CN113702559A

CN113702559A - Method for separating and identifying active substances of lactobacillus plantarum source inhibiting double-bacterium biological membrane

Info

Publication number: CN113702559A
Application number: CN202110936771.1A
Authority: CN
Inventors: 张秋香; 陈卫; 李佳珣; 陆文伟; 赵建新; 张灏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-11-26
Anticipated expiration: 2041-08-16
Also published as: CN113702559B

Abstract

The invention discloses a method for separating and identifying active substances of a lactobacillus plantarum source for inhibiting a double-bacterium biofilm, belonging to the technical field of microorganisms. According to the method, ethyl acetate is used for effectively extracting active ingredients in the lactobacillus plantarum fermentation liquor, the extract is detected through LC-MS so as to comprehensively understand the ingredients in the extract, then the ethyl acetate extract is further separated through silica gel column chromatography, the components with the obvious inhibition effect are obtained through screening, the components are subjected to derivatization and non-derivatization treatment respectively, then the detection is carried out through GC-MS, and the small molecular components can be effectively separated through sectional heating. The invention can comprehensively and accurately measure the components and the content of the small molecular compounds in the effective parts of the lactobacillus plantarum fermentation liquor.

Description

Method for separating and identifying active substances of lactobacillus plantarum source inhibiting double-bacterium biological membrane

Technical Field

The invention relates to a method for separating and identifying active substances of a lactobacillus plantarum source for inhibiting a double-bacterium biofilm, belonging to the technical field of microorganisms.

Background

Dental caries is one of the most common oral bacterial infectious diseases, and is troubled not only for adults but also for children of low age. Caries (ECC) in young children seriously harms the health of preschool children, and the incidence of the caries is the first disease of children. Clinical studies in recent years have shown that large numbers of candida albicans and streptococcus mutans are present in the plaque biofilm in the oral cavity of children with ECC. Mutans streptococci are the main cariogenic bacteria, and utilize sucrose to rapidly produce acid, create local low pH environment to assist the colonization of other cariogenic bacteria, form cariogenic biomembranes and further cause dental caries. And candida albicans can interact with streptococcus mutans on the tooth surface and form a dual-bacterial biofilm with stronger cariogenicity. Thus controlling biofilm formation, especially double-bacterial biofilm, can prevent dental caries.

The method for preventing and treating the decayed teeth is to brush teeth and fill teeth, while children usually have low brushing will and insufficient cleaning degree. With the increasing popularity of probiotics, there have been some studies and attempts to apply probiotic therapies in the oral medical field, such as lactobacillus paracasei NTU101 and its fermented milk (Lin and Pan 2014) and lactobacillus salivarius (krzyscik et al 2017). However, these studies have focused on the use and effect of lactobacillus, and have not analyzed specific effective substances. CN102533618A discloses a Lactobacillus plantarum CCFM8724 with good probiotic properties, which can significantly reduce the biofilm amount of Streptococcus mutans and Candida albicans in vitro and significantly reduce the dental caries score and tooth demineralization condition of a rat in a dental caries model in vivo. However, it is unknown what substance exerts an inhibitory effect on the biofilm of two bacteria.

At present, the following methods are mainly used for extracting active substances for inhibiting the biological membrane by lactic acid bacteria: firstly, separating protein substances by using an ammonium sulfate precipitation method; secondly, separating the effective components of the lactobacillus by using a single method such as an organic solvent extraction method, for example, a separation method of bacteriostatic active substances from the lactobacillus casei in patent CN 103937837 discloses that the bacteriostatic substances of the lactobacillus casei are separated by using an ethyl acetate multiple extraction method, and the result shows that the bacteriostatic substances exist in an ethyl acetate phase, but the bacteriostatic substances are not identified; or, the effective components of the lactobacillus plantarum are researched by changing pH, temperature, enzyme treatment and the like, for example, CN 104561227A, an analysis method for the inhibition of the lactobacillus plantarum metabolite and application thereof disclose that the lactobacillus plantarum metabolite capable of inhibiting the streptococcus mutans biofilm is analyzed, and the result shows that the effective substances have thermal stability and pH dependence and are sensitive to protease, but the substances are not identified.

In general, the following technical difficulties exist for separating, analyzing and identifying the active substances inhibiting the biological membrane:

(1) the ability of lactic acid bacteria to inhibit biofilms is related to their metabolites, but the active metabolite components of lactic acid bacteria are complex and commonly include organic acids, bacteriocins, 7-amino-4-methylcoumarin, and the like. This presents difficulties in the isolation of active metabolites that inhibit biofilms. And the secondary metabolites produced by different strains are different, and the production amount is also different.

(2) The method for extracting the biofilm inhibiting substance produced by lactic acid bacteria needs to be determined in accordance with the site where the substance exists, specific properties such as different structures, properties, molecular weights, and the like, and there are few methods that can take these influencing factors into consideration.

Disclosure of Invention

[ problem ] to

The invention aims to solve the technical problems that components playing a role in inhibiting double-bacterium biofilms in lactobacillus plantarum metabolites are unknown, and a corresponding separation and identification method is lacked.

[ solution ]

The invention provides a method for separating and identifying a substance which is derived from lactobacillus plantarum and inhibits a double-bacterium biofilm, wherein the double-bacterium biofilm is a biofilm inhibiting the interaction formation of streptococcus mutans and candida albicans, and the biofilm inhibiting 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, and the method comprises the following steps:

step (1): extraction of lactobacillus plantarum fermentation liquor by ethyl acetate

Fermenting and culturing lactobacillus plantarum CCFM8724, collecting fermentation liquor, extracting for multiple times by using ethyl acetate, respectively collecting raffinate phase and extract phase, concentrating the extract phase to obtain ethyl acetate extract, and detecting components in the ethyl acetate extract by using LC-MS;

step (2): separating and detecting components in the ethyl acetate extract by silica gel column chromatography and TLC

Separating and purifying the components in the ethyl acetate extract by silica gel column chromatography, using a mixture of chloroform and methanol as an eluent, concentrating the eluate, detecting by TLC, concentrating and drying the same components; the volume ratio of chloroform to methanol is 95: 5. 90: 10. 85: 15. 80: 20. 70: 30. 60: 40 or 50: 50;

and (3): detecting the effect of each component obtained in the step (2) on inhibiting the formation of the biological membrane

Mixing bacterial suspensions of streptococcus mutans and candida albicans, then respectively adding the components obtained in the step (2), standing and culturing for a period of time, detecting the film forming condition of a biological film, and replacing the components obtained in the step (2) with 5% DMSO water with the same volume as that of the biological film to be used as a negative control group; selecting components with obvious inhibition effect, and identifying by using GC-MS;

and (4): GC-MS identification of the composition of matter

Derivatizing the components with obvious inhibition effect obtained in the step (3), and analyzing the derivatized components and the components without derivatization by GC-MS respectively;

and (5): data processing

And (3) carrying out peak extraction, retention time correction, peak alignment and other treatments on the original data file obtained by GC-MS analysis to obtain related data matrixes of mass spectrum information, peak area, retention time and the like of the metabolite, and matching the data with data in a database to obtain a corresponding compound.

In one embodiment of the invention, in step (1), extraction is performed 3 times with ethyl acetate.

In one embodiment of the present invention, in step (1), the concentration is performed by rotary evaporation.

In one embodiment of the present invention, in step (3), 75. mu.L of each of Streptococcus mutans and Candida albicans suspension is added to a 96-well plate, followed by 50. mu.L of each of the components obtained in step (2), and the mixture is incubated at 37 ℃ for 24 hours.

In one embodiment of the invention, the derivatization in step (4) is to add 100 μ L of 0.1mg/mL methoxylamine pyridine hydrochloride solution to the dried sample obtained in step (2), vortex and shake for 30s, dry and constant temperature metal bath at 37 ℃ for 90min, then add 40 μ L of MSTFA reagent (containing 1% TMCS), metal bath at 37 ℃ for 30min, centrifuge at 12000g at room temperature for 15min, absorb 100 μ L of supernatant to the inner lining tube, and perform GC-MS identification.

In one embodiment of the present invention, the non-derivatization in step (4) is to add 100 μ L of ethanol to the dried sample in step (2), transfer the mixture to a lining tube, and perform GC-MS identification.

In one embodiment of the present invention, the GC-MS used in step (4) is programmed as follows: the mass number range of scanning is 40-450 m/z, the type of ion source is EI source, the temperature is programmed: the initial temperature is 150 ℃, the temperature is kept for 1min, the temperature is increased to 180 ℃ at the heating rate of 10 ℃/min, the temperature is kept for 4min, then the temperature is increased to 300 ℃ at the heating rate of 15 ℃/min, and the temperature is maintained for 17min under the condition; the flow rate of carrier gas (He) is 1 mL/min; electron ionization energy: 70 eV; the column model was RTX-5MS (30 m. times.0.25 mm. times.0.25 μm).

The invention also provides application of phenylalanine-proline (phe-pro) cyclic dipeptide or 3-phenyllactic acid in preparing a product for inhibiting a double-bacterium biofilm, wherein the double-bacterium biofilm is a biofilm formed by interaction of streptococcus mutans and candida albicans, and the product comprises the following components: medicine, daily chemical product, and snack. The daily chemical article comprises: tooth cleaning articles, for example: toothpaste, mouthwash, tooth powder and tooth washing liquid. The snack comprises a candy.

[ advantageous effects ]

According to the method, firstly, ethyl acetate (high extraction efficiency and low toxicity) is utilized to effectively extract active ingredients in the lactobacillus plantarum fermentation liquor, then the extract is detected through LC-MS so as to comprehensively understand the ingredients in the extract, then, the ethyl acetate extract is further separated through silica gel column chromatography, a component with a remarkable inhibition effect is obtained through screening, the component is subjected to derivatization and non-derivatization treatment respectively, and then, the component is detected through GC-MS, and the small molecular components can be effectively separated through sectional heating. The invention can comprehensively and accurately measure the components and the content of the small molecular compounds in the effective parts of the lactobacillus plantarum fermentation liquor.

The phenylalanine-proline (phe-pro) cyclic dipeptide or 3-phenyllactic acid obtained by the invention has the effect of inhibiting streptococcus mutans and candida albicans from forming a double-bacteria biological membrane, and can be used for preparing medicines, daily chemical products and snacks.

Drawings

FIG. 1 is a graph showing the effect of ethyl acetate extract and raffinate phases on the formation of a double-bacterial biofilm

FIG. 2 shows TLC exploration of ethyl acetate extract at different ratios of developing solvent

FIG. 3 shows the effect of chloroform-methanol elution on biofilm formation in silica gel column chromatography

FIG. 4 shows the results of the 3-phenyllactic acid-inhibiting biofilm test on two bacteria

FIG. 5 is a total ion flow diagram of the Cycloleu-pro standard

FIG. 6 is a total ion flow diagram of the ring phe-pro standard

FIG. 7 is a total ion flow graph of a sample

FIG. 8 is a cycloleu-pro mass spectrum

FIG. 9 is a mass spectrum of sample F10 at a retention time of 10.36min

FIG. 10 is a ring phe-pro mass spectrum

FIG. 11 is a mass spectrum of sample F10 at a retention time of 14.42min

FIG. 12 shows the results of the experiment for inhibiting two-bacterium biofilm by cyclic dipeptide

Detailed Description

The preparation method of the lactobacillus plantarum CCFM8724 fermentation liquid comprises the following steps: the overnight culture of Lactobacillus plantarum CCFM8724 in MRS medium was centrifuged at 8000r/min at 4 ℃ for 10min, and the supernatant was sterilized by filtration through a 0.22 μm filter and stored in a refrigerator at 4 ℃ for further use.

TSB liquid Medium (g/L): 17.0 parts of tryptone, 6.0 parts of yeast powder, 5.0 parts of sodium chloride, 3.0 parts of soybean peptone, 2.5 parts of glucose, 2.5 parts of dipotassium hydrogen phosphate and pH 7.0-7.4.

YPD liquid medium (g/L): peptone 20.0, glucose 20.0 and yeast powder 10.0.

MRS liquid medium (g/L): 10.0 parts of peptone, 10.0 parts of beef extract, 5.0 parts of yeast powder, 20.0 parts of glucose, 5.0 parts of sodium acetate, 2.0 parts of diammonium hydrogen citrate, 0.4 part of dipotassium hydrogen phosphate, 0.58 part of magnesium sulfate, 0.25 part of manganese sulfate, 801.0 mL of tween-801.0 and pH 6.2-6.4.

Example 1: method for separating and identifying substance derived from lactobacillus plantarum and capable of inhibiting double-bacterium biofilm

(1) Extracting the fermentation broth with ethyl acetate and preliminarily judging the composition by LC-MS

Adding equal volume of ethyl acetate into lactobacillus plantarum CCFM8724 fermentation liquor, extracting for 3 times, fully mixing uniformly, standing, collecting raffinate phase and extract phase respectively, combining extract phases extracted for 3 times, performing evaporation concentration by using a rotary evaporator at 50 ℃ and 100r/min to obtain ethyl acetate extract, and storing in a refrigerator at 4 ℃ for later use.

The raffinate phase and the extract phase are used for carrying out an experiment for inhibiting double-bacterium film formation, and the experimental method comprises the following steps: 75 mu L of streptococcus mutans and Candida albicans suspension are respectively added into a 96-well plate, then 50 mu L of ethyl acetate extract phase and 50 mu L of raffinate phase are respectively added, and standing culture is carried out for 24h at 37 ℃. The negative control group replaced the extract phase with the same volume of MRS raffinate phase and 5% DMSO aqueous solution. 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 reader₆₀₀ _nmAn absorbance value. Each set was set to 6 replicates. The experimental result is shown in fig. 1, the ethyl acetate extract phase significantly inhibits the formation of the double-bacteria biofilm, and the raffinate phase has no inhibitory effect. This indicates that the active ingredient is mostly of medium polarity.

Re-dissolving part of the ethyl acetate extract in 0.1% formic acid water solution, filtering with 0.22 μm filter membrane, and detecting 100 μ L filtrate in sample injection bottle liner by LC-MS. The LC-MS detection conditions are as follows: separating the components in the ethyl acetate extract by using a UPLC-Q-active Orbitrap/MS system, and keeping a column incubator at 30 ℃. Data acquisition was performed in positive ion mode using a mobile phase a composition of: 10mM HCOONH₄0.1% formic acid, 95:5 acetonitrile-water, and 10mM HCOONH as mobile phase B₄0.1% formic acid, acetonitrile-water 50:50, gradient elution procedure: 98% of A at 0-1min, 98-50% of A at 1-17min, 50% of A at 17-21min, 50-98% of A at 21-22min, and 98% of A at 22-25.1 min. Mass spectrometry data acquisition sets the primary scan range to 50-1050 m/z. The results of LC-MS measurements are shown in Table 1.

TABLE 1 LC-MS identification of the first ten compounds in the ethyl acetate fraction extract content

(2): separating and detecting components in the ethyl acetate extract by silica gel column chromatography and TLC

First, TLC determined the eluent ratio

Diluting 200 mu L of ethyl acetate extract to 1mL by using ethyl acetate, sucking a half tube by using a capillary tube, and spotting the half tube on a silica gel plate, wherein the volume ratio of the half tube to the half tube is 9: 1. 5:5 or 1: 9 chloroform: developing an ethyl acetate mixture; or, the volume ratio of 9: 1. 5:5 or 1: ethyl acetate of 9: developing a methanol mixture; or, the volume ratio of 9: 1. 5:5 or 1: 9 chloroform: the methanol mixture developed. As shown in fig. 2, the first 6 developers developed with a large amount of substance remaining at the origin and not suitable as an eluent, and chloroform: it is preferable that the Rf of the spots is at the midpoint when methanol is developed.

Then separating each component in the ethyl acetate extract by silica gel column chromatography

Performing wet column packing by using chloroform, adding ethyl acetate on a silica gel column to extract an extract, and performing wet column packing by using chloroform: methanol mixture (volume ratio 95:5, 90: 10, 85: 15, 80: 20, 70: 30, 60: 40 or 50: 50) as eluent. One tube was collected every 2mL, concentrated in vacuo, and the same fractions were combined by TLC to give 13 fractions F1-F13. The 13 fractions were separately purged to 1mL with nitrogen, each fraction was packed into 3 tubes and placed in a vacuum concentrator and dried at 45 ℃ under 0.1Kpa, wherein 1 tube was reconstituted with 5% DMSO water for evaluation of biofilm inhibition effect, and 2 tubes were subjected to GC-MS identification of the material components.

(3): detecting the effect of each component obtained in the step (2) on inhibiting the formation of the biological membrane

The strong membrane forming strains streptococcus mutans ATCC25175 and candida albicans ATCC18804 are respectively inoculated in 5mL of TSB liquid culture medium and YPD liquid culture medium in the inoculation amount of 2%, and are respectively subjected to static culture at 37 ℃ for 18h and shaking culture at 37 ℃ for 18 h. Each 75. mu.L of Streptococcus mutans and Candida albicans suspension was added to a 96-well plate, followed by silica gel column chromatography to obtain fractions F1-F1350. mu.L, and the mixture was incubated at 37 ℃ for 24 hours to form a biofilm. The negative control group replaced the F1-F13 component with an equivalent volume of 5% DMSO water. 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, and OD-labeled with microplate reader₆₀₀ _nmThe absorbance value is read. As shown in fig. 3, F10 had a significant effect of inhibiting biofilm formation.

(4): GC-MS identification of the composition of matter

The F10 fraction was identified by different pretreatment methods (derivatization and non-derivatization) to more fully reflect the composition of the active ingredient.

And (3) subjecting the component F10 obtained in the step (2) to derivatization and non-derivatization pretreatment, and performing GC-MS identification. For derivatization, one tube of the dried sample from step (2) was added to 100. mu.L of 0.1mg/mL methoxylamine pyridine hydrochloride solution, vortexed for 30s, and then subjected to dry isothermal metal bath at 37 ℃ for 90min, followed by addition of 40. mu.L of MSTFA reagent (containing 1% TMCS) and metal bath at 37 ℃ for 30 min. Centrifuging at 12000g for 15min at room temperature, sucking 100 μ L of supernatant into a lining tube, and performing GC-MS identification. For non-derivatization treatment, another tube dried sample in step (2) was added with 100. mu.L of ethanol, transferred to a lined tube, and subjected to GC-MS identification. A1 mg/mL standard solution of ring phe-pro and ring leu-pro was prepared simultaneously (ethanol dissolution).

The procedure for GC-MS was: the mass number range of scanning is 40-450 m/z, the type of ion source is EI source, the temperature is programmed: the initial temperature is 150 ℃, the temperature is kept for 1min, the temperature is increased to 180 ℃ at the heating rate of 10 ℃/min, the temperature is kept for 4min, then the temperature is increased to 300 ℃ at the heating rate of 15 ℃/min, and the temperature is maintained for 17min under the condition; the flow rate of carrier gas (He) is 1 mL/min; electron ionization energy: 70 eV; the column model was RTX-5MS (30 m. times.0.25 mm. times.0.25 μm).

The detection results of the derivatized samples are shown in Table 2, and the substances are mainly organic acids. The main bacteriostatic mechanism of organic acid is: h produced by dissociation thereof⁺After accumulation, the pH value in the environment can be reduced, and the growth of pathogenic bacteria is inhibited; the undissociated acid can damage the cell membrane of some bacteria, thus destroying energy metabolism and leading to cell death. The organic acid produced by lactobacillus, which has the effect of inhibiting biofilm formation, is mainly a moderately polar organic acid dissolved in ethyl acetate, and we speculate that it mainly acts by damaging the cell membrane of microorganisms. The experiment of inhibiting the biofilm is carried out on the organic acid 3-phenyllactic acid with the highest content detected by GC-MS, as shown in figure 4, the result shows that the 3-phenyllactic acid can obviously inhibit the formation of the double-bacterium biofilm.

TABLE 2 identification of chloroform-methanol column chromatography component F10 component

The detection results of the non-derivatized samples are shown in FIGS. 5-7, wherein FIG. 5 shows the leucine-proline cyclic dipeptide (cyclodeu-pro) standard, which peaks at 10.47 min; FIG. 6 is a phenylalanine-proline cyclic dipeptide (cyclophe-pro) standard, which peaks at 14.39 min; FIG. 7 shows the results of the sample, showing peaks at retention times of 10.36min and 14.42min, indicating that both leucine-proline cyclic dipeptide and phenylalanine-proline cyclic dipeptide may be contained in the sample. Further, the mass spectrograms of the samples at about 10.36min (FIG. 9) and 14.42min (FIG. 11) were analyzed, and compared with the mass spectrograms of the leucine-proline cyclic dipeptide standard (FIG. 8) and the phenylalanine-proline cyclic dipeptide standard (FIG. 10), it was confirmed that the samples did contain both of the leucine-proline cyclic dipeptide and the phenylalanine-proline cyclic dipeptide.

We speculate that cyclopeptide compounds can inhibit the formation of streptococcus mutans and candida albicans double-bacterial biofilms by regulating quorum sensing. Two cyclic dipeptides are subjected to biofilm inhibition experiments, as shown in figure 12, the results show that leucine-proline cyclic dipeptide and phenylalanine-proline cyclic dipeptide both can significantly inhibit the formation of double-bacterial biofilms, the leucine-proline cyclic dipeptide has better inhibition effect than the phenylalanine-proline cyclic dipeptide, and the leucine-proline cyclic dipeptide can still significantly inhibit the formation of the double-bacterial biofilms at 50 ppm.

TABLE 3 Cyclo-dipeptide content in chloroform methanol column chromatography component F10

Claims

1. A method for separating and identifying a substance inhibiting a biofilm of two bacteria derived from Lactobacillus plantarum, comprising the steps of:

Separating and purifying the components in the ethyl acetate extract by silica gel column chromatography, using a mixture of chloroform and methanol as an eluent, concentrating the eluate, detecting by TLC, concentrating and drying the same components; the volume ratio of chloroform to methanol in the chloroform-methanol mixture is 95: 5. 90: 10. 85: 15. 80: 20. 70: 30. 60: 40 or 50: 50;

Mixing bacterial suspensions of streptococcus mutans and candida albicans, respectively adding the components obtained by concentration and drying in the step (2), standing and culturing for a period of time, detecting the film forming condition of a biological film, selecting the components with obvious inhibition effect compared with negative control, and identifying by using GC-MS;

and (4): GC-MS identification of the composition of matter

Derivatizing the components with obvious inhibition effect obtained in the step (3), and analyzing the components which are subjected to derivatization treatment and are not subjected to derivatization treatment by using GC-MS respectively;

and (5): data processing

And (3) carrying out peak extraction, retention time correction, peak alignment and other treatment on the original data file obtained by GC-MS analysis to obtain related data matrixes including mass spectrum information, peak area and retention time of the metabolite, and matching the data matrixes with data in a database to obtain corresponding structural information of the compound.

2. The method according to claim 1, wherein in step (1), the extraction is performed 3 times by using ethyl acetate.

3. The method according to claim 1 or 2, wherein in step (1), the concentration is performed by rotary evaporation.

4. The method according to claim 1, wherein in step (3), 75 μ L of each of Streptococcus mutans and Candida albicans suspension is added to a 96-well plate, followed by 50 μ L of each of the components obtained in step (2), and the mixture is subjected to static culture at 37 ℃ for 24 hours.

5. The method of claim 1, wherein the derivatization in step (4) is performed by adding 100 μ L of 0.1mg/mL methoxylamine pyridine hydrochloride solution to the dried sample obtained in step (2), vortexing and shaking for 30s, performing a dry constant temperature metal bath at 37 ℃ for 90min, adding 40 μ L of 1% TMCS-containing MSTFA reagent, performing a metal bath at 37 ℃ for 30min, centrifuging, and sucking 100 μ L of supernatant into the inner lining tube for GC-MS identification.

6. The method of claim 1, wherein the underivation in step (4) is performed by adding 100 μ L of ethanol to the dried sample in step (2), transferring the sample to a lining tube, and performing GC-MS identification.

7. The method as claimed in claim 1, 5 or 6, wherein the GC-MS used in step (4) is programmed to: the mass number range of scanning is 40-450 m/z, the type of ion source is EI source, the temperature is programmed: the initial temperature is 150 ℃, the temperature is kept for 1min, the temperature is increased to 180 ℃ at the heating rate of 10 ℃/min, the temperature is kept for 4min, then the temperature is increased to 300 ℃ at the heating rate of 15 ℃/min, and the temperature is maintained for 17min under the condition; the carrier gas is helium, and the flow rate is 1 mL/min; electron ionization energy: 70 eV; the column model was RTX-5MS (30 m. times.0.25 mm. times.0.25 μm).

8. Use of phenylalanine-proline cyclic dipeptide or 3-phenyllactic acid in the manufacture of a product for inhibiting a biofilm of two bacteria.

9. Use according to claim 8, characterized in that the product comprises: medicine, daily chemical product, and snack.

10. The use according to claim 9, wherein the commodity comprises: a tooth cleaning article.