Application of soyasaponin Bb in preparation of anti-caries product

Abstract

The invention relates to application of soyasaponin Bb in preparation of an anti-caries product, and belongs to the technical field of biology. The invention provides an application of soyasaponin Bb in preparation of an anti-caries product. In-vitro experiments find that the soyasaponin Bb has the effects of inhibiting cariogenic bacterium biofilm formation and acid production, reducing enamel demineralization degree and regulating dental plaque microbial community structure and metabolic function so as to improve oral micro-ecology, meanwhile, the soyasaponin Bb has good biological safety and caries prevention potential in animal bodies, and the soyasaponin Bb can be used for preparing dental caries. The soybean saponin Bb is expected to be developed into a novel natural anti-caries product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to application of soyasaponin Bb in preparation of caries-preventing products. BACKGROUND

[0002] Legumes are rich in bioactive compounds and are a low-cost and nutrient-rich food source in human diets and animal feeds. Plant proteins, represented by legume proteins, have more advantages than animal proteins in terms of environmental sustainability, production cost, nutritional and health benefits, and moral ethics, and show better application prospects. Soybean is one of the main food crops in China, and has advantages such as high yield, reasonable price, and well-established extraction process of health-related components, and is therefore attracting attention from researchers at home and abroad. Previous studies have confirmed that soybean-derived compounds such as isoflavones, saponins, flavonoids, peptides, proteins, lipids, vitamins, and minerals have immunomodulatory effects. Among them, soyasaponin (Ss) is an important secondary metabolite of soybean, and has various biological functions, including antibacterial, antifungal, antiviral, antioxidant, and immunomodulatory effects. Ss can inhibit a variety of pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and play a role in disease prevention and treatment by regulating the host microbiota. However, the preventive and therapeutic effects of soyasaponin on the microecology of dental caries and its mechanism are not clear, and therefore it is urgent to develop the potential of soyasaponin for preventing and treating dental caries and provide a new direction for the development of natural anti-caries drugs. SUMMARY

[0003] The present application aims to overcome the deficiencies of the prior art and provide application of soyasaponin Bb in preparation of caries-preventing products.

[0004] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows:

[0005] In a first aspect, the present application provides application of soyasaponin Bb in preparation of caries-preventing products.

[0006] In a second aspect, the present application provides application of soyasaponin Bb in preparation of products against cariogenic bacteria.

[0007] The present application found through in vitro experiments that soyasaponin Bb has the effects of inhibiting the formation of biofilm and acid production of cariogenic bacteria, reducing the degree of enamel demineralization, regulating the structure and metabolic function of dental plaque microbiota, and thus improving the oral microecology. Meanwhile, soyasaponin Bb has good biological safety and caries-preventing potential in animals, suggesting that soyasaponin Bb is expected to develop into a new type of natural caries-preventing product.

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

[0009] As a preferred embodiment of the application, the Lactobacillus comprises Lactobacillus casei.

[0010] As a preferred embodiment of the application, the anti-cariogenic product and / or anti-cariogenic bacteria product comprises at least one of, but not limited to, a pharmaceutical product, a daily chemical product, and a food product.

[0011] As a preferred embodiment of the application, the anti-cariogenic product and / or anti-cariogenic bacteria product comprises at least one of, but not limited to, an oral product. The application proves by experiments that soy saponin Bb has good anti-cariogenic effect and can be added to oral products such as gargling solution, sustained-release gel, and toothpaste to play an anti-cariogenic effect.

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

[0013] As a preferred embodiment of the application, when the dosage form of the anti-cariogenic product and / or anti-cariogenic bacteria product is a liquid dosage form, the content of soy saponin Bb in the anti-cariogenic product and / or anti-cariogenic bacteria product is 100-200 mg / L. When the concentration of soy saponin Bb is 100 mg / L, not only can it effectively prevent caries, but also has good biological safety.

[0014] As a preferred embodiment of the application, the anti-cariogenic product and / or anti-cariogenic bacteria product further comprises an excipient.

[0015] As a preferred embodiment of the application, the excipient comprises at least one of, but not limited to, a filler, a binder, a humectant, a disintegrant, a lubricant, a glidant, a preservative, a flavoring agent, a coloring agent, a solvent, and a cosolvent.

[0016] As a preferred embodiment of the application, the filler comprises at least one of, but not limited to, starch, dextrin, glucose, mannitol, hydroxybenzoic fiber, and microcrystalline cellulose.

[0017] As a preferred embodiment of the application, the binder and the humectant comprise at least one of, but not limited to, water, gelatin, starch paste, polyethylene glycol, and ethanol.

[0018] As a preferred embodiment of the application, the disintegrant comprises at least one of, but not limited to, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, and cross-linked carboxymethyl cellulose.

[0019] As a preferred embodiment of the application, the lubricant and the glidant comprise at least one of, but not limited to, gum arabic, polyethylene glycol, magnesium stearate, micro-powder silica, and talc.

[0020] As a preferred embodiment of the application, the preservatives include, but are not limited to, at least one of domiphen bromide, benzoic acid, sodium benzoate, propylene glycol, ethyl lactate, and sorbic acid.

[0021] As a preferred embodiment of the application, the flavoring agents and coloring agents include, but are not limited to, at least one of sucrose, sorbitol, mannitol, orange oil, carmine, and malic acid,

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

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The application finds through in-vitro experiments that soyasaponin Bb has the effects of inhibiting the formation of cariogenic bacteria biofilm and acid production, reducing the degree of enamel demineralization, regulating the structure and metabolic function of dental plaque microbial community, and thus improving the oral microecology. Meanwhile, soyasaponin Bb has good biological safety and caries prevention potential in animals, indicating that soyasaponin Bb is expected to develop into a new type of natural caries prevention product. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 2 is the effect of different concentrations of Ss-Bb on the formation of Sm-Lc biofilm in Example 1 of the application, wherein A is the biofilm morphology after crystal violet staining, and B is the statistical result of the biofilm formation amount of each group. The figure with “****” indicates that there is a statistical difference (p<0.0001) between the group and the PBS group.

[0026] Figure 2 Figure 3 is the effect of different concentrations of Ss-Bb on the 24h mature Sm-Lc biofilm in Example 1 of the application, wherein A is the biofilm morphology after crystal violet staining, and B is the statistical result of the biofilm formation amount of each group. The figure with “****” indicates that there is a statistical difference (p<0.0001) between the group and the PBS group.

[0027] Figure 3 Figure 4 is the demineralization depth and mineral loss of the enamel blocks after different treatments in Example 1 of the application, wherein A is the micro-CT image of the enamel blocks in different treatment groups, B is the statistical result of the enamel demineralization depth in different treatment groups, and C is the statistical result of the enamel mineral loss in different treatment groups. The figure with “*” indicates that there is a statistical difference (p<0.05) between the group and the Sm-Lc group, and the figure with “****” indicates that there is a statistical difference (p<0.0001) between the group and the Sm-Lc group.

[0028] Figure 4Cell viability after treatment of human oral epithelial cells (HOEC) with different concentrations of Ss-Bb in Example 2 of the present application, wherein A is the result of cell viability assay after 1 day of co-culture, B is the result of cell viability assay after 3 days of co-culture, C is the result of cell viability assay after 5 days of co-culture, and D is the result of cell viability assay after 7 days of co-culture, and "*" in the figure indicates a statistically significant difference compared with the Control group (p < 0.05), "**" indicates a statistically significant difference compared with the Control group (p < 0.01), "***" indicates a statistically significant difference compared with the Control group (p < 0.001), and "****" indicates a statistically significant difference compared with the Control group (p < 0.0001);

[0029] Figure 5 Effect of Ss-Bb on the formation of plaque biofilm and acid production in caries-active and caries-free populations in Example 3 of the present application, wherein A is the result of biofilm formation measurement, and B is the result of acid production concentration measurement, and "*" in the figure indicates a statistically significant difference between the two groups (p < 0.05), "**" indicates a statistically significant difference between the two groups (p < 0.01), and "****" indicates a statistically significant difference between the two groups (p < 0.0001);

[0030] Figure 6 Relative abundance distribution chart of microorganisms in different treatment groups in Example 3 of the present application, and "Others" in the figure represents the species ranked after the 20th in terms of relative abundance;

[0031] Figure 7 KEGG functional abundance clustering heat map of different treatment groups in Example 3 of the present application, wherein A is the 1st level function, B is the 2nd level function, and C is the 3rd level function;

[0032] Figure 8 Micro-CT image of rat molar and analysis of enamel volume and density in different treatment groups in Example 4 of the present application, wherein A is a representative micro-CT image of rat molar in each group (the yellow arrow indicates caries lesion), B is the statistical result of rat molar enamel volume in each group, and C is the statistical result of rat molar enamel density in each group, and "*" in the figure 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), and "****" indicates a statistically significant difference between the two groups (p < 0.0001);

[0033] Figure 9 HE staining results of heart, liver, spleen, lung, and kidney of rats in Ss-Bb group and Con group in Example 5 of the present application. DETAILED DESCRIPTION

[0034] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in combination with specific examples.

[0035] The other materials, reagents and the like used in the examples, comparative examples and effect examples can be obtained from commercial channels unless otherwise specified.

[0036] The soyasaponin Bb described in the present application can be obtained from various commercial channels. The soyasaponin Bb used in the following examples was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., with a purity of 98%, a product code of S873374 and a CAS number of 51330-27-9. The soyasaponin Bb was mixed with anhydrous ethanol to prepare a 5 mg / mL stock solution for standby.

[0037] Streptococcus mutans (Sm) was purchased from the American Type Culture Collection (ATCC) with an ATCC number of 25175, and Lactobacillus casei (Lc) was purchased from the American Type Culture Collection (ATCC) with an ATCC number of 393.

[0038] The Sm and Lc strains were stored in BHI liquid medium containing 30 v / v% glycerol and stored at -80℃. When used, the frozen Sm and Lc were thawed at room temperature, and a sterile inoculation loop was used to take a loop of bacterial liquid to streak inoculate on BHI solid medium (Sm) and MRS solid medium (Lc), respectively. After 24 h of culture at 37℃ under a 6% oxygen concentration, single colonies of Sm and Lc were picked and inoculated in 5 mL of BHI liquid medium, and cultured at 37℃ under a 6% oxygen concentration for 24 h. The bacterial liquid concentration was adjusted to 5×10 5 CFU / mL, and an equal amount of mixed bacterial liquid was prepared as Sm-Lc mixed bacterial liquid.

[0039] BHI liquid medium: 37 g of BHI medium dry powder was dissolved in 1 L of pure water, high-pressure steam sterilized, and stored in a 4℃ refrigerator.

[0040] BHI solid medium: 37 g of BHI medium dry powder and 15 g / L of agar powder were dissolved in 1 L of pure water, completely dissolved, high-pressure steam sterilized, and prepared as a plate.

[0041] Neutral BHIS: 37 g of BHI medium dry powder was dissolved in 1 L of pure water, high-pressure steam sterilized, and added with sterile sucrose solution to prepare BHI liquid medium containing 0.2 wt% sucrose, and stored in a 4℃ refrigerator.

[0042] Acidic BHIS: 37 g of BHI medium dry powder was dissolved in 1 L of pure water, and glacial acetic acid was added to adjust the pH to 5.5. High-pressure steam sterilization was performed, and a sterile sucrose solution was added to prepare a BHI liquid medium containing 0.2wt% sucrose. It was stored in a refrigerator at 4°C.

[0043] BHI medium dry powder was purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd., with the item number 028360.

[0044] MRS liquid medium: MRS medium dry powder was dissolved in pure water according to the instructions, high-pressure steam sterilization was performed to prepare, and it was stored in a refrigerator at 4°C. If it is prepared as MRS solid medium, 15 g / L agar powder is added before high-pressure steam sterilization, and after complete dissolution, high-pressure steam sterilization is performed to prepare a plate.

[0045] MRS medium dry powder was purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd., with the item number 027312.

[0046] CCK-8 detection kit was purchased from GLPBIO, with the item number GK10001.

[0047] The macro-genome sequencing of the plaque biofilm bacterial community structure and functional metabolism was entrusted to Beijing Nuowoziyuan Science and Technology Co., Ltd.

[0048] Example 1

[0049] In order to explore the anti-cariogenic bacteria effect of soybean saponin Bb (Ss-Bb), the cariogenic bacteria Sm and Lc were treated with soybean saponin Bb, and the specific scheme is as follows:

[0050] 1. The effect of Ss-Bb on the formation of Sm-Lc mixed bacteria liquid biofilm.

[0051] Take 96-well plates, add 100 μL of Sm-Lc mixed bacteria liquid, 100 μL of Ss-Bb liquid (500 mg / L, 400 mg / L, 300 mg / L, 200 mg / L, 150 mg / L, 100 mg / L, 50 mg / L) to each well, and the final concentration of Ss-Bb liquid is 250 mg / L, 200 mg / L, 150 mg / L, 100 mg / L, 75 mg / L, 50 mg / L, 25 mg / L, and the final concentration of Sm-Lc mixed bacteria liquid is 5×10 5 CFU / ml. The positive control group added the same amount of 100 μL of 1wt% chlorhexidine solution and 100 μL of Sm-Lc mixed bacteria liquid, the negative control group added 100 μL of PBS and 100 μL of Sm-Lc mixed bacteria liquid, and the blank control group added 200 μL of neutral BHIS without Ss-Bb and Sm-Lc bacteria liquid. Each treatment has 3 replicates, and the volume of each treatment and each replicate well is the same.

[0052] After 24h incubation at 37℃ with 6% oxygen concentration, remove the culture medium, rinse with PBS for 3 times, fix with methanol for 15min, dry for 15min after removing methanol, stain with 0.1wt% crystal violet solution for 5min, remove crystal violet solution, rinse with PBS for 2 times, add 200μL deoxycholate sodium into each well and mix, measure the optical density at 600nm to evaluate the biofilm amount of each group, calculate the minimum biofilm inhibition concentration (MBIC), the drug concentration at which the biofilm inhibition rate reaches 50% (MBIC 50 ), the results are shown in Figure 1 .

[0053] Meanwhile, according to the above grouping, first mix Sm-Lc mixed bacteria solution with neutral BHI liquid medium and incubate for 24h, then add Ss-Bb mother liquor according to the above Ss-Bb final concentration, determine the minimum biofilm reduction concentration (MBRC) by crystal violet staining, the results are shown in Figure 2 .

[0054] The meaning of MBIC is (OD treated group-OD blank group) / (OD negative control-OD blank group) x 100%≤10%, the minimum concentration in which is MBIC.

[0055] The meaning of MBRC is (OD treated group-OD blank group) / (OD negative control-OD blank group) x 100%≤10%, the minimum concentration in which is MBRC.

[0056] As shown in Figures 1-2 , the biomass of Sm-Lc dual-species biofilm after co-culturing with Ss-Bb at a concentration of ≥75mg / L for 24h was significantly lower than that of the negative control group (p<0.0001, Figure 1 A and 1B), the MBIC was 100mg / L and the MBIC 50 was 75mg / L; after 24h treatment with Ss-Bb at a concentration of ≥100mg / L, the biomass of Sm-Lc dual-species biofilm was significantly lower than that of the negative control group (p<0.0001, Figure 2 A and 2B), the MBRC was 200mg / L. The above results show that Ss-Bb at a concentration of ≥75mg / L can effectively inhibit the formation of cariogenic bacteria biofilm, thereby playing a role in preventing dental caries.

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

[0058] Sterile bovine incisors were selected under stereomicroscope (×20) without fracture, crack, caries and complete root development. A 4mm×4mm×3mm block was prepared on the buccal surface of the bovine incisor using diamond saw under running water. The surface was polished using 400-2000 grit silicon carbide paper. The sample was ultrasonically cleaned for 10 min at 40 Hz to remove debris and contamination layer. The sample was naturally dried. The enamel block was covered with two layers of acid-resistant nail polish on all surfaces except the experimental surface. The enamel block was sterilized to obtain the enamel block.

[0059] Ss-Bb treatment groups (200mg / L, 100mg / L and 75mg / L Ss-Bb solution were added respectively), positive control (0.5wt% CHX), caries model group (Sm-Lc mixed bacteria solution without Ss-Bb solution) and blank control (BHIS without solution and bacteria) were set up. The experiment was divided into two parts:

[0060] Part A: Sm-Lc mixed bacteria solution, Ss-Bb solution and CHX solution were added to the 24-well sterile culture plate according to the above settings, and the total volume of each well was 2mL. After 24h of anaerobic culture at 37℃, the culture supernatant was removed, and the wells were washed twice with sterile PBS. 2mL of neutral BHIS was added to each well for 8h of culture. The culture supernatant was removed, and the wells were washed twice with sterile PBS. 2mL of acidic BHIS was added to each well for 16h of culture. The culture supernatant was removed, and the wells were washed twice with sterile PBS. The biofilm was cultured under pH cycling conditions (8h neutral, 16h acidic) for 14 days.

[0061] Part B: The effect of Ss-Bb on the demineralization of tooth blocks after acting on mature Sm-Lc dual-species biofilm. In the 24-well sterile culture plate, the soybean saponin treatment group and the positive control group were added with 2mL of Sm-Lc mixed bacteria solution, and the blank control group was added with 2mL of neutral BHIS without solution and bacteria. After 24h of anaerobic culture at 37℃, the culture supernatant was removed, and the wells were washed twice with sterile PBS. 2mL of neutral BHIS containing Ss-Bb solution was added to each well of the soybean saponin treatment group, 2mL of neutral BHIS containing 0.5wt% CHX was added to each well of the positive control group, and 2mL of neutral BHIS without solution and bacteria was added to the blank control group. The culture was continued for 24h. The culture supernatant was removed, and the wells were washed twice with sterile PBS. 2mL of neutral BHIS was added to each well for 8h of culture. The culture supernatant was removed, and the wells were washed twice with sterile PBS. 2mL of acidic BHIS was added to each well for 16h of culture. The biofilm was cultured under pH cycling conditions (8h neutral, 16h acidic) for 14 days.

[0062] The above pH cycling conditions were that neutral BHIS was added for 8h and acidic BHIS was added for 16h every 24h to simulate the oral environment, and no additional reagents were needed 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 significantly reduced the demineralization depth and mineral loss of enamel fragments, whether co-cultured with Sm-Lc bacterial solutions or applied to existing Sm-Lc biofilms (p < 0.0001). In contrast, 75 mg / L Ss-Bb only reduced the demineralization depth of enamel by the biofilm when co-cultured with bacterial solutions (p < 0.05). These results indicate that Ss-Bb at concentrations above 100 mg / L can reduce the risk of enamel demineralization caused by cariogenic bacterial biofilms, thus playing a role in caries prevention.

[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 culture 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, as well as treatment with 200 mg / L Ss-Bb for 1 day, had no significant effect on the cell viability of HOECs (p>0.05); however, 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 the 100 mg / L Ss-Bb concentration has the best safety 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 18-55 years were recruited and divided into caries-active group (group C) and healthy group (group H).

[0073] The screening criteria for caries-active group (group C) were as follows:

[0074] 1) DMFT ≥ 6, with at least 3 teeth with caries;

[0075] 2) No other oral diseases and systemic diseases, no smoking, no use of orthodontic appliances;

[0076] 3) No use of antibiotics and hormones in the past 3 months;

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

[0078] The screening criteria for healthy group (group H) were as follows:

[0079] 1) DMFT = 0;

[0080] 2) No use of antibiotics and hormones in the past 3 months;

[0081] 3) No oral diseases such as periodontal disease, oral mucosa disease, oral and maxillofacial tumor and history of radiotherapy and chemotherapy;

[0082] 4) No systemic diseases.

[0083] All volunteers signed the informed consent form, and the study was reviewed and approved by the ethics committee. Clinical dental plaque samples were collected from the subjects, resuspended with PBS, diluted with 60 v / v% glycerol solution at a volume ratio of 1:1, and used for subsequent in vitro studies.

[0084] According to the foregoing 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 reduce its demineralization effect on enamel, and the concentration of Ss-Bb has no obvious cytotoxicity, so it is used as the working concentration for clinical plaque detection and rat molar caries model.

[0085] The two groups of clinical dental plaque samples were mixed, diluted with neutral BHIS (plaque: BHIS = 1:20), and 100 mg / L Ss-Bb solution or neutral BHIS (negative control) was mixed with the plaque diluent and cultured for 48 h, followed by crystal violet staining and lactic acid production detection. Figure 5 .

[0086] The above experiment was done with two plates, and the plaque biofilm of the other plate was collected with a sterile swab. Macro-genome sequencing was used to detect changes in the structure and metabolic function of the flora, and the relative abundance of species at the species level and KEGG functional annotation were analyzed. The results are shown in Table 1. Figures 6-7 and Table 1.

[0087] Table 1: Bacterial species with significant changes in relative abundance

[0088] Strain C C + Bb H H + Bb Streptococcus oralis 3.522％ 14.153％ 5.149％ 4.543％ Streptococcus salivarius 0.253％ 0.423％ 0.669％ 6.310％ Veillonella parvula 3.852％ 0.005％ 2.950％ 0.008％ Streptococcus vestibularis 0.376％ 0.568％ 0.493％ 3.382％ Veillonella sp. 2.208％ 0.002％ 1.637％ 0.002％ uncultured Veillonella sp. 1.723％ 0.005％ 2.105％ 0.006％ Veillonella dispar 0.629％ 0.000％ 0.530％ 0.000％ Veillonella atypica 0.428％ 0.000％ 0.258％ 0.000％ Veillonella tobetsuensis 0.558％ 0.000％ 0.116％ 0.000％

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

[0090] As shown in Figure 6 and Table 1, the relative abundance of Veillonella parvula, Veillonella atypica, and Veillonella dispar, which are associated with caries, decreased in the caries-active group plaque with Ss-Bb added (group C+Bb), and the relative abundance of Streptococcus oralis, Streptococcus salivarius, and Streptococcus vestibularis, which are probiotic species or low-caries-susceptibility symbiotic bacteria, increased. The relative abundance of Veillonella also significantly decreased in the caries-free group plaque with Ss-Bb added (group H+Bb), and the relative abundance of Streptococcus salivarius and Streptococcus vestibularis significantly increased, but the abundance of Streptococcus oralis did not change significantly between group H and group H+Bb.

[0091] As shown in Figure 7 , the Ss-Bb-enriched plaque biofilm in the caries-active group and the caries-free group was enriched in biological system, cell process, and environmental information processing at the primary functional level (A), and was mainly enriched in prokaryotic cell colony formation, membrane transport, signal transduction, and carbohydrate metabolism at the secondary functional level (B). Figure 7 A), and the secondary function was mainly enriched in prokaryotic cell colony formation, membrane transport, signal transduction, and carbohydrate metabolism (B). Figure 7B), the third function is enriched in quorum sensing (ko02024: Quorumsensing), starch and sucrose metabolism (ko00500: Starch and sucrose metabolism), two-component system (ko02020: Two-component system) and ATP-binding cassette transporters (ko02010: ATP-binding cassette transporters) and the like Figure 7 C), wherein the caries-active group with Ss-Bb added is also enriched in amino sugar and nucleotide sugar metabolism (ko00520: Amino sugar and nucleotide sugar metabolism), purine metabolism (ko00230: purine metabolism) and biosynthesis of aminoacyl-tRNA (ko00970: Aminoacyl-tRNA biosynthesis) and the like metabolic processes.

[0092] The above results show that Ss-Bb has the effects of reducing the formation of dental plaque biofilm and acid production of caries-active people, acid production of dental plaque biofilm of caries-free people, and improving the flora ratio and metabolic function of dental plaque biofilm of caries-active people and caries-free people, thereby playing a role in preventing caries.

[0093] Example 4

[0094] In order to evaluate whether soy saponin Bb (Ss-Bb) also has the effect of preventing caries in animals, a rat molar caries model was constructed and Ss-Bb was given for evaluation, and the specific scheme is as follows:

[0095] 25 male Sprague-Dawley rats of 21 days of age (body weight 100±10 g) not carrying specific pathogens (Specific Pathogen Free, SPF) were given 200 mg / L ampicillin in drinking water after a 7-day quarantine period for 3 days to inhibit endogenous flora and promote subsequent bacterial colonization.

[0096] The above rats were randomly divided into 5 groups (5 rats per group):

[0097] ① Cariogenic model group (Cario): inoculated with 5×10 5 CFU / mL of Sm-Lc mixed bacterial solution for 5 days;

[0098] ② Low-frequency treatment group (L-Tre): inoculated with 5×10 5 CFU / mL of Sm-Lc mixed bacterial solution for 5 days, and then coated with 100 mg / 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, then apply 100mg / L Ss-Bb solution daily 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 hours post-inoculation. All three groups were given cariogenic feed (2000#) and their drinking water was replaced with a 5 wt% 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 dose 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 dose 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 ( Figure 8 In the high-frequency treatment group (B and 8C), there were no statistically significant differences in enamel volume and density between the high-frequency treatment group, the Ss-Bb control group, and the blank Con group (p>0.05), but the enamel volume and density were significantly higher in the high-frequency treatment group than in the caries model group (p<0.01) and the low-frequency treatment group (p<0.05). The enamel volume and density in the low-frequency treatment group were also higher than those in the caries model group (p<0.05). These results indicate that Ss-Bb can reduce enamel damage to molars caused by caries in animals and has a good caries-preventing effect.

[0106] Example 5

[0107] In order to explore the safety of soyasaponin Bb (Ss-Bb), the in vivo safety test of soyasaponin Bb was carried out, and the specific scheme was as follows:

[0108] The heart, liver, spleen, lung and kidney of the rats in the Ss-Bb group and the Con group in Example 4 were subjected to hematoxylin and eosin (HE) staining, and the tissue sections after staining were observed under an optical microscope to evaluate the biological safety of Ss-Bb, and the results are shown in Figure 9 .

[0109] As shown in Figure 9 , the tissue structures of the heart, liver, spleen, lung and kidney of the rats in the Ss-Bb group and the Con group were complete, and the cell morphology was normal, and no pathological changes such as inflammatory cell infiltration, tissue edema or swelling, cell degeneration or necrosis, and fibrous tissue hyperplasia were observed.

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

[0111] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

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

2. Application of soybean saponin Bb in the preparation of anti-cariogenic products.

3. The application as described in claim 1 or 2, characterized in that, The cariogenic bacteria include at least one of Streptococcus mutans, Lactobacillus, and Actinomycetes.

4. The application as described in claim 1 or 2, characterized in that, The caries prevention products and / or anti-cariogenic bacteria products include at least one of pharmaceuticals, daily chemical products, and food.

5. The application as described in claim 1 or 2, characterized in that, The caries-preventing products and / or anti-cariogenic products include oral care products.

6. The application as described in claim 1 or 2, characterized in that, The dosage forms of the caries prevention products and / or anti-cariogenic bacteria products include at least one of solid dosage forms, liquid dosage forms, semi-solid dosage forms, and gaseous dosage forms.

7. The application as described in claim 6, characterized in that, 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.

8. The application as described in claim 1 or 2, characterized in that, The caries-preventing products and / or anti-cariogenic bacteria products also contain excipients.

9. The application as described in claim 8, characterized in that, 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.

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