A new actinomycete strain with high-yield quorum sensing inhibitory activity and its application

Abstract

The invention discloses a new actinomycete Streptomyces sp.D67 with high yield of quorum sensing inhibitory activity product and its application. An actinomycete Streptomyces sp.D67 isolated from the soil of Kumtag Desert in Xinjiang belongs to a new bacterium of the genus Streptomyces. The strain is subjected to metabolic fermentation to obtain a crude extract of a high yield of quorum sensing inhibitory activity product, with the highest reaching 540 mg / L. The crude extract of the strain has a strong quorum sensing inhibitory activity against purple bacillus CV026, can significantly inhibit the clustering swimming characteristics of Pseudomonas aeruginosa, and has significant inhibitory activity against the production of its biofilm, pyocyanin and rhamnolipid. When the concentration of the crude extract is 5 mg / mL, the highest inhibition rates can reach 47%, 40.9% and 27% respectively. The TLC biological development and antibacterial activity results of the crude extract of Streptomyces D67 show that the crude extract has a variety of quorum sensing inhibitory activity molecules and antibacterial compounds, has a good application prospect for being developed to prepare functional drugs and agricultural biological control, and can be widely used in the pharmaceutical and agricultural industries.

Description

Technical field:

[0001] The invention relates to the technical field of microbial strains and applications thereof, and specifically to the technical field of a new actinomycete strain with high-yield quorum sensing inhibitory activity and application of a crude extract thereof. Background technology:

[0002] Quorum sensing (QS) refers to the ability of microorganisms to spontaneously produce and release certain specific signal molecules, sense changes in their concentration, and regulate the group behavior of microorganisms. Quorum sensing inhibitor (QSI) is a chemical substance that can block the quorum sensing pathway. Existing studies have shown that quorum sensing can regulate gene expression and participate in many important biological processes, such as spore formation, virulence, and biofilm formation, which can cause animal and plant diseases and food spoilage. Quorum sensing inhibitors can block the quorum sensing pathway by inhibiting the synthase of quorum sensing signal molecules or producing signal molecule analogs to compete for binding to transcription protein receptors, without causing significant survival pressure on pathogens, thereby delaying the increase of pathogen resistance and providing new ideas for pathogen prevention and control and the development of related new drugs. Therefore, screening safe, efficient, and relatively specific quorum sensing inhibitors has also become an important topic and research hotspot for research related to human and animal health, agricultural plant protection, food preservation, and other national and people's livelihood research.

[0003] Among animal pathogens, Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that is widely found in soil, water, air, human skin, respiratory tract and intestines. It can cause human wound infection, keratitis, pulmonary cystic fibrosis and other diseases; it is also a member of the ESKAPE group of multi- and pan-antibiotic-resistant bacteria, which is related to the production of a large number of biofilms, pyocyanin, rhamnolipids and proteases in its quorum sensing pathway, making the cure of related diseases a great challenge. Among plant pathogens, Erwinia amylovora can cause apricot fire blight, which has a wide host range and can harm more than 220 species of plants in more than 40 genera, such as pears, apples, hawthorns, kiwanos, plums, etc. It can also produce biofilms and other pathogenic factors, all of which are related to quorum sensing. Related studies have shown that the quorum sensing of related plant pathogenic strains is blocked, the pathogenicity of the pathogens is reduced or even lost, providing new ideas for the prevention and control of related pathogens.

[0004] Actinomycetes have always been an important source of natural active metabolites. Since the British Fleming discovered penicillin in 1929, according to incomplete statistics, there are 33,500 natural active products from microorganisms, of which 13,700 are from actinomycetes, accounting for about 41%, and about 80% are from Streptomyces. The extreme environment of the desert is characterized by extreme drought, strong solar ultraviolet radiation, and extreme temperature changes in the diurnal cycle. It breeds a rich variety of extreme environmental microorganisms, among which actinomycetes are one of its important components. Relevant studies have shown that the metabolites of actinomycetes in arid areas exhibit the characteristics of high temperature resistance and easy solubility, and have broad application prospects in the fields of medicine and health, agricultural and animal husbandry production, and food processing. The Kumtag Desert is located in the eastern part of Xinjiang. It is hot and dry in summer, with the highest temperature reaching 49.6℃ and the highest surface temperature reaching 82.5℃. In winter, the lowest temperature is generally -10℃, and the extreme low temperature can reach -29℃. Its ecological environment is extremely special, which provides a theoretical basis for the excavation of relevant actinomycetes and the development of relevant quorum sensing inhibitory active products. However, there are few reports in China on actinomycetes in this region and the development of related quorum sensing inhibitory activity products, and there is a need to continuously explore and find better and more excellent strains and applications. Summary of the invention:

[0005] In view of the technical problems that the existing quorum sensing inhibitors have few new bacteria that can be used and the efficiency is not high, the present invention aims to provide a new actinomycete Streptomyces sp.D67 with high-yield quorum sensing inhibitory activity and its application in quorum sensing inhibition. The actinomycete Streptomyces sp.D67 isolated from the soil of the Kumtag Desert in Xinjiang belongs to a new bacterium in the genus Streptomyces. The strain has quorum sensing inhibitor activity and has quorum sensing capabilities such as inhibiting Erwinia from producing biofilms, Pseudomonas aeruginosa from producing biofilms, pyocyanin, rhamnolipids, and proteases. The strain is subjected to metabolic fermentation to obtain a crude extract of a high-yield quorum sensing inhibitory activity product, with a maximum yield of 540 mg / L. The strain was verified by antibacterial activity, quorum sensing inhibition activity, and the characteristics of Pseudomonas aeruginosa cluster swimming characteristics, biofilm, pyocyanin and rhamnolipid metabolism. The crude extract of the strain has a strong quorum sensing inhibition activity against purple bacillus CV026, can significantly inhibit the cluster swimming characteristics of Pseudomonas aeruginosa, and has significant inhibitory activity on the production of Pseudomonas aeruginosa biofilm, pyocyanin and rhamnolipid, and is obviously concentration-dependent. When the concentration of Streptomyces sp.D67 crude extract is 5 mg / mL, its maximum inhibition rate on Pseudomonas aeruginosa biofilm, pyocyanin and rhamnolipid can reach 47%, 40.9% and 27%, respectively. The TLC biodevelopment and antibacterial activity results of the new strain Streptomyces sp.D67 crude extract show that the crude extract contains a variety of quorum sensing inhibitory active molecules and antibacterial compounds, and has a good application prospect for the development and preparation of functional drugs and agricultural biological control, and can be widely used in the pharmaceutical and agricultural industries.

[0006] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0007] The invention provides a new actinomycete Streptomyces sp.D67 with high-yield quorum sensing inhibition activity. The Streptomyces sp.D67 belongs to a new strain of the genus Streptomyces, and the strain is preserved in China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCC NO: M 20221542.

[0008] The new actinomycete Streptomyces sp.D67 provided by the present invention is screened and separated from soil samples in the Kumtag region of Xinjiang. Through 16S rRNA gene sequence phylogenetic analysis and morphological analysis, Streptomyces D67 belongs to the genus Streptomyces. By sequencing the gene of the strain, the obtained sequence is subjected to BLAST comparison analysis on the NCBI website. The 16S rRNA gene sequence of Streptomyces sp.D67 is consistent with the standard model bacteria of the genus, Streptomyces ardesiacus NBRC 15402.T , StreptomycesheliomyciniNBRC 15899 T and Streptomyces pluripotens MUSC 135 T The highest homology was 98.39%, 98.11% and 98.03% respectively. The sequences with higher homology were selected to construct the 16S rRNA phylogenetic tree. Streptomyces sp.D67 and Streptomyces pluripotens MUSC 135 T (CP021080) are more closely related and clustered in one branch. After a series of polyphasic classification identifications, Streptomyces sp.D67 and Streptomyces ardesiacusNBRC 15402 T , Streptomyces heliomyciniNBRC 15899 T and Streptomyces pluripotens MUSC135 T There are obvious differences, and it can be determined that Streptomyces sp.D67 is a new species of Streptomyces, which has the typical characteristics of new species.

[0009] The Streptomyces D67 provided by the present invention is identified at the molecular level by the above-mentioned strain system well-known and recognized in the art, combined with colony morphology identification and physiological and biochemical characteristics identification analysis. The Streptomyces sp. D67 provided by the present invention is Gram-staining positive, with a colony size of 0.2 cm-0.6 cm×0.2 cm-0.6 cm, is tightly bound to the culture medium, has a dense texture, has white velvety aerial hyphae, white spores, and light yellowish intrabasal hyphae.

[0010] The Streptomyces D67 provided by the invention has positive protease, amylase and cellulase activities; the sugar carbon sources that can be utilized by Streptomyces ardesiacus D67 are dextrin, xylose, turanose, sucrose, inositol, L-arabinose, D-arabitol, D-fructose, D-galactose, D-glucose and L-rhamnose; and the physiological and biochemical characteristics of the strain show that the Streptomyces ardesiacus NBRC 15402 and the adjacent strains are homologous to each other. T , Streptomyces heliomycini NBRC 15899 T and Streptomycespluripotens MUSC 135 T There are clear differences.

[0011] After the above-mentioned strain identification, as well as the well-known and recognized strain system molecular level identification and strain physiological and biochemical system test verification in the art, it was confirmed that the strain numbered D67 is a typical new strain Streptomyces sp.D67 within the Streptomyces category. The strain has been deposited in the Budapest Treaty International Microbiological Depository: China Type Culture Collection (CCTCC) before the application date, address: Wuhan University, Wuhan, China, Postal Code: 430072, Deposit Number: CCTCCNO: M 20221542, and the deposit date is October 7, 2022.

[0012] The gene sequence of the novel actinomycete Streptomyces sp. D67 with high quorum sensing inhibitory activity is shown in SEQ ID NO: 1.

[0013] In the present invention, the isolation medium of the new actinomycete Streptomyces sp.D67 is: soluble starch 20 g / L, KNO31 g / L, K2HPO40.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, agar 18 g / L, 10% soil extract 1000 mL, pH 7.5.

[0014] In the present invention, the purification culture medium of the new actinomycete Streptomyces sp.D67 is: soluble starch 10.0 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 1.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 2.0 g / L, CaCl2 2.0 g / L, FeSO4·7H2O 0.001 g / L, MnSO4 0.001 g / L, ZnSO4·7H2O 0.001 g / L, agar 15.0 g / L, distilled water 1.0 L, pH 7.2.

[0015] The invention provides a method for preparing a crude extract of a new actinomycete Streptomyces sp.D67 with high-yield quorum sensing inhibitory activity. The preparation method specifically comprises the following steps: inoculating a single colony of Streptomyces sp.D67 into an ISP2 culture medium, culturing at 30°C and 150rpm for 2 days to obtain a Streptomyces sp.D67 seed liquid; inoculating the Streptomyces sp.D67 seed liquid into Gao's No. 1 liquid culture medium at an inoculation rate of 1% by volume, fermenting and culturing at 30°C and 150rpm for 7 days to obtain a Streptomyces sp.D67 fermentation liquid; centrifuging the Streptomyces sp.D67 fermentation liquid, taking a supernatant, extracting with an equal volume of ethyl acetate for 3h, taking an organic phase, evaporating to dryness in a vacuum rotary evaporator at 40°C, weighing, and obtaining a Streptomyces sp.D67 extract. sp.D67 crude extract; weigh an appropriate amount of the crude extract and dissolve it in dimethyl sulfoxide, filter it with a 0.22 μm sterile filter membrane to obtain the Streptomyces sp.D67 crude extract, and store it at -20°C for future use.

[0016] Furthermore, the present invention provides the use of a crude extract of a novel actinomycete Streptomyces sp. D67 with high-yield quorum sensing inhibitory activity in the preparation of quorum sensing inhibitory drugs.

[0017] By implementing the above specific technical solutions provided by the present invention to implement the content of the present invention, the following can be obtained:

[0018] Beneficial effects:

[0019] (1) The present invention provides a new actinomycete, Streptomyces sp. D67, which is identified as a new actinomycete belonging to the genus Streptomyces, and thus needs to be deposited in accordance with legal requirements.

[0020] (2) The crude metabolic extract obtained by fermentation extraction of the new actinomycete Streptomyces sp.D67 provided by the present invention was subjected to preliminary analysis and functional identification. The crude extract of Streptomyces sp.D67 has a strong quorum sensing inhibitory activity against Bacillus violaceus CV026, can significantly inhibit the clustering swimming characteristics of Pseudomonas aeruginosa, and has significant inhibitory activity against the production of Pseudomonas aeruginosa biofilm, pyocyanin and rhamnolipid, and is significantly concentration-dependent. When the concentration of the crude extract of Streptomyces sp.D67 is 5 mg / mL, the maximum inhibition rates can reach 47%, 40.9% and 27%, respectively.

[0021] (3) The TLC biodevelopment and antibacterial activity results of the crude extract of the new actinomycete Streptomyces sp. D67 provided by the present invention indicate that the crude extract contains quorum sensing inhibitory activity molecules and antibacterial compounds, and has good application prospects for the development and preparation of functional drugs and agricultural biological control, and can be widely used in the pharmaceutical and agricultural industries. Description of the drawings:

[0022] Figure 1 Shown is the phylogenetic tree of Streptomyces sp. D67 constructed based on 16S rRNA sequences.

[0023] Figure 2 Shown is the colony morphology of Streptomyces sp. D67.

[0024] Figure 3 The display shows the screening plate for quorum sensing inhibitory activity of Streptomyces sp. D67 fermentation broth.

[0025] Figure 4 Shown are the TLC chart of the crude extract of Streptomyces sp.D67 and the bio-development chart of the active ingredients.

[0026] Figure 5 Shown is the effect of different concentrations of crude extract of Streptomyces sp.D67 on the content of purple pigment produced by Bacillus violaceus CV026.

[0027] Figure 6 Shown is the effect of different concentrations of Streptomyces sp.D67 crude extract on Erwinia biofilm

[0028] Figure 7 Shown is the effect of 5 mg / mL of Streptomyces sp. D67 crude extract on the colonization and motility of Pseudomonas aeruginosa.

[0029] Figure 8 Shown is the effect of different concentrations of Streptomyces sp.D67 crude extract on the biofilm produced by Pseudomonas aeruginosa

[0030] Fig. 9 Shown is the effect of different concentrations of Streptomyces sp.D67 crude extract on the production of pyocyanin by Pseudomonas aeruginosa

[0031] Fig.10 Shown is the effect of different concentrations of Streptomyces sp.D67 crude extract on the production of rhamnolipids by Pseudomonas aeruginosa

[0032] Fig.11Shown is the effect of different concentrations of Streptomyces sp.D67 crude extract on protease production by Pseudomonas aeruginosa. Specific implementation method:

[0033] The present invention is described below with reference to the following examples, but the present invention is not limited to the following examples. All raw and auxiliary materials used in the present invention, as well as the selected bacterial strain culture methods are well known in the art, and the % involved in the present invention are all mass volume percentages, unless otherwise specified.

[0034] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

[0035] The Gao's No. 1 culture medium of the present invention comprises: 20 g / L soluble starch, 1.0 g / L KNO3, 0.5 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.01 g / L FeSO4·7H2O, 15 g / L agar, 1000 mL pure water, and pH 7.4-7.6.

[0036] The LB medium of the present invention (g / L): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH 7.0.

[0037] The cluster detection culture medium of the present invention includes: 10 g / L peptone, 5 g / L glucose, 5 g / L NaCl and 5 g / L agar.

[0038] The electrophoresis detection culture medium of the present invention comprises 10 g / L tryptone, 3 g / L NaCl and 5 g / L agar.

[0039] The protease detection culture of the present invention includes 20 g / L skim milk powder and 15 g / L agar.

[0040] Example 1 Isolation, purification and identification of strain Streptomyces sp. D67

[0041] (I) Screening, separation and purification

[0042] The plate dilution method was used to separate actinomycetes for the separation and purification of microorganisms. 5 g of soil sample from the Kumtag Desert in Xinjiang was placed in 45 mL of sterile water and incubated at 150 r / min and 30 °C for 12 h. Standard gradient dilution was used to dilute with sterile water to make 10 -1 -10 -3concentration dilution; take 100 μL of each concentration dilution, spread it on the separation culture medium plate, and place it in a constant temperature incubator at 30°C for culture; after one week, after colonies grow on the plate, pick a single colony on the plate for purification culture, and transfer the purified single colony to the slant of Gao's No. 1 culture medium for storage.

[0043] (II) 16S rRNA gene identification

[0044] 1. Extraction of PCR Template DNA

[0045] The purified Streptomyces sp. D67 was inoculated into Gao's No. 1 liquid culture medium and cultured at 37°C in a shaking incubator for 2 days. The bacteria were collected and the total genomic DNA was extracted using a DNA extraction kit.

[0046] 2. PCR Amplification

[0047] 16S rRNA gene sequence primers:

[0048] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0049] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0050] The total volume of the PCR reaction system was 25 μL, and the PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min 30 s, 30 cycles; and extension at 72°C for 7 min.

[0051] 3. Sequence Determination

[0052] The PCR amplification product was sequenced after electrophoresis detection and purification, and the sequence length was 1421 bp. The test results are shown in SEQ ID No: 1. BLAST homologous sequence search was performed on NCBI, and the phylogenetic tree was established by the Neighbor-Joining method using MEGA 7.0 software commonly used in the field (repeated sampling 1000 times). The results are shown in the attached Figure 1 The obtained sequences were compared and analyzed on the NCBI website and it was found that Streptomyces sp.D67 and Streptomyces ardesiacus NBRC15402 T , Streptomyces heliomyciniNBRC 15899 T and Streptomycespluripotens MUSC 135 TThe highest homology was 98.39%, 98.11% and 98.03% respectively. The sequences with higher homology were selected to construct the 16SrRNA phylogenetic tree. Streptomyces sp.D67 and Streptomyces pluripotens MUSC 135 T (CP021080) are more closely related and clustered in one branch. After a series of polyphasic taxonomic identifications, Streptomyces sp.D67 and Streptomyces ardesiacus NBRC 15402 T , Streptomyces heliomycini NBRC15899 T and Streptomyces pluripotens MUSC 135 T There are obvious differences, confirming that the obtained species numbered D67 within the genus Streptomyces is a typical new species.

[0053] Example 2: Physiological and biochemical assays of Streptomyces sp. D67

[0054] (I) Analysis of colony morphology characteristics

[0055] Streptomyces D67 can grow well on ISP2, Gao's No. 1, Bennett's agar and other media. Inoculate the Streptomyces D67 to be observed on Gao's No. 1 plate and culture it at 30℃ for 4 days. When the colony grows all over the plate, observe the characteristics of the colony, record it and take photos for preservation. At the same time, record the scanning electron microscope photos of the colony. The recording results are shown in the attached Figure 2 shown.

[0056] By the attached Figure 2 The results show that the Streptomyces D67 provided by the present invention is a Gram-positive bacterium with a colony size of 0.2cm-0.6cm×0.2cm-0.6cm. It is tightly bound to the culture medium and has a dense texture. The aerial hyphae are white and velvety, the spores are white, and the hyphae in the base are light yellow.

[0057] Based on the above biological characteristics, the above Streptomyces D67 was identified as a Streptomyces genus. The strain has been deposited in the Budapest Treaty International Microbiological Depository: China Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, postal code: 430072, deposit number: CCTCCNO: M 20221542, deposit date is October 7, 2022.

[0058] (II) Analysis of physiological and biochemical characteristics of strains

[0059] Streptomyces D67 was inoculated on Gao's medium No. 1, and the physiological and biochemical characteristics were detected. The growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen peroxide production, urease, amylase, esterase, cellulose decomposition, carbon source utilization and other characteristic analysis were mainly based on the "Common Bacteria System Identification Manual". Streptomyces D67 protease, amylase and cellulase activities were all positive; the sugar carbon sources that Streptomyces D67 can utilize are dextrin, xylose, turanose, sucrose, inositol, L-arabinose, D-arabitol, D-fructose, D-galactose, D-glucose and L-rhamnose. In the antibacterial test of pathogens, Streptomyces D67 showed a certain resistance to Escherichia coli, Pseudomonas aeruginosa, Erwinia and Staphylococcus aureus.

[0060] Comprehensive 16Sr RNA gene sequence homology comparison analysis, colony morphology identification, physiological and biochemical characteristics comparison and other aspects have obvious differences from the standard model strain, and it is determined that Streptomyces D67 is a new species of Streptomyces with the characteristics of a new species in the same genus Streptomyces.

[0061] Example 3: Preparation of Streptomyces sp. D67 crude extract

[0062] In this example, on the basis of Example 1-2, the crude extract of the strain Streptomyces sp.D67 is prepared as follows: a single colony of Streptomyces D67 is inoculated into ISP2 liquid culture medium, and cultured at 30°C and 150rpm for 2 days to obtain Streptomyces sp.D67 seed solution; the Streptomyces sp.D67 seed solution is inoculated into a 100mL Erlenmeyer flask containing 50mL Gao's No. 1 liquid culture medium at a volume ratio of 1%, and fermented and cultured at 30°C and 150r / min for 7 days to obtain D67 fermentation liquid, and the fermentation liquid is centrifuged at 1000r / min at room temperature for 8min, the supernatant is taken, and extracted with an equal volume of ethyl acetate for 3h, the organic phase is evaporated to dryness in a vacuum rotary evaporator at 40°C, and weighed to obtain a crude extract of Streptomyces sp.D67, the maximum yield of which can reach 540mg / L; an appropriate amount of Streptomyces sp.D67 is weighed. The crude extract of Streptomyces sp. D67 was dissolved in dimethyl sulfoxide and filtered with a 0.22 μm sterile filter membrane to obtain a crude extract of Streptomyces sp. D67, which was stored at -20°C for future use.

[0063] Example 4: Application of Streptomyces sp.D67 crude extract

[0064] Based on Examples 1-3, this Example provides the use of a crude extract of a new bacterium, Streptomyces p.D67, with high-yield quorum sensing inhibitory activity in the preparation of quorum sensing inhibitory drugs, and mainly examines its effects on the quorum sensing activity of Bacillus violaceus CV026, the clustering swimming characteristics of Pseudomonas aeruginosa, the biofilm activity of Pseudomonas aeruginosa, the activity of pyocyanin and rhamnolipid, etc.

[0065] (1) Effect of crude extract of Streptomyces sp. D67 on quorum sensing of Bacillus violaceus CV026

[0066] Take a loop of fresh bacterial moss of Bacillus sp. CV026 and inoculate it into LB medium, and culture it at 30℃, 150r / min constant temperature shaking for 16h; take 1mL of CV026 bacterial liquid and 20μL of 100μmol / mL C6-HSL and add them to the plate, then add 25mL of LB solid medium, mix them quickly at 50℃, and after it solidifies, use a 9mm diameter puncher to punch holes for later use; take 100μL Treptomyces sp. D67 crude extract and add it to the hole, and culture it at 30℃ for 24h, observe the color change of the plate, and record the strains with fuzzy transparent circles to preliminarily determine that the strain has quorum sensing inhibition activity. The statistical results are shown in the attached Figure 3 shown.

[0067] By the attached Figure 3 The results showed that crude extracts of Streptomyces sp. D67 at different dilution multiples could inhibit the production of purple pigment by Bacillus violaceus CV026 to varying degrees, preliminarily proving that the strain had an obvious quorum sensing inhibitory effect.

[0068] (2) Verification of quorum sensing activity of crude extract of Streptomyces sp. D67

[0069] This experiment was based on experiment (1). Thin layer chromatography (TLC)-bioautography was used. 5 μL of crude extract of Treptomyces sp. D67 was spotted on an aluminum thin layer plate. The developing solvent system was methanol-water with a volume ratio of 60:40. The plate was sealed to 0.5 cm above the top and dried with cold air. One of the strips was observed for fluorescent spots under an ultraviolet lamp with a wavelength of 254 nm and photographed. The other strip was placed in a plate. LB agar mixed with purple bacillus CV026 and C6-HSL was spread on the TLC plate. After solidification, it was incubated at 30°C for 36 h. The color change of the plate was observed and photographed to determine the location of the active substance. For specific results, see the attached Figure 4 shown.

[0070] By the attached Figure 4The results showed that an obvious dark spot appeared in the crude extract of Streptomyces sp.D67 under 245nm ultraviolet irradiation, with an Rf value of 0.77; through biological development, there was an obvious strain growth above the TLC plate, but the strain had no transparent circle of purple pigment accumulation, proving that the crude extract of Streptomyces sp.D67 contained strong quorum sensing inhibitory active ingredients.

[0071] (3) Effect of crude extract of Streptomyces sp. D67 on purple pigment production by Bacillus violaceus

[0072] Using the 96-well plate method, 100 μL of LB liquid culture medium (containing 0.1 μmol C6-HSL) was taken and placed in the wells, and the purple bacillus CV026 bacterial liquid was inoculated with a 2% inoculation amount, and the Streptomyces sp.D67 crude extracts with concentrations of 0.005 mg / mL, 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL and 2 mg / mL were added, and an equal amount of dimethyl sulfoxide was added as a control, and the plate was placed in an ELISA instrument and shaken at 30°C for 24 hours; 100 μL of the above crude extracts were respectively taken and placed in 1.5 mL centrifuge tubes, centrifuged at 10000 r / min for 5 minutes, the supernatant was discarded, 100 μL of DMSO was added, vortexed to fully dissolve the purple pigment of purple bacillus, centrifuged again, and the supernatant was taken, and its OD was measured by an ELISA instrument 600 The values ​​were used to determine the effect of the crude extract on the production of purple pigment by Bacillus violaceus. The results are shown in the attached Figure 5 shown.

[0073] By the attached Figure 5 The results showed that different concentrations of Streptomyces sp.D67 crude extract had a significant effect on the purple pigment production of Bacillus violaceus CV026. As the concentration of Streptomyces sp.D67 crude extract increased, the purple pigment content decreased significantly, indicating that the quorum sensing inhibitor in the Streptomyces sp.D67 crude extract could significantly inhibit the production of purple pigment of Bacillus violaceus CV026. Even a low concentration of 0.005 mg / mL of Streptomyces sp.D67 crude extract could still effectively reduce its purple pigment production, and the purple pigment production was 27% at this time, indicating that the Streptomyces sp.D67 crude extract had a strong quorum sensing inhibitor activity.

[0074] (4) Effect of Streptomyces sp. D67 crude extract on Erwinia biofilm

[0075] Using the 96-well plate method, 100 μL of LB liquid culture medium was taken into the wells, and the Erwinia bacterial solution was inoculated at a mass volume ratio of 2%. A certain amount of crude extract was added to the final concentrations of 0.06 mg / mL, 0.125 mg / mL, 0.2 mg / mL and 0.5 mg / mL, respectively. An equal amount of dimethyl sulfoxide was added as a control, and the plate was placed in an ELISA instrument and shaken and cultured at 30°C for 24 hours; the bacterial solution was discarded, and the biofilm was rinsed 3 times with distilled water to obtain the biofilm, which was naturally air-dried and stained with 1% crystal violet for 15 minutes, rinsed and dried, eluted with 95% ethanol, and the OD590 value was determined. The statistical results are shown in the attached Figure 6 shown.

[0076] By the attached Figure 6 The data show that compared with the control group, all concentrations of Streptomyces sp.D67 crude extract have a significant effect on the production of Erwinia biofilm. As the concentration of Streptomyces sp.D67 crude extract increases, its inhibitory effect on Erwinia biofilm gradually increases. When the concentration of Streptomyces sp.D67 crude extract is 0.5 mg / mL, its inhibitory effect on Erwinia biofilm is the most significant, and the inhibition rate can reach 37%.

[0077] (4) Effects of Streptomyces sp. D67 crude extract on the clustering and swimming properties of Pseudomonas aeruginosa

[0078] Prepare cluster agar medium and swimming agar medium plates containing 5 mg / mL of Streptomyces sp.D67 crude extract, spot-inoculate Pseudomonas aeruginosa, and culture them upright at 30°C for 24 hours to confirm the migration, clustering or swimming of the strains. Add an equal volume of dimethyl sulfoxide as a control. The statistical results are shown in the attached Figure 7 shown.

[0079] By the attached Figure 7 The data show that the clustering and swimming of Pseudomonas aeruginosa are obvious without the addition of crude extract. The addition of 5 mg / mL of Streptomyces sp. D67 crude extract can significantly inhibit the clustering and swimming of Pseudomonas aeruginosa.

[0080] (5) Effect of Streptomyces sp. D67 crude extract on Pseudomonas aeruginosa biofilm

[0081] Using the 96-well plate method, 100 μL of LB liquid culture medium was taken into the wells, and the Pseudomonas aeruginosa bacterial solution was inoculated with a 2% inoculation volume. A certain amount of Streptomyces sp. D67 crude extract was added to the final concentration of 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 5 mg / mL, and an equal amount of dimethyl sulfoxide was added as a control. The plates were placed in a microplate reader and shaken at 30°C for 24 hours. The bacterial solution was discarded, and the biofilm was washed three times with distilled water. After natural air drying, it was stained with 1% crystal violet for 15 minutes, rinsed and dried, eluted with 95% ethanol, and the OD590 value was measured to determine the effect of the Streptomyces sp. D67 crude extract on the Pseudomonas aeruginosa biofilm. For specific results, see the attached Figure 8 shown.

[0082] By the attached Figure 8 The data show that compared with the control group, all concentrations of Streptomyces sp.D67 crude extract have a significant effect on the production of Pseudomonas aeruginosa biofilm. As the concentration of Streptomyces sp.D67 crude extract increases, its inhibitory effect on the biofilm of Pseudomonas aeruginosa gradually increases. When the concentration of Streptomyces sp.D67 crude extract is 5 mg / mL, its inhibitory effect on the production of Pseudomonas aeruginosa biofilm is most significant, and the inhibition rate can reach 53%.

[0083] (6) Effect of Streptomyces sp. D67 crude extract on pyocyanin of Pseudomonas aeruginosa

[0084] In this experiment, based on experiment (5), 100 μL of bacterial solution was centrifuged at 8000 r / min for 5 min, 100 μL of chloroform was added to the supernatant to extract pyocyanin, and 100 μL of 0.2 mol / L HCl was used to back-extract pyocyanin in the chloroform. When the HCl layer solution changed from colorless to red to dark red, its OD was measured. 520 For specific results, see the attached Fig. 9 shown.

[0085] By the attached Fig. 9 The data show that within the experimental concentrations, the crude extract of Streptomyces sp. D67 showed a certain inhibitory effect on Pseudomonas aeruginosa pyocyanin, with an inhibition rate of 12.3%-40.9%. When the concentration of the crude extract of Streptomyces sp. D67 was 5 mg / mL, the inhibition rate was the most significant, reaching 40.9%.

[0086] (7) Effect of crude extract of Streptomyces sp. D67 on rhamnolipids of Pseudomonas aeruginosa

[0087] In this experiment, based on experiment (5), 100 μL of bacterial supernatant was extracted twice with ethyl acetate, evaporated to dryness at 40°C, dissolved with 100 μL of ultrapure water, and then color-developed with 300 μL of 2% anthrone-sulfuric acid solution. The reaction was carried out in boiling water for 15 min and cooled. A blank control group was set up at the same time. The experiment was repeated three times. The OD625 values ​​of the sample and the blank control group were measured to determine the effect of the crude extract of Streptomyces sp. D67 on rhamnolipids of Pseudomonas aeruginosa. For specific results, see the attached Fig.10 shown.

[0088] By the attached Fig.10 The data show that adding different concentrations of Streptomyces sp.D67 crude extract has different effects on the rhamnolipid production of Pseudomonas aeruginosa strains. When the concentration of Streptomyces sp.D67 crude extract is 5 mg / mL and 2 mg / mL, the inhibitory effect is most significant, with inhibition rates of 27% and 19%, respectively.

[0089] (8) Effect of crude extract of Streptomyces sp. D67 on total protease production of Pseudomonas aeruginosa

[0090] In this experiment, based on experiment (5), 100 μL of bacterial solution was centrifuged at 10,000 r for 8 min at room temperature. A 6 mm diameter hole was punched in the center of a skim milk plate culture dish and 50 μL of supernatant was added. The culture was carried out at 30°C for 8 h. The size of the transparent circle was observed and measured. For specific results, see the attached Fig.11 shown.

[0091] By the attached Fig.11 The data results show that when different concentrations of Streptomyces sp. D67 crude extract are added, the inhibition rate of Pseudomonas aeruginosa protease is about 6%, indicating that the Streptomyces sp. D67 crude extract has no obvious inhibitory effect on the total protease production of Pseudomonas aeruginosa.

[0092] From the above data, it can be seen that the crude extract obtained by fermentation extraction of the new bacterium Streptomyces sp.D67 provided by the present invention was subjected to preliminary analysis and functional identification tests. The crude extract of Streptomyces sp.D67 has a strong quorum sensing inhibitory activity against purple bacillus CV026, can significantly inhibit the clustering swimming characteristics of Pseudomonas aeruginosa, and has significant inhibitory activity on the production of Pseudomonas aeruginosa biofilm, pyocyanin and rhamnolipid, and is significantly concentration-dependent, with the highest inhibition rates reaching 47%, 40.9% and 27%, respectively; the TLC biological development and antibacterial activity results of the crude extract of Streptomyces sp.D67 show that the crude extract contains a variety of quorum sensing inhibitory active molecules and antibacterial compounds, laying a solid foundation for the application of quorum sensing inhibitors.

[0093] In summary, the above embodiments are merely descriptions of the preferred implementation methods of this test, and do not limit the scope of this test. Without departing from the spirit of the design of this test, various modifications and improvements made to the technical solutions of this test by ordinary technicians in this field should fall within the protection scope determined by this test.

Claims

1. A new strain of Streptomyces spp. D67 with high quorum sensing inhibitory activity, characterized in that: The strain Streptomyces cess p.D67 belongs to the genus Streptomyces, and the strain is preserved in the China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCCNO: M20221542.

2. A method for preparing a crude extract of the novel bacterium Streptomyces p.D67 with high quorum sensing inhibitory activity as claimed in claim 1, characterized in that: A single colony of Streptomyces p.D67 was inoculated into ISP2 medium, and cultured at 30°C and 150 rpm for 2 days to obtain Streptomyces p.D67 seed solution; the Streptomyces p.D67 seed solution was inoculated into Gao's No. 1 liquid medium at a volume ratio of 1%, and fermented and cultured at 30°C and 150 rpm for 7 days to obtain Streptomyces p.D67 fermentation liquid; the Streptomyces p.D67 fermentation liquid was centrifuged, the supernatant was taken, and an equal volume of ethyl acetate was extracted for 3 hours, the organic phase was evaporated to dryness in a vacuum rotary evaporator at 40°C, and weighed to obtain a crude extract of Streptomyces p.D67; an appropriate amount of the crude extract was weighed and dissolved in dimethyl sulfoxide, and filtered with a 0.22 μm sterile filter membrane to obtain a crude extract of Streptomyces p.D67, which was stored at -20°C for later use.

3. Use of a crude extract of a novel bacterium Streptomyces p.D67 with high quorum sensing inhibitory activity as claimed in claim 1 in the preparation of inhibitory drugs for Pseudomonas aeruginosa, Bacillus violaceus and Erwinia.

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