CN114099413A

CN114099413A - Probiotic and tea polyphenol compound composition, preparation and application thereof

Info

Publication number: CN114099413A
Application number: CN202111453744.5A
Authority: CN
Inventors: 陈健芬; 郑雪峰; 郑根大
Original assignee: Suzhou Jinmao Daily Chemicals Co ltd
Current assignee: Suzhou Jinmao Daily Chemicals Co ltd
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-03-01

Abstract

The occurrence of oral diseases is closely related to the adhesion and colonization of oral pathogenic bacteria and the formation of a biological membrane, the application of probiotics to prevent and treat the oral diseases is concerned more and more, and meanwhile, the tea polyphenol is widely applied to food as a natural bacteriostatic agent. The invention provides a composition compounded by probiotics and tea polyphenol, a preparation and application thereof, and the application of methods such as a Scanning Electron Microscope (SEM), an Atomic Force Microscope (AFM), a Fourier infrared spectrum (FT-IR), surface charge analysis and the like proves that the composition realizes a synergistic antibacterial effect through co-coagulation and sterilization effects, is used for preventing and treating oral diseases, and is applied to oral cleaning and nursing products such as toothpaste and the like.

Description

Probiotic and tea polyphenol compound composition, preparation and application thereof

Technical Field

The invention relates to the technical field of medical biological preparations or oral health products, in particular to a composition compounded by probiotics and tea polyphenol, a preparation and application thereof.

Background

The occurrence of oral diseases is closely related to the adhesive colonization of oral pathogens and the formation of biofilms. Especially the biomembrane formed by pathogenic bacteria has strong adaptability to the external environment. The oral pathogenic bacteria can not only cause oral microecological imbalance so as to induce oral diseases, but also be one of the disease inducing factors such as Alzheimer's disease, pneumonia, tumor, diabetes and the like. Actinobacillus actinomycetemcomitans is a gram-negative oral pathogenic bacterium with high pathogenic potential, and is easy to breed and colonize in the oral cavity to form dental plaque biomembranes with complex structures to cause periodontal diseases. In addition, actinobacillus actinomycetemcomitans can secrete and synthesize various high toxicity factors to cause the occurrence and development of periodontitis, and further cause symptoms such as gingival bleeding, tooth loosening, halitosis and the like. Research reports that actinobacillus actinomycetemcomitans has certain correlation with diseases such as gastric cancer, leukemia and the like.

In recent years, the use of probiotics for the prevention and treatment of oral diseases has received increasing attention. Compared with the traditional mechanical therapy and pharmaceutical therapy, the probiotics can play roles in regulating the oral micro-ecological balance, inhibiting a biological membrane, destroying the virulence of pathogenic bacteria and the like. Probiotics improve oral health by competing with oral pathogens for binding sites, hindering colonization by the pathogens, thereby allowing the pathogens to be excreted from the oral cavity. Metabolites such as organic acids, bacteriocins and the like generated by probiotics can effectively inhibit the growth of pathogenic bacteria and the formation of a biological membrane. Research shows that after the probiotic is inactivated, the probiotic still can exert corresponding effects on organisms, and the effects are superior to those of live bacteria. The lactobacillus paracasei is the most common lactobacillus in oral microbiota, and the inactivated bacteria powder can generate coagulation precipitation with oral pathogenic bacteria. Research shows that the co-aggregation capacity of probiotics and pathogenic bacteria has great relation with the hydrophobicity of probiotics, probably because the surface protein of thallus influences the surface property of the thallus. However, the co-aggregation mechanism of probiotic inactivated bacteria powder against oral pathogens is currently unknown.

The tea polyphenol is widely applied to food as a green pollution-free natural bacteriostatic agent. The tea polyphenol can inhibit the formation of an oral cavity pathogenic bacteria biomembrane, kill pathogenic bacteria by destroying bacterial membranes and bacterial walls, inhibiting nucleic acid synthesis and the like, and inhibit the pathogenic bacteria from generating harmful metabolites and proinflammatory related gene expression. The existing research shows that the tea polyphenol has the inhibiting effect on oral pathogenic bacteria, and is widely used in toothpaste products at present.

The current research shows that although probiotics and tea polyphenol are common active ingredients for preventing and treating oral diseases in the current market, no report is found in the research on the synergistic bacteriostasis mechanism of inhibiting the oral pathogenic bacteria by compounding the tea polyphenol and the probiotics. Because the development of oral diseases has the characteristics of long-term and slow development, no safe and effective medicine or oral health-care product is developed for preventing or treating the oral diseases in the market at present. Meanwhile, medicines or oral health products for preventing and treating oral diseases in the market generally have the problems of poor antibacterial effect and unclear antibacterial mechanism. Therefore, in view of the above-mentioned drawbacks, there is a need to develop a drug or an oral health product for preventing and/or treating oral diseases, which has a better antibacterial effect and a clear mechanism, and is of great significance for preventing and treating oral diseases.

Disclosure of Invention

The invention mainly aims to provide a composition compounded by probiotics and tea polyphenol, a preparation and application thereof, and aims to solve the problems of poor antibacterial effect and undefined antibacterial mechanism of common oral disease prevention and treatment medicines or oral health products in the prior art.

In order to achieve the above objects, one of the objects of the present invention is to provide a composition of probiotic bacteria compounded with tea polyphenol, the composition comprising: probiotics and tea polyphenol.

Further, the composition consists of the following raw materials in parts by weight: 1-100 parts of probiotics and 1-100 parts of tea polyphenol.

Further, the composition consists of the following raw materials in parts by weight: 1-50 parts of probiotics and 1-50 parts of tea polyphenol.

Further, the composition consists of the following raw materials in parts by weight: 1-10 parts of probiotics and 1-10 parts of tea polyphenol.

Further, the composition consists of the following raw materials in parts by weight: 4-8 parts of probiotics and 1-6 parts of tea polyphenol.

Further, the composition consists of the following raw materials in parts by weight: 8 parts of probiotics and 1 part of tea polyphenol; or, the composition is composed of the following raw materials in parts by weight: 4 parts of probiotics and 1 part of tea polyphenol; or, the composition is composed of the following raw materials in parts by weight: 2 parts of probiotics and 1 part of tea polyphenol; or, the composition is composed of the following raw materials in parts by weight: 4 parts of probiotics and 3 parts of tea polyphenol.

Further, in the composition compounded by the probiotics and the tea polyphenol, the probiotics are one or more of saccharomycetes, probiotics bacillus, clostridium butyricum, lactobacillus, bifidobacteria or actinomycetes.

Further, in the composition compounded by the probiotics and the tea polyphenol, the probiotics is lactobacillus.

Further, in the composition compounded by the probiotics and the tea polyphenol, the probiotics is lactobacillus paracasei.

Furthermore, in the composition compounded by the probiotics and the tea polyphenol, the concentration of the probiotics is 0.001 g/mL-0.05 g/mL.

Furthermore, in the composition compounded by the probiotics and the tea polyphenol, the concentration of the probiotics is 0.01 g/mL-0.03 g/mL.

Further, in the composition compounded by the probiotics and the tea polyphenol, the concentration of the probiotics is 0.01 g/mL.

Further, in the composition compounded by the probiotics and the tea polyphenol, the concentration of the tea polyphenol is 0.001 g/mL-0.05 g/mL.

Further, in the composition compounded by the probiotics and the tea polyphenol, the concentration of the tea polyphenol is 0.001 g/mL-0.01 g/mL.

Further, in the composition compounded by the probiotics and the tea polyphenol, the concentration of the tea polyphenol is 0.0025 g/mL.

Further, in the composition compounded by the probiotics and the tea polyphenol, the weight ratio of the probiotics to the tea polyphenol is 1: 1-8: 1, preferably the weight ratio of the probiotics to the tea polyphenol is 4: 3-8: 1, and preferably the weight ratio of the probiotics to the tea polyphenol is 8: 1.

According to another aspect of the invention, a preparation prepared by compounding probiotics and tea polyphenol is provided, and the preparation is used for preparing toothpaste, mouthwash, disinfectant, oral spray or buccal tablets.

Further, in the preparation compounded by the probiotics and the tea polyphenol, the preparation is toothpaste.

According to another aspect of the invention, an oral health product compounded by probiotics and tea polyphenol is provided, and the oral health product is prepared into toothpaste, mouthwash, disinfectant, oral spray or buccal tablets.

Further, in the oral health product compounded by the probiotics and the tea polyphenol, the oral health product is toothpaste.

According to another aspect of the present invention, there is provided a use of the above composition for the preparation of an agent for preventing and/or treating oral diseases or an oral health product.

Further, the oral disease is one or more of periodontitis, dental caries, tartar, dental plaque, gingival bleeding, tooth loosening, dentin sensitivity, tooth whitening or halitosis.

According to another aspect of the invention, the application of the composition compounded by probiotics and tea polyphenol in preparing bacteriostatic preparations or oral health products is provided.

Further, the bacteriostasis is inhibition of one or more of actinobacillus actinomycetemcomitans, streptococcus mutans and fusobacterium nucleatum.

Further, the bacteriostasis is through co-aggregation and/or bactericidal action.

The invention establishes a quantifiable probiotic and tea polyphenol compound model and determines the bacteriostasis mechanism of the compound model on the oral pathogenic bacteria and the efficacy of the compound model in toothpaste. The optimal ratio of the probiotics to the tea polyphenol is determined through experiments, and characteristic indexes such as biofilm inhibition amount and the like, thallus morphology observation and bacteria surface component and roughness analysis are carried out by using various material science and biochemical analysis means such as a Scanning Electron Microscope (SEM), an Atomic Force Microscope (AFM), a Fourier infrared spectrum (FT-IR) and surface charge analysis, so that the synergistic bacteriostasis mechanism of the probiotics and the tea polyphenol to actinobacillus actinomycetemcomitans is explored. Experimental results show that the optimal ratio of probiotics to tea polyphenol is 2:1, the inhibition rate of the probiotics to a pathogenic bacteria biofilm is improved to 71.7%, and a compound model inhibits the growth of actinobacillus actinomycetemcomitans through the double effects of coagglomeration and sterilization. Compared with the difference of the bacteriostatic action results of the probiotic and tea polyphenol separately added and the probiotic and tea polyphenol compound bacteriostatic model on three common pathogenic bacteria (actinobacillus actinomycetemcomitans, streptococcus mutans and fusobacterium nucleatum) in the toothpaste, the probiotic dry powder compound tea polyphenol bacteriostatic model provides a sufficient theoretical basis and application technology support for the application of the oral cleaning and nursing products such as toothpaste and the like. In conclusion, the invention clarifies the synergistic bacteriostasis mechanism of the probiotics and the tea polyphenol and verifies the bacteriostasis function of the probiotic and tea polyphenol compound bacteriostasis model in the toothpaste.

Compared with the prior art, the composition has the beneficial technical effects as follows:

1. the typically selected lactobacillus paracasei can generate a co-coagulation effect with actinobacillus actinomycetemcomitans, so that the colonization of pathogenic bacteria in the oral cavity is reduced. When the tea polyphenol is synergistically acted, the tea polyphenol not only can generate a co-coagulation effect with pathogenic bacteria, but also can play a role in killing the pathogenic bacteria. Meanwhile, a synergistic antibacterial model of probiotics and tea polyphenol is established, so that the inhibition effect on the formation of pathogenic bacteria biofilm is greatly improved.

2. The bacteriostasis mechanism of the probiotics and the tea polyphenol to actinobacillus actinomycetemcomitans is researched. The invention can quantitatively observe the surface morphology, chemical components and surface roughness of the probiotics and pathogenic bacteria by applying various material science and biochemical means, and researches and clarifies the bacteriostasis mechanism of the probiotics and tea polyphenol on actinobacillus actinomycetemcomitans in multiple dimensions.

3. The probiotic and tea polyphenol compound antibacterial model which is researched and established is applied to toothpaste, and compared with toothpaste which is independently added with probiotics or tea polyphenol, the antibacterial effect of the probiotic and tea polyphenol compound antibacterial model on three common oral pathogenic bacteria (actinobacillus actinomycetemcomitans, streptococcus mutans and fusobacterium nucleatum) is enhanced. Therefore, the probiotics and the tea polyphenol have obvious oral cavity probiotic characteristics and have wide application prospects in the aspect of oral cavity cleaning and nursing products.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.

FIG. 1 shows the decrease of Actinobacillus actinomycetemcomitans after probiotic action (different lower case letters indicate significant differences between data, P < 0.05).

FIG. 2 shows the decrease of Actinomyces actinomycetemcomitans at different probiotic addition levels (different lower case letters indicate significant differences between data, P < 0.05).

FIG. 3 shows the decrease of Actinobacillus actinomycetemcomitans after different ratios of probiotics to tea polyphenols (different lower case letters indicate significant difference between data, P < 0.05).

FIG. 4 shows the inhibition of Actinobacillus actinomycetemcomitans biofilm in different treatments (different lower case letters indicate significant differences between data, P < 0.05).

FIG. 5 is a scanning electron micrograph (a, Actinobacillus actinomycetemcomitans; b, probiotic; c, probiotic + Actinobacillus actinomycetemcomitans; d, tea polyphenol + Actinobacillus actinomycetemcomitans; e, probiotic: tea polyphenol (2:1) + Actinobacillus actinomycetemcomitans).

FIG. 6 shows Fourier near infrared spectra of different bacteria (a, co-aggregation of probiotic bacteria, b, pathogenic bacteria, c, co-aggregation of probiotic bacteria and pathogenic bacteria, d, co-aggregation of tea polyphenols and pathogenic bacteria)

FIG. 7 is a 2D atomic force microscope optical picture of the bacteria (a, pathogen; b, probiotic) and 3D roughness profile (c, pathogen; D, probiotic).

Fig. 8 is a schematic diagram of the synergistic bacteriostatic mechanism of probiotics and tea polyphenol.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application, and it should be apparent that the described embodiments are some, but not all, embodiments of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.

Reagents and instrumentation:

two types of probiotics commonly used in the existing toothpaste are selected: lactobacillus paracasei dry powder and composite probiotic dry powder; the blood plate and 0.5% crystal violet staining solution were purchased from Hedgerman Biotech, Inc., and the 2.5L anaerobic canister C-31, carbon dioxide gas bag C-3 and anaerobic gas bag were purchased from Shanghai Chuangling Biotech, Inc.;

BacLight^TMthe bacterial activity detection kit 7012 is purchased from Shanxi Jieke Kodak science and technology GmbH; phosphate buffer (pH7.2-7.4), 2.5% glutaraldehyde stationary solution (analytical grade), sodium chloride (analytical grade), and tea polyphenols (95%) were purchased from national drug group chemical reagent, Inc., and toothpaste samples were provided by Chongo chemical, Inc., Suzhou.

Preparing bacterial liquid: actinobacillus actinomycetemcomitans ATCC 29523 selected in the experiment was purchased from institute of microbiology, academy of sciences, Guangdong province, and cultured in a blood plate medium (TSA + 5% defibrinated sheep blood) at 37 ℃ with 5% CO₂Culturing for 48 hr, adjusting the cell concentration to 1 × 10 with phosphate buffer solution (PBS, pH7.2-7.4)⁹CFU/mL is ready for use.

The main equipment is shown in table 1:

TABLE 1 Main Instrument

And (3) data analysis: in the experiments, statistical analysis was performed on the data using SPSS 18.0 software at a significance level of 5%, the experimental data was expressed as mean ± standard deviation, and 3 replicates were set for all experiments.

Example 1 determination of optimal effective addition of probiotic

The probiotics with superior bacteriostatic effect are selected by measuring the bacteriostatic performance of the two probiotic dry powder products. Lv et al (Xin, Lv., Pan, Hu., Ying Dang., etc. purification and partial characterization of a novel bacterial process by Lactobacillus casei TN-2isolated from transformed media mill (Shubat) of Xinjiang uv autonomous region, China [ J]The Oxford cup double-layer plate method used by Food Control,2014,43(5):276-283.) firstly prepares the lactobacillus paracasei and the compound probiotic bacteria liquid with the concentration of 0.02 g/mL. 100. mu.L of actinobacillus actinomycetemcomitans-containing bacterial solution (concentration about 1X 10) was prepared⁹CFU/mL) blood plate (TSA + 5% defibrinated sheep blood), sucking 100 μ l of the prepared probiotic bacteria liquid, adding into Oxford cup, and placing into a culture dish at 3%And (4) measuring the diameter of the inhibition zone after culturing for 48 hours at 7 ℃.

Meanwhile, the effect of the probiotics on actinobacillus actinomycetemcomitans is further determined by a co-aggregation test method. The methods used in the methods of engineering-Ayala (K, engineering-Ayala, F, J, Ascencio-Valle, P, Gutierrez-Gonzalez, etc., hybrid and additive patterns of lactic acid bacteria and the anti-agglutination agent pattern on substrate surface (solar competent L.) [ J ]. Journal of Applied Microbiology,2020,129: 876. 891.) and Fadare (O, S, Fadare, V, Singh., O, I., Enablel, C. in vitro evaluation of the synthetic effect of strain and strain) are described in detail by the methods of engineering-2022,153,112439: preparing a bacterium solution with the concentration of 0.02g/mL from lactobacillus paracasei probiotic dry powder and composite probiotic dry powder, respectively mixing the bacterium solution with the prepared actinomyces bacterial suspension according to the volume of 1:1, standing for 0.5h and 1h, then gently sucking 100 mu l of supernatant, diluting by a tenfold dilution method, sucking 100 mu l of diluent with different proportions, coating, culturing at 37 ℃ for 48h, and then counting. Finally, by comparing the diameter of the inhibition zone and the reduction amount of actinobacillus actinomycetemcomitans, probiotics with good effect is selected for the next test, and the test is repeated for three times.

The results show that: under the same concentration, the inhibition effect of the lactobacillus paracasei and the composite probiotics on actinobacillus actinomycetemcomitans is not obviously different (table 2), but the inhibition effect is obviously smaller than that of tea polyphenol (P)<0.05). As shown in figure 1, after the lactobacillus paracasei and the composite probiotics with the concentration of 0.02g/ml and the actinobacillus actinomycetemcomitans suspension are mixed and placed for 0.5h in equal proportion, the reduction amount of the actinobacillus actinomycetemcomitans is not obviously different (P)>0.05). After standing for 1h, the reduction amount of actinobacillus actinomycetemcomitans treated by lactobacillus paracasei is 0.22 +/-0.02 Log₁₀CFU/mL is obviously higher than the reduction amount (0.17 plus or minus 0.02 Log) of actinobacillus actinomycetemcomitans after the action of the composite probiotics₁₀CFU/mL), according to which Lactobacillus paracasei was selected for further study.

TABLE 2 inhibition of Actinobacillus actinomycetemcomitans by different substances

Note: mean ± standard deviation are the results of three replicates, with each different lower case (a, b) indicating significant difference between data (P < 0.05).

The screened probiotic dry powder is prepared into probiotic solutions with different concentrations by PBS buffer solution according to the addition amount of 0.3%, 0.5%, 0.7%, 1%, 1.5%, 2%, 2.5%, 3% and 4% (m/v). According to the operation of the method, the appropriate probiotic adding amount is selected by comparing the colony reduction amount of actinobacillus actinomycetemcomitans after standing for 1h, and the test is repeated three times.

The results show that: after mixing different concentrations of lactobacillus paracasei and actinobacillus actinomycetemcomitans for 1h, when the probiotic addition amount is less than 1.0% (0.01g/mL), the decrease of actinobacillus actinomycetemcomitans is not significantly different (P >0.05), and the decrease of actinobacillus increases with the increase of the probiotic concentration (figure 2). However, when the probiotic addition amount is more than 3.0% (0.03g/mL), the decrease amount of Actinomyces actinomycetemcomitans gradually decreases. Therefore, the optimum amount of Lactobacillus paracasei to be added is 1.0% to 3.0%.

Example 2 establishment of optimal probiotic and tea polyphenol bacteriostatic model

The Minimum Inhibitory Concentration (MIC) of tea polyphenol to actinobacillus actinomycetemcomitans is determined, a tea polyphenol solution with the concentration of 0.02g/mL is prepared, diluted according to a double dilution method (L, Zhang, L, Wang, L, H, Yi., etc. A novel antibacterial biological substrate produced by Y Lactobacillus rhamnous LS8[ J ]. Food Control,2017,73:754 and 760.), and the mixture is mixed with the bacterial suspension of the actinobacillus actinomycetemcomitans according to the proportion of 1:1, and is placed in a 96-well plate for culturing for 48H at 37 ℃, and the growth of bacteria is observed by naked eyes to be the minimum inhibitory concentration. The results show that: the Minimum Inhibitory Concentration (MIC) of tea polyphenols is 2.5 mg/mL.

In addition, a probiotic solution is prepared according to the addition of 1% of lactobacillus paracasei, a tea polyphenol solution with the minimum inhibitory concentration is prepared, the probiotic solution and the probiotic solution are mixed according to the volume ratio of 2:1, 1:2 and 1:3, namely the weight ratio of probiotics to tea polyphenol is 8:1, 4:1, 2:1 and 4:3, then the mixture is mixed with a bacterial suspension of actinobacillus actinomycetemcomitans according to the volume ratio of 1:1, the mixture is stood for 1h, supernatant is slightly sucked up for counting, the positive control is formed by adding the same amount of probiotics or tea polyphenol, and the negative control is formed by adding the same amount of PBS buffer solution. The optimum ratio of probiotic to tea polyphenol was determined by comparing the reduction of actinobacillus actinomycetemcomitans and the experiment was repeated three times.

The results show that: when the volume ratio of the probiotics (0.01g/mL) to the tea polyphenol with the minimum inhibitory concentration is 2:1, namely the weight ratio is 8:1, the reduction amount of actinobacillus actinomycetemcomitans under the synergistic effect of the tea polyphenol and the probiotics is obviously higher than that of the probiotics or the tea polyphenol (figure 3). In addition, as the amount of tea polyphenol increases, the decrease of actinobacillus actinomycetemcomitans after the action of tea polyphenol increases significantly, because tea polyphenol has bactericidal action (Y,Li.,x, G, Jiang, J, Q, Hao, etc. tea polyphenols: application in the control of organic microbial infection diseases of organic Biology,2019,102:74-82.) with increasing tea polyphenol concentration, the bacteriostatic effect after compounding tea polyphenol and probiotic is lower than that of the tea polyphenol alone. The antibacterial effect of the probiotics is mainly dependent on the co-coagulation effect, and the tea polyphenol is dependent on the sterilization effect to inhibit the growth of actinobacillus actinomycetemcomitans. Therefore, the optimal antibacterial model of the probiotics and the tea polyphenol is that the ratio of the probiotics (0.01g/mL) to the tea polyphenol is 2: 1.

Example 3 antibacterial mechanism study of probiotic and tea polyphenol Compound antibacterial model-analysis and determination of Actinobacillus actinomycetemcomitans biofilm amount

Refer to the inhibition of Qinshuojia (Qinshuojia, Xuyangtong, Zhang Qixiang, etc. Lactobacillus plantarum CCFM8724 on cariogenic double-bacteria biofilm [ J]The food and fermentation industry 2020, 46(13): 127-132.) andPhumatetc. (P,Phumat.,S, Khongkhunthian.,P,Wanachantararak.,etc.Comparative inhibitory effects of 4-acetylpyrocatechol isolated from needle on Streptococcus intermedia, Streptococcus mutans, and Candida albicans, Archives of Oral Biology,2020,113,104690.) the amount of the biofilm of Actinobacillus actinomycetemcomitans was determined analytically. Preparing probiotic and tea polyphenol solution with optimal proportion by using TSB liquid culture medium, sucking 100 mul of the prepared probiotic, tea polyphenol and optimal compound model solution of the probiotic and the tea polyphenol, placing the sucked 100 mul of the prepared probiotic, tea polyphenol and optimal compound model solution of the probiotic and the tea polyphenol in a 96-well plate, and mixing the sucked 100 mul of the optimal compound model solution with actinobacillus actinomycete suspension in equal proportion. The negative control is that the TSB culture medium and actinobacillus actinomycetemcomitans suspension are mixed according to equal proportion and cultured for 48h at 37 ℃. Then the culture solution is sucked out, washed twice by PBS, added with 50 mul of crystal violet with the concentration of 0.5 percent and dyed for 15min in a dark place, washed and dried, dissolved with 200 mul of 95 percent ethanol, and then the crystal violet is dissolved at OD by an enzyme-linked immunosorbent assay (ELISA) instrument_595nmThe absorbance was measured and the test was repeated three times. The formula for calculating the biofilm inhibition rate is as follows:

dental plaque biofilm is the initiating factor in periodontitis, inhibiting the formation of pathogenic biofilm is effective in treating periodontal disease (W, J, Kim, Y, Soh, S, M, Heo. recent advances of therapeutic targets for the treatment of periodontal disease. biologicals & Therapeutics,2021,29: 263-. Through the measurement of the biofilm formation amount, the probiotics and the tea polyphenol can effectively inhibit the formation of pathogenic bacteria biofilm, and the inhibition rates respectively reach 62.3% and 61.4% (figure 4). The inhibition rate of the optimal ratio of probiotics to tea polyphenol for the antibacterial model to the actinomycete biofilm amount is increased to 71.7 percent, which is obviously higher than the inhibition rate of single probiotics or tea polyphenol (figure 4).

Example 4 antibacterial mechanism study-scanning electron microscope morphology analysis of probiotic and tea polyphenol compound antibacterial model

Observing the surface morphology of actinobacillus cells treated by probiotics and tea polyphenol by using a scanning electron microscope. With reference to the method described by Song et al (Y, Y, Song., L, H, Zhang., L, M, Dong., etc. pH-responsive smart surface with dual bacterial and recycling properties. ACS Applied Materials & Interfaces,2021,13:46065-46075.), the probiotic and tea polyphenol optimal ratio bacteriostatic model was mixed with the actinobacillus actinomycete suspension in equal proportion, and the supernatant was discarded after standing for 1H. The positive control is added with probiotic bacteria or tea polyphenol with the same quantity, and the negative control is added with PBS buffer with the same quantity. The cell pellets of the treated and control groups were fixed with 2.5% glutaraldehyde overnight at 4 ℃, followed by washing twice with PBS (0.1mol/L, pH 7.2), followed by dehydration with 30%, 50%, 70%, 80%, 90% and 100% ethanol, and finally drying and spraying gold and morphological observation.

As shown in FIG. 5a, the cells of Actinobacillus actinomycetemcomitans alone were relatively intact and had a relatively flat surface. Most of the probiotics have complete thallus, and a small part of the probiotics have slight depressions (5b), and the analysis is that the probiotics partially damage the thallus when being prepared into dry powder. However, the individual probiotic and actinobacillus actinomycetemcomitans colonies dispersed or simply stacked together, with no co-aggregation. After probiotic treatment, the probiotic and the pathogenic bacteria were adhered together, indicating that co-aggregation occurred between the probiotic and the pathogenic bacteria (fig. 5 c). After the tea polyphenol treatment, the surface of the thallus becomes uneven, a part of the thallus is broken, and intracellular substances flow out (figure 5d), which shows that the tea polyphenol has the bactericidal effect on pathogenic bacteria and destroys the integrity of the bacterial cells (Y, Han, J, Ding, J.T, Zhang, etc. the purification and characterization of the porous acidic microorganisms embedded with the cellular impurities/tea polyphenol with the food packaging potential. the International Journal of Biological Macromolecules,2021,184: 739-. When probiotic bacteria and tea polyphenols act synergistically, both co-aggregation and bacterial rupture were observed (fig. 5 e). The results of the scanning electron microscope further prove that the antibacterial test results show that the probiotics mainly have a co-coagulation effect on actinobacillus actinomycetemcomitans, and the antibacterial effect of the probiotics on actinobacillus actinomycetemcomitans can be improved by the bactericidal effect of the probiotics after the tea polyphenol is added.

Example 5 antibacterial mechanism study of probiotic and tea polyphenol Compound antibacterial model-determination of surface charges of thallus

Mixing the optimal probiotic bacteria and tea polyphenol bacteriostasis model with actinobacillus actinomycetemcomitans suspension in equal proportion, standing for 1 hr, discarding supernatant, re-dissolving the obtained thallus precipitate with PBS (pH7.2-7.4), and measuring the surface charge of the thallus with zeta potential instrument. The positive control is added with probiotic bacteria or tea polyphenol with the same amount, and the negative control is added with probiotic bacteria and actinobacillus actinomycetemcomitans with the same amount of PBS buffer solution.

The higher the surface charge of the bacteria, the repulsive force is provided, and the stability of the cells is increased. When the absolute value of zeta potential is reduced, the cells are subjected to condensation and deposition due to the reduction of repulsive force (consider Yuliei. the research on the antibacterial mechanism of the moringa essential oil on listeria monocytogenes and the application thereof in cheese storage and fresh-keeping [ D ]. Jiangsu university, 2019). Table 3 the results show that: after the probiotics and actinobacillus actinomycetemcomitans act, the zeta potential absolute value is reduced, which indicates that the thalli have co-aggregation effect. In addition, after the probiotics and the tea polyphenol have synergistic effect, the zeta potential absolute value is reduced, the reduction amount is smaller than the effect of single probiotics, and the influence on the surface potential value of the thallus is related to the fact that actinobacillus actinomycetemcomitans cells are broken under the sterilization effect of the tea polyphenol, and substances such as cytoplasm and intracellular ions flow out.

TABLE 3 variation of surface charge of cells under different treatments

Example 6 antibacterial mechanism study of probiotic and tea polyphenol Compound antibacterial model-Fourier near Infrared Spectroscopy for determining surface ingredients of thallus

The changes in the surface functional groups of the bacteria are measured by an infrared spectroscopy technique employed with reference to Song et al (Y, Y, Song., Z, P, Yu., Y, Liu., etc. A. theoretical physical array with controlled addition and drop boundary ability for reducing the residual reactive non-newtonian lipids [ J ]. Journal of biological Engineering,2021,18(3): 637-648): mixing the optimal probiotic bacteria and tea polyphenol bacteriostasis model with actinobacillus actinomycetemcomitans suspension in equal proportion, standing for 1h, discarding supernatant, re-dissolving the obtained thallus precipitate with PBS (pH7.2-7.4), and measuring with Fourier near infrared spectrometer. The positive control is added with probiotic bacteria or tea polyphenol with the same quantity, and the negative control is added with PBS buffer with the same quantity.

From the above results, it is understood that probiotic bacteria inhibit actinobacillus actinomycetemcomitans mainly by means of co-aggregation. The probiotic bacteria are measured by Fourier infrared spectroscopy (figure 6) at 2855cm^-1The peak observed at (C) belongs to-CH₂-a functional group, -CH₃The functional group is 2920cm^-1Producing a peak. It is known that the surface energy is 24mJ m^-2Of (C-CH)₃Group and surface energy of 31mJ m^-2Of (C-CH)₂The groups all have a low surface energy, which is very abundant in hydrophobic polymers. Actinobacillus actinomycetemcomitans also has-CH on the surface₂-and-CH₃A functional group. Thus, co-aggregation of probiotic bacteria having similar low surface energy groups with Actinobacillus actinomycetemcomitans occurred, and no-CH was detected after co-aggregation₂-and-CH₃The groups indicate that sites containing low surface energy groups are shielded due to the co-condensation of the substance, infrared spectroscopy can only detect surface functional groups, and the accurate determination of internal groups of the substance is difficult. but-CH can still be detected after the tea polyphenol and actinobacillus actinomycetemcomitans are co-coagulated₂-and-CH₃The groups indicate that the co-coagulation of the two is not due to low surface energy functional groups, but is electrostatic adsorption.

Example 7 antibacterial mechanism study of probiotic and tea polyphenol Compound antibacterial model-determination of surface roughness of bacteria

The surface roughness of bacteria is related to the amount of adhesion of the bacteria. In addition to the surface composition, the present study also analyzed the co-agglomeration mechanism from the surface structure. Probiotic solutions were prepared at a concentration of 0.02g/mL, referred to Sandin (J, N, Sandin, S, P, aryal, T, wilkop, etc. near atomic laser scanning confocal and atomic force microscopy on live cells, Journal of visual Experiments 2020,162, e 61433), etc. and the webet (webet atomic force microscopy for silver nitrate and nano-particles)Bacteriostatic mechanism of silver on bacteria [ D]University of Guangxi, 2019.) was used to determine bacterial surface roughness. The specific method comprises the following steps: mixing probiotic solution with Actinobacillus actinomycetemcomitans (10)⁹CFU/mL) bacterial liquid is respectively fixed by 2.5% glutaraldehyde for 30min, then 5 mu l of the bacterial liquid is dropped on a mica sheet, and the surface roughness of the two strains of bacteria is measured by an atomic force microscope after the two strains of bacteria are naturally dried.

Fig. 7a and c are 2D atomic force microscope optical pictures and 3D roughness profile images of pathogens. The average surface roughness Ra of the pathogenic bacteria surface is 11.5 nm. Fig. 7b and D are 2D atomic force microscope optical pictures and 3D roughness profile of probiotics. The average surface roughness Ra of the probiotic surface was 19.5 nm. The surface of the probiotics is rough compared with the surface of actinobacillus actinomycetemcomitans, and the result shows that the probiotics has stronger adhesion effect, can compete with pathogenic bacteria for adhesion sites in the oral cavity, and reduces the adhesion of the pathogenic bacteria.

Therefore, by combining the analysis, the synergistic bacteriostasis mechanism of the probiotics and the tea polyphenol is shown in fig. 8, the common hydrophobic groups cause the probiotics and the pathogenic bacteria to be co-agglomerated, the pathogenic bacteria are taken away from the oral cavity, meanwhile, the natural bacteriostasis substance tea polyphenol can also assist in killing the pathogenic bacteria, and the synergistic bacteriostasis strengthening and stacking effect with the probiotics is achieved.

Example 8 evaluation of antibacterial action of probiotic and tea polyphenol compound antibacterial model in toothpaste

8.1 test methods

In order to evaluate the bacteriostatic effect of probiotics and tea polyphenol on pathogenic bacteria in the toothpaste, the probiotics and the tea polyphenol are added into the toothpaste according to the optimal proportion. Selecting Streptococcus mutans CGMCC 12499 and Fusobacterium nucleatum ATCC 25586 in addition to Actinobacillus actinomycetemcomitans ATCC 29523, inoculating the two strains provided by food institute of Jiangsu university, inoculating the two strains on blood plate, and placing the blood plate containing 80% N₂、10％H₂、10％CO₂Culturing at 37 deg.C for 48h in anaerobic bag, and adjusting thallus concentration to 10⁹CFU/mL. Adopts the selective inhibition effect of the lactobacillus paracasei on the pathogenic bacteria in the oral cavity [ J]In the oral care product industry, 2016 (26) (5): 26-28), a toothpaste sample is diluted by a method comprising the following steps: 0.5g of toothpaste was weighed out and dissolved in 1mL of PBS (pH)7.2-7.4), fully shaking and dissolving, and removing air bubbles in the solution. And then mixing the three strains of pathogenic bacteria suspensions according to a ratio of 1:1, standing and culturing for 1h, and then slightly sucking the supernatant for coating and counting, wherein the positive control is toothpaste added with the same amount of probiotics or tea polyphenol, and the negative control is toothpaste not added with the probiotics and the tea polyphenol. The antibacterial efficacy of the probiotics and the tea polyphenol in the toothpaste is evaluated by comparing the pathogenic bacteria reduction amount, and the test is repeated for three times.

8.2 test results

Table 4 shows the effect of probiotics and tea polyphenol added separately and in toothpaste together on three common oral pathogenic bacteria. After the probiotics and the tea polyphenol are added, the inhibition effect of the toothpaste on pathogenic bacteria is enhanced (table 4). Meanwhile, the probiotics and tea polyphenol compound antibacterial model is applied to toothpaste, and compared with toothpaste with probiotics or tea polyphenol added independently, the reduction of pathogenic bacteria can be further improved. Thereby more effectively playing the role of inhibiting pathogenic bacteria such as periodontitis common in the oral cavity.

TABLE 4 inhibition of Actinobacillus actinomycetemcomitans by different substances in toothpaste

The foregoing detailed description of the embodiments of the present application has been presented to illustrate the principles and implementations of the present application, and the description of the embodiments is only intended to facilitate the understanding of the methods and their core concepts of the present application. Meanwhile, a person skilled in the art should, according to the idea of the present application, change or modify the embodiments and applications of the present application based on the scope of the present application. In view of the above, the description should not be taken as limiting the application.

Claims

1. The composition is characterized by comprising the following raw materials in parts by weight: 1-100 parts of probiotics and 1-100 parts of tea polyphenol.

2. The composition according to claim 1, wherein the composition is composed of the following raw materials in parts by weight: 1-50 parts of probiotics and 1-50 parts of tea polyphenol; preferably, the composition consists of the following raw materials in parts by weight: 1-10 parts of probiotics and 1-10 parts of tea polyphenol.

3. The composition according to claim 2, wherein the composition comprises the following raw materials in parts by weight: 4-8 parts of probiotics and 1-6 parts of tea polyphenol; preferably, the composition consists of the following raw materials in parts by weight: 8 parts of probiotics and 1 part of tea polyphenol; preferably, the composition consists of the following raw materials in parts by weight: 4 parts of probiotics and 1 part of tea polyphenol; preferably, the composition consists of the following raw materials in parts by weight: 2 parts of probiotics and 1 part of tea polyphenol; preferably, the composition consists of the following raw materials in parts by weight: 4 parts of probiotics and 3 parts of tea polyphenol.

4. The composition according to any one of claims 1 to 3, wherein the probiotic bacteria are one or more of yeast, probiotic bacillus, Clostridium butyricum, Lactobacillus, Bifidobacterium or Actinomycetes; preferably, the probiotic is lactobacillus; preferably, the probiotic is lactobacillus paracasei.

5. The composition of any one of claims 1 to 3, wherein the concentration of the probiotic is 0.001g/mL to 0.05g/mL, the concentration of the tea polyphenol is 0.001g/mL to 0.05 g/mL; preferably, the concentration of the probiotics is 0.01 g/mL-0.03 g/mL, and the concentration of the tea polyphenol is 0.001 g/mL-0.01 g/mL; preferably, the concentration of the probiotics is 0.01g/mL, and the concentration of the tea polyphenol is 0.0025 g/mL.

6. A formulation or oral care product comprising a composition according to any one of claims 1 to 5, wherein the formulation or oral care product is a toothpaste, mouthwash, disinfectant, oral spray or lozenge; preferably, the formulation or oral care product is a toothpaste.

7. Use of a composition according to any one of claims 1 to 5 or a formulation or oral care product according to claim 6 in the manufacture of a product for the prevention and/or treatment of an oral disease; preferably, the oral disease is one or more of periodontitis, dental caries, tartar, dental plaque, gingival bleeding, tooth loosening, dentinal hypersensitivity, tooth whitening or halitosis.

8. Use of a composition according to any one of claims 1 to 5 or a formulation or oral care product according to claim 6 in the manufacture of a bacteriostatic product.

9. Bacteriostatic product according to claim 8, wherein the bacteriostatic action is one or more of actinobacillus actinomycetemcomitans, streptococcus mutans and fusobacterium nucleatum.

10. Bacteriostatic product according to claim 8, wherein the bacteriostatic action is by co-aggregation and/or bactericidal action.