A whitening toothpaste containing natural plant extracts and its preparation method

Abstract

This invention discloses a synergistic anti-caries oral care composition and its application. The composition comprises: (a) a single-domain antibody targeting the Streptococcus mutans surface protein PAc; (b) a plant extract complex containing kudzu root extract and phellodendron bark extract; and (c) gallic acid liposomes. This invention also provides a toothpaste containing this composition and a method for its preparation. Experiments have shown that the single-domain antibody can specifically bind to and disturb the Streptococcus mutans biofilm, increasing cell membrane permeability and synergistically promoting the entry of plant extracts into the cell to exert a bactericidal effect, achieving a clearance rate of over 98% for mature biofilms. Through formulation and process optimization, this invention solves the stability problem of antibodies in toothpaste; the product retains more than 90% of its activity after two years of storage at 25°C. The composition and product provided by this invention have the advantages of highly efficient removal of mature biofilms, synergistic effect, safety with low damage, and excellent stability, and have broad application prospects in the field of oral care.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and oral care, specifically relating to a whitening toothpaste containing natural plant extracts and its preparation method. Background Technology

[0002] Tooth discoloration is a common problem affecting oral aesthetics, and it can generally be divided into two categories based on its cause: extrinsic staining and intrinsic staining. Extrinsic staining mainly originates from daily diet and lifestyle habits, such as polyphenols and tar-like substances in tea, coffee, red wine, and tobacco. These pigments easily deposit on the tooth surface and combine with acquired enamel membranes and dental plaque to form stable stains. Over time, the pigments gradually embed themselves in the microporous structure of the enamel surface and the plaque biofilm, causing the teeth to appear yellow or brown. Intrinsic staining is mostly related to factors such as drug deposition during tooth development, fluorosis, and tooth aging.

[0003] Currently, whitening toothpaste is one of the most common teeth whitening products. Existing whitening toothpastes mainly achieve teeth whitening through two main technological approaches. The first is physical abrasion stain removal technology, which involves adding abrasives such as calcium carbonate, hydrated silica, or alumina to the toothpaste. During brushing, these abrasives remove stains from the tooth surface through mechanical friction. This method can remove extrinsic pigments attached to the tooth surface to a certain extent, but when the abrasive value of the toothpaste is high, long-term use may lead to enamel wear, increasing the roughness of the tooth surface and making it easier for new pigments to adhere. The second is chemical oxidation bleaching technology, which involves adding oxidants such as hydrogen peroxide or urea peroxide to the toothpaste. This decomposes colored organic molecules through an oxidation reaction. However, peroxides are somewhat irritating and may cause gum irritation or tooth sensitivity, thus limiting their long-term daily use.

[0004] Studies have shown that dental plaque biofilm on the tooth surface is a crucial structural element for extrinsic pigment deposition. Dental plaque biofilm is a complex three-dimensional network structure composed of various oral bacteria and their secreted extracellular polysaccharides. This network not only provides a stable living environment for bacteria but also offers attachment sites for food pigments. Therefore, mechanical abrasion or oxidative bleaching alone is often insufficient to completely remove mature plaque biofilm, thus limiting the durability of teeth whitening effects.

[0005] Existing technologies also include research on improving oral health using antibodies or natural plant extracts. For example, some studies report the use of antibodies targeting enzymes or adhesion proteins associated with cariogenic bacteria in the oral cavity to inhibit bacterial adhesion, thereby reducing plaque formation. Furthermore, some plant extracts (such as those containing alkaloids or polyphenols) are also used in oral care products to exert certain antibacterial or antioxidant effects. However, these technologies primarily focus on inhibiting plaque formation or providing broad-spectrum antibacterial effects, and their effectiveness in efficiently removing already formed, mature plaque biofilms remains insufficient.

[0006] Therefore, developing a teeth whitening composition that can target key structures of dental plaque and improve the efficiency of active ingredients in the plaque biofilm is of great significance for improving tooth discoloration problems. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an innovative oral care composition and product thereof that can efficiently remove mature biofilms of Streptococcus mutans, has a synergistic effect, and exhibits excellent stability.

[0008] To achieve the above objectives, the first aspect of the present invention provides an oral care composition, the core of which comprises three synergistic active ingredients:

[0009] 1. Targeted antibody component: A single-domain antibody capable of specifically binding to the surface protein PAc of Streptococcus mutans. This antibody effectively penetrates and disrupts established biofilms by targeting structural antigens on the bacterial surface, thereby damaging the integrity of the bacterial cell membrane.

[0010] 2. Plant-derived antibacterial components: a complex containing kudzu root extract and phellodendron bark extract. Kudzu root extract is rich in puerarin, and phellodendron bark extract is rich in berberine. Both have broad-spectrum antibacterial activity, but their intracellular accumulation is often limited due to the barrier effect of bacterial cell membranes.

[0011] 3. Long-lasting sustained-release / antioxidant component: Liposomes containing gallic acid. Gallic acid has antibacterial and antioxidant activities. Encapsulation with liposomes enables its long-lasting release in the oral environment and protects its activity.

[0012] In a preferred embodiment of the present invention, the single-domain antibody is VHH-PAc17, which has the amino acid sequence shown in SEQ ID NO:1. This antibody is obtained by immunizing alpacas with recombinant PAc protein, constructing a phage display library, and then performing affinity maturation and screening. Its affinity (KD) for PAc protein reaches the nanomolar level.

[0013] In another preferred embodiment of the present invention, the kudzu root extract is obtained by alcohol extraction, concentration, and drying, wherein the content of puerarin is not less than 40% (w / w). The phellodendron bark extract is obtained by alcohol extraction, purification with macroporous adsorption resin, and drying, wherein the content of berberine is not less than 25% (w / w). Mixing the two at a mass ratio of 1-5:1 (preferably 3:1) can produce the best synergistic antibacterial effect.

[0014] In another preferred embodiment of the present invention, the gallic acid liposomes are prepared by ethanol injection-high pressure homogenization, with an average particle size of 100-200 nm, a PDI < 0.3, a Zeta potential of -15 to -25 mV, and an encapsulation efficiency of gallic acid greater than 75%. These nanoliposomes can effectively protect gallic acid and achieve its adhesion and sustained release on oral mucosa and biomembranes.

[0015] A second aspect of the present invention provides an oral care product comprising the above-described oral care composition, particularly toothpaste. To address the technical challenge of antibody activity loss in complex multiphase systems such as toothpaste, the present invention systematically optimizes the toothpaste formulation and preparation process. The toothpaste comprises the above-described three active components in specific proportions, as well as excipients such as pH buffers (e.g., histidine), stabilizers (e.g., trehalose), mild abrasives (e.g., hydrated silica), humectants, binders, and surfactants. By precisely maintaining the pH value of the toothpaste within a mild range of 5.5-6.8 and employing a "low-temperature phase-separation addition method," i.e., mixing the antibody and liposomes with the cooled paste matrix at room temperature or low temperature, the activity of heat-sensitive components such as the antibody is protected to the greatest extent.

[0016] Compared with the prior art, the present invention has the following significant advantages:

[0017] 1. This invention reveals for the first time a novel synergistic mechanism between anti-PAc single-domain antibodies and plant extracts (especially berberine). As shown in Experiment 1, after the anti-PAc antibody binds to the bacterial surface, it significantly increases the permeability of the bacterial cell membrane (PI staining positivity rate reaches 28.5%), thereby opening a channel for berberine to enter the cell and increasing its intracellular accumulation by nearly 6 times. This synergistic strategy of "opening the gates" and "annihilating the enemy from within" achieves a clearance rate of over 98% against mature Streptococcus mutans biofilms (Experiment 3), far exceeding the effect of single components.

[0018] 2. Compared with antibodies targeting glucosyltransferase (GTF) in the prior art, the anti-PAc antibody of the present invention has an overwhelming advantage in clearing mature biofilms (92.3%), breaking through the inherent technical prejudice in the art that antibodies are difficult to clear mature biofilms (Experimental Example 2).

[0019] 3. The product not only has excellent anti-biofilm (anti-caries) ability, but also achieves significant whitening effect (ΔE up to 10.58) through the synergy of plant extracts and liposomes, and causes almost no damage to the enamel surface (ΔRa only 0.07 μm), achieving multiple functions of "anti-caries + whitening + safety" (Experimental Example 4).

[0020] 4. Addressing the industry pain point of poor stability of biomolecules in daily chemical products, this invention successfully integrated single-domain antibodies into the toothpaste matrix through lyophilization protectant screening (activity retention rate >97%), buffer system construction, and a unique low-temperature phase-separation addition process. Accelerated and long-term stability experiments show that after 24 months of storage at 25°C, the optimized toothpaste still retains more than 90% of antibody activity, fully meeting the shelf-life requirements of commercial products (Experimental Example 5), paving the way for the practical application of antibody-based oral care products.

[0021] 5. The single-domain antibody preparation technology, industrial extraction process of plant extracts, and liposome preparation technology used in this invention are all relatively mature, easy to achieve large-scale production, and have good prospects for industrialization and market application.

[0022] In summary, this invention provides a novel oral care solution that integrates targeting, synergy, stability, safety, and high efficiency, which is of great significance for improving the prevention and control of dental caries and developing high-performance oral care products. Attached Figure Description

[0023] Figure 1 This image shows the purification and identification results of the anti-PAc single-domain antibody VHH-PAc17 of this invention. In the image, A is an SDS-PAGE electrophoresis diagram, lane M represents the protein molecular weight standard, and lane 1 shows the purified VHH-PAc17 sample; B is a Western blotting diagram, with lane 1 showing a single target band that specifically reacts with the anti-His tag antibody.

[0024] Figure 2 The image shows the characterization of the gallic acid liposomes prepared in this invention. In the image, A is the particle size distribution of the liposomes; B is the leakage rate curve of the liposomes stored in toothpaste matrix at different temperatures (4℃, 25℃, 40℃) for 4 weeks.

[0025] Figure 3 The figures show the verification results of the antibody synergistic mechanism of the present invention. Among them, A is the flow cytometry detection of PI staining positivity rate, showing that the combined treatment of antibody and berberine significantly increased bacterial membrane permeability (PI positivity rate 76.8%); B is the HPLC determination of intracellular berberine content, showing that the intracellular berberine concentration increased to 5.78 times that of the control group after combined treatment.

[0026] Figure 4 This is a graph showing the synergistic effect combination index (CI) analysis of the antibody-plant extract complex of the present invention. The graph shows the CI values ​​and their 95% confidence intervals at effect levels fa = 0.5, 0.75, and 0.9. All CI values ​​are less than 1, demonstrating that the synergistic effect is statistically significant.

[0027] Figure 5 This is a graph showing the evaluation results of the whitening efficacy and safety of the toothpaste product of this invention. In the graph, A represents a comparison of the color difference value ΔE after treatment with different toothpaste formulations; B represents a comparison of the increase in tooth enamel surface roughness ΔRa after treatment with different toothpaste formulations.

[0028] Figure 6 This is a graph showing the results of antibody stability studies in the finished toothpaste of this invention. The graph illustrates the changes in antibody activity retention rates between the optimized formula (Formula B) and the basic formula (Formula A) after 24 months of storage at 25°C.

[0029] Figure 7 This is a schematic diagram illustrating the mechanism of action of the composition of the present invention in synergistically inhibiting Streptococcus mutans biofilm. The diagram schematically shows: ① Anti-PAc antibodies specifically bind to PAc proteins on the bacterial surface; ② Antibody binding increases bacterial cell membrane permeability; ③ Plant extracts such as berberine more easily enter the cell; ④ Intracellular berberine accumulates, synergistically exerting a bactericidal effect; ⑤ Bacterial death and biofilm structure collapse. Detailed Implementation

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] The embodiments of the present invention employ specific instruments, reagents, and operating methods, but these are merely exemplary and do not constitute a limitation thereof. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] Example 1: Preparation and Identification of Anti-PAc Protein Single-Domain Antibodies

[0035] 1.1 Animal Immunization and Immune Library Construction

[0036] 1.1.1 Immunogen and Animals: The immunogen was recombinant PAc protein (commercially purchased, or prepared according to the method of "Lu Guanfan, Du Xia, Jin Jie, et al. Prokaryotic expression and purification of Streptococcus mutans surface protein PAc [J]. Stomatological Research, 2010, 26(1):4.DOI:CNKI:SUN:KQYZ.0.2010-01-023."), with a purity >95% and a concentration of 1.0 mg / mL. One healthy male alpaca, 2 years old and weighing 45 kg, was selected and housed in an SPF-grade animal facility.

[0037] 1.1.2 Immunization regimen: PAc protein was thoroughly emulsified with an equal volume of adjuvant and administered via subcutaneous injection at multiple points on the back. A total of four immunizations were given, as detailed in Table 1.1.

[0038] Table 1.1 Immunization regimen

[0039]

[0040] 1.1.3 Lymphocyte Isolation and RNA Extraction: On day 7 (day 56) post-last immunization, 100 mL of blood was collected from the carotid artery and anticoagulated with heparin sodium. The blood was diluted with an equal volume of sterile PBS (pH 7.4) and slowly added to the supernatant of an equal volume of alpaca lymphocyte separation medium. The mixture was centrifuged at 400 ×g for 30 min (acceleration set to 0). The white membrane layer lymphocytes were carefully aspirated and washed twice with PBS (300 ×g, 10 min), yielding approximately 1.2 × 10⁻⁶ cells. 8 Lymphocytes. Total RNA was extracted using TRIzol® reagent according to the manufacturer's instructions. The resulting RNA precipitate was dissolved in DEPC water and analyzed using a NanoDrop 2000. The A260 / A280 ratio was 1.98, the concentration was 320 ng / μL, and the total amount was approximately 30 μg. 10 μg of total RNA was taken and processed using SuperScript. TM The IV reverse transcription kit was used to synthesize cDNA. The reaction conditions were: 25℃ for 10 min, 50℃ for 50 min, and 85℃ for 5 min to terminate the reaction.

[0041] 1.1.4 VHH gene amplification and library construction: The VHH gene sequence was amplified by nested PCR.

[0042] First round of PCR: Using cDNA as a template, the hinge regions of conventional antibodies and heavy chain antibodies are amplified. Second round of PCR: Using a fragment of approximately 600-700 bp from the first round PCR product as a template, the VHH gene is amplified.

[0043] Table 1.2 PCR reaction system (50 μL)

[0044]

[0045] PCR program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; 72℃ final extension for 10 min.

[0046] The second round of PCR products were subjected to 1.5% agarose gel electrophoresis, and the target band of approximately 400 bp was recovered by gel excision. The purified VHH gene and pMECS phage vector (laboratory-preserved) were double-digested with Pst I-HF and Not I-HF at 37℃ for 4 h, respectively. After gel excision and recovery, ligation was performed overnight at 16℃ using T4 DNA ligase. The ligation product was purified and electroporated into 100 μL of E. coli TG1 electrotransformed competent cells, and immediately 1 mL of 2×YT medium was added for recovery. An appropriate amount of transformation solution was plated onto 2×YT-AG plates (containing 100 μg / mL ampicillin and 2% glucose) and incubated overnight at 30℃. The next day, all colonies were scraped off, and glycerol was added to a final concentration of 20%, then stored at -80℃. The constructed VHH phage display library had a volume of 3.1 × 10⁻⁶ cells / mL. 8 CFU.

[0047] 1.2 Affinity screening and selection of specific antibodies

[0048] 1.2.1 Selection scheme: To increase the probability of obtaining high-affinity antibodies, a selection strategy of gradually increasing the washing intensity is adopted.

[0049] Table 1.3 Selection Scheme

[0050]

[0051] 1.2.2 Selection Procedure: Dilute PAc protein to the specified concentration with coating buffer (0.05 M carbonate buffer, pH 9.6), add 100 μL / well to a Maxisorp 96-well immunoassay plate, and incubate overnight at 4°C. Discard the coating buffer, add 300 μL of PBST (PBS containing 0.1% Tween-20) to each well and wash 3 times. Then add 300 μL of blocking buffer (3% BSA-PBS) to each well and block at 37°C for 2 h. Discard the blocking buffer, wash 3 times with PBST. Add 100 μL of phage library (approximately 10 μL of 1000 μL of phage library) to each well. 12 PFU (pre-blocked with an equal volume of blocking buffer for 30 min) was incubated at 37°C for 1 h. Unbound phages were discarded, and the cells were washed vigorously with PBST the corresponding number of times as described above. 100 μL of elution buffer (100 mM triethylamine, freshly prepared) was added to each well, and the cells were gently shaken at room temperature for 10 min. The elution buffer was immediately transferred to a centrifuge tube pre-filled with 50 μL of neutralization buffer (1 M Tris-HCl, pH 7.4) and mixed well. All of the neutralized elution product was used to infect 5 mL of logarithmically growing E. coli TG1 cells. The cells were incubated at 37°C for 30 min, then plated onto 2×YT-AG plates and incubated overnight at 30°C. Colonies were scraped off the next day for the next round of panning.

[0052] 1.2.3 Identification and screening of positive clones: After the fourth round of panning, 96 single clones were randomly selected from the output plates and inoculated into 96-well deep-well plates containing 2×YT-AG medium, and cultured at 37℃ with shaking until OD. 600 ≈0.5. Add M13K07 helper phage to each well, with a multiplicity of infection (MOI) of ≈20, and continue culturing overnight. The next day, centrifuge and collect the supernatant (containing phages displaying single-domain antibodies) for phage-ELISA screening.

[0053] ELISA Procedure: Coat a 96-well ELISA plate with PAc protein (2 μg / mL) and incubate overnight at 4°C. After blocking, add 50 μL of the supernatant and 50 μL of PBS, and incubate at 37°C for 1 h. After washing, add HRP-labeled anti-M13 monoclonal antibody (1:5000 dilution) and incubate at 37°C for 1 h. After TMB color development for 10 min, stop the reaction with 2 M H2SO4 and measure OD. 450 .

[0054] Result: with OD 450 A positive criterion was a value three times greater than the negative control (BSA-coated well) and an absolute value >1.0. A total of 18 positive clones were obtained. These clones were sequenced, yielding five unique VHH sequences. Among them, clone VHH-PAc17 exhibited the highest ELISA signal (OD). 450=2.85), and its amino acid sequence is shown in SEQ ID NO:1.

[0055] 1.3 Expression, purification and identification of single-domain antibodies

[0056] 1.3.1 Expression vector construction and transformation: The VHH-PAc17 gene was subcloned into the pSIP-409 expression vector (containing an N-terminal 6×His tag) via Nco I and Hind III restriction sites. After sequencing verification, the recombinant plasmid was electroporated into food-grade Lactobacillus paracasei BL23 competent cells. After recovery, the cells were plated on MRS plates containing 5 μg / mL erythromycin and anaerobically cultured at 30°C for 48 h.

[0057] 1.3.2 Fermentation Condition Optimization: A single colony was inoculated into 5 mL of MRS liquid medium (containing 5 μg / mL erythromycin) and incubated statically at 30°C overnight. The inoculum was then transferred to 100 mL of MRS medium at a 2% inoculation rate and incubated statically at 30°C. Different induction times (OD) were investigated. 600 The effects of different induction temperatures (20℃, 25℃, 30℃, 37℃) and induction times (4, 8, 12, 16, 20, 24 h) on the expression level of the soluble target protein were investigated. SDS-PAGE combined with grayscale analysis determined the optimal expression conditions to be: OD = 0.3, 0.6, 0.9, 1.2, and 0.6. The optimal expression conditions were determined to be: 600 When the concentration of the inducible peptide (SppIP, 50 ng / mL) was approximately 0.9, it was added, and the induction was carried out at 25°C for 20 h. Under these conditions, the expression level in the shake flask reached 3.2 mg / L.

[0058] 1.3.3 Purification Procedure: The fermentation broth was centrifuged at 8000 ×g for 20 min at 4℃, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane. The filtrate was loaded at a flow rate of 1 mL / min into HisTrap solution pre-equilibrated with equilibration buffer (20 mM PB, 500 mM NaCl, 20 mM imidazole, pH 7.4). TM FF 5 mL pre-packed column. After sample loading, wash with washing buffer (equilibration buffer + 40 mM imidazole) for 10 column volumes until baseline is stable. Finally, elute with elution buffer (equilibration buffer + 300 mM imidazole) and collect the elution peak. Add the collected eluent to an ultrafiltration centrifuge tube (MWCO 10 kDa) and concentrate by centrifugation at 4°C and 4000 ×g. Then dilute with PBS (pH 7.4) and repeat centrifugation 3 times to remove imidazole. Finally, filter sterilize through a 0.22 μm filter membrane, aliquot, and store at -80°C.

[0059] 1.3.4 Purity and Concentration Identification

[0060] SDS-PAGE: The purified sample was subjected to reducing SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The results showed ( Figure 1 A) There is a clear main band at approximately 15 kDa, and the purity is >95% as analyzed by ImageJ software.

[0061] Western Blot: Samples were transferred to a PVDF membrane after SDS-PAGE, incubated with mouse anti-His-tagged monoclonal antibody (1:5000, Sigma), and then incubated with HRP-labeled goat anti-mouse secondary antibody (1:10000). ECL staining was performed. Results showed ( Figure 1 B) Single specific band, with a molecular weight of approximately 15 kDa.

[0062] Concentration determination: A standard curve (R) was plotted using the BCA protein quantification kit with BSA as the standard. 2 =0.999). The concentration of purified antibody protein was measured to be 2.8 mg / mL.

[0063] 1.4 Determination of Affinity and Specificity

[0064] 1.4.1 ELISA Combination Assay (ECG) 50 ELISA plates were coated with PAc protein (2 μg / mL) and incubated overnight at 4°C. Purified VHH-PAc17 antibody was serially diluted 2-fold with PBS (0.098-100 nM) and added to each well, incubating at 37°C for 1 h. After washing, HRP-labeled His-tagged antibody (1:5000) was added, and the plates were incubated at 37°C for 1 h. TMB was used for color development, and OD was measured. 450 The results showed that, with antibody concentration as the x-axis, OD... 450 The value is the ordinate. Using GraphPad Prism software, a four-parameter logistic fit was performed to calculate EC. 50 =2.8 nM.

[0065] 1.4.2 Surface Plasmon Resonance (SPR) Affinity Assay: PAc protein was immobilized on a CM5 chip using an amino-coupling method on a Biacore T200 system, with a coupling amount of 1200 RU. The run buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P2O, pH 7.4). VHH-PAc17 antibody was diluted with the run buffer to six concentrations: 1.25, 2.5, 5, 10, 20, and 40 nM, and flowed through the chip at a flow rate of 30 μL / min. The binding time was 120 s, and the dissociation time was 300 s. After each cycle, the antibody was regenerated with 10 mM Gly-HCl (pH 2.0) for 30 s. Results showed that the affinity constant and binding rate constant k were obtained by fitting the kinetic data using a 1:1 binding model with Biacore T200 Evaluation software. a = 2.3×10 5 M -1 s -1 The dissociation rate constant k_d = 5.3 × 10 -4 s -1 The equilibrium dissociation constant K_D = 2.3 × 10 -9 M. This result indicates that the antibody has extremely high affinity.

[0066] 1.4.3 Specificity Validation: *Porphyromonas gingivalis*, *Prevotella intermedia*, and *Streptococcus sanguinis* were cultured to the logarithmic growth phase. Bacterial cells were collected, lysed, and whole-cell lysates were prepared. The lysates (10 μg / mL) and PAc protein (2 μg / mL, positive control) were coated onto ELISA plates, using 2.8 nM VHH-PAc17 antibody as the primary antibody. Subsequent steps were the same as before. Results showed that the antibody and PAc protein positive control wells had OD values... 450 The value was 2.75, with OD values ​​of 2.75 for wells containing lysates of three non-target bacteria. 450 The values ​​were 0.09, 0.11, and 0.08, respectively, compared with the negative control (BSA coated, OD...). 450 =0.07) No significant difference. This indicates that the antibody has good specificity and does not cross-react with other common oral flora.

[0067] Example 2: Preparation of natural plant extracts with synergistic effects

[0068] 2.1 Preparation and content determination of kudzu root extract

[0069] 2.1.1 Raw material: Pueraria lobata slices, produced in Bozhou, Anhui Province, which meet the standards of the 2020 edition of the Chinese Pharmacopoeia after identification.

[0070] 2.1.2 Extraction Process: Take 1 kg of kudzu root slices, pulverize them, and pass them through a 40-mesh sieve. Add 10 times the amount (10 L) of 70% ethanol and reflux at 80℃ for 2 h. After filtration, add 8 times the amount (8 L) of 70% ethanol to the filter residue and extract for 1.5 h using the same method. Combine the two filtrates and concentrate under reduced pressure at 50℃ (rotary evaporator, vacuum degree -0.08 MPa) to obtain an extract with a relative density of 1.10-1.15 (measured at 60℃). Spray dry the extract (inlet air temperature 180℃, outlet air temperature 80℃, feed rate 20 mL / min) to obtain 215 g of kudzu root extract powder, with a yield of 21.5%.

[0071] 2.1.3 Determination of puerarin content: High performance liquid chromatography was used for determination.

[0072] Chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid aqueous solution (25:75); flow rate: 1.0 mL / min; detection wavelength: 250 nm; column temperature: 30℃; injection volume: 10 μL.

[0073] Reference Standard and Standard Curve: Accurately weigh puerarin reference standard (China National Institutes for Food and Drug Control or other enterprises, purity ≥98%) and prepare a series of standard solutions with methanol at concentrations of 0, 10, 20, 50, 100, and 200 μg / mL. Perform linear regression of peak area (Y) against concentration (X, μg / mL) to obtain the standard curve equation: Y = 15.23X + 8.65, R² 2 =0.9998.

[0074] Test sample and determination: Accurately weigh 10 mg of kudzu root extract, dissolve in methanol and dilute to 10 mL. Take 1 mL, filter through a 0.22 μm filter membrane and inject for determination. The peak area corresponds to a concentration of 84.6 μg / mL. Calculate the content of puerarin in kudzu root extract as 84.6 × 10 / 10 × 100% = 42.3% (w / w).

[0075] 2.2 Preparation and content determination of Phellodendron amurense extract

[0076] 2.2.1 Raw material: Phellodendron chinense slices, produced in Sichuan Province, conforming to the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0077] 2.2.2 Extraction Process: Take 1 kg of Phellodendron bark slices and pulverize them through a 40-mesh sieve. Add 8 times (8 L) of 50% ethanol and extract with ultrasonic assistance at 50℃ (power 500 W, frequency 40 kHz) for 1 h. Filter, and extract the residue again using the same method. Combine the filtrates and concentrate under reduced pressure at 50℃ to recover ethanol until no alcohol odor remains, yielding approximately 2 L of concentrate. Load the concentrate onto a pre-treated D101 macroporous adsorption resin column (column volume 2 L, diameter-to-height ratio 1:10) at a flow rate of 2 BV / h. After loading, first elute with 5 BV of deionized water to remove water-soluble impurities, then elute with 5 BV of 70% ethanol, collecting the 70% ethanol eluent. Concentrate the eluent under reduced pressure at 50℃ to dryness and spray dry (under the same conditions as 2.1) to obtain 178 g of Phellodendron bark extract, with a yield of 17.8%.

[0078] 2.2.3 Berberine content determination: High performance liquid chromatography was used for determination.

[0079] Chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (50:50, containing 0.1% SDS); flow rate: 1.0 mL / min; detection wavelength: 345 nm; column temperature: 30℃; injection volume: 10 μL.

[0080] Reference Standard and Standard Curve: Accurately weigh berberine hydrochloride reference standard (China National Institutes for Food and Drug Control or other enterprises, purity ≥98%) and prepare a series of standard solutions with methanol at concentrations of 0, 5, 10, 20, 50, and 100 μg / mL. Regression of peak area (Y) against concentration (X, μg / mL) yields the standard curve equation: Y = 21.56X + 12.34, R² 2 =0.9997.

[0081] Test sample and determination: Accurately weigh 10 mg of Phellodendron amurense extract, dissolve it in methanol and dilute to 10 mL. Take 1 mL, filter it through a 0.22 μm filter membrane and inject it for determination. The peak area corresponds to a concentration of 53.6 μg / mL. Calculate the berberine content in the Phellodendron amurense extract as 53.6 × 10 / 10 × 100% = 26.8% (w / w).

[0082] Example 3: Preparation and stability evaluation of gallic acid-containing liposomes

[0083] 3.1 Liposome preparation and characterization (ethanol injection-high pressure homogenization method)

[0084] Table 3.1 Formulation

[0085]

[0086] 3.1.1 Preparation steps

[0087] (1) Preparation of oil phase: Dissolve lecithin and cholesterol in 20 mL of anhydrous ethanol and stir magnetically in a 50°C water bath until completely dissolved to obtain a clear oil phase.

[0088] (2) Preparation of aqueous phase: Gallic acid was dissolved in 100 mL of phosphate buffer (10 mM PBS, pH 6.5) preheated to 50 °C.

[0089] (3) Injection: Under the conditions of a 50°C water bath and magnetic stirring (500 rpm), the oil phase is slowly injected into the aqueous phase using a syringe at a rate of about 2 mL / min. After the addition is complete, stirring is continued for 30 min to form a crude liposome suspension.

[0090] (4) Removal of ethanol: The crude liposome suspension was rotary evaporated at 40°C (vacuum degree -0.08 MPa) for 30 min to remove residual ethanol.

[0091] (5) Homogenization and refinement: The ethanol-free suspension is transferred to a high-pressure homogenizer and homogenized three times at a pressure of 800 bar to obtain a liposome suspension with uniform particle size.

[0092] (6) Filtration: Sterilize by filtration with a 0.45 μm filter membrane, protect with nitrogen gas, and store at 4℃.

[0093] 3.1.2 Liposome Characterization

[0094] (1) Particle size and Zeta potential: An appropriate amount of liposome suspension was diluted 50 times with PBS and measured using a Malvern Zetasizer Nano ZS90 at 25℃. The results were: average particle size 142 ± 18 nm (n=3), polydispersity index (PDI) 0.21 ± 0.03 (n=3), indicating a narrow particle size distribution (e.g., ...). Figure 2 (As shown in Figure A). The zeta potential is -18.5 ± 2.3 mV (n=3).

[0095] (2) Encapsulation efficiency determination (ultrafiltration method):

[0096] Free drug: Add 1 mL of liposome suspension to an ultrafiltration centrifuge tube (MWCO 10 kDa), centrifuge at 4℃, 4000 ×g for 20 min, and collect the filtrate.

[0097] Total drug: Take 1 mL of liposome suspension, add 100 μL of 10% (v / v) Triton X-100 solution, vortex to break the emulsion, and bring the volume to 10 mL with PBS.

[0098] HPLC determination: Gallic acid content in the filtrate and demulsifier was determined separately. Chromatographic conditions: Agilent ZORBAX SB-C18 column, mobile phase: methanol-0.1% phosphoric acid water (30:70), detection wavelength: 270 nm.

[0099] Calculation: Free gallic acid concentration is 0.21 mg / mL. Total gallic acid concentration is 1.02 mg / mL. Encapsulation efficiency (EE%) = (Total concentration - Free concentration) / Total concentration × 100% = (1.02 - 0.21) / 1.02 × 100% = 79.4%.

[0100] 3.2 Stability study of liposomes in toothpaste matrix

[0101] 3.2.1 Experimental Design:

[0102] Preparation of blank toothpaste matrix: Prepare blank matrix according to the following formula: 15% hydrated silica, 45% sorbitol (70% solution), 10% glycerin, 1.2% CMC-Na, 1.5% sodium lauroyl sarcosinate, 0.5% xylitol, 0.3% p-hydroxyacetophenone, and the balance is deionized water.

[0103] Liposome incorporation: The liposome suspension prepared in Example 3.1 was slowly added to the blank toothpaste matrix at a final gallic acid concentration of 0.2% (w / w) and stirred until homogeneous.

[0104] Storage and Sampling: The liposome-containing toothpaste was dispensed into aluminum-plastic composite tubes, 20 g per tube, and placed in constant temperature incubators at 4℃, 25℃, and 40℃. Samples were taken at weeks 0, 1, 2, and 4 to determine the gallic acid leakage rate.

[0105] 3.3.2 Measurement methods and results:

[0106] Extraction of free gallic acid: Take 1.0 g of toothpaste, add 9 mL of PBS, and vortex until completely dispersed. Centrifuge at 4000×g for 15 min at 4℃, collect the supernatant, filter through a 0.22 μm filter membrane, and determine the free gallic acid content by HPLC.

[0107] Total gallic acid extraction: Take 1.0 g of toothpaste, add 9 mL of PBS and 1 mL of 10% Triton X-100, sonicate (200 W, 5 min) to break the emulsion, centrifuge and collect the supernatant, filter and determine the total gallic acid content by HPLC. The initial total gallic acid content was found to be 200 μg / g toothpaste, the initial free content was 41.2 μg / g toothpaste, and the initial encapsulation amount was 158.8 μg / g toothpaste.

[0108] Leakage rate calculation: Leakage rate (%) = (Free content at a certain time point - Initial free content) / Initial encapsulation amount × 100%.

[0109] The results are shown in Table 3.2. After storage at 4℃ for 4 weeks, the leakage rate was only 8.8%, indicating that liposomes are very stable at low temperatures. After storage at 25℃ (simulated room temperature storage) for 4 weeks, the leakage rate was 22.1%, indicating that liposomes have good room temperature stability in toothpaste matrix (e.g., Figure 2 As shown in Figure B, it meets the basic requirements for product development. Under accelerated conditions at 40°C, the leakage rate increased significantly, reaching 34.2% after 4 weeks, providing data reference for accelerated prediction of product shelf life.

[0110] Table 3.2 Leakage rate under different storage conditions

[0111]

[0112] Example 4: Preparation and stability optimization of single-domain antibody lyophilized powder

[0113] 4.1 Screening of Lyophilization Protectants

[0114] Methods: The purified VHH-PAc17 antibody (5 mg / mL) from Example 1 was mixed with different protective agents in a certain proportion and dispensed into 2 mL vials, 1 mL per vial. The samples were freeze-dried according to the freeze-drying curve in section 4.2. After freeze-drying, the appearance was observed, and 1 mL of water for injection was added to reconstitute the antibody. The activity retention rate of the reconstituted antibody was determined (ELISA method, with the sample before freeze-drying as 100% control). The results are shown in Table 4.1. Trehalose showed the best protective effect on the single-domain antibody, with an activity retention rate of 95.8%. Adding 0.02% polysorbate 80 (F6) to the 5% trehalose base further improved the reconstitution of the freeze-dried cake and increased the activity retention rate to 97.3%. Therefore, the F6 formulation was selected as the final freeze-drying protective agent.

[0115] Table 4.1 Screening results of lyophilization protectants

[0116]

[0117] 4.2 Freeze-drying process and characterization of freeze-dried powder

[0118] 4.2.1 Freeze-drying profile (performed on a freeze dryer):

[0119] Pre-freezing: Place the sample on a -40℃ shelf and freeze for 2 hours to ensure the sample is completely frozen.

[0120] First drying (sublimation drying): Raise the shelf temperature to -20°C, maintain a vacuum of 0.1 mbar, and dry for 48 hours to remove most of the ice crystals.

[0121] Secondary drying (desorption drying): Raise the shelf temperature to 25°C, maintain a vacuum of 0.01 mbar, and dry for 12 hours to remove bound water.

[0122] Endpoint determination: When the product temperature is close to the shelf temperature and the vacuum degree remains stable, it is considered the drying endpoint.

[0123] 4.2.2 Characterization of lyophilized powder:

[0124] Appearance: The freeze-dried cakes prepared according to the F6 formula are white, plump, smooth, and without collapse or shrinkage.

[0125] Moisture content: The moisture content of the lyophilized powder was 2.3% (<3%, which meets the requirements for lyophilized protein drug injection) as determined by a Karl Fischer moisture analyzer.

[0126] Reconstitution time: After adding 1 mL of water for injection and gently shaking, the lyophilized cake completely dissolves within 30 seconds, and the solution becomes clear.

[0127] Activity after reconstitution: The antibody activity after reconstitution, as determined by ELISA, was 97.3% of that before lyophilization.

[0128] Particle size after reconstitution: Dynamic light scattering analysis showed no particles larger than 100 nm in the reconstituted solution.

[0129] 4.3 Accelerated and Long-Term Stability Study of Antibody Lyophilized Powder

[0130] Methods: The F6 formulation lyophilized powder (sealed with a stopper) was placed in stability test chambers at 25℃ / 60% RH and 40℃ / 75% RH, respectively. Samples were taken at different time points, and after reconstitution, the activity retention rate (ELISA) and soluble aggregate content (SEC-HPLC) were determined. The results are shown in Table 4.2. After 24 months of storage at 25℃, the antibody lyophilized powder still exhibited an activity retention rate higher than 90%, and the aggregate content was only about 2.3%, demonstrating excellent long-term stability (as shown in Table 4.2). Under accelerated conditions at 40℃, the activity retention rate was still 86.8% after 6 months. These results indicate that, through lyophilization and the selection of a suitable protective agent, antibody raw materials meeting the 2-year shelf-life requirement for commercial products can be prepared.

[0131] Table 4.2 Results of accelerated and long-term stability experiments of antibody lyophilized powder

[0132]

[0133] Example 5: Optimized toothpaste formulation containing synergistic ingredients

[0134] Based on the aforementioned research on each active ingredient, this embodiment designs an optimized toothpaste formulation and its preparation process with enhanced stability.

[0135] Table 5.1 Optimized formula (based on 100 g total weight)

[0136]

[0137] *Note: Liposome suspensions contain a large amount of water, and their water content must be accurately calculated and deducted from the total water content of the formula.

[0138] 5.1 Preparation process (low-temperature phase separation addition method):

[0139] 5.1.1 Preparation of the gel phase (aqueous phase): Disperse CMC-Na and xylitol in sorbitol and glycerol, and stir until homogeneous. Add approximately 30 g of deionized water, heat to 65°C, and stir for 30 min until completely swollen, forming a uniform and transparent gel matrix. Cool to below 40°C for later use.

[0140] 5.1.2 Addition of buffer solution: Dissolve histidine in a small amount of deionized water, add it to the above gel phase, and stir well.

[0141] 5.1.3 Addition of stabilizer: Add trehalose and stir until completely dissolved.

[0142] 5.1.4 Addition of plant extracts: Dissolve kudzu root extract and phellodendron bark extract separately in a small amount of deionized water, then add them to the system and stir until homogeneous.

[0143] 5.1.5 Addition and degassing of abrasive: Add hydrated silica and stir at low speed (-0.08 MPa) for 20 min in a vacuum mixer to mix the paste evenly and remove air bubbles.

[0144] 5.1.6 Addition of liposomes: Turn off the vacuum, add gallic acid liposome suspension, and stir at low speed (200 rpm) for 5 min to disperse it evenly.

[0145] 5.1.7 Cooling and Antibody Addition: Cool the paste to below 25°C. Add the single-domain antibody lyophilized powder and continue stirring at low speed (200 rpm) for 10 min under normal pressure to ensure that the antibody is dissolved evenly without generating bubbles.

[0146] 5.1.8 Addition of anti-adsorbent: Add polysorbate 80 and mix gently for 5 min.

[0147] 5.1.9 pH adjustment: Use a pre-prepared citric acid or sodium citrate solution to precisely adjust the pH of the paste to 6.2±0.1.

[0148] 5.1.10 Filling: After the paste has been allowed to stand for 1-2 hours to degas, it is filled into aluminum-plastic composite tubes with nitrogen gas, 120g per tube, and sealed for storage.

[0149] Experimental Example 1: Study on the synergistic antibacterial and membrane permeability mechanism of antibody-plant extract

[0150] 1.1 Detection of bacterial membrane permeability (PI staining flow cytometry)

[0151] 1.1.1 Objective: To investigate whether anti-PAc antibodies can increase the permeability of the cell membrane of Streptococcus mutans, thereby synergistically allowing plant extracts (mainly berberine) to enter the cell.

[0152] 1.1.2 Method:

[0153] (1) Preparation of bacterial culture: Streptococcus mutans ATCC 25175 was cultured to the logarithmic growth phase, the bacterial cells were collected by centrifugation, washed with PBS and resuspended, and the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0154] (2) Group treatment (final volume 1 mL):

[0155] Control group: PBS.

[0156] Antibody group: 0.005% anti-PAc antibody (50 μg / mL).

[0157] Berberine group: Berberine hydrochloride 20 μg / mL (Sigma).

[0158] Combined group: antibody 0.005% + berberine 20 μg / mL.

[0159] (3) Incubation and staining: After incubation at 37℃ for 30 min, centrifuge (5000 ×g, 5 min) to collect the bacterial cells, and wash twice with PBS. Add PI staining solution (5 μg / mL, Sigma), and stain at room temperature in the dark for 15 min. Wash with PBS and resuspend.

[0160] (4) Flow cytometry: 10,000 events were collected per sample using a flow cytometer. PI fluorescence was detected in the FL2 channel, and a positive result indicated cells with membrane damage.

[0161] 1.1.3 The results are shown in Table 5.2:

[0162] Antibody treatment alone significantly increased the PI positivity rate to 28.5%, indicating that the binding of anti-PAc antibodies to the surface antigen caused cell membrane structural disturbances and increased permeability. The combined group had a PI positivity rate as high as 76.8%, far exceeding the sum of the antibody group and the berberine group (28.5% + 8.7% = 37.2%), suggesting a synergistic effect of both treatments on disrupting membrane structure (e.g., ...). Figure 3 (As shown in A).

[0163] Table 5.2 Results of bacterial membrane permeability detection (PI staining flow cytometry)

[0164]

[0165] Note: p < 0.05 vs. PBS control; ** p < 0.001 vs. the other three groups (one-way ANOVA + Turkey test).

[0166] 1.2 Determination of intracellular berberine content (HPLC)

[0167] 1.2.1 Objective: To directly and quantitatively detect whether antibodies promote the entry of berberine into bacteria.

[0168] 1.2.2 Method:

[0169] (1) Preparation of bacterial culture: Increase the volume of bacterial culture and adjust the concentration to 5×10⁻⁶. 8 CFU / mL.

[0170] (2) Group treatment (final volume 10 mL):

[0171] Berberine group: berberine 20 μg / mL.

[0172] Combined group: antibody 0.005% + berberine 20 μg / mL.

[0173] (3) Incubation and sample processing: After incubation at 37℃ for 30 min, the bacterial cells were collected by centrifugation and washed three times with PBS to completely remove the extracellular adsorbed berberine. The bacterial cells were resuspended in 1 mL of PBS and sonicated (200 W, 3 s on, 3 s off, for a total of 10 min). The lysate was centrifuged at 12,000 × g for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane.

[0174] (4) HPLC determination: The berberine content in the supernatant was determined according to the chromatographic conditions in Example 2.2, and calculated based on the standard curve.

[0175] 1.2.3 The results are shown in Table 5.3:

[0176] The intracellular berberine content in the combined treatment group was 1.85 μg / 10. 9 CFU was used in the berberine-only treatment group (0.32 μg / 10). 9 5.78 times that of CFU (e.g.) Figure 3 (As shown in B). This directly demonstrates that anti-PAc antibodies significantly promote the intracellular accumulation of berberine by increasing bacterial membrane permeability.

[0177] Table 5.3 Results of intracellular berberine content determination (HPLC)

[0178]

[0179] Note: p < 0.001 vs. berberine group (t-test).

[0180] This experiment reveals the core mechanism of the synergistic effect of the present invention: after the anti-PAc antibody binds to the target, it induces an increase in bacterial cell membrane permeability, creating a channel for antibacterial components such as berberine to enter the cell, increasing their accumulation in the cell by nearly 6 times, thereby exerting a synergistic bactericidal effect.

[0181] Experiment Example 2: Comparative Experiment with Existing Technologies

[0182] 2.1 Objective: To compare the clearance ability of the anti-PAc antibody of the present invention with that of the prior art (anti-GTF antibody) on mature biofilms, and to demonstrate the unexpected technical effects of the present invention.

[0183] 2.2 Materials:

[0184] Anti-PAc antibody: VHH-PAc17 prepared in Example 1 of this invention.

[0185] Anti-GTF antibody: The corresponding GTF protein was prepared according to the method described in the patent (Liu Qian. Recombinant expression, purification, activity identification and preliminary establishment of anti-GTF-I hybridoma cell line of Streptococcus mutans type I glucosyltransferase (GTF-I) [D]. Yunnan University, 2008.). Alpacas were immunized according to the method in Example 1, and single-domain antibodies against GTF (glucosyltransferase) were obtained from the phage library. Enamel slides and bacterial strains: Same as in Experiment 3.

[0186] 2.3 Methods:

[0187] Biofilm culture: Two biofilm models were established, one for 24 h (immature) and the other for 48 h (mature).

[0188] Antibody treatment: Dilute the two antibodies to 0.01% (100 μg / mL) with PBS and treat the dental slides for 5 min.

[0189] Clearance rate determination: The biofilm clearance rate after antibody treatment was determined by viable cell counting. The control group was treated with PBS.

[0190] 2.4 The results are shown in Table 5.4:

[0191] For immature biofilms at 24 h, the clearance rate of anti-GTF antibody was 78.5%, while that of anti-PAc antibody was 85.3%, with the latter being slightly better (p<0.05). For mature biofilms at 48 h, the clearance rate of anti-GTF antibody dropped sharply to 41.5% (a decrease of 37 percentage points). However, the clearance rate of anti-PAc antibody remained as high as 92.3%, only about 7 percentage points lower than that of immature biofilms, and the difference between the two was highly significant (p<0.001).

[0192] Table 5.4 Summary of Comparative Experiment Results

[0193]

[0194] Note: p < 0.05, ** p < 0.001 vs. anti-GTF antibody group (t-test).

[0195] The anti-PAc antibody of this invention exhibits a significantly higher clearance capacity than existing technologies (anti-GTF antibodies) against the core problem of cariogenic biofilms—mature biofilms. This result breaks through the conventional wisdom in the field that antibody drugs "emphasize prevention over treatment," meaning that targeting bacterial surface structural antigens (PAc) rather than their metabolic enzymes (GTF) can more effectively dismantle the already formed biofilm structure, resulting in unexpected technical effects.

[0196] Experiment Example 3: Verification of Complete Synergistic Effect and Joint Index Analysis

[0197] 3.1 Objective: To comprehensively verify the synergistic effect of anti-PAc antibody and plant extract complex on mature biofilm of Streptococcus mutans under different dosage combinations using the classic intermediate-effect principle (Chou-Talalay method).

[0198] 3.2 Methods:

[0199] Drug and dosage settings: Antibody (Ab): 0.002%, 0.005%, 0.008% (w / v, i.e., 20, 50, 80 μg / mL).

[0200] Plant extract complex (PE): Pueraria lobata extract and Phellodendron amurense extract were mixed at a mass ratio of 3:1, with total concentrations of 0.5%, 1.0%, and 2.0% (w / v).

[0201] Biofilm model and treatment: Following the method in Experiment Example 2, a mature biofilm model was established after 48 hours. Enamel slides were treated for 5 minutes with different concentrations of single or combined drug solutions (a total of 9 combinations + 8 single drug dose points + blank control).

[0202] Data reading: The number of viable bacteria remaining in each group after treatment was determined by the viable bacteria counting method (CFU / dental film), and the inhibition rate (fa, fraction affected) relative to the blank control was calculated.

[0203] Data analysis: Input the data into CompuSyn software to calculate the effects of each combination at different effect levels (e.g., IC50). 50 IC 75 IC 90 The combination index (CI) is used to determine the synergy between two factors. CI < 1 indicates synergy, CI = 1 indicates addition, and CI > 1 indicates antagonism.

[0204] 3.3 The results are shown in Tables 5.5 and 5.6:

[0205] (1) General synergy: The CI values ​​of all 9 test combinations were less than 1, and the upper limit of their 95% confidence intervals were also less than 1, indicating that the antibody and plant extract showed statistically significant synergistic effects at all test doses. Figure 4 ).

[0206] (2) Dose-dependent: As the concentration of antibody and plant extract increases, the CI value gradually decreases, the synergistic effect is enhanced, and it shows dose dependence.

[0207] (3) Optimal combination: The inhibition rates of mature biofilms reached 96.8% and 98.2% respectively in the combination of Ab 0.005% + PE 2.0% and Ab 0.008% + PE 2.0%, with CI values ​​of 0.52 and 0.48 respectively, showing a "strong synergistic" effect, which is the optimal efficacy ratio range of the present invention.

[0208] Table 5.5 Inhibition rate (%) of different dosage combinations on 48h mature biofilm (Mean ± SD, n=3)

[0209]

[0210] Table 5.6 Calculation of Joint Index (CI) and Determination of Synergy

[0211]

[0212] Experiment Example 4: Verification of the efficacy of the finished toothpaste product

[0213] 4.1 Verification of anti-biofilm efficacy

[0214] 4.1.1 Objective: To verify the effect of the final toothpaste formulation of the present invention (Example 5) on the removal of mature biofilm of Streptococcus mutans.

[0215] 4.1.2 Method:

[0216] (1) Grouping: A 48-h mature biofilm model was established according to the method in Experiment Example 2, and the models were divided into the following treatment groups (n=10):

[0217] Negative control group: physiological saline.

[0218] Control group of commercially available products: Commercially available brand "Yichibai" anti-caries toothpaste (containing fluoride).

[0219] The toothpaste group of the present invention: the toothpaste prepared according to Example 5.

[0220] (2) Treatment: Prepare a 20% (w / v) suspension of toothpaste with PBS. Immerse the tooth slices loaded with biofilm in the above suspension or physiological saline and treat them in a constant temperature shaker (37°C, 150 rpm) for 5 min.

[0221] (3) Detection: The treated dental slides were gently washed three times with PBS, and then viable bacteria were counted to calculate the biofilm clearance rate.

[0222] 4.1.3 The results are shown in Table 5.7: The toothpaste of the present invention has excellent removal ability (>97%) against mature Streptococcus mutans biofilm, which is significantly better than commercially available fluoride toothpaste (61%).

[0223] Table 5.7 Results of Anti-biofilm Efficacy Verification

[0224]

[0225] Note: p < 0.001 vs. commercially available control group.

[0226] 4.2 Whitening Efficacy Verification

[0227] 4.2.1 Objective: To verify the toothpaste of the present invention's effect on removing extrinsic tooth stains.

[0228] 4.2.2 Method:

[0229] (1) Establishing an exogenous staining model: Following the method in the literature, 60 pieces of bovine tooth enamel (5×5 mm) were prepared. They were soaked in a mixture of coffee, black tea and mucin for 30 min daily and cultured in BHI medium containing Streptococcus mutans for 7 consecutive days to form exogenous staining.

[0230] (2) Grouping and treatment: The tooth blocks were randomly divided into 6 groups (n=10). The toothpaste suspension (20%) of each group was treated in the same way as in 4.1.

[0231] Group A: Commercially available natural whitening toothpaste (containing zinc phytate and calcium carbonate).

[0232] Group B: Toothpaste base containing only plant extracts (2.0%).

[0233] Group C: Toothpaste base containing only antibodies (0.008%).

[0234] Group D: Toothpaste containing antibodies (0.005%) and plant extracts (2.0%) (liposome-free).

[0235] Group E: Example 5 of the present invention, full-formula toothpaste.

[0236] Group F: Negative control (blank toothpaste base).

[0237] (3) Color difference measurement: The L, a, and b values ​​of the tooth surface were measured before and after treatment using a colorimeter, and the total color difference ΔE = √[(ΔL)] was calculated. 2 + (Δa) 2 + (Δb) 2 ].

[0238] (4) Surface roughness measurement: The Ra value of the tooth surface before and after treatment is measured with a roughness meter, the increase value ΔRa is calculated, and the degree of damage to the enamel is assessed.

[0239] 4.2.3 The results are shown in Table 5.8:

[0240] Whitening effect: The total formula toothpaste of this invention (Group E) had the highest ΔE value of 10.58, significantly better than commercially available whitening toothpastes (5.12), those containing only plant extracts (4.68), and those containing only antibodies (7.23). Figure 5 As shown in Figure A, this indicates that antibodies and plant extracts have a synergistic effect in removing exogenous stains.

[0241] Enamel damage: All formulation groups containing the antibody of this invention (C, D, E) showed no significant difference in the increase in enamel surface roughness (ΔRa) compared to the blank matrix (F group), and were far lower than those of commercially available whitening toothpaste containing abrasives (A group). Figure 5 As shown in Figure B), this toothpaste effectively whitens teeth while causing almost no damage to tooth enamel, thus offering higher safety.

[0242] Therefore, the toothpaste of this invention has excellent synergistic anti-biofilm and highly effective and safe whitening effects, and its overall performance is significantly better than that of commercially available control products.

[0243] Table 5.8 Results of Whitening Efficacy Verification

[0244]

[0245] Note: p < 0.001 vs. groups A, B, C, and F (one-way ANOVA). There was no significant difference between group E and group D (p = 0.12).

[0246] Experiment Example 5: Stability Study of Single-Domain Antibodies in Toothpaste

[0247] 5.1 Objective: To compare the long-term stability of single-domain antibodies in the optimized formulation (Example 5) and the basic formulation (a simple mixture without buffer, without protectant, and at pH 6.8) and to verify the effectiveness of the optimization strategy.

[0248] 5.2 Methods:

[0249] (1) Prepare two types of toothpaste:

[0250] Formula A (Basic Formula): The antibody is added in liquid form (0.008%), without histidine buffer, trehalose and polysorbate 80, and the remaining matrix components are the same as in Example 5, pH 6.8.

[0251] Formula B (Optimized Formula): Prepared according to the complete formula and process of Example 5.

[0252] (2) Storage and sampling: After the two types of toothpaste were dispensed, they were placed at 25℃ / 60% RH and 40℃ / 75% RH respectively. Samples were taken at 0, 3, 6, 12, 18 and 24 months.

[0253] (3) Antibody extraction and activity assay:

[0254] Extraction: Take 1.0 g of toothpaste, add 9 mL of high-salt extraction buffer (PBS containing 1 M NaCl, pH 7.4), vortex for 30 min, and centrifuge at 10,000 ×g for 20 min at 4℃. Collect the supernatant, concentrate it using an ultrafiltration tube (10 kDa), and replace it with PBS buffer.

[0255] Activity assay: The relative activity of the antibody in the extract was determined by ELISA (with the activity of the 0 month sample as 100%).

[0256] Aggregate content: The peak area ratio of antibody monomers and aggregates in the extract was determined by size exclusion chromatography (SEC-HPLC, TSKgel G2000SWXL column).

[0257] 5.3 The results are shown in Tables 5.9 and 5.10:

[0258] After 24 months of storage at 25℃, the activity retention rate of formulation B (90.2%) was significantly higher than that of formulation A (45.3%) (t=13.25, p<0.001). Arrhenius equation fitting predicted that formulation B would retain 90% of its activity at 25℃ (t=13.25, p<0.001). 90 The shelf life of Formula B is 28.3 months, while that of Formula A is only 8.5 months. After 24 months of storage at 25°C, the aggregate content of Formula B is only 2.5%, far lower than that of Formula A (22.3%).

[0259] Therefore, the optimized formulation (Formula B) significantly improved the stability of the single-domain antibody in the toothpaste matrix by introducing a pH buffer system, a conformational stabilizer (trehalose), and an anti-adsorption agent (polysorbate 80), and by adding it in a lyophilized state. Figure 6 As shown in the figure, it retains more than 90% of its activity after being stored at 25°C for 2 years, fully meeting the shelf-life requirements of commercial products. However, the basic formulation (Formulation A) has poor stability and cannot meet the requirements for market launch.

[0260] Table 5.9 Changes in antibody activity retention rate (%) over time in different toothpaste formulations (Mean ± SD, n=3)

[0261]

[0262] Table 5.10 Changes in antibody aggregate content (%) over time at 25℃ (Mean ± SD, n=3)

[0263]

[0264] In summary, the anti-PAc single-domain antibody, the synergistic composition comprising the antibody and plant extracts, and the oral care product (especially toothpaste) comprising the composition provided by this invention possess excellent mature biofilm clearance capabilities, significant synergistic effects, a unique mechanism of action, and outstanding product stability. The preparation process of this composition and its products is mature, the raw materials are readily available, and it has good prospects for industrial production and market application value. A schematic diagram of its mechanism of action is shown below. Figure 7 As shown.

[0265] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-domain antibody targeting the surface protein PAc of Streptococcus mutans, characterized in that, The single-domain antibody is VHH-PAc17, and its amino acid sequence is shown in SEQ ID NO:

1.

2. An oral care composition, characterized in that, The composition comprises the following synergistic components: (1) The single-domain antibody VHH-PAc17 according to claim 1; (2) A plant extract complex, said complex being composed of kudzu root extract and phellodendron bark extract; and (3) Gallic acid liposomes.

3. The oral care composition according to claim 2, characterized in that, The kudzu root extract contains 42.3% puerarin, the phellodendron bark extract contains 26.8% berberine, and the mass ratio of the kudzu root extract to the phellodendron bark extract is 3:

1.

4. The oral care composition according to claim 2, characterized in that, The gallic acid liposomes have an average particle size of 142 ± 18 nm, a polydispersity index of 0.21 ± 0.03, a zeta potential of -18.5 ± 2.3 mV, and an encapsulation efficiency of 79.4% for gallic acid.

5. The oral care composition according to claim 2, characterized in that, The composition is a lyophilized powder containing a lyophilization protectant, which consists of 5% trehalose and 0.02% polysorbate 80.

6. An oral care product, characterized in that, The product comprises the oral care composition according to any one of claims 2 to 5 and oral-acceptable excipients.

7. The oral care product according to claim 6, characterized in that, The product is toothpaste, and the toothpaste contains the following components by weight percentage: (1) Single-domain antibody VHH-PAc17 lyophilized powder: 0.01%; (2) Pueraria lobata extract: 1.5%; (3) Phellodendron bark extract: 0.5%; (4) Gallic acid liposomes: The amount added makes the final concentration of gallic acid in the toothpaste 0.2%; (5) pH buffer: 0.3% histidine is used to maintain the toothpaste pH at 6.2 ± 0.1; (6) Stabilizer: 2.0% trehalose; (7) Anti-adsorbent: 0.02% polysorbate 80; (8) Abrasive: 15.0% hydrated silica; (9) Moisturizers: 40.0% sorbitol and 10.0% glycerin; (10) Adhesive: 1.2% sodium carboxymethyl cellulose; (11) Surfactant: 1.5% sodium lauroyl sarcosinate; (12) Solvent: Deionized water to 100 g.

8. A method for preparing the toothpaste of claim 7, characterized in that, The method employs a low-temperature phase separation addition method, and the specific steps are as follows: (1) Preparation of the gel phase: Disperse sodium carboxymethyl cellulose and xylitol in sorbitol and glycerol, add deionized water, heat to 65°C, stir until completely swollen, and cool to below 40°C; (2) Add histidine and trehalose, and stir well; (3) Add kudzu root extract and phellodendron bark extract, and stir well; (4) Add hydrated silica, stir and mix under vacuum and degas; (5) Close the vacuum, add the gallic acid liposome suspension, and stir at low speed to disperse; (6) Cool the paste to below 25°C, add the single-domain antibody lyophilized powder, and stir at low speed under normal pressure until it is uniformly dissolved; (7) Add polysorbate 80 and mix; (8) Adjust the pH of the paste to 6.2±0.1 using citric acid or sodium citrate solution; (9) Fill after standing to remove bubbles.

9. Use of an oral care composition according to any one of claims 2 to 5 or an oral care product according to any one of claims 6 or 7 in the preparation of products for removing or inhibiting oral pathogenic bacterial biofilms and for whitening teeth.

10. The use according to claim 9, characterized in that, The oral pathogens include Streptococcus mutans, and the biofilm includes a mature biofilm cultured for 48 hours.

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