CN114404563B

CN114404563B - Composition for inhibiting formation of biological film of cariogenic bacteria

Info

Publication number: CN114404563B
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: 2023-09-08
Anticipated expiration: 2042-02-28
Also published as: CN114404563A

Abstract

The invention discloses a composition for inhibiting formation of a cariogenic bacteria biological film, and belongs to the technical field of microorganisms. The composition provided by the invention is a mixture of 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro, and can inhibit a biological film formed by interaction of streptococcus mutans and candida albicans. And the ability of the mixture to inhibit the double-bacteria biological membrane of streptococcus mutans and candida albicans is stronger than the individual inhibition ability of 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro, which is similar to the inhibition ability of lactobacillus plantarum CCFM8724 fermentation supernatant.

Description

Composition for inhibiting formation of biological film of cariogenic bacteria

Technical Field

The invention relates to a composition for inhibiting formation of a cariogenic bacteria biological film, and belongs to the technical field of microorganisms.

Background

Caries is one of the most common oral bacterial infectious diseases and does not occur in a short period of time, and the process of demineralization and enamel decomposition is often accompanied by the life of one person, thus afflicting not only adults but also young children. Wherein, the incidence rate of caries of preschool children, namely caries of low-age children (early childhood caries, ECC), is in the first place of children diseases, and seriously endangers the health of children. Caries occurrence is closely related to factors such as microorganisms, ingestion of foods and beverages containing sugar, oral hygiene habits of the host, and the like, wherein the formation of plaque biofilm by microorganisms is an initiating factor for caries.

Streptococcus mutans is a gram-positive spherical bacterium, has strong ability to metabolize sucrose to produce acid and form cariogenic biofilms, and is a major colonic bacterium early in the biofilm formation process, and is recognized as a major pathogenic bacterium for caries. Clinical studies have also found that candida albicans participates in the caries process, and a large amount of candida albicans and streptococcus mutans are detected in plaque biofilms of the oral cavity of children suffering from ECC, and interaction of the candida albicans and streptococcus mutans can form a biofilm with stronger caries. Candida albicans is a conditionally pathogenic fungus that usually colonizes the oral cavity with other conditionally pathogenic bacteria. Depending on the oral and systemic health conditions, these opportunistic pathogens may be converted from commensal to pathogenic bacteria. Candida albicans has the ability to adhere to a hydroxyapatite substrate and dissolve hydroxyapatite by releasing calcium ions, which invade dentin and secrete acids, promoting enamel demineralization. Thus, the interaction of bacteria and fungi is involved in the formation of dental plaque biomembrane, and brings a brand new view and greater challenges for preventing and treating dental caries. Therefore, inhibition of biofilm formation is particularly important to obtain an effective means of preventing dental caries in children.

In general, caries prevention methods are tooth brushing, dietary sugar intake reduction, dental floss and mouthwash use, tooth filling, and dental plaque biofilm removal using fluoride and other different points of entry, so it is of great importance that a set of compositions can be applied to oral products. IN201747009062a discloses an inhibitory effect of a small molecule inhibitor on oral plaque biofilm, KR1020210092052a discloses that a composition containing nanoemulsion cinnamon oil as an active ingredient can inhibit oral biofilm formation, but it requires nanoemulsification of cinnamon oil, which is costly.

Studies by Strom et al show that the loop (L-Phe-L-Pro) and 3-phenyllactic acid produced by Lactobacillus plantarum MiLAB393 have synergistic inhibition effects on Fusarium, aspergillus fumigatus and other fungi, but the required concentration is higher, and the Minimum Inhibitory Concentration (MIC) is 7.5mg/mL and 20mg/mL respectively. CN104739742a discloses a traditional Chinese medicine mouthwash for preventing and treating gingival bleeding, but the use of a Chinese herbal medicine product also risks pigmentation of teeth. CN113702559a (method for separating and identifying active substances of lactobacillus plantarum source for inhibiting double-bacteria biological membrane) discloses that lactobacillus plantarum metabolite 3-phenyllactic acid and cycloleu-pro can inhibit double-bacteria biological membrane, but the effect of the metabolite for inhibiting biological membrane alone does not reach the same effect as lactobacillus plantarum CCFM8724 fermentation liquor.

Disclosure of Invention

[ technical problem ]

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

Technical scheme

The invention provides a composition for inhibiting the formation of a biological film of cariogenic bacteria, which contains 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 biological film is a streptococcus mutans and candida albicans double bacteria biological film. Inhibition of the double bacterial biofilm means inhibition of the formation of a biofilm by interaction of Streptococcus mutans and Candida albicans, and a significant decrease in the formation of the biofilm (p < 0.05) compared with the control group as measured by crystal violet staining is judged to have an inhibitory effect.

In certain embodiments, the composition is dissolved in a solvent to form a mixed solution, optionally water, ethanol. In certain embodiments, the concentration of 3-phenyllactic acid in the solution is 50 to 200 μg/mL, the concentration of phenyllactic acid is 25 to 200 μg/mL, and the concentration of leucine-proline cyclic dipeptide is 50 to 100 μg/mL. In certain embodiments, the concentration of 3-phenyllactic acid in the solution is 150 to 200 μg/mL, the concentration of phenyllactic acid is 150 to 200 μg/mL, and the concentration of leucine-proline cyclic dipeptide is 50 to 60 μg/mL. In certain embodiments, the concentration of 3-phenyllactic acid in the solution is 200 μg/mL, the concentration of phenyllactic acid is 200 μg/mL, and the concentration of leucine-proline cyclic dipeptide is 50 μg/mL.

The invention provides a product which is obtained by applying the composition and can inhibit interaction of streptococcus mutans and candida albicans to form a biological film, wherein the product can be mouthwash or toothpaste. Of course, the composition may also be used to prepare other oral care products.

[ advantageous effects ]

The mixture of 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro provided by the invention has the effect of inhibiting streptococcus mutans and candida albicans from forming a double-bacterial biological film. Wherein the concentration of the lowest inhibition biological film of 3-phenyllactic acid is 50 mug/mL, the concentration of the lowest inhibition biological film of phenylpropionic acid is 25 mug/mL, and the concentration of the lowest inhibition biological film of cycloleu-pro is 50 mug/mL; compared with 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro, the inhibition capability of the mixture is improved, the effect of the mixture on inhibiting biological membranes is reduced by 1.2 compared with the OD of the mixture when the cyclic peptide is singly used at the same concentration, the OD of the mixture is reduced by about 0.3 compared with the OD of the mixture when the organic acid is singly used at the same concentration, and the inhibition capability of the mixture is similar compared with Lactobacillus plantarum CCFM8724, and the OD of the mixture is lower than 1.

The mixtures provided by the present invention can be used to make oral care products.

Drawings

FIG. 1 checkerboard dilution method determines the effect of the complex metabolites on biofilm formation. (A) The effect of the compounding of 3-phenyllactic acid and cycloleu-pro at different concentrations on biofilm formation; (B) The effect of different concentrations of phenylpropionic acid and cycloleu-pro complexation on biofilm formation; (C) The effect of the compounding of 3-phenyllactic acid and phenylpropionic acid at different concentrations on biofilm formation.

FIG. 2 effects of complex metabolites on biofilm formation.

FIG. 3 standard curve of extracellular polysaccharide assay.

FIG. 4 effect of complex metabolites on extracellular polysaccharide production.

FIG. 5 standard curve of extracellular protein assay.

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

FIG. 7 effects of the complex metabolites on the quorum sensing system.

FIG. 8 thermogram analysis of the effect of the complex metabolites on various indicators.

FIG. 9 is a scanning electron microscope observation of the effect of the negative control group on the structure of the double-bacteria biofilm. (A) 7000×; (B) 20000×.

FIG. 10 is a scanning electron microscope observation of the effect of positive control group Lactobacillus plantarum CCFM8724 on the structure of double bacterial biofilms. (A) 7000×; (B) 20000×.

FIG. 11 scanning electron microscopy observes the effect of the composition on the structure of the dual bacterial biofilm. (A) 7000×; (B) 20000×.

Detailed Description

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

The preparation method of the streptococcus mutans bacterial suspension comprises the following steps: the streptococcus mutans ATCC25175 deposited at-80℃was inoculated into 5mL of TSB liquid medium at an inoculum size of 2%, and the mixture was allowed to stand at 37℃for 18 hours. After 3 generations of activation, the test can be used. The concentration of the bacterial suspension is regulated to 10 when the biological film experiment is carried out ⁷ cfu/mL。

The preparation method of candida albicans suspension comprises the following steps: candida albicans ATCC18804 was inoculated into YPD liquid medium and shake-cultured at 37℃for 18 hours at 200 r/min. After 3 generations of activation, the test can be used. The concentration of the bacterial suspension is regulated to 10 when the biological film experiment is carried out ⁶ cfu/mL。

1: determination of biological film by crystal violet method

The streptococcus mutans suspension and candida albicans suspension are added into a 96-well plate respectively, 75 mu L of the streptococcus mutans suspension and the candida albicans suspension are added into the well plate by a chessboard dilution method, and then the mixture is subjected to stationary culture for 24 hours at 37 ℃. The negative control group uses 5% DMSO water solution with the same volume to replace the compound, and the positive control group uses lactobacillus plantarum CCFM8724 (CGMCC No. 5492) supernatant with the same volume to replace the compound. After the culture is finished, the culture solution is removed, the biological film is carefully washed by PBS for 2 times, and the biological film is stood at room temperature for airing. Adding 100 mu L of methanol into each hole to fix the biological film, removing the methanol after 10min, naturally airing, adding 100 mu L of crystal violet solution with the mass fraction of 0.1%, and dyeing the biological film for 30min. After the dyeing is finished, the solution is washed by PBS for 2 times, each hole is dissolved by 100 mu L of 33 percent glacial acetic acid, and OD is read by an enzyme-labeled instrument _600nm Absorbance values. Each set was set up with 6 parallels.

Wherein, for the compound, 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro are compounded in pairs by adopting a chessboard dilution method, so that the final concentrations of the compound are respectively 0, 12.5, 25, 50, 100 and 200 mug/mL. As a result, as shown in FIG. 1, the effects of inhibiting the biofilm were strongest when 3-phenyllactic acid and cycloleu-pro were 200, 50. Mu.g/mL, respectively, or when phenylpropionic acid and cycloleu-pro were 200, 50. Mu.g/mL, respectively, or when 3-phenyllactic acid and phenylpropionic acid were 200, 200. Mu.g/mL, respectively. Therefore, 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro are respectively 200, 200 and 50 mug/mL for compounding, and the inhibition effect of the compound on the double-bacteria biological membrane is detected.

As shown in fig. 2, the formulation (composition) significantly reduced the formation of the double bacterial biofilm compared to Control (i.e., negative Control, with the same volume of 5% dmso aqueous solution instead of the formulation), CCFM8724 (i.e., positive Control, with the same volume of CCFM8724 fermentation supernatant instead of the formulation).

2: determination of extracellular polysaccharide content

Besides determining the amount of the biological film by a crystal violet staining method, the detection of macromolecular substances in the biological film is also an important index in the research of the biological film. We measured the effect of the mixture on extracellular polysaccharide in the biofilm 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 ensure that the final concentration of the standard solution is 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 at room temperature for 30min, measuring the absorbance value at 490nm, taking the glucose concentration as the abscissa, and the absorbance OD _490nm On the ordinate, a glucose standard curve is obtained as shown in FIG. 3.

Adding Streptococcus mutans and Candida albicans suspension into 96-well plate respectively at 75 μl, adding 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro to final concentrations of 200, 50 μg/mL respectively, carefully washing with PBS after culturing, eluting with 100 μl PBS, collecting biological membrane in 1.5mL centrifuge tube, centrifuging 12000r/min for 10min, and discardingThe supernatant was washed three times repeatedly, ensuring removal of the water-soluble polysaccharide. 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 alkali extract of water insoluble polysaccharide into test tube containing 1mL 6% phenol, adding concentrated sulfuric acid 5mL, standing at room temperature for 30min, and standing at OD _490nm And measuring absorbance, and substituting the absorbance into a standard curve to calculate the content of the extracellular polysaccharide. The results are shown in fig. 4, which shows that the mixture also significantly reduced the synthesis of extracellular polysaccharide, thereby blocking the production of extracellular polymer 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.

Firstly, a protein standard curve is drawn, a BSA protein standard solution with the concentration of 0.05mg/mL is prepared, and the standard solution is added into a 96-well plate to ensure that the final concentration is 0.05, 0.10, 0.20, 0.40, 0.80, 1.20, 1.60 and 2.00 mug/mL. According to the operation of BCA protein determination kit, mixing BCA reagent A and BCA reagent B according to the volume ratio of 50:1 to obtain BCA working solution, adding 200 μl working solution into each well, standing at 37deg.C for 30min, and standing at OD _562nm Measuring absorbance at the position, and measuring absorbance OD by taking protein concentration as an abscissa _562nm On the ordinate, BSA protein standard curves were obtained as shown in FIG. 5.

A double-bacterial biofilm was obtained as in example 2, carefully rinsed with PBS, and then eluted with 100. Mu.L of PBS, collected in a 1.5mL centrifuge tube, added with an appropriate amount of small magnetic beads, placed in a pre-chilled module and broken with a high throughput tissue breaker. The high throughput tissue disruptor parameters were set to disrupt for 45s, stop for 15s, and cycle 10 times. Centrifuging at 12000g/min for 10min after crushing, and collecting supernatant. 200 mu L of working solution is added to each well of a 96-well plate, 20 mu L of supernatant is added, the mixture is placed at 37 ℃ for reaction for 30min, and the mixture is cooled to OD _562nm Absorbance was measured and substituted into the standard curve to calculate extracellular protein content. The results are shown in fig. 6, which shows that the mixture also significantly reduced synthesis of extracellular proteins, thereby blocking production of extracellular polymers and reducing biofilm formation.

4: determination of quorum sensing Signal molecule AI-2

The complex oral environment is usually multi-bacterial symbiosis, different strains transmit information through signal molecules to mutually regulate, dental plaque biomembrane formed by cariogenic bacteria is closely related to the regulation and control of Quorum Sensing (QS) signal molecules, wherein the signal molecules of AI-2 widely existing in the communication among microorganism species play an important role in the communication among microorganisms. The QS system is involved in the regulation of a number of gene expressions including various phenotypes such as luminescence, biofilm formation, motor capacity, etc., and by means of the QS system the biofilm formation can be reflected from another point of view. We examined the effect of the composition on the activity of the self-inducing molecule (AI-2) between Streptococcus mutans and Candida albicans species by the Vibrio harveyi BB170 bioluminescence method.

375. Mu.L of each of Streptococcus mutans and Candida albicans suspension was added to the 24-well plate, and 250. Mu.L of the composition was added thereto, and the negative control group was replaced with an equivalent volume of 5% DMSO aqueous solution, and the positive control group was replaced with Lactobacillus plantarum CCFM8724 supernatant. After 24h of culture, collecting and filtering to obtain sterile supernatant for later use. Inoculating activated Vibrio harveyi BB170 into AB culture medium, culturing at 30deg.C and 180r/min for 12 hr, and regulating bacterial liquid OD _600nm About 0.8, diluting the bacterial liquid by using a sterile fresh AB culture medium according to a ratio of 1:2 000, and uniformly mixing for later use. The supernatant collected above was mixed with the diluted bacterial suspension of Vibrio harveyi BB170 at a ratio of 1:50. Shake-flask culturing at 30deg.C and 100r/min for 5 hr, absorbing 200 μL in black opaque ELISA plate under dark condition, and measuring OD with multifunctional ELISA _500nm Detecting the chemiluminescent condition of the vibrio harveyi BB 170. The composition also significantly reduces the production of inter-species AI-2 signaling molecules, affecting their quorum sensing and thus affecting biofilm formation.

5: structure for observing biological film by scanning electron microscope

Mature biological film is a three-dimensional structure composed of a large amount of extracellular polymers such as the above-mentioned extracellular polysaccharide, protein and other macromolecular substances, and a scanning electron microscope with high-power imaging is an excellent tool for observing the formation of biological film and extracellular matrix of biological film, and is widely applied in the research of biological film structure. We analyzed the effect of scanning electron microscope compositions on the structure of the biofilm.

A sterile cover slip is placed on a 6-well plate, 1.5mL of each pathogenic bacterium is added, 3-phenyllactic acid, phenylpropionic acid and cycloleu-pro are added to make the final concentration of each pathogenic bacterium be 200, 200 and 50 mug/mL respectively, after culturing a double-bacterial biological membrane for 24 hours, the cover slip is taken out, 1mL of 2.5% glutaraldehyde is used for fixing for 1 hour, PBS is used for washing, and ethanol gradients (concentration 10%, 25%, 50%, 75% and 90% v/v) are used for dehydration, and each concentration is 20 minutes. Subsequently, the coverslip was immersed in 100% ethanol for 1h and dried at room temperature for one day. The coverslip was transferred to a copper bar for metal spraying (160 s,40 mA) and then observed by a scanning electron microscope.

The results are shown in FIGS. 9-11. The negative double-bacteria control group biological film of FIG. 9 is formed densely and has a certain thickness and is in a remarkable wavy shape; while the biological film interfered by the positive control lactobacillus plantarum CCFM8724 of fig. 10 and the compound metabolite of fig. 11 is obviously broken, obvious invagination can be observed under 20000 times, the biological film structure is obviously loosened, the thickness of the matrix is obviously reduced, and the physiological activity is influenced.

EXAMPLE 2 preparation of mouthwashes containing compositions

3-6 parts of 3-phenyllactic acid, 3-6 parts of phenylpropionic acid and 2-5 parts of cycloleu-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 250-350 parts of water. And (5) filling the mixture into small bottles after preparation.

EXAMPLE 3 preparation of toothpaste containing composition

100 parts of toothpaste base stock, 3 parts of natural mineral salt, 7 parts of composition and 40 parts of cranberry extract (concentrated to 1000 mug/mL) are added into a stirrer to be stirred to obtain a mixture, the mixture is vacuumized and then degassed to obtain paste, and the paste is ground to prepare the probiotic toothpaste, and the cranberry extract added in the example has a good inhibition effect on cariogenic bacteria.

The mouthwash prepared according to the method of example 2 above was applied to children suffering from caries by the following experimental method: 30 children 8-12 years old, diagnosed with caries, were selected and randomly divided into 2 groups, and the commercial mouthwash was given to the control group, and the mouthwash with the composition added was given to the experimental group. All children were orally closed for 3 minutes prior to the experiment, followed by collection of at least 2mL of non-irritating saliva and statistics of mutans streptococcus numbers using qPCR; the children in the experimental group and the control group brush teeth once a day in the morning and evening, and mouthwash is used for gargling after meals. After 6 weeks, saliva samples were collected as described previously and the streptococcus mutans was counted using qPCR. The experimental results are shown in table 1.

TABLE 1

Note that: * P <0.05 is indicated with significant differences.

From the experimental results, after the mouthwash is used, the quantity of the mutans streptococcus in the oral cavity of the children suffering from caries is obviously reduced, which proves that the application product of the bacteria can effectively resist the growth condition of the mutans streptococcus in the oral cavity and relieve the caries of the children.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 a biofilm of a cariogenic bacterium is characterized by comprising 3-phenyllactic acid, phenylpropionic acid and leucine-proline cyclic dipeptide, wherein the mass ratio of the 3-phenyllactic acid, the phenylpropionic acid and the leucine-proline cyclic dipeptide is (50-200): (25-200): (50-100).

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

3. A composition according to claim 1 or claim 2, wherein the composition is dissolved in a solvent to form a mixed solution, the solvent being ethanol or water.

4. The composition of claim 3, wherein the concentration of 3-phenyllactic acid is 50-200 μg/mL, the concentration of phenylacrylic acid is 25-200 μg/mL, and the concentration of leucine-proline cyclic dipeptide is 50-100 μg/mL.

5. The composition of claim 3, wherein the concentration of 3-phenyllactic acid is 150-200 μg/mL, the concentration of phenylacrylic acid is 150-200 μg/mL, and the concentration of leucine-proline cyclic dipeptide is 50-60 μg/mL.

6. A composition according to claim 3, characterized in that the concentration of 3-phenyllactic acid is 200 μg/mL, the concentration of phenylacrylic acid is 200 μg/mL, and the concentration of leucine-proline cyclic dipeptide is 50 μg/mL.

7. A product comprising the composition of 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 preparation of an oral care product.

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