An actinomycete strain, Nonomuraea jiliniensis DL99, and its applications

Abstract

The present invention discloses an actinomycete strain Jilin Nonomura DL99 and its application, named Nonomuraea Jilinensis The DL99 strain has significant antibacterial properties when used as an antibacterial agent. It has good application prospects in biological pesticides, microbial preparations and other antibacterial-related fields, providing a new resource for the development and production of new antibacterial drugs. It can also be used for antibacterial purposes in the food, medicine and feed industries.

Description

Technical Field

[0001] The present invention discloses an actinomycete strain Jilin Yenomura DL99 and its application, which is a new strain named: Nonomuraea Jilinensis DL99 can be used to prepare antibacterial agents in medicine and agriculture, and relates to the technical field of microbial strains and their applications. Background Art

[0002] Of the over 2,000 antibiotics currently discovered worldwide, approximately 56% are produced by actinomycetes (primarily Streptomyces). Examples include streptomycin, oxytetracycline, tetracycline, and gentamicin. Furthermore, some agricultural bacteriostatics and vitamins for plants are extracted and purified from actinomycetes. These antibiotics are widely used in human society. Some species of actinomycetes also produce enzymes (such as proteases, amylases, and cellulases), vitamins, and organic acids. Furthermore, actinomycetes are used in steroid conversion, hydrocarbon fermentation, petroleum dewaxing, and wastewater treatment. However, a small number of actinomycetes can pose a threat to humans, causing diseases in humans, animals, and plants. Therefore, actinomycetes have a close relationship with humans and are of great significance in medicine and agriculture.

[0003] Actinomycete strains are commonly found in terrestrial soils but have also been isolated from marine sediments and marine sponges. These actinomycete metabolites have high structural diversity and a wide range of activities and are often used to produce antibacterial agents, antiprotozoal drugs, antiparasitic drugs, antitumor drugs and antimalarial drugs. Summary of the Invention

[0004] The present invention discloses an actinomycete strain Jilin Nonomura DL99 ( Nonomuraea Jilinensis DL99, a new strain, exhibits significant antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes. It can be used to prepare microbial antibacterial agents for agricultural or medical use, providing a new resource for the research of antibacterial drugs.

[0005] The present invention discloses an actinomycete strain Jilin Nonomura DL99, which was deposited in China Center for Type Culture Collection on August 10, 2023, and the deposit location is Wuhan University, Wuhan, China, and is named Nonomuraea Jilinensis DL99, the strain deposit number is CCTCCNO.M20231421.

[0006] The present invention describes a Nonomuraea JilinensisThe application of DL99 in the preparation of an antibacterial agent, wherein the antibacterial spectrum of the antibacterial agent includes methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes.

[0007] The present invention describes a Nonomuraea Jilinensis DL99 is used in the preparation of an antibacterial agent in the pharmaceutical industry. The antibacterial spectrum of the antibacterial agent includes methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes.

[0008] The present invention describes a Nonomuraea Jilinensis The method for preparing DL99 antibacterial agent is characterized by:

[0009] Will Nonomuraea Jilinensis A single colony of DL99 was inoculated into IPS2 liquid medium and cultured at 37°C and 200 rpm for 3 days to obtain Nonomuraea Jilinensis DL99 seed solution; Nonomuraea Jilinensis DL99 seed solution was inoculated into the fermentation medium at a volume ratio of 5%, and fermented at 37°C and 200 rpm for 7 days to obtain Nonomuraea Jilinensis DL99 fermentation broth, after centrifugation and filtration sterilization, the Nonomuraea Jilinensis DL99 antibacterial agent, stored at -20℃ for future use.

[0010] The present invention adopts Nonomuraea Jilinensis DL99 The IM fermentation medium was as follows: glucose 20 g / L, peptone 10 g / L, yeast extract 10 g / L, NaCl 2 g / L, CaCO3 5 g / L, distilled water 1 L, pH = 7.2.

[0011] The present invention Nonomuraea Jilinensis The DL99 strain is Gram-positive and can grow at temperatures between 10 and 40°C, with an optimum temperature of 28 to 37°C. It forms single, round, pale yellow colonies with a diameter of 1 to 2 mm on ISP2 agar plates, and a wrinkled surface. The (G+C) mol% content of its genome is 71.64%.

[0012] The deposited strains used in the present invention Nonomuraea Jilinensis DL99 was characterized by 16S rRNA gene sequence phylogenetic analysis and morphological analysis. Nonomuraea Jilinensis DL99 belongs to the genus Nonomura, Nonomuraea Jilinensis The 16S rRNA gene sequence of DL99 is shown as SEQ. NO 1.

[0013] The strain gene was sequenced and the obtained sequence was compared with the BLAST analysis on the NCBI website. The phylogenetic tree constructed by the software MEGA11 using the maximum likelihood (ML) and neighbor-joining (NJ) methods showed that Nonomuraea Jilinensis The 16S rRNA gene sequence of DL99 is similar to that of the closest relative strain of the genus Nonomuraea fastidiosa 104 (NR025995.1), Nonomuraea coxensis JCM 13931 (NR 112924.1) and Nonomuraea wenchangensis 210417 (NR 116598.1) has 98.17%, 97.73% and 97.31% similarities. The genomic relatedness index showed that the average nucleotide similarity and DNA-DNA hybridization values between DL99 and other species of Nonomura were between 76.42% and 87.48% and 20.23% and 30%, respectively, which are far below the ANI 95% and DDH 70% thresholds for identifying new bacterial species. After a series of polyphasic classification identifications, Nonomuraea Jilinensis DL99 and Nonomuraea fastidiosa 104 There are obvious differences, it can be determined Nonomuraea Jilinensis DL99 is a new species of the genus Nonomura, and has the typical characteristics of a new species.

[0014] The present invention Nonomuraea Jilinensis The DL99 strain can grow in a pH environment of 4.0-10.0, with the most suitable pH for growth being 7.0-8.0; the highest NaCl concentration (%w / v) it can tolerate is 5%. Hydrogen sulfide production, gelatin liquefaction test, cellulose hydrolysis, and milk peptone test were negative. Urea production, ONPG beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylation, and citrate utilization were positive. It can utilize glucose but not sucrose. Analysis of the strain's physiological and biochemical characteristics revealed that it is similar to neighboring strains. Nonomuraea fastidiosa 104 There are obvious differences.

[0015] The present invention Nonomuraea Jilinensis The DL99 strain exhibits antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes. The strain exhibits excellent antibacterial properties against the aforementioned bacteria, with inhibition zone diameters reaching between 10 and 20 mm.

[0016] A new strain was disclosed, named Nonomuraea JilinensisThe DL99 strain has significant antibacterial properties when used as an antibacterial agent. It has good application prospects in biological pesticides, microbial preparations and other antibacterial related fields, providing a new resource for the development and production of new antibacterial drugs, and can also be used for antibacterial purposes in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This invention Nonomuraea Jilinensis Colony morphology of DL99 strain (A is ISP1 medium, B is ISP2 medium, C is ISP3 medium, D is ISP4 medium, E is ISP5 medium, F is ISP6 medium, and G is ISP7 medium);

[0018] Figure 2 This invention Nonomuraea Jilinensis Gram staining of strain DL99;

[0019] Figure 3 This invention Nonomuraea Jilinensis Phylogenetic tree of DL99 strain based on 16S rRNA sequence (A is the maximum likelihood (ML) method, B is the neighbor-joining (NJ) method);

[0020] Figure 4 yes Nonomuraea Jilinensis Effects of DL99 antibacterial agent on methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes. DETAILED DESCRIPTION

[0021] 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 bacterial culture methods used, are well known in the art, and all percentages mentioned in the present invention are by weight and volume, unless otherwise specified.

[0022] In order to better explain the present invention, the main content of the present invention is further illustrated below in conjunction with specific examples, but the content of the present invention is 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 products.

[0023] The ISP2 culture medium of the present invention comprises: 4 g / L yeast extract, 10 g / L malt extract, 4 g / L glucose, 20 g / L agar powder, 1 L distilled water, and a pH of 7.2. Example 1

[0024] Collected fecal samples were placed in sterile culture dishes, mixed and diluted with 10 mL of PBS, dried, and incubated at 80°C for 1 hour. The suspension, 10-fold dilution, and 100-fold dilution of the fecal suspension were plated and incubated aerobically and anaerobically. The suspension was placed in an incubator at 37°C and inverted for culture, with observations made every few days. Based on colony morphology, size, color, and stage of incubation, individual colonies were selected and purified onto isolation medium. Selected colonies were labeled, inoculated into appropriate liquid culture medium, and frozen after growth to the logarithmic phase. Spores or cells of pure cultured strains were scraped and stored in 50% glycerol at -80°C. Simultaneously, individual colonies were purified a second time and a third time, and the strains were preserved and sequenced for identification.

[0025] The strain was deposited in the China Center for Type Culture Collection on August 10, 2023, at Wuhan University, Wuhan, China, and named Nonomuraea Jilinensis DL99, the strain deposit number is CCTCCNO.M20231421. Example 2

[0026] Nonomuraea Jilinensis Preparation of DL99 antibacterial agent

[0027] The new strain of actinomycetes Nonomuraea Jilinensis A single colony of DL99 was inoculated into ISP2 liquid medium and cultured at 37°C and 200 rpm for 3 days. Nonomuraea Jilinensis DL99 seed solution; Nonomuraea Jilinensis DL99 seed solution was inoculated into IM liquid culture medium at a volume ratio of 1%, and fermented at 37°C and 200 rpm for 7 days to obtain Nonomuraea Jilinensis DL99 fermentation broth, after centrifugation and filtration sterilization, the Nonomuraea Jilinensis DL99 antibacterial agent, stored at -20℃ for future use.

[0028] Experimental Example 1

[0029] Nonomuraea Jilinensis Morphological and physiological and biochemical identification of DL99 strain

[0030] 1. Nonomuraea Jilinensis Colony morphology characteristics of DL99

[0031] Jilin Nonomura DL99 can grow well on the International Streptomyces Plan Medium ISP1-7 agar medium. Inoculate the Jilin Nonomura DL99 to be observed on the International Streptomyces Plan Medium ISP1-7 agar medium and culture at 37℃ for 7 days. When the colony has grown all over the plate, observe the characteristics of the colony, record it, and take photos for preservation. The recording results are shown in the attached Figure 1 shown.

[0032] By the attached Figure 1 The results show that the Jilin Nonomura DL99 provided by the present invention is a Gram-positive bacterium with a colony diameter of 1-2 mm. It is tightly bound to the culture medium, has a dense texture, is round, and presents different single colony morphologies on different culture media.

[0033] 2. Nonomuraea Jilinensis Gram staining of DL99

[0034] The activated Jilin Nonomura DL99 strain was inoculated into ISP2 liquid medium and incubated at 37°C and 200 rpm for 3 days. A small amount of bacterial suspension was picked up with an inoculating loop near an alcohol burner and evenly spread onto a glass slide. Heat was then applied over the burner to kill the bacteria and fix them. The slide was then stained with ammonium oxalate crystal violet for 1 minute, rinsed with distilled water to remove any excess stain. Mordanted with iodine-potassium iodide solution for 1 minute, and the excess was discarded. Decolorization with 95% ethanol (a neutral decolorant) for 30 seconds resulted in Gram-positive bacteria remaining purple, while Gram-negative bacteria were colorless. The slide was then counterstained with safranin solution for 30 seconds; Gram-positive bacteria remained purple, while Gram-negative bacteria appeared red.

[0035] 3. Nonomuraea Jilinensis Physiological and biochemical characteristics of DL99

[0036] Jilin Nonomura DL99 was inoculated on ISP2 medium and its physiological and biochemical characteristics were tested. The growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen sulfide production, milk peptone, urea, ONPG beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylation, citrate utilization, and cellulose decomposition were analyzed with reference to the "Common Bacterial System Identification Manual". Jilin Nonomura DL99 tested positive for urea production, ONPG beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylation, and citrate utilization. Lin Nonomura DL99 can utilize glucose but not sucrose. The identification results are shown in Table 1 below:

[0037]

[0038] Experimental Example 2

[0039] Nonomuraea Jilinensis Molecular Biological Studies on Strain DL99

[0040] (I) Extraction of PCR template DNA

[0041] The purified Nonomuraea Jilinensis DL99 was inoculated into ISP2 liquid medium and cultured at 37°C in a shaking incubator for 7 days. The bacteria were collected and the total genomic DNA was extracted using the CTAB method.

[0042] 1. Collect 1 mL of bacterial suspension in the logarithmic growth phase, centrifuge at 12000 r / min for 1 min, collect the bacteria, add TE buffer, gently wash, centrifuge at 4000 r / min for 10 min, discard the supernatant, and add 400 μL of TE buffer to resuspend.

[0043] 2. Add 20 μL of 50 mg / mL lysozyme to the suspension, mix well, and incubate in a 37°C water bath overnight to remove the cell walls.

[0044] 3. Add 70 μL of 10% SDS and 10 μL of 20 mg / mL proteinase K, mix gently, and place in a 68°C water bath for 30 min to remove protein.

[0045] 4. Add 100 μL of 5 mol / L sodium chloride and 100 μL of 10% CTAB preheated at 68°C, mix well, and place in a 68°C water bath for 15 min.

[0046] 5. Add an equal volume of chloroform / isoamyl alcohol (24:1), mix well, centrifuge at 13,000 rpm for 5 min, and transfer the supernatant to a new EP tube.

[0047] 6. Add an equal volume of DNA extraction buffer (phenol / chloroform / isoamyl alcohol = 25:24:1) to the supernatant, mix well, and centrifuge at 13,000 rpm for 5 minutes. Keep the supernatant.

[0048] 7. Add 0.6-0.8 times the volume of isopropanol to the supernatant, mix thoroughly, and place at -20°C for at least 2 hours. Centrifuge at 13,000 rpm for 15 minutes to collect DNA.

[0049] 8. Wash the DNA with 1 mL of 70% ethanol and centrifuge at 12,000 rpm / min for 5 minutes. Repeat twice.

[0050] 9. After the EP tube is inverted and dried, resuspend it with pure water and store it at -20℃.

[0051] (2) PCR amplification

[0052] 16S rRNA gene sequence primers:

[0053] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0054] 1492R: 5'-AAGGAGGTGATCCAGCCGCA-3';

[0055] The total volume of the PCR reaction system was 50 μL, and the PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min 30 s, for 30 cycles; and extension at 72°C for 7 min.

[0056] (III) Sequence determination

[0057] The PCR amplification product was detected by electrophoresis and purified before sequencing. The sequence length was 1517 bp. The test results are shown in the attached sequence table. BLAST homologous sequence search was performed on NCBI. The phylogenetic tree was established using the maximum likelihood (ML) and neighbor-joining (NJ) methods using MEGA11 software commonly used in this field (repeated sampling 1000 times). The results are shown in the attached Figure 3 As shown, the obtained sequence was compared and analyzed on the NCBI website and found that Nonomuraea Jilinensis The 16S rRNA gene sequence of DL99 is similar to that of the closest relative strain of this genus and fungus Nonomuraea fastidiosa 104 (NR 025995.1), Nonomuraea coxensis JCM 13931 (NR 112924.1) and Nonomuraea wenchangensis 210417 (NR 116598.1) has 98.17%, 97.73% and 97.31% similarity. The genomic relatedness index is shown in Table 2 below. The average nucleotide similarity and DNA-DNA hybridization values between DL99 and other species of Nonomura are between 76.42%-87.48% and 20.23-30%, respectively, which are far below the ANI 95% and DDH 70% thresholds for identifying new bacterial species. After a series of polyphasic taxonomic identification, Nonomuraea Jilinensis DL99 and Nonomuraea fastidiosa 104 There are obvious differences, it can be determined Nonomuraea Jilinensis DL99 is a new species of the genus Nonomura, and has the typical characteristics of a new species.

[0058] Table 2.

[0059]

[0060] Comprehensive 16SrRNA gene sequence homology analysis, morphological identification, physiological and biochemical characteristics comparison, etc. show Nonomuraea Jilinensis DL99 is obviously different from the standard model strain, and it is determined that Jilin Nonomura DL99 is a new species of the genus Nonomura, which has the characteristics of a new species within the same genus Nonomura.

[0061] Based on the above biological characteristics, the above-mentioned Jilin Nonomura DL99 was identified as a new strain of the genus Nonomura.

[0062] Nonomuraea Jilinensis Antibacterial activity of DL99 antibacterial agent

[0063] Methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes were cultured in 5 mL LB liquid medium test tubes at 37°C until the logarithmic growth phase; the indicator bacteria were diluted to 10 6 CFU / mL, then draw 100 μL and evenly spread it on LB solid medium, put it into an Oxford cup with an outer diameter of 8 mm, and Nonomuraea Jilinensis DL99 antibacterial agent was added to the cup and cultured at 37℃ for 18 h. Its antibacterial diameter was calculated to determine its antibacterial activity. For specific results, see the attached Figure 4 shown.

[0064] By the attached Figure 4 As a result, the present invention provides Nonomuraea Jilinensis The DL99 antibacterial agent has a significant inhibitory effect on methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes. Among them, the inhibitory effect on Listeria monocytogenes is stronger, with an inhibition diameter of 20 mm. The inhibition diameters of methicillin-resistant Staphylococcus aureus are 13.51 mm, the inhibition diameters of vancomycin-resistant Enterococcus faecium are 16.3 mm, the inhibition diameters of Staphylococcus aureus are 14.55 mm, the inhibition diameters of Bacillus subtilis are 10.23 mm, and the inhibition diameters of Salmonella typhimurium are 11 mm. This shows that the antibacterial agent provided by the present invention has a significant inhibitory effect on methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium are 16.3 mm, the inhibition diameters of Staphylococcus aureus are 14.55 mm, the inhibition diameters of Bacillus subtilis are 10.23 mm, and the inhibition diameters of Salmonella typhimurium are 11 mm. Nonomuraea Jilinensis DL99 antibacterial agent has a wide range of antibacterial activity. Its application as an antibacterial agent has excellent application value and has good development and application prospects in biopesticides, microbial preparations and other antibacterial related fields.

[0065] In summary, the above embodiments are merely descriptions of 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 scope of protection determined by this test.

Claims

1. An actinomycete strain Jilin Nonomura DL99 was deposited in the China Center for Type Culture Collection on August 10, 2023, at Wuhan University, Wuhan, China, and named Nonomuraea Jilinensis DL99, the strain deposit number is CCTCCNO.M20231421.

2. The bacterial strain according to claim 1 Nonomuraea Jilinensis The use of DL99 in the preparation of an antibacterial agent, wherein the antibacterial spectrum of the antibacterial agent includes: methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes.

3. The bacterial strain according to claim 1 Nonomuraea Jilinensis DL99 is used in the preparation of antibacterial agents in the pharmaceutical industry. The antibacterial spectrum of the antibacterial agent includes: methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes.

4. The bacterial strain according to claim 1 Nonomuraea Jilinensis DL99 is a method for preparing an antibacterial agent, characterized in that: Will Nonomuraea Jilinensis A single colony of DL99 was inoculated into IPS2 liquid medium and cultured at 37°C and 200 rpm for 3 days to obtain Nonomuraea Jilinensis DL99 seed solution; Nonomuraea Jilinensis DL99 seed solution was inoculated into the fermentation medium at a volume ratio of 5%, and fermented at 37°C and 200 rpm for 7 days to obtain Nonomuraea Jilinensis DL99 fermentation broth is centrifuged and filtered for sterilization to obtain the fermentation broth; the antibacterial agent has an antibacterial spectrum including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes.

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