CN114574386A

CN114574386A - Bacillus licheniformis and application thereof

Info

Publication number: CN114574386A
Application number: CN202210198047.8A
Authority: CN
Inventors: 徐波; 邱胡林
Original assignee: Guangdong University of Petrochemical Technology
Current assignee: Guangdong University of Petrochemical Technology
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2022-06-03
Anticipated expiration: 2042-03-01
Also published as: CN114574386B

Abstract

The invention relates to the field of microorganisms, and particularly relates to bacillus licheniformis and application thereof. The deposited number of the bacillus licheniformis is GDMCC No. 62062. The invention also discloses a product composition which contains the thallus substance and/or metabolite of the bacillus licheniformis. The bacillus licheniformis provided by the invention has killing effect on staphylococcus aureus, escherichia coli, clostridium, salmonella enteritidis and pseudomonas aeruginosa and shows dose dependence. The QX8 purified product inhibited the response of purple bacilli to the signal factor acetylhomoserine lactone, and in another test, the QX8 purified product produced a significant inhibitory effect on purpurin. Compounds produced by bacillus licheniformis QX8 at low concentrations are also able to interfere with cellular responses to signaling factors and inhibit the production of virulence factors, preventing biofilm formation.

Description

Bacillus licheniformis and application thereof

Technical Field

The invention relates to the field of microorganisms, and particularly relates to bacillus licheniformis and application thereof.

Background

The number of microbial species is enormous, and a wide variety of biologically active substances can be produced under great intermediate competition and evolutionary pressure, and the active substances play an important role in the aspect of drug development. The vigorous development of the microbial pharmaceutical industry also brings great problems that the drug resistance of pathogenic microorganisms is continuously enhanced, and the emergence of drug-resistant bacteria and super bacteria urgently needs to find new and high-activity antibacterial substances. Thermophilic bacteria are used as a microorganism in an extreme environment, and the mechanism of the thermophilic bacteria is different from that of conventional terrestrial organisms to adapt to the extreme environment, so that the thermophilic bacteria have the potential of generating new antibacterial active substances, but the research on the thermophilic bacteria antibacterial activity in China is less at present. The microorganism can generate a large amount of bioactive substances in the metabolic process, and the bioactive substances have the characteristics of high activity, wide action, novel structure, good safety and the like, and have greater biological adaptability compared with chemical synthesis. The microbial resource has important significance for the research and development of new drugs and is an indispensable resource. Microorganisms are an important source of research-oriented compounds, and most of the antibiotics used in agriculture, animal husbandry and modern medicine are metabolites screened from microorganisms or derivatives of the metabolites. Therefore, the method for breeding the microorganism with the antibacterial activity, separating the antibacterial compound, identifying and researching the metabolic mechanism of the microorganism and analyzing the chemical structure and the biological activity is a research hotspot for developing new microbial medicaments. Under the conditions of huge population base and huge interspecific competition, a plurality of microorganisms can produce various bioactive substances, one strain can produce a large number of active substances with different structures, and some strains even produce the same active substance, such as bacillus licheniformis, paenibacillus, erwinia, pseudomonas and the like, can try different antagonistic substances, wherein the bacillus can produce substances with stable physical properties and chemical properties and bacteriostatic activity, and has great application potential in biological and industrial aspects. At present, many achievements in the aspect of microbial pharmacy mainly comprise antibacterial drugs, antifungal drugs and macrolide drugs, and in addition, some anti-cancer drugs, immunosuppressant drugs, enzyme inhibitors and other drugs have obtained certain research achievements. The microbial reserves are huge, the hot spring resources in China are rich, the research on the microbes in special habitats is beneficial to finding out novel bioactive compounds with special functions, and the research is necessary to find out novel compounds with antibacterial action.

Disclosure of Invention

The first purpose of the invention is to provide a strain of Bacillus licheniformis (Bacillus licheniformis), and the preservation number of the Bacillus licheniformis is GDMCC No. 62062.

The strain is isolated from a hot spring sample in the geological park of the biological reef country in Mianyang county of southwest China.

Bacillus licheniformis GDMCC No.62062 has antibacterial activity, has broad-spectrum antibacterial activity to common pathogenic bacteria, QX8 is gram-positive, no movement and no hemolysis, and catalase, protease and phospholipase are negative when measured.

It is a second object of the present invention to provide a product composition comprising the bacterial material and/or metabolite of Bacillus licheniformis as described above.

Preferably, the microbial substance may be a live microbial cell, a dead microbial cell or a mixed microbial cell of a live microbial cell and a dead microbial cell of the Bacillus licheniformis.

Preferably, the metabolite contains 3-isopropylhexahydro-4H-pyridine [1,2-a ] pyrazine-1,4(6H) -diketone (3-isopropylhexahydro-4H-pyrido [1,2-a ] pyrazine-1,4(6H) -dion).

The molecular mass of this compound was 211.14410, which was the first compound to be isolated from microbial metabolism. The research result of the invention shows that the MIC and MBC of the compound aiming at the P.aeruginosa are respectively between 13 plus or minus 0.17mg/L and 22 plus or minus 0.7mg/L, and the compound has strong antibacterial effect on the P.aeruginosa at high concentration. Further antibacterial tests show that the compound can interfere with the signal transduction of pseudomonas aeruginosa and the release of virulence factors at 0.5 × MIC. Can inhibit the growth of the pseudomonas aeruginosa at 1 × MIC, and can rapidly kill the pseudomonas aeruginosa at 1 × MBC according to the mode of dosage and time.

Characterization assay profile of this compound:

high performance liquid chromatography analysis spectrum: see 6A;

analyzing the spectrum by a Fourier infrared spectrometer: see 6B;

GC-MS analysis spectrum: see 6C;

high resolution mass spectrometry spectrum: see 6D;

nuclear magnetic resonance separation spectrum: see 6E.

Wherein, the form of the metabolite is not particularly limited as long as it contains 3-isopropylhexahydro-4H-pyrido [1,2-a ] pyrazine-1,4(6H) -dione. For example, it may be a fermentation broth, a fermentation supernatant, an extract of a fermentation supernatant, or the like of the Bacillus licheniformis.

According to a particular embodiment of the invention, the extract can be either a crude or a purified extract.

Wherein, the preparation method of the extract preferably comprises the following steps:

(1) culturing the microorganism as described above in a medium to obtain a fermentation product containing the compound;

(2) isolating and optionally purifying the compound from the fermentation product.

In the present invention, the kind of the medium is not particularly limited as long as the microorganism can be allowed to grow and reproduce and the compound can be efficiently synthesized. According to a preferred embodiment of the invention, the culture medium is selected from BHI broth. These media are commercially available and the specific composition thereof will not be described herein.

In the present invention, the conditions of the culture are not particularly limited as long as the microorganisms can grow and reproduce and the compounds can be efficiently synthesized. According to a preferred embodiment of the invention, the culture conditions are such that the compound synthesized by the microorganism is secreted into the fermentation broth. More preferably, the conditions of the culture include: the temperature is from 30 to 90 ℃, preferably from 50 to 60 ℃, for example 55 ℃; the time is 24 to 100 hours, preferably 60 to 80 hours, for example 72 hours. Further preferably, the cultivation is carried out aerobically, for example, by means of shake cultivation.

In the present invention, the amount of the microorganism to be inoculated is also not particularly limited, and for example, the microorganism may be inoculated into a culture medium at a ratio of 8 to 12% by volume.

In the present invention, preferably, the method for isolating and optionally purifying the compound from the fermentation product comprises:

(21) carrying out solid-liquid separation on the fermentation product to obtain fermentation supernatant;

(22) contacting the fermentation supernatant with ethyl acetate, and extracting the compound at low temperature of 0-10 ℃ to obtain a crude extract containing the compound;

optionally, the method further comprises removing ethyl acetate from the crude extract;

further optionally, the method further comprises removing ethyl acetate in the crude extract, and then drying the obtained material to obtain crude powder of the compound;

(23) and sequentially carrying out silica gel column chromatography and reverse C18 silica gel column elution on the crude extract or the solution of the crude extract powder to obtain the compound.

In step (21), the term "fermentation product" is a fermentation broth including a fermentation cell and a fermentation supernatant, unless otherwise specified.

The method for solid-liquid separation of the fermentation product is not particularly limited as long as the fermentation cells can be efficiently separated from the fermentation solution, and for example, a conventional method such as centrifugation or filtration can be used. According to a specific embodiment of the present invention, the fermentation supernatant may be obtained by centrifugation at 10,000-15,000rpm for 5-20 min.

The amount of ethyl acetate used in step (22) may vary widely, provided that the compound is efficiently extracted. Preferably, ethyl acetate is used in an amount of 0.5 to 2 parts by volume, more preferably 0.8 to 1.2 parts by volume, and further preferably mixed in equal volumes, relative to 1 part by volume of the fermentation broth.

The extraction may be performed in an extraction tank.

Wherein the extraction time is not particularly limited, and preferably, the extraction time is 8 to 20 hours, more preferably 10 to 15 hours.

Wherein, for the convenience of subsequent operation, the purity and yield of the compound are increased, preferably, the method further comprises a step of removing the ethyl acetate after the extraction is finished, for example, the ethyl acetate can be removed by evaporation in a rotary evaporator.

Wherein, for the convenience of preservation, preferably, after removing the ethyl acetate, the method further comprises concentrating and drying the obtained material to obtain crude extract powder. The method of concentration may be vacuum concentration. The drying may be freeze drying, for example, in a freeze dryer.

In the step (23), preferably, the method of silica gel column chromatography comprises:

(i) loading the sample by wet column filling method, and mixing silica gel and CHCl₃Mixing, adding into chromatographic column (slowly) to make the silica gel precipitate in the column without bubbles, and adding CHCl₃Washing the column for multiple times for the mobile phase to make the silica gel precipitate uniformly;

(ii) adding 50-100 mesh silica gel powder into the crude extract or the solution of the crude extract powder to obtain clear liquid, performing rotary evaporation to obtain dry powder, and uniformly filling the dry powder in a column head of a silica gel column;

(iii) using CHCl₃And methanol as eluent to obtain different fractions, and testing antibacterial activity.

In step (ii), a solution of the crude extract powder is preferably used as a sample, and a methanol solution of the crude extract powder is more preferably used. It will be appreciated that some non-solution may be formed upon dissolution of the crude powder, which may be separated by centrifugation or filtration.

The addition amount of the silica gel powder is not particularly limited, and is preferably 10 to 20g, preferably 12 to 18g, and more preferably 15g, relative to 10g of the clarified liquid.

In step (iii), the mobile phase as the eluent can be prepared by conventional procedures, for example, CHCl is used for eluting the active ingredient₃And the methanol is matched with the flowing phase to obtain different fractional fractions by gradient elution.

Wherein, by testing the antibacterial activity, the fraction with the highest antibacterial activity is selected for next separation.

In some embodiments, the method of reverse C18 silica gel column elution comprises:

(a) wet loading by using a wet column filling method, soaking C1818-30 h in methanol to completely expand carbon chains, adding the solution along a chromatographic column to ensure that C18 is uniformly precipitated in the column body without bubbles, and washing the column body for multiple times by using the methanol as a mobile phase to ensure that silica gel is uniformly precipitated;

(b) dissolving the crude extract obtained from forward silica gel in eluent (methanol: water: 1), discarding insoluble substances, and uniformly filling the obtained clear liquid in column head of silica gel column;

(c) different fractions were obtained by elution using water and methanol as mobile phases and tested for antibacterial activity.

Similarly, the mobile phase as the eluent can be used for elution of the effective components by a conventional procedure, for example, gradient elution using water and methanol as the mobile phase ratio to obtain different fractions.

Wherein, by testing the antibacterial activity, the fraction with the highest antibacterial activity is selected as the purified compound.

The third purpose of the invention is to provide the application of the bacillus licheniformis or the product composition in bacteriostasis.

For example, the bacillus licheniformis bacteria or the product composition as described above may be used in vitro for bacteriostasis.

According to one embodiment of the invention, the application is a non-therapeutic purpose application.

The bacteria that can be inhibited are not particularly limited, provided that the growth, reproduction, metabolism or other vital activity of the bacteria can be interfered by the bacillus licheniformis or the product composition as described above. According to a preferred embodiment of the invention, the bacterial cells are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Clostridium violaceum, Salmonella enteritidis and Pseudomonas aeruginosa.

A fourth object of the present invention is to provide the use of a bacillus licheniformis strain as described above or a product composition as described above in the manufacture of a product for bacteriostasis.

It is a fifth object of the present invention to provide the use of a bacillus licheniformis bacteria as described above or a product composition as described above for inhibiting biofilm formation.

Studies have shown that the compounds provided by the invention are also capable of interfering with the response of cells to signaling factors and inhibiting the production of virulence factors to prevent the formation of biofilms. Specifically, 0.5XMIC extract inhibits PA biofilm formation, SEM and AFM images show that compared with a control group, the topological structure of the biofilm added with the compound provided by the invention is obviously changed, the biofilm in an experimental group becomes loose, the control group is flat and smooth, and for the formed biofilm, the formed biofilm can be torn and perforated after the extract is added, and finally the biofilm is damaged.

It is a sixth object of the present invention to provide the use of a bacillus licheniformis bacterium as described above or a combination of products as described above for inhibiting the response of violobacterium to the signal factor acetylhomoserine lactone.

A seventh object of the present invention is to provide the use of bacillus licheniformis as described above or a product composition as described above for inhibiting purpurin.

The invention can obtain the following beneficial effects:

under high concentration, QX8 metabolite shows killing effect on staphylococcus aureus, escherichia coli, clostridium, salmonella enteritidis and pseudomonas aeruginosa and shows dose dependence. The QX8 purified product inhibited the response of purple bacilli to the signal factor acetylhomoserine lactone, and in another test, the QX8 purified product produced a significant inhibitory effect on purpurin. The highest inhibitory concentration tested reached 96%. In this study, the compound was identified as 3-isopropylhexahydro-4H-pyrido [1,2-a ] pyrazine-1,4(6H) -dione by high resolution mass spectrometry, nmr, gc, fourier-ir, hplc after purification, having a molecular mass of 211.14410, the first compound isolated from microbial metabolism. The research result of the invention shows that MIC and MBC aiming at P.aeruginosa are respectively between 13 plus or minus 0.17mg/L and 22 plus or minus 0.7mg/L, and the invention has strong antibacterial effect on P.aeruginosa at high concentration. Further antibacterial tests showed that QX8 purified product could interfere with Pseudomonas aeruginosa signalling, the release of virulence factors at 0.5 × MIC. Can inhibit the growth of pseudomonas aeruginosa at 1 × MIC, and can rapidly kill the pseudomonas aeruginosa at 1 × MBC according to the dosage and time mode.

Compounds produced by bacillus licheniformis QX8 at low concentrations are also capable of interfering with cell responses to signaling factors and inhibiting the production of virulence factors preventing biofilm formation. The research result shows that the extract can block the QS regulation cascade reaction of the pseudomonas aeruginosa. As described above, the 0.5XMIC extract inhibited PA biofilm formation. SEM and AFM images show that, compared with a control group, the topological structure of the biomembrane added with the QX8 purified substance is obviously changed, the biomembrane in the experimental group becomes loose, the control group is flat and smooth, and the formed biomembrane can be torn and perforated after the extract is added, and finally the membrane is damaged. Disruption of biofilm structure is a promising strategy to inhibit the formation of drug-resistant P-biofilms. The surge fine movements play an important role in biofilm formation in pseudomonas aeruginosa, and in order for bacteria to form biofilms, they need to adhere to surfaces, and once adhered to surfaces, the bacteria spread through swarm and swimming type activities, eventually leading to biofilm formation on biological or non-biological surfaces, inducing persistent infections. Both colony and swimming activity of PA was observed to be inhibited in the presence of QX8 purified extract. SJ16 extract may also have the ability to block the original attachment of P. By preventing bacteria from moving towards the surface. This process has the potential to open up new pathways for the prevention of bacterial transmission, thereby minimizing the ability of the microorganisms to form biofilms.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

Bacillus licheniformis

Bacillus licheniformis (Bacillus licheniformis) of the invention is preserved in Guangdong province microorganism culture collection center (address: No. 59 building 5 of Middleya 100, Md., Guangdong province scientific microbiological research institute, zip code 510070) (the preservation unit is abbreviated as GDMCC) in 11-15.11-2021, and the preservation number is GDMCC number 62062, which is abbreviated as QX 8.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 shows the cell morphology of QX8 under an electron microscope;

FIG. 2 is a phylogenetic tree established by 16S rRNA sequence alignment;

FIG. 3 shows the antibacterial activity of the extract obtained by extracting QX8 culture medium with ethyl acetate and washing with water;

FIG. 4 shows the variation of the thermostability of crude QX8 extract with increasing temperature;

FIG. 5(a) is a photograph after recrystallization after purification of a crude extract;

FIG. 5(b) is a picture of silica gel plate showing iodine color development;

FIG. 6 is a graph of a QX8 extract characterization assay; (A) high performance liquid chromatography, (B) Fourier infrared spectrometer analysis (C), GC-MS analysis, (D) high resolution mass spectrometry analysis, and (E) nuclear magnetic resonance separation;

FIG. 7 is the anti-population effect of QX8 extract; (A) the extract interfered with the response of chromobacterium violaceum CV026 to homoserine lactone at a concentration of 0.2 xMIC; (B) positive control cinnamaldehyde and negative control methanol; (C) effect of different concentrations of QX928 extract on the concentration of lac-genin;

FIG. 8 is the effect of QX8 extract on Pseudomonas aeruginosa cell viability and biofilm topology (A) biofilm architecture under microscopic microscope; (B) biofilm structure under atomic force microscopy; (C) biofilm structure under an electron microscope;

FIG. 9 is a graph of the effect of QX8 extracts on Pseudomonas aeruginosa; wherein, (a) the effect of QX8 extract (MIC 13 ± 0.17mg/L) on pseudomonas aeruginosa cell motility and surge; (B) interference of QX8 extract (MIC 13 + -0.17 mg/L) on PA virulence factor; (C) interference of QX8 extract (MIC 13 + -0.17 mg/L) on Pseudomonas aeruginosa energy metabolism;

FIG. 10 shows the effect of QX8 extract on cell membrane permeability.

Detailed Description

The following preparation examples, examples and comparative examples will further illustrate the present invention, but do not limit the present invention accordingly.

Example 1

This example illustrates the obtaining of Bacillus licheniformis (Bacillus licheniformis) provided by the present invention

1.1 enrichment and isolation of Thermus spa

Five main hot springs (N104 degrees 35 '09.9 degrees in northern latitude, E31 degrees 58' 83.6 degrees in east longitude) in the national geological park of biological reef in Miyangan county in southwest China are collected, and water samples of the hot springs are collected by using a sterile container. The sample was 2m from the shore and 40cm deep. Away from the edges to become representative samples. The sample is stored at 4 ℃ and transported to a laboratory, after physicochemical parameters such as the salinity and the pH value of a hot spring are measured in the laboratory, the sample is filtered by a 0.45mm membrane, and the filter membrane is placed in Tryptic Soy Broth (TSB) for culture at 50-90 ℃. Setting a gradient at every 10 ℃ for 5 groups, and incubating for 3d at the rotating speed of a shaking table of 160 r/min. Diluting 1mL of the enriched solution with sterile water to 10%^-2、10^-3、10^-4、10^-5、10^-6The concentration gradient is evenly coated on an LB/TSA/BHI/MA solid plate, the temperature is between 50 and 90 ℃, and the culture is continuedCulturing for 3-5 days, observing and classifying the colony morphology, selecting representative colony, purifying by streaking, labeling and preserving to obtain 247 strains.

1.2 screening of bacteria having antibacterial Activity

The purified single strain was inoculated into BHI broth, shake-cultured at 50-90 deg.C for 72h at 160prm, and a gradient was set at every 10 deg.C. Meanwhile, pseudomonas aeruginosa, clostridium, salmonella enteritidis, staphylococcus aureus and escherichia coli which are stored in a laboratory are respectively inoculated into an LB liquid culture medium for activation culture for 12 hours, 50 mu L of activated pseudomonas aeruginosa, clostridium, salmonella enteritidis, staphylococcus aureus and escherichia coli are coated on an LB plate, and cell-free supernatant (CFS) of thermophilic bacteria zymocyte liquid which is 10,000 Xg and centrifuged for 15min is added into an Oxford cup for analysis and screening of antibacterial activity. Putting the oxford cup into a plate coated with pathogenic bacteria, adding 50 mu L of cell-free supernatant into the plate, incubating the plate at 37 ℃ for 24h, observing the inhibition zone, and preliminarily deducing that the bacteria can generate antibacterial active substances if the inhibition zone is generated (repeating each group of experiments for three times).

13 strains with antibacterial activity are screened out, and the supernatant fluid shows single or broad-spectrum antibacterial activity, and the antibacterial activity is shown in table 1.

TABLE 1 bacterial Strain numbering with antibacterial Activity

The strains QX8, QX13, QX31, QX51 and QX137 show positive antibacterial effect on 5 experimental bacteria, wherein the inhibition zone of the strain QX8 is the largest, so that the strain identification and subsequent research are carried out by taking the strain QX8 as a target strain.

1.3 Metabolic identification of strains

The metabolism identification is carried out on QX8, experiments show that QX8 has antibacterial activity and broad-spectrum antibacterial activity on common pathogenic bacteria, QX8 is gram-positive, does not move and does not cause hemolysis, catalase, protease and phospholipase of the QX8 are determined to be negative, and the thallus morphology is shown in figure 1 under an electron microscope.

1.4 preliminary characterization of 16S RNA of Strain

The strain with antibacterial activity isolated from hot spring was inoculated into TSA medium, shaken at 160R/min and cultured at 55 ℃ for 24h, 16S RNA sequence amplification primers were 1492R (5 'ACGGHTACCTTGTTTACGACTT-3', SEQ ID NO:1) and 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2), and PCR amplification systems are shown in Table 2.

TABLE 2 PCR reaction amplification System

The PCR reaction conditions are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 40s for 30 cycles; finally extending for 5min at 72 ℃, detecting the amplified product by 1.0% agarose gel electrophoresis, and entrusting the Beijing Bomaide company Limited to sequence, wherein the expected size of the product is about 1500 bp.

Analysis of the phylogenetic tree created by 16S rRNA sequence alignment (as shown in figure 2) revealed that strain QX8 was most closely related to b.tequilensis with 99% similarity, indicating that strain QX8 is bacillus licheniformis and that the 16S rRNA sequence of QX8 was submitted to GenBank under accession number MK 994992.

1.5 Strain preservation

The strain QX8 was deposited at 11/15.2021 at the Guangdong province culture Collection center (address: building No. 59, building No. 5, Middleyao No. 100, Md., Guangdong province scientific microbiological research institute, zip code 510070) (the deposit is abbreviated as GDMCC), with the deposit number GDMCC No.62062, QX8 for short.

Example 2

This example illustrates the preparation of a crude extract of the strain QX8

Inoculating activated Bacillus licheniformis QX8 into the basic fermentation medium, shake culturing at 55 deg.C for 72h, and centrifuging at 12,000r/min for 10min to obtain cell-free supernatant. The supernatant was mixed with an equal volume of ethyl acetate and incubated overnight at 4 ℃ in a separate extraction pot. The ethyl acetate is then evaporated in a rotary evaporator and the remaining residue is concentrated in a vacuum concentrator and lyophilized in a lyophilizer to obtain the crude extract.

Example 3

This example illustrates the performance of the selected strain QX8

3.1 evaluation of antibacterial Activity

Diluting Pseudomonas aeruginosa, Salmonella enteritidis, Clostridium, Staphylococcus aureus, and Escherichia coli to OD₆₀₀Each strain was added to a 96-well plate (one row of wells per strain), 20mg/mL of crude extract (dissolved in distilled water) was added to the first well of each row and each well was serially diluted in a gradient, and the strain was incubated at 37 ℃ for 24 hours to observe the growth of the strain, which was repeated three times per strain. The results are shown in FIG. 3.

As can be seen from FIG. 3, the QX8 culture medium extracted by ethyl acetate and washed by water has broad-spectrum antibacterial activity, and can obviously inhibit the growth of Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis and Staphylococcus aureus.

3.2 evaluation of the stability of the antibacterial active substances

The stability of the antibacterial active substance plays an important role in application, and the heat stability of the crude QX8 extract is evaluated by different temperature conditions. The method comprises the following specific steps:

(1) dissolving the extracted QX8 in methanol again for secondary purification, removing insoluble solid particles, and centrifuging to obtain dark red transparent liquid;

(2) rotary evaporating to obtain solid particles, treating the solid at 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C for 24h, dissolving 20mg in 1,000mL distilled water every 30min, and measuring its antibacterial activity;

(3) pseudomonas aeruginosa experimental bacteria are diluted in LB culture medium at a ratio of 1:300 after overnight culture. Coating 50 mu L of the solution on an LB flat plate, placing an oxford cup on the flat plate, and adding the solution prepared from the crude extract in the step (2) into the oxford cup;

(4) the plates were incubated at 37 ℃ for 24h, and the heat stability of the crude QX8 extract was evaluated by the inhibition zone size, and the results are shown in FIG. 4A.

As can be seen from FIG. 4A, the thermal stability of the crude QX8 extract decreased with increasing temperature, and the extract treated at 80 ℃ within 3.5h still had effective antibacterial activity, indicating that the crude QX8 extract had good thermal stability.

Example 4

This example illustrates the purification of an antibacterial active substance

4.1 determination of fractions of crude extracts of antibacterial Activity

After the crude extract obtained in example 2 after aqueous lyophilization was dissolved in methanol, the crude extract was measured by GC-MS using a Chemstation column under column conditions set to a column box temperature of 150 ℃ (4min), a temperature rise program of 4 ℃/min, a sample inlet temperature of 250 ℃ and a detector inlet temperature of 280 ℃. And (3) analyzing a spectrum peak by mass spectrometry, and determining and preliminarily estimating the components, the molecular structure and the molecular weight of the compound by GC-MS (gas chromatography-mass spectrometry), thereby providing necessary data support for later-stage purification and separation.

After comparing the GC-MS detection result of the crude extract of QX8 with a proton library, the antibacterial compound of the strain is mainly a pyrrole compound structure, as shown in Table 3, the literature shows that pyrrole is well known for various therapeutic uses and has been used as antibiotics, anti-tumor agents, antifungal agents, anti-inflammatory agents and cholesterol-lowering agents. Azoles also have the ability to inhibit the activity of HIV-1 virus, DNA polymerase and protein kinase. Pyrrole and its derivatives pyrrole protects against seizures and is useful as an effective anticonvulsant. But the similarity of the compound and the library comparison is about 80 percent, the fact that a new functional group exists on the bacteriostatic compound molecule of the strain is inferred, and the compound is separated and purified for characterization identification and molecular structure analysis of the antibacterial compound in subsequent research.

TABLE 3 GC-MS hit Structure

4.2 silica gel column elution

Silica gel column chromatography is a method for separating each component of a mixture according to the difference of adsorption capacity of silica gel to different substances. Silica gel has stronger adsorption capacity to large polar substances, and substances with small polarity are not easy to be adsorbed by the silica gel and are easy to be eluted. When solvent elution is adopted, a series of processes of adsorption → desorption → re-adsorption → re-desorption occur on silica gel, substances with larger polarity move on the silica gel for a small distance, and then are discharged from a column; the components with small polarity move on the silica gel for a long distance, and are firstly separated out of the column, a target sample is separated by utilizing the principle that different substances have different affinities for the silica gel, and the currently most common chromatographic separation method of the silica gel column chromatography is widely applied to the aspects of medicines, water quality treatment, microbial fermentation and the like. Laboratories typically select the appropriate ion exchanger based on the isoelectric point of the protein or by reviewing relevant literature data, and this study selects CHCl based on a review of the literature₃And methanol as eluent. On the basis of the optimized thermophilic anaerobic bacillus optimal fermentation condition, antibacterial active substances are collected.

The silica gel column separation is a common method for separating the mixture at present, and comprises the following specific steps:

(1) loading the sample by wet column filling method, and mixing silica gel and CHCl₃Mixing well, slowly adding along the chromatographic column to make the silica gel precipitate in the column body without bubbles. Using CHCl₃Washing the column for multiple times for the mobile phase to make the silica gel precipitate uniformly;

(2) dissolving crude extract obtained after evaporating ethyl acetate to dryness in methanol, removing insoluble substances, adding 15g of 50-100 mesh silica gel powder into the obtained clear liquid, performing rotary evaporation to obtain dry powder, and uniformly filling the dry powder in a column head of a silica gel column;

(3) use of CHCl in mobile phase₃And gradient elution with methanol as flow matching ratio to obtain different fractions, and testing antibacterial activity.

4.3 reverse C18 silica gel column elution

The C18 reverse phase column, principle is the same as hydrophobic chromatography, the sample will be adsorbed because of hydrophobic interaction, when the polarity is reduced, the force is reduced and eluted, the whole chromatography process is adsorption, desorption, re-adsorption, re-desorption process, the concrete steps are as follows:

(1) wet loading by using a wet column filling method, soaking C1824 h in methanol to completely spread carbon chains, and slowly adding the solution along a chromatographic column to ensure that C18 is uniformly precipitated in the column without bubbles. Washing the column for multiple times by using methanol as a mobile phase to ensure that the silica gel is uniformly precipitated;

(2) dissolving the crude extract obtained from the forward silica gel in a small amount of eluent, discarding insoluble substances, and uniformly filling the obtained clear liquid in the column head of the silica gel column;

(3) using water and methanol as mobile phase, gradient eluting according to proportion to obtain different fraction, and testing antibacterial activity.

4.4TCL Board verification

The antibacterial activity of each elution grade is verified by experimental bacteria, the grades with the antibacterial activity are subjected to silica gel plate sample loading verification by using methanol and CHCl₃As a mobile phase, a TCL plate was developed with elementary iodine, and single-point fractionation was obtained to prove that the fractionation was a purified product. As shown in fig. 5, fig. 5(a) is a picture of crude extract after purification and recrystallization, and fig. 5(b) is a picture of silica gel plate iodine color development verification.

4.5 Fourier Infrared analysis

Dissolving the purified antibacterial active substance in methanol, and performing infrared spectrum scanning by using a Fourier transform infrared spectrometer to obtain an infrared spectrogram for characteristic analysis of compound groups.

4.6 high performance liquid chromatography analysis

The detection sensitivity of the high performance liquid chromatograph is higher compared with that of a thin layer chromatograph. Because the active substance is unknown compound, the early-stage separation product of the compound is found to have strong fluorescence reaction on 210nm purple laser, and C18 is adopted for classification according to the separation, a C18 column (C18) is selected in high performance liquid chromatography analysis

C18 5μ

) And (6) carrying out verification. Detection using high performance liquid chromatographyMetabolite component, high performance liquid chromatography mobile phase is 20% methanol flow rate 1mL/min, ultraviolet detector wavelength is 210 nm.

4.7GC-MS analysis

GC-MS is commonly used for the study of natural metabolites, and is generally suitable for relatively small molecular mass substances, and the composition of QX8 metabolites is analyzed by GC-MS in the study. The Chemstation column conditions of the GC-MS were set to a column box temperature of 150 ℃ (4min), a temperature program of 4 ℃/min, a sample inlet temperature of 250 ℃ and a detector port of 280 ℃. And analyzing the gas chromatographic peak after the GC-MS test is finished.

4.8 nuclear magnetic resonance analysis

CNMR1H and C13 spectra of 10mg/mL purified compound dissolved in deuterated chloroform test compound and the peaks analyzed.

4.9 results

Dissolving the collected crude extract, adsorbing with macroporous adsorbent resin, eluting with 40% ethanol, lyophilizing, subjecting to silica gel column chromatography with chloroform and methanol at ratio of 6:4v/v as fluidity, collecting the fraction, and rotary evaporating to obtain solid. Using a carbon 18 column, the fraction collected from the C18 column (containing 40% methanol) showed bacteriostatic activity (fig. 5). After freeze-drying, detection was performed by hplc, and the maximum elution peak was shown at a retention time of 8.732 minutes (fig. 6A). GC-MS analysis was performed to identify the structure of the bioactive molecule, and compared to the NIST library, a major compound was present in the sample based on molecular weight, retention time and molecular formula, and the spectrum showed a major peak at 16.9 minutes, with the peak area corresponding to the amount of compound present in the sample. Mass spectrometry showed a compound mass of 210Da and a molecular formula of C11H18N2O2 (FIG. 6C), and HR-MS analysis of the active moiety showed a major spectral peak at m/z 211.14338. Thus, the experimental molecular weight of the active compound was determined to be 210.14338 (fig. 6D), which corresponds to the theoretical mass of the active moiety. The absorption peaks of the compounds were further investigated by FTIR spectroscopy. The FTIR spectrum showed that the characteristic peak located at (FIG. 6B)3,400-3,140 cm-1 corresponds to the amide N-H group, 2,940 cm-1 corresponds to the alkyl C-H group, and the characteristic peak at 1,638 cm-1 corresponds to the C-O amide and C ═ O stretches at 1450-1244 cm-1, respectively. The spectrum of H NMR (400MHz, CDCl3) in nuclear magnetic resonance (fig. 6E) showed signals at 1H NMR (400MHz, CDCl3) δ 6.28(s,1H), corresponding to secondary amide protons, at 4.03(dd, J ═ 9.7,3.9Hz,1H), 2.42-2.30 (m,1H) and 0.96(d, J ═ 6.5Hz, 3H), corresponding to methine protons, at 3.63-3.51 (m,2H), 2.13-1.99 (m,3H), 1.98-1.85 (m,1H) and 1.86-1.72 (m,2H), corresponding to piperidine protons. 13C NMR (101MHz, CDCl3) spectra showed signals at δ 170.33, 166.24 for the amide carbon atom, δ 58.99, δ 53.40 for the cyclohexane lipid aliphatic carbon atom, δ 45.51, δ 23.30 δ 24.66, δ 22.75 δ 28.10 for the piperidine carbon atom, δ 38.57, δ 21.22 for the aliphatic carbon atom. The mass spectrum data shows that the detected molecular ion peak is 211.14338, which is consistent with the theoretical value of [ M + H + ] -211.14410. The compound of formula C11H18N2O2, combined with carbon-hydrogen spectroscopy and high resolution mass spectroscopy, was identified as 3-isophytahydro-4H-pyrido [1,2-a ] pyrazine-1,4(6H) -dione, a molecule with two chiral centers (asterisks) with possibly a small amount of the diastereomer. In addition, some impurity peaks are observed in nuclear magnetism, particularly a plurality of small peaks accompanied in a carbon spectrum, and the material source has better non-corresponding selectivity with microbial fermentation. This was the first compound synthesized by the microorganism to have antibacterial and anti-QS activity, as queried by SCIFinder.

4.10 Heat stability and kill time determination

The compounds obtained after freeze-drying were treated at different temperatures. Pseudomonas aeruginosa was used as a test bacterium to verify the thermostability of the purified compounds. The compound is obtained by fermenting and separating thermophilic bacteria, the stability of a six-membered ring in a molecule is good, the compound still has good antibacterial activity after being treated for 21 hours at 60 ℃, and the inhibition zone can still reach 8mm after being treated for 21 hours at 80 ℃, which shows that the compound QX8 has good thermal stability and good application prospect.

By determining the minimum inhibitory concentration of CGT928 to Pseudomonas aeruginosa to be 17 +/-0.072 mg L-1, the killing time of QX8 is increased along with the increase of MIC concentration in the sterilization time determination. Pseudomonas aeruginosa was completely killed within 14 hours. When the concentration of the crude extract is 4 times of MIC, the pseudomonas aeruginosa can be completely killed within 1 hour. CFU was reduced by 7 log units (fig. 4B). The results show that the QX8 has a rapid bactericidal effect, and the rapid death of the pseudomonas aeruginosa when the dosage is increased shows that the crude extract QX8 has a strong bactericidal effect.

Example 5

This example illustrates the effect of QX8 extract on P.aeruginosa

5.1 Effect of QX8 extracts on cell morphology and Membrane Permeability

The overnight cultured pseudomonas was diluted to OD600nm ═ 1.0 and inoculated in LB broth for 12 hours. The collected bacteria were diluted to OD600 nm-1.0. The QX8 extract at a concentration of 1XMIC was added to the culture well, and the QX8 extract was used as a positive control and the water-added well as a negative control, and incubated for 30 minutes, and the morphology of the cells was observed under a microscope. The effect of QX8 on cell permeability was evaluated by electroporation, and the collected bacterial solutions were washed 3 times with PBS and diluted to OD600nm 2. Equal volume of PBS containing extracts of QX8 at concentrations of 1XMIC, 2XMIC, and 3 XMIC was added to the control, equal volume of PBS was added to the control, conductivity was measured at 20 ℃ ambient temperature for 0.15.30.60.120min, and all experiments were performed in triplicate and repeated several times

5.2 Effect of QX8 extracts on the metabolism of Pseudomonas aeruginosa cells

Activated P.aeruginosa was diluted with TSA 1:300 and cultured at 37 ℃ for 12 hours, and then QX8 extract was added to the medium to achieve a concentration of 1XMIC and cultured at 37 ℃ for 3 hours. The cells were collected and washed 3 times with PBS to remove water and medium from the cell surface. 0.6g of the cells were accurately weighed on an analytical balance, placed in a mortar, and 1g of sterilized quartz sand and 2mL of phosphate buffer were added. The slurry was ground in a mortar on ice, centrifuged at 12000 rpm for 10 minutes in a precooled centrifuge and the supernatant collected for use.

0.1mL of the collected supernatant was added with 5mL of Coomassie brilliant blue working solution, and then the volume was adjusted to 6mL with distilled water. Adding 1mL of distilled water and 5mL of Coomassie brilliant blue working solution as a control, respectively measuring the absorbance value of OD595nm, and calculating the content of the soluble protein after each treatment: and (3) taking 2mL of solution to be detected and an equivalent DNS reagent, measuring the absorbance value after treatment, and calculating the content of reducing sugar in the mycelium according to the standard curve.

1mL of the collected supernatant was taken, 2mL of trichloroacetic acid (8%) was added, 1mL of 2, 4-dinitrophenylhydrazine solution (0.1%) was added, shaking was performed, 5mL of NaOH solution (1.5mol/L) was finally added, each absorbance value was measured and then 540nm of the treatment solution with a wavelength of 540nm was used, and the pyruvic acid content per unit mass of the bacteria was calculated from the standard curve.

5.3 anti-quorum sensing and anti-biofilm Activity

Detection of anti-QS activity takes violet CV026 ATCC31532 as a report strain, cinnamaldehyde as a positive control and methanol as a negative control, and anti-biofilm activity is evaluated by quantifying the yield of viologen and the diameter of a zone of inhibition. Biofilm formation tests were performed using different concentrations of bacterial (QX8 strain) extracts (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mg/ml), using pseudomonas aeruginosa strains to dilute overnight cultured pseudomonas aeruginosa into 96-well plates of different concentrations of QX8 crude extract, incubated at 37 ℃ for 24 hours, and biofilm formation was detected by crystal violet staining. All experiments were performed in triplicate. The results are shown in FIG. 7.

As can be seen in fig. 7, QX8 exhibited anti-quorum sensing and anti-biofilm activity while inhibiting plankton growth. Specifically, chromobacterium violaceum CV026 was grown on a medium containing acyl homoserine lactone. After addition of 0.2XMIC purified material, white and purple areas were observed. In the white area, the bacteria grew normally but did not change color, indicating that this compound interrupted the response of chromobacterium violaceum CV026 to acyl-homoserine lactones (fig. 7A). As a result, 1XMBC showed significant inhibition compared to the previous QS inhibitor cinnamon, and no inhibition zone was detected using the extraction solvent methanol as a negative control (FIG. 7B). The lac-cine production was positively correlated to the activity of QS. Therefore, different concentrations of QX8 extract were used to assess the level of QSI. The research shows that the extract has dosage dependence on the inhibition rate of the lac-genin. As the concentration of the extract increased, the lac content decreased, with a 94% decrease in the 0.7x MIC lac content (fig. 7C). The above results indicate that the purified product of QX928 can interrupt intercellular quorum sensing by inhibiting the bacterial response to the signal factor acyl homoserine lactone.

5.4 biofilm formation assay

Pseudomonas aeruginosa were grown in 24-well polystyrene plates with different concentrations of qx8 extract to assess biofilm development on sterile glass coverslips (11 mm). In 24-well polystyrene plates, 1ml of LB medium containing a pseudomonas aeruginosa culture with an OD600nm ═ 0.1 was added to each well, with different densities of QX8 extract. Immersing a glass cover glass, standing the culture plate at 37 ℃, adding certain 16mg/L QX8 crude extract into the culture solution for incubation for 3 hours when another group of pseudomonas aeruginosa is cultured for 24 hours to form a normal membrane, and then dyeing the biological membrane by using fluorescent dye. Bacterial cells within the biofilm were labeled with fluorescent dyes (propidium iodide and fluorescein diacetate), further processed according to the manufacturer's instructions, and observed under an epifluorescence microscope. Viable cells are green and dead cells are red. The effect of the crude QX8 extract on membrane formation was evaluated using equal volumes of methanol as a negative control in the experiment.

FIG. 8 shows the effect of compounds under fluorescence microscopy on the structure of Pseudomonas aeruginosa biofilms. From figure 8 it can be seen that QX928 was used to assess the inhibitory and killing activity of biofilms. Pseudomonas aeruginosa was labelled with propidium iodide and fluorescein oxalate. The cells were observed under confocal laser fluorescence microscope, and the dead cells showed red color and the live cells showed green fluorescence. The experimental groups were treated with 0.5 × MIC for 24 hours (panel a, trial B). Compared with the control group, the biomembrane treated by the QX8 crude extract has loose structure, development defect and loose cell connection. The normally developed control group had intact membrane structures. There was a significant difference between the control and experimental groups. The death number of the microorganisms in the experimental group is far higher than that in the control group.

When the membrane structure develops normally, compared with the experimental group, 1x MIC QX8 crude extract is added, the membrane structure is damaged, and perforation and tearing occur. A number of cells die. The control group had normal structure and tight intercellular junction. The result shows that the QX8 crude extract has an inhibiting effect on the growth of pseudomonas aeruginosa at low concentration, can effectively kill reference bacteria at high concentration, and destroys formed biological membranes.

5.5 scanning Electron and atomic force microscopy

The effect of QX8 extract on the biofilm topology formed by pseudomonas aeruginosa was observed using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). For SEM, biofilms of pseudomonas aeruginosa were grown on cover slips immersed in BHI. Broth on 24 well polystyrene plate. The plates and coverslips were left at 37 ℃ for 24 hours. Different concentrations (12mg/L and 18mg/L) of cgt928 extract were added, incubated at 37 ℃ for 6 hours to remove plankton, and the coverslips were gently rinsed with 0.9% PBS. The samples were treated with 2.5% glutaraldehyde for 20 minutes and dehydrated with an ethanol gradient (10% to 95%) for 10 minutes. The dehydrated and dried biofilm was covered with gold and observed under a scanning electron microscope.

For AFM, pseudomonas aeruginosa grown overnight in BHI broth was diluted to OD600nm of 0.1, sterile coverslips were immersed in 24-well polystyrene plates, and the plates were held at 37 ℃ for 24 hours. Different concentrations of QX8 were added to the medium and incubated for 6 hours. After this incubation period, the biofilm formed on the coverslip was gently rinsed with phosphate buffer (pH 7.4) and then placed under a desiccator until completely dried. Finally, the biofilms were scanned in semi-contact mode under AFM. Equal volume of methanol was used as a negative control in the experiment.

As can be seen in fig. 9, the QX8 extract disrupted the topology of the biofilm. Specifically, we can see the effect of the crude QX8 extract on the biofilm topology formed by pseudomonas aeruginosa under SEM and AFM. Scattered cells and immature cell membranes were observed by scanning electron microscopy in the presence of crude extract compared to control. The change of the structure and the surface appearance of the film, the unevenness of the surface and the poor adhesion of the biological film to the surface of the glass plate are also observed under AFM. The height distribution indicates that the normal film structure is flat and compact. The experimental set of membrane structures was perforated. The membrane thickness significantly reduces the QX8 extract to down-regulate the motility of Pseudomonas aeruginosa.

5.6 bee colony motility assay and swimming motility assay

Bee sting movements were tested in the presence of QX8 extract (1.0 mg/ml). Overnight grown pseudomonas aeruginosa was diluted to OD600nm 1.0 and spotted in a colony culture medium containing BM2 (62 mM PBS at pH 7, 2mM MgSO4, 10 μ M FeSO4, 0.4% glucose, 0.1% casamino acids and 0.5% agar) supplemented with 10.0mg/L SJ16 extract supplement. The plates were incubated at 37 ℃ for 24 hours and the colony area was observed. Equal volume of methanol was used as a negative control in the experiment.

Overnight pseudomonas aeruginosa was diluted to OD600nm 1.0, spotted in trypsin broth supplemented with 12.0mg/L QX8 extract (10 g/L trypsin, 5g/L NaCl, and 0.3% agar), and the plates incubated for 24 hours at 37 ℃ for analysis. Equal volume of methanol was used as a negative control in the experiment.

5.7 virulence factor analysis

The effect of QX8 bacterial extract (12.0mg/L) on the production of a reference P.aeruginosa virulence factor was studied by measuring the levels of pyocyanin and rhamnolipids, and analyzing elastase and protease activities. For the pyocyanin assay, P.aeruginosa strains were grown overnight and diluted to OD600nm ═ 0.1 in PB medium (5 ml; 20g/l peptone, 1.4g/l MgCl2 and 10g/l K2SO 4). The tubes were sterilized, 10ml PB medium supplemented with QX8 crude extract and incubated at 37 ℃ for 24 h. The control group was negative by adding an equal volume of methanol. The supernatant containing 5ml PB medium (OD600nm ═ 0.1) was collected and centrifuged at 10,000 × g for 10 min; pyocyanin was extracted in 3ml chloroform, then 1ml 0.2N HCl was added and quantified in a 520nm spectrophotometer. For the rhamnolipid assay, pseudomonas aeruginosa strains (OD600nm ═ 0.1) were grown overnight at 37 ℃ in NB medium and supplemented with a control group supplemented with 10mg/L of QX8 extract and an equal volume of methanol as a negative control. The culture was centrifuged (10,000 Xg 10min), the supernatant collected and acidified to pH 2 (with HCl), and the absorbance measured at 570 nm.

The QX8 extract has effect in inhibiting virulence factor

The virulence activity of pseudomonas aeruginosa mainly comprises elastase and protease activities, and the formation of virulence factors accelerates the formation of a biological membrane. The virulence factors of P.aeruginosa were significantly reduced after QX8 treatment. Compared with the control group, the pyocyanin in the presence of QX8 is reduced by 14.7%; the rhamnolipid content is reduced by 58.2%. Shows a strong inhibitory effect on the activity of the enzyme. Compared with the control group, the elastase activity is reduced by 34.3 percent, and the protease activity is reduced by 87.1 percent. The results show that the crude extract of QX8 strongly inhibits intercellular signal channels and inhibits the formation of biological membranes.

5.8 Elastase assay

To assess elastase activity, P.aeruginosa containing QX8 purified product was cultured overnight and the culture supernatant (750. mu.l) and elastin Congo solution (250. mu.l; 5mg/in 0.1M Tris-HCl pH 8; 1 mM CaCl2) ml) were incubated at 37 ℃ for 16h, the control group was added with an equal volume of methanol as a negative control, the reaction mixture was centrifuged (3,000 Xg, 10 minutes), the supernatant was collected, absorbance was measured at 495nm, the control group was added with an equal volume of methanol as a negative control, and the experiment was run in parallel 3 times.

5.9QX8 extract has effect in inhibiting cell metabolism

For the metabolism of pseudomonas aeruginosa, we mainly determined the content of pyruvate, soluble protein and reducing sugar. Pyruvate is one of the basic products of the participating organisms. The change in pyruvate content laterally reflects the effect of the extract on bacterial metabolism. After QX8 treatment, the pyruvic acid content is reduced by 16.9% compared with that of a control group; the content of reducing sugar is reduced by 31 percent compared with that of a control group; the soluble protein in the experimental group was significantly affected by 41.5% compared to the control group. Crude extracts of QX8 may affect the synthesis of soluble proteins or disrupt cell membranes, resulting in protein loss and reduced soluble proteins. The results show that the crude extract of QX8 has strong inhibition effect on cell metabolism, and can inhibit the formation of biomembrane and cell growth.

5.10 Effect of QX8 extracts on the ion permeability of cell membranes

Cells are surrounded by cell membranes, which change permeability when damaged. The protoplasm will leak and penetrate into the liquid causing the conductivity of the solution to increase increasing conductivity. After the QX8 compound is added, the conductivity of water rises with time compared with the control group, the conductivity of the solution and the dosage of the compound are dependent on the tolerance, and the conductivity of the control group is basically kept unchanged. The results are shown in FIG. 10, which shows that the crude extract destroys the cell membrane and changes its permeability.

The preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the foregoing embodiments may be combined in any suitable manner without contradiction. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

<110> Guangdong institute of petrochemical industry

<120> bacillus licheniformis and application thereof

<130> P2022F0084-GDSH

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> 16S RNA sequence amplification primer 1492R ()

<400> 1

acgghtacct tgtttacgac tt 22

<210> 2

<211> 20

<212> DNA

<213> 16S RNA sequence amplification primer 27F ()

<400> 2

agagtttgat cctggctcag 20

Claims

1. A strain of Bacillus licheniformis (Bacillus licheniformis), characterized in that the preservation number of the Bacillus licheniformis is GDMCC No. 62062.

2. A product composition comprising biomass and/or metabolites of bacillus licheniformis according to claim 1.

3. The product composition of claim 2, wherein the bacterial material is live and/or dead bacteria of the bacillus licheniformis; and/or

The metabolite contains 3-isopropylhexahydro-4H-pyridine [1,2-a ] pyrazine-1,4(6H) -diketone.

4. Use of a bacillus licheniformis strain according to claim 1 or a product composition according to claim 2 or 3 for bacteriostasis.

5. Use of a bacillus licheniformis according to claim 1 or a product composition according to claim 2 or 3 for the preparation of a product for bacteriostasis.

6. The use of claim 4 or 5, wherein the Bacillus licheniformis or the product composition is capable of inhibiting bacteria selected from at least one of Staphylococcus aureus, Escherichia coli, Bacillus violaceus, Clostridium clostridia, Salmonella enteritidis and Pseudomonas aeruginosa.

7. Use of a bacillus licheniformis according to claim 1 or a product composition according to claim 2 or 3 for inhibiting biofilm formation.

8. The use of claim 7, wherein the inhibiting biofilm formation inhibits biofilm formation by P.

9. Use of a bacillus licheniformis according to claim 1 or a product combination according to claim 2 or 3 for inhibiting the response of violobacterium to the signal factor acetyl homoserine lactone.

10. Use of a bacillus licheniformis strain according to claim 1 or a product composition according to claim 2 or 3 for inhibiting purpurin.