PBS, and add 2 mL of acidic BHIS for 16 h each time; continue to culture the biofilm under pH cycling conditions (8 h neutral, 16 h acidic) for 14 days.

[0062] The pH cycling conditions described above are: every 24 hours, add neutral BHIS for 8 hours of culture and add acidic BHIS for 16 hours of culture to simulate the oral environment. No additional reagents are required at this time.

[0063] Micro-CT analysis was used to analyze the demineralization depth and mineral loss of bovine tooth enamel fragments. The results are shown in [Figure 1]. Figure 3 .

[0064] like Figure 3 As shown, 200 mg / L and 100 mg / L Ss-Bb, whether co-cultured with Sm-Lc bacterial solution or applied to existing Sm-Lc biofilms, significantly reduced the demineralization depth and mineral loss of enamel fragments. p <0.0001), while 75 mg / L Ss-Bb only reduced the demineralization depth of tooth enamel by the biofilm when co-cultured with bacterial solution ( p <0.05). The above results indicate that Ss-Bb at concentrations above 100 mg / L can reduce the risk of enamel demineralization caused by cariogenic bacterial biofilms, thereby playing a role in preventing tooth decay.

[0065] In summary, Ss-Bb achieves its caries-preventing effect by inhibiting the formation of cariogenic bacterial biofilms and reducing enamel demineralization damage caused by these biofilms.

[0066] Example 2

[0067] To investigate the safety of soy saponin Bb (Ss-Bb), an in vitro safety test was conducted. The specific protocol is as follows:

[0068] Ss-Bb, with MBIC, MBIC50, and MBRC concentrations as measured in Example 1, was co-cultured with human oral epithelial cells (HOEC) in 96-well cell plates. Cell viability was assessed using the CCK-8 assay after 1, 3, 5, and 7 days of culture. The CCK-8 assay was performed according to the kit instructions. The results are shown in [Figure 1]. Figure 4 .

[0069] like Figure 4 As shown, treatment with 100 mg / L and 75 mg / L Ss-Bb for 1, 3, 5, and 7 days, and treatment with 200 mg / L Ss-Bb for 1 day, had no significant effect on the cell viability of HOECs. p >0.05); while treatment with 200 mg / L Ss-Bb for 3, 5, and 7 days had an inhibitory effect on HOEC cells ( p (<0.05), therefore it can be determined that a concentration of 100 mg / L of Ss-Bb is the safest for human cells.

[0070] Example 3

[0071] To investigate the effect of Ss-Bb on dental plaque, dental plaque samples were collected from human oral cavity and co-cultured with Ss-Bb. Analysis was then performed using metagenomic sequencing. The specific protocol is as follows:

[0072] Eighteen volunteers aged 18 to 55 were recruited and divided into an active caries group (Group C) and a healthy group (Group H).

[0073] The selection criteria for the caries-active group (Group C) are as follows:

[0074] 1) Decayed-missing-filled tooth (DMFT) ≥ 6, with at least 3 teeth having caries;

[0075] 2) No other oral or systemic diseases, no smoking, and not currently using orthodontic appliances;

[0076] 3) Has not taken antibiotics or hormones in the past 3 months;

[0077] 4) No history of oral and maxillofacial tumors or radiotherapy / chemotherapy.

[0078] The selection criteria for the healthy group (Group H) are as follows:

[0079] 1) DMFT=0;

[0080] 2) No antibiotics or hormones have been used in the past 3 months;

[0081] 3) No oral-related diseases, such as periodontal disease, oral mucosal disease, oral and maxillofacial tumors, or history of radiotherapy and chemotherapy;

[0082] 4) No systemic diseases.

[0083] All volunteers signed informed consent forms, and the study was reviewed and approved by the ethics committee. Clinical dental plaque samples were collected from the participants' mouths, resuspended in PBS, and diluted 1:1 by volume with 60 v / v glycerol solution for subsequent in vitro studies.

[0084] Based on the aforementioned results, 100 mg / L of Ss-Bb can inhibit the formation of Sm-Lc dual-species biofilm, reduce mature Sm-Lc dual-species biofilm, and decrease its demineralization effect on tooth enamel. At the same time, this concentration of Ss-Bb has no obvious cytotoxicity. Therefore, it can be used as a working concentration for clinical plaque detection and rat molar caries model.

[0085] Two groups of clinical dental plaque samples were mixed separately and diluted with neutral BHIS (plaque:BHIS = 1:20). 100 mg / L Ss-Bb solution or neutral BHIS (negative control) was mixed with the plaque dilution and incubated for 48 h. Crystal violet staining and lactic acid production were then performed. Results are shown below. Figure 5 .

[0086] The above experiments were performed simultaneously on two plates. Sterile swabs were used to collect biofilm from the second plate. Metagenomic sequencing was employed to detect changes in bacterial community structure and metabolic function. The relative abundance of species at the species level and KEGG functional annotation were analyzed. Results are shown in […]. Figure 6-7 See Table 1.

[0087] Table 1. Facts with significant changes in relative abundance.

[0088]

[0089] like Figure 5 As shown, 100 mg / L Ss-Bb has an effect on the formation of plaque biofilm in the caries-active group (Group C). p <0.05) and acid production ( p All samples <0.001 showed significant inhibitory effects, but had no significant inhibitory effect on the formation of plaque biofilm in the caries-free group (Group H). p>0.05), but can significantly reduce acid production ( p <0.01).

[0090] like Figure 6 As shown in Table 1, the presence of *Virginia valerate* (Virginia valerate) in dental plaque of the caries-active group (C+Bb group) after the addition of Ss-Bb... Veillonella parvula Atypical Veillonella ( Veillonella atypica ) and Veillonella severae ( Veillonella dispar The relative abundance of caries-causing bacteria such as Veillonella (Veillonella) is reduced, while that of oral streptococci (Veillonella) is reduced. Streptococcus oralis ), Streptococcus salivarius ( Streptococcus salivarius ), vestibular streptococci ( Streptococcus vestibularis The relative abundance of probiotics or low cariogenic commensal bacteria increased; in the caries-free dental plaque group (H+Bb group) with added Ss-Bb, the relative abundance of Veillonella was significantly reduced, while the relative abundance of Salicylic Streptococcus and Vestibular Streptococcus was significantly increased, but the abundance of oral Streptococcus did not change significantly between the H group and the H+Bb group.

[0091] like Figure 7 As shown, the caries-active and caries-free plaque biofilms of Ss-Bb are enriched in primary functions such as organismal systems, cellular processes, and environmental information processing. Figure 7 A), secondary functions are mainly enriched in prokaryotic cell community formation, membrane transport, signal transduction, and carbohydrate metabolism, etc. Figure 7 B), its tertiary functions are largely concentrated in quorum sensing (ko02024), starch and sucrose metabolism (ko00500), two-component systems (ko02020), and ATP-binding cassette transporters (ko02010), etc. Figure 7 C), among which the caries-active group with added Ss-Bb is also enriched with metabolic processes such as amino sugar and nucleotide sugar metabolism (ko00520), purine metabolism (ko00230), and aminoacyl-tRNA biosynthesis (ko00970).

[0092] The above results indicate that Ss-Bb can reduce the formation and acid production of dental plaque biofilm in caries-active individuals, reduce the acid production of dental plaque biofilm in caries-free individuals, and improve the bacterial flora ratio and metabolic function of dental plaque biofilm in both caries-active and caries-free individuals, thereby playing a role in preventing dental caries.

[0093] Example 4

[0094] To evaluate whether soy saponin Bb (Ss-Bb) also has anti-caries effects in animals, a rat molar caries model was constructed and Ss-Bb was administered for evaluation. The specific protocol is as follows:

[0095] Twenty-five 21-day-old (100 ± 10 g) male Sprague-Dawley rats that were not carrying the specific pathogen (SPF) were given 200 mg / L ampicillin in their drinking water for 3 days after a 7-day quarantine period to suppress endogenous flora and promote subsequent bacterial colonization.

[0096] The rats were randomly divided into 5 groups (n=5 in each group):

[0097] ① Caries model group (Cario): 5 × 10⁵ cells / day for 5 days 5 Sm-Lc mixed bacterial culture at CFU / mL;

[0098] ②Low-frequency treatment group (L-Tre): 5×10 doses administered over 5 days 5 CFU / mL of Sm-Lc mixed bacterial solution, then coated with 100mg / L Ss-Bb solution for 5 days;

[0099] ③ High-frequency treatment group (H-Tre): 5 days of vaccination with 5×10 5 CFU / mL of Sm-Lc mixed bacterial suspension was then applied daily with 100 mg / L Ss-Bb solution until the end of the experiment.

[0100] ④ Soybean saponin control group (Bb): Only 100 mg / L Ss-Bb solution was applied for 5 days;

[0101] ⑤ Control group (Con): No bacteria were inoculated and no Ss-Bb solution was applied.

[0102] From day 1 to day 5, all three groups—the caries model group, the low-frequency treatment group, and the high-frequency treatment group—used sterile cotton swabs to apply a solution with a concentration of 5×10⁻⁶. 5 A CFU / mL Sm-Lc mixed bacterial solution was applied to the surface of rat molars, with 200 μL applied to each quadrant for 15 seconds per rat. Rats were kept from drinking or eating for 0.5 h post-inoculation. All three groups were given cariogenic feed (2000#) and their drinking water was replaced with a 5wt% sucrose solution. The control group received no bacterial inoculation and a normal diet.

[0103] From day 6 to day 10, the low-frequency treatment group and the soybean saponin control group applied 100 mg / L Ss-Bb solution to the molar surfaces of rats using sterile cotton swabs at a rate of 200 μL / rat. The high-frequency treatment group applied 100 mg / L Ss-Bb solution to the molar surfaces of rats using sterile cotton swabs daily, applying 15 s to each quadrant, at a rate of 200 μL / rat. After inoculation, rats were kept from drinking or eating for 0.5 h.

[0104] Six weeks later, the rats were anesthetized and euthanized by cervical dislocation. The intact maxillae and mandibles were separated, and the soft tissue attached to the detached surface was removed. The rats were stored in 4% paraformaldehyde, and the enamel volume and density were quantitatively analyzed by Micro-CT. The results are shown in […]. Figure 8 .

[0105] like Figure 8 As shown, no low-density caries lesions were observed in the Ss-Bb control group, blank Con group, and high-frequency treatment group; however, low-density caries lesions of varying degrees appeared in the caries model group and the low-frequency treatment group. Further quantitative analysis showed that ( Figure 8 B and 8C), there was no statistically significant difference in enamel volume and density of molars between the high-frequency treatment group, the Ss-Bb control group, and the blank Con group. p >0.05), and significantly higher than the caries model group ( p <0.01) and low-frequency treatment group ( p <0.05), the enamel volume and density of the molars in the low-frequency treatment group were higher than those in the caries model group ( p <0.05). The above results indicate that Ss-Bb can also reduce enamel damage to molars caused by dental caries in animals, and has a good anti-caries effect.

[0106] Example 5

[0107] To investigate the safety of soy saponin Bb (Ss-Bb), an in vivo safety test was conducted. The specific protocol is as follows:

[0108] Heart, liver, spleen, lung, and kidney tissues from rats in the Ss-Bb and Con groups of Example 4 were stained with hematoxylin and eosin (HE). The stained tissue sections were observed under an optical microscope to assess the biosafety of Ss-Bb. Results are shown in […]. Figure 9 .

[0109] like Figure 9 As shown, the heart, liver, spleen, lungs, and kidneys of rats in the Ss-Bb group and Con group had intact tissue structures and normal cell morphology. No pathological changes such as inflammatory cell infiltration, tissue edema or swelling, cell degeneration or necrosis, or fibrous tissue hyperplasia were observed.

[0110] The above results indicate that Ss-Bb at a concentration of 100 mg / L has good biocompatibility.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of soybean saponin Bb in the preparation of anti-caries products, characterized in that, The caries prevention products are pharmaceuticals or daily chemical products.

2. The application of soybean saponin Bb in the preparation of anti-cariogenic bacteria products, characterized in that, The cariogenic bacteria are Streptococcus mutans and Lactobacillus casei; the anti-cariogenic bacteria products are pharmaceuticals or daily chemical products.

3. The application as described in claim 1 or 2, characterized in that, The daily chemical products mentioned include oral care products.

4. The application as described in claim 1 or 2, characterized in that, The dosage form of the medicine or daily chemical product includes at least one of solid dosage form, liquid dosage form, semi-solid dosage form and gas dosage form.

5. The application as described in claim 4, characterized in that, When the dosage form of a pharmaceutical or daily chemical product is a liquid dosage form, the content of soybean saponin Bb in the pharmaceutical or daily chemical product is 100-200 mg / L.

6. The application as described in claim 1 or 2, characterized in that, The medicines or daily chemical products mentioned also contain excipients.

7. The application as described in claim 6, characterized in that, The excipients include at least one of the following: fillers, binders, wetting agents, disintegrants, lubricants, flow aids, preservatives, flavoring agents, colorants, solvents, and cosolvents.

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