CN114404563A

CN114404563A - Composition for inhibiting formation of cariogenic bacteria biofilm

Info

Publication number: CN114404563A
Application number: CN202210188495.XA
Authority: CN
Inventors: 张秋香; 赵建新; 李佳珣; 崔树茂; 毛丙永; 唐鑫; 陈卫; 张灏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2022-04-29
Anticipated expiration: 2042-02-28
Also published as: CN114404563B

Abstract

The invention discloses a composition for inhibiting the formation of cariogenic bacteria biofilm, belonging to the technical field of microorganisms. The composition provided by the invention is a mixture of 3-phenyllactic acid, phenylpropionic acid and cyclodeu-pro, and can inhibit a biofilm formed by interaction of streptococcus mutans and candida albicans. Moreover, the capability of the mixture for inhibiting the biofilm of the streptococcus mutans and the candida albicans is higher than the independent inhibition capability of the 3-phenyllactic acid, the phenylpropionic acid and the cyclodeu-pro, and is similar to the inhibition capability of the fermentation supernatant of the lactobacillus plantarum CCFM 8724.

Description

Composition for inhibiting formation of cariogenic bacteria biofilm

Technical Field

The invention relates to a composition for inhibiting the formation of cariogenic bacteria biofilm, belonging to the technical field of microorganisms.

Background

Dental caries is one of the most common oral bacterial infectious diseases and does not occur in a short time, and the process of demineralization and enamel decomposition is often accompanied by one's lifetime, thus bothering not only adults but also children of low age. Among them, the incidence of pre-school children suffering from caries, i.e., caries in young children (ECC), is the first of the childhood diseases and seriously harms the health of children. Dental caries is closely related to factors such as microorganisms, food and drink intake such as sugar-containing food and drink, and host oral hygiene habits, wherein the formation of a microbial-dominated dental plaque biofilm is a causative factor of dental caries.

The streptococcus mutans is a gram-positive coccoid bacterium, has strong capacity of metabolizing sucrose to produce acid and forming cariogenic biomembranes, is a main colonizing bacterium in the early stage of the biomembrane forming process, and is known as a main pathogenic bacterium of dental caries. In recent years, clinical researches show that candida albicans participates in the process of dental caries, and a large amount of candida albicans and streptococcus mutans are detected in the dental plaque biomembrane of the oral cavity of children suffering from ECC, and the interaction of the candida albicans and the streptococcus mutans can form a biomembrane with stronger cariogenicity. Candida albicans is a conditionally pathogenic fungus that normally colonizes the oral cavity with other conditionally pathogenic bacteria. Depending on the oral and general health, these opportunistic pathogens may switch from commensal to pathogenic. Candida albicans has the ability to adhere to hydroxyapatite substrates and dissolve hydroxyapatite by releasing calcium ions, which invade dentin and secrete acid, promoting enamel demineralization. Therefore, the interaction of bacteria and fungi is commonly involved in the formation of dental plaque biomembrane, which brings a brand-new visual angle and greater challenge to the prevention and treatment of dental caries. Therefore, it is important to inhibit the formation of biofilm and to obtain an effective means for preventing and treating dental caries in children.

Generally, the method for preventing and treating dental caries is to brush teeth, reduce the intake of dietary sugar, use dental floss and mouthwash, fill teeth, use different entry points such as fluoride to remove plaque biofilm, so that obtaining a group of compositions that can be applied to oral products is of great significance. IN201747009062A discloses the inhibitory effect of a small molecule inhibitor on oral plaque biofilm, and KR1020210092052A discloses that a composition containing nano-emulsified cinnamon oil as an active ingredient can inhibit the formation of oral biofilm, but requires nano-emulsification of cinnamon oil, which is costly.

Studies by Strom et al indicate that the loop (L-Phe-L-Pro) and 3-phenyllactic acid produced by Lactobacillus plantarum MiLAB393 have synergistic inhibitory effects on fungi such as fusarium and Aspergillus fumigatus, but the required concentrations are high, and the Minimum Inhibitory Concentrations (MIC) are 7.5mg/mL and 20mg/mL respectively. CN104739742A discloses a traditional Chinese medicine mouth wash for preventing and treating gingival bleeding, but the use of Chinese herbal medicine products also has the risk of dental pigmentation. CN113702559A (method for separating and identifying active substances of lactobacillus plantarum source for inhibiting double-bacterial biofilms) discloses that lactobacillus plantarum metabolites 3-phenyllactic acid and cyclodeu-pro can inhibit double-bacterial biofilms, but the effect of the metabolites on inhibiting the biofilms alone does not achieve the same effect as that of lactobacillus plantarum CCFM8724 fermentation liquor.

Disclosure of Invention

[ problem ] to

The invention aims to solve the technical problems that the existing substances for inhibiting the formation of the double-bacterium biofilm have poor effects or are inconvenient to develop into oral products.

[ solution ]

The invention provides a composition for inhibiting the formation of cariogenic bacteria biofilm, which comprises a composition of 3-phenyllactic acid, phenylpropionic acid and leucine-proline cyclic dipeptide, wherein the mass ratio of the 3-phenyllactic acid to the phenylpropionic acid to the leucine-proline cyclic dipeptide is (50-200): (25-200): (50-100). The cariogenic bacteria biomembrane is a streptococcus mutans and candida albicans biomembrane. The inhibition of the double-bacteria biofilm refers to a biofilm inhibiting the interaction formation of streptococcus mutans and candida albicans, and the inhibition effect is judged if the biofilm formation amount measured by crystal violet staining is remarkably reduced (p is less than 0.05) compared with that of a control group.

In some embodiments, the composition is dissolved in a solvent to form a mixed solution, and the solvent is selected from water and ethanol. In some embodiments, the concentration of 3-phenyllactic acid in the solution is 50-200. mu.g/mL, the concentration of phenylpropionic acid is 25-200. mu.g/mL, and the concentration of leucine-proline cyclodipeptide is 50-100. mu.g/mL. In some embodiments, the concentration of 3-phenyllactic acid in the solution is 150-200. mu.g/mL, the concentration of phenylpropionic acid is 150-200. mu.g/mL, and the concentration of leucine-proline cyclodipeptide is 50-60. mu.g/mL. In certain embodiments, the concentration of 3-phenyllactic acid in the solution is 200. mu.g/mL, the concentration of phenylpropionic acid is 200. mu.g/mL, and the concentration of leucine-proline cyclodipeptide is 50. mu.g/mL.

The invention provides a product for inhibiting streptococcus mutans and candida albicans from interacting to form a biofilm, which can be mouthwash or toothpaste, and is prepared by applying the composition. Of course, the compositions may also be used to prepare other oral care products.

[ advantageous effects ]

The mixture of the 3-phenyllactic acid, the phenylpropionic acid and the cyclodeu-pro has the function of inhibiting streptococcus mutans and candida albicans from forming a double-bacterium biofilm. Wherein the concentration of the 3-phenyllactic acid minimum inhibition biomembrane is 50 mug/mL, the concentration of the phenylpropionic acid minimum inhibition biomembrane is 25 mug/mL, and the concentration of the cyclo leu-pro minimum inhibition biomembrane is 50 mug/mL; compared with 3-phenyllactic acid, phenylpropionic acid and cyclodeu-pro, the inhibition ability of the mixture is improved, the effect of the mixture on inhibiting the biological membrane is reduced by 1.2 compared with that of the mixture using cyclopeptide with the same concentration alone, the effect of the mixture on inhibiting the biological membrane is reduced by about 0.3 compared with that of the mixture using organic acid with the same concentration alone, and compared with lactobacillus plantarum CCFM8724, the inhibition ability of the mixture is similar, and the OD of the mixture is lower than 1.

The mixture provided by the invention can be used for preparing oral care products.

Drawings

FIG. 1A chessboard dilution method was used to determine the effect of complex metabolites on biofilm formation. (A) The influence of the compound of 3-phenyllactic acid and cyclic leu-pro with different concentrations on the formation of the biological membrane; (B) the influence of different concentrations of phenylpropionic acid and cyclodeu-pro combination on biofilm formation; (C) the influence of different concentrations of 3-phenyllactic acid and phenylpropionic acid on biofilm formation.

FIG. 2 Effect of re-formulated metabolites on biofilm formation.

FIG. 3 Standard Curve of exopolysaccharide assay.

FIG. 4 effect of complex metabolites on exopolysaccharide production.

FIG. 5 Standard Curve for extracellular protein assay.

FIG. 6 effect of complex metabolites on extracellular protein production.

FIG. 7 Effect of complex metabolites on quorum sensing systems.

FIG. 8 is a heat map analysis of the effect of the complex metabolites on each index.

FIG. 9 scanning electron microscope observation of the negative control group on the structure of the double-bacterium biofilm. (A)7000 x; (B)20000 x.

FIG. 10 scanning electron microscope observation of the effect of positive control group Lactobacillus plantarum CCFM8724 on the structure of the double-bacterial biofilm. (A)7000 x; (B)20000 x.

FIG. 11 is a scanning electron microscope for observing the effect of the composition on the structure of the biological membrane of the double bacteria. (A)7000 x; (B)20000 x.

Detailed Description

EXAMPLE 1 inhibition of biofilm formation by the combination of Streptococcus mutans and Candida albicans

The preparation method of the streptococcus mutans bacterial suspension comprises the following steps: the Streptococcus mutans ATCC25175 was collected at-80 ℃ and inoculated in 5mL of TSB liquid medium at an inoculum size of 2%, and then subjected to static culture at 37 ℃ for 18 hours. Activation 3 generations were used for experiments. Adjusting the concentration of the bacterial suspension to 10 during the biological membrane experiment⁷cfu/mL。

The preparation method of the candida albicans suspension comprises the following steps:candida albicans ATCC18804 was inoculated into YPD liquid medium, and shake-cultured at 37 deg.C and 200r/min for 18 h. Activation 3 generations were used for experiments. Adjusting the concentration of the bacterial suspension to 10 during the biological membrane experiment⁶cfu/mL。

1: determination of biofilm amount by crystal violet method

And respectively adding 75 mu L of each of the streptococcus mutans bacterial suspension and the candida albicans bacterial suspension into a 96-well plate, then adding the compound into the well plate by a chessboard dilution method, and standing and culturing for 24h at 37 ℃. The negative control group replaces the compound with 5 percent DMSO aqueous solution with the same volume, and the positive control group replaces the compound with supernatant of lactobacillus plantarum CCFM8724(CGMCC No.5492) with the same volume. After the completion of the culture, the culture solution was removed, the biofilm was carefully washed with PBS for 2 times, and then allowed to stand at room temperature and air-dried. Adding 100 μ L of methanol into each well to fix the biological membrane, removing the methanol after 10min, naturally drying, adding 100 μ L of crystal violet solution with mass fraction of 0.1%, and dyeing the biological membrane for 30 min. After staining, the cells were washed 2 times with PBS, dissolved in 100. mu.L of 33% glacial acetic acid in each well, and OD was read with microplate reader_600nmAn absorbance value. Each set was set to 6 replicates.

Wherein, regarding the compound, the 3-phenyllactic acid, the phenylpropionic acid and the cyclodeu-pro are compounded in pairs by adopting a chessboard dilution method, so that the final concentrations are respectively 0, 12.5, 25, 50, 100 and 200 mu g/mL. As shown in FIG. 1, the most potent biofilm-inhibiting effect was observed when 3-phenyllactic acid and cyclodeu-pro were 200. mu.g/mL and 50. mu.g/mL, respectively, or phenylpropionic acid and cyclodeu-pro were 200. mu.g/mL and 50. mu.g/mL, respectively, or 3-phenyllactic acid and phenylpropionic acid were 200. mu.g/mL and 200. mu.g/mL, respectively. Therefore, the 3-phenyllactic acid, the phenylpropionic acid and the cyclodeu-pro are compounded respectively at 200, 200 and 50 mu g/mL, and the inhibition effect of the compounds on the biological membrane of the double bacteria is detected.

As shown in fig. 2, the formulation (composition) significantly reduced the formation of a dual-bacterial biofilm compared to Control (i.e., negative Control, which replaced the formulation with an equal volume of 5% DMSO aqueous solution), CCFM8724 (i.e., positive Control, which replaced the formulation with an equal volume of CCFM8724 fermentation supernatant).

2: determination of extracellular polysaccharide content

Besides the crystal violet staining method for determining the quantity of the biological membrane, the detection of macromolecular substances in the biological membrane is also an important index in the research of the biological membrane. We measured the effect of the mixture on exopolysaccharides in biofilms by the phenol-sulfuric acid method.

Firstly, drawing a glucose standard curve, preparing a glucose standard solution with the concentration of 200 mug/mL, adding the standard solution into a test tube to enable the final concentration to be 0, 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 mug/mL, then adding 1.0mL of 6% phenol, quickly shaking up, adding 5mL of concentrated sulfuric acid, standing for 30min at room temperature, measuring the light absorption value at 490nm, taking the glucose concentration as the abscissa and the absorbance OD_490nmAs an ordinate, a glucose standard curve was obtained as shown in FIG. 3.

Respectively adding 75 mu L of streptococcus mutans and Candida albicans suspension into a 96-well plate, then respectively adding 3-phenyllactic acid, phenylpropionic acid and cyclodeu-pro to make the final concentrations of the streptococcus mutans and the Candida albicans suspension be 200 mu g/mL, respectively, carefully washing by PBS after the culture is finished, then eluting the biological membrane by 100 mu L of PBS, collecting the biological membrane into a 1.5mL centrifugal tube, centrifuging at 12000r/min for 10min, then removing supernatant, and repeatedly washing for three times to ensure that water-soluble polysaccharide is removed. The water-insoluble polysaccharide was extracted with 500. mu.L of 1.0M sodium hydroxide at 37 ℃ for 2h with stirring. Centrifuging at 12000r/min for 10min to obtain supernatant, transferring the water insoluble polysaccharide alkali extractive solution into test tube containing 1mL 6% phenol, adding concentrated sulfuric acid 5mL, standing at room temperature for 30min, and adjusting OD_490nmAnd measuring the absorbance, and substituting the absorbance into a standard curve to calculate the content of the exopolysaccharide. The results are shown in fig. 4, which indicates that the mixture also significantly reduces the synthesis of exopolysaccharides, thereby blocking the production of exopolymers and reducing the formation of biofilm.

3: determination of extracellular protein content

Extracellular proteins are also important components of the biofilm matrix, and we determined the effect of the mixture on extracellular protein synthesis by BCA method.

First, a protein standard curve was plotted, a BSA protein standard solution was prepared at a concentration of 0.05mg/mL, and the standard solution was added to a 96-well plate to give final concentrations of 0.05, 0.10, 0.20, 0.40, 0.80, 1.20, 1.60, and 2.00. mu.g/mL. The BCA reagent A and the BCA reagent were prepared according to the BCA protein assay kitB, mixing according to the volume ratio of 50:1 to obtain BCA working solution, adding 200 mu L of working solution into each hole, placing at 37 ℃ for reaction for 30min, and reacting at OD_562nmMeasuring the absorbance, with the protein concentration as abscissa and the absorbance OD_562nmAs an ordinate, a BSA protein standard curve was obtained as shown in FIG. 5.

The double-bacterial biofilm obtained according to the method of example 2 was washed with PBS, then the biofilm was eluted with 100. mu.L PBS and collected in a 1.5mL centrifuge tube, and a suitable amount of small magnetic beads was added and placed in a pre-cooled module and disrupted with a high-throughput tissue disruption apparatus. The high throughput tissue disruptor parameters were set to disrupt 45s, stop 15s, and cycle 10 times. After crushing, the mixture is centrifuged at 12000g/min for 10min to take the supernatant. Adding 200 μ L of the working solution into each well of a 96-well plate, adding 20 μ L of the supernatant, reacting at 37 deg.C for 30min, and adjusting the OD_562nmAnd (4) measuring the absorbance, and substituting the absorbance into a standard curve to calculate the content of the extracellular protein. The results are shown in fig. 6, which indicates that the mixture also significantly reduces the synthesis of extracellular proteins, thereby blocking the production of extracellular polymers and reducing the formation of biofilms.

4: determination of the quorum-sensing Signal molecule AI-2

The oral complex environment is usually symbiotic with multiple bacteria, different bacteria transmit information through signal molecules, and are mutually regulated, dental plaque biomembranes formed by cariogenic bacteria are closely related to regulation and control of Quorum Sensing (QS) signal molecules, and the signal molecules of AI-2 which are widely communicated among various microbial species play an important role in microbial interaction. The QS system is involved in the regulation of a large number of gene expressions, including various phenotypes such as luminescence effect, biofilm formation, motility, etc., and the formation of biofilms can be reflected from another angle by the QS system. We examined the effect of the composition on the activity of the self-inducible molecule (AI-2) between Streptococcus mutans and Candida albicans by Vibrio harveyi BB170 bioluminescence.

mu.L of each of Streptococcus mutans and Candida albicans suspensions was added to a 24-well plate, and 250. mu.L of the composition was added, with the negative control group being replaced with an equivalent volume of 5% DMSO aqueous solution, and the positive control group being replaced with a supernatant of Lactobacillus plantarum CCFM 8724. After 24h of culture, sterile supernatant was collected and filtered for use.Inoculating the activated Vibrio harveyi BB170 to an AB culture medium, culturing at 30 ℃ and 180r/min for 12h, and adjusting the OD of a bacterial liquid_600nmDiluting the bacterial liquid with sterile fresh AB culture medium at a ratio of 1: 2000, and mixing well for later use. Mixing the collected supernatant with a Vibrio harveyi BB170 diluted bacterial suspension according to a ratio of 1: 50. Shaking-culturing at 30 deg.C and 100r/min for 5h, sucking 200 μ L in black opaque enzyme-labeled plate in dark condition, and placing in OD with multifunctional enzyme-labeled instrument_500nmDetecting the chemiluminescence condition of the Vibrio harveyi BB 170. The compositions also significantly reduce the production of interspecies AI-2 signal molecules, affecting their quorum sensing and thus biofilm formation.

5: scanning electron microscope observation biological film structure

The mature biological membrane is a three-dimensional structure composed of a large number of extracellular polymers such as the above extracellular polysaccharide, protein and other macromolecular substances, and the scanning electron microscope with high-power imaging is an excellent tool for observing the formation of the biological membrane and the extracellular matrix of the biological membrane, and is widely applied to the research of the structure of the biological membrane. We analyzed the effect of the scanning electron microscopy composition on biofilm structure.

Placing a sterile cover glass on a 6-hole plate, adding 1.5mL of pathogenic bacteria, adding 3-phenyllactic acid, phenylpropionic acid and cyclodeu-pro to make the final concentrations of the pathogenic bacteria respectively 200, 200 and 50 mu g/mL, culturing the double-bacteria biomembrane for 24h, taking out the cover glass, fixing the cover glass for 1h by 1mL of 2.5% glutaraldehyde, washing by PBS, and dehydrating by ethanol gradients (the concentrations are 10%, 25%, 50%, 75% and 90% v/v) for 20 min. Subsequently, the coverslip was immersed in 100% ethanol for 1h and dried at room temperature for one day. The cover glass was transferred to a copper stud and subjected to gold spraying (160s, 40mA) and then observed by a scanning electron microscope.

The results are shown in FIGS. 9-11. The negative double-bacterium control group biofilm in the fig. 9 is formed compactly, has a certain thickness and is in a remarkable wavy shape; and the biomembrane intervened by the positive control lactobacillus plantarum CCFM8724 in the figure 10 and the compound metabolite in the figure 11 is obviously crushed, obvious invagination can be observed under 20000 times, the structure of the biomembrane is obviously loosened, the thickness of the matrix is obviously reduced, and the physiological activity is influenced.

Example 2 preparation of mouthwash containing composition

3-6 parts of 3-phenyllactic acid, 3-6 parts of phenylpropionic acid and 2-5 parts of cyclodeu-pro, 4-9 parts of tween-60, 3-6 parts of span-40, 4-8 parts of glycerol, 5-10 parts of sodium carboxymethylcellulose and 350 parts of water. After being prepared, the mixture is filled into small bottles.

Example 3 preparation of toothpaste containing the composition

100 parts of toothpaste base material, 3 parts of natural mineral salt, 7 parts of composition and 40 parts of cranberry extract (concentrated to 1000 mu g/mL) are added into a stirrer to be stirred to obtain a mixture, the mixture is vacuumized and degassed to obtain paste, and the paste is ground to obtain the probiotic toothpaste.

The mouthwash prepared according to the method of example 2 above was applied to carious children, the experimental method being: 30 children aged 8-12 years, diagnosed with dental caries, were selected and randomized into 2 groups, and the control group was given a commercial mouthwash, and the experimental group was given a mouthwash supplemented with the composition. All children had their mouth closed for 3 minutes before the experiment, followed by collection of at least 2mL of non-irritating saliva and counting the number of streptococcus mutans by qPCR; the children in the experimental group and the children in the control group brush teeth once every morning and evening, and the mouth wash is used for rinsing after meals. After 6 weeks, saliva samples were collected as described before and counted for S.mutans using qPCR. The results of the experiment are shown in table 1.

TABLE 1

Note: denotes p <0.05 with significant differences.

The experimental results show that after the mouthwash is used, the number of the streptococcus mutans in the oral cavity of the children suffering from dental caries is obviously reduced, and the application product of the streptococcus mutans can effectively resist the growth condition of the streptococcus mutans in the oral cavity and relieve the dental caries of the children.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The composition for inhibiting the formation of cariogenic bacteria biofilm is characterized by comprising 3-phenyllactic acid, phenylpropionic acid and leucine-proline cyclic dipeptide, wherein the mass ratio of the 3-phenyllactic acid to the phenylpropionic acid to the leucine-proline cyclic dipeptide is (50-200): (25-200): (50-100).

2. The composition according to claim 1, wherein the cariogenic biofilm is a streptococcus mutans and candida albicans biofilm.

3. The composition of claim 1 or 2, wherein the solvent is ethanol or water.

4. The composition of claim 3, wherein the concentration of 3-phenyllactic acid is 50 to 200 μ g/mL, the concentration of phenylpropionic acid is 25 to 200 μ g/mL, and the concentration of leucine-proline cyclodipeptide is 50 to 100 μ g/mL.

5. The composition of claim 3, wherein the concentration of 3-phenyllactic acid is 150 to 200 μ g/mL, the concentration of phenylpropionic acid is 150 to 200 μ g/mL, and the concentration of leucine-proline cyclodipeptide is 50 to 60 μ g/mL.

6. The composition of claim 3, wherein the concentration of 3-phenyllactic acid is 200 μ g/mL, the concentration of phenylpropionic acid is 200 μ g/mL, and the concentration of leucine-proline cyclodipeptide is 50 μ g/mL.

7. A product comprising a composition according to any one of claims 1 to 6.

8. The product of claim 7, wherein the product is a mouthwash, toothpaste.

9. Use of a composition according to any one of claims 1 to 6 in the manufacture of an oral care product.

10. Use of the composition of any one of claims 1 to 6 for inhibiting the formation of a two-bacterial biofilm by streptococcus mutans and candida albicans.